CERTIFICATE

This is to certify that Ms. Anita has worked for her dissertation entitled

"Investigations on the proteases in Thermoactinomyces vulgaris". This

dissertation being submitted to the University of Delhi for the award of the

Degree of Master of Philosophy in Botany, is an original record of the work

done by the candidate herself and has not been submitted in part or full to

this or any other university for any other degree or diploma.

October 2010 Anita

(Candidate)

Prof. I. Usha Rao Prof. Ved Pal Singh

(Head of the Department) (Supervisor)

ACKNOWLEDGEMENTS

I would like to show my heartfelt gratitude to my supervisor Prof. Ved Pal Singh for the

innumerable help, valuable suggestions and meaningful advices given by him. The freedom

to design and conduct experiments and his multidimensional attitude has taught me a lot.

He has always been very cooperative, be it selecting the research problem, purchasing the

chemicals, troubleshooting the numerous problems, checking this thesis, interpretation of

results.

I would like to sincerely thank Prof. I. Usha Rao, Head, Department of Botany for providing

me the necessary facilities in the Department.

My sincere thanks are also for Mr. S. K. Das for photography. I would also like to thank

Mr. Raheja, Mrs. Neena and Mr. Satish for their assistance in the Central Instrumentation

Facility and other Laboratory staff for their kind and timely cooperation.

I am thankful to the University of Delhi for the financial assistance provided during my M.

Phil. course.

I would like to thank my seniors Dr. Nellie Laisram, Ms. Archana, Mr. Rajesh and Dr.

Vivek Kumar Kedia for their full support and cooperation in the laboratory.

My friends and colleagues, Meenakshi, Alok, Anita, Mahalakshmi, Rupam, Sashi, Sachin,

Dr. Kuldeep Sharma and Yamal, all deserves my sincere gratitude for their full support,

motivation and the healthy and conducive atmosphere. I am thankful to Sameer for his

assistance in the laboratory.

Last but not the least, I wish to extend my thanks to my husband, Dr. Sanjay, my cute

daughter, Bhavya and my lovely son, Labhansh for their patience and all kinds of adjustments,

motivation and support.

ii

CONTENTS

1 INTRODUCTION 1

2 INTRODUCTION TO THERMOACTINOMYCES VULGARIS AND PROTEASES 2

2.1 Taxonomy of Thermoactinomyces vulgaris . . . . . . . . . . . . . . . . . . . 3

2.2 Importance of Thermoactinomyces vulgaris . . . . . . . . . . . . . . . . . . 4

2.3 Introduction to Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.4 Different Sources of Proteases and Significance of Microbial Proteases . . . 7

2.5 Classification of Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.6 Mechanism of Catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.7 Thermophilic Proteases and Thermitase . . . . . . . . . . . . . . . . . . . . . 22

2.8 Industrial Applications of Microbial Proteases . . . . . . . . . . . . . . . . . 26

2.9 General Consideration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3 MATERIALS AND METHODS 37

3.1 Microorganism Used and Culture Conditions . . . . . . . . . . . . . . . . . . 37

3.2 Assay for Protease Activity in T. vulgaris Strains . . . . . . . . . . . . . . . 38

3.3 Screening of Thermoactinomyces vulgaris (1227) for the Production of

Extracellular Protease with respect to Substrate Utilization . . . . . . . . . . 39

3.4 Effect of Incubation Period on the Production of Extracellular Protease . . . 40

3.5 Effect of pH on the Production of Extracellular Protease . . . . . . . . . . . 40

3.6 Effect of Temperature on the Production of Extracellular Protease . . . . . . 40

3.7 Effect of Carbon Sources on the Production of Extracellular Protease . . . . 40

3.8 Effect of Metal Ions on the Production of Extracellular Protease . . . . . . . 40

3.9 Comparison of Static and Shake Culture Conditions for the Production of

Protease in Five Strains of T. vulgaris . . . . . . . . . . . . . . . . . . . . . 40

i

4 RESULTS AND DISCUSSION 41

4.1 Screening of T. vulgaris (strain 1227) for the Production of Extracellular

Protease with respect to Substrate Utilization . . . . . . . . . . . . . . . . . 41

4.2 Standard Curve of Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

4.3 Effect of Incubation Period on the Production of Extracellular Protease . . . 42

4.4 Effect of pH on the Production of Extracellular Protease . . . . . . . . . . . 44

4.5 Effect of Temperature on the Production of Extracellular Protease . . . . . . 45

4.6 Effect of Carbon Sources on the Production of Extracellular Protease . . . . 46

4.7 Effect of Metal Ions on the Production of Extracellular Protease . . . . . . . 48

4.8 Comparison of Static and Shake Culture Conditions for the Production of

Protease in Different Strains of T. vulgaris . . . . . . . . . . . . . . . . . . . 49

5 CONCLUDING REMARKS 51

REFERENCES 53

ii

INTRODUCTION

The genus Thermoactinomyces and its type species Thermoactinomyces vulgaris was first

described in 1899 by Tsiklinsky (Tsiklinsky, 1899). T. vulgaris, a thermophilic, spore

forming bacterium has been well characterized in our research laboratory with respect to its

morphology (Singh, 1997), growth and metabolism (Singh, 1984; Singh and Sinha, 1982a),

genetics (Bhatnagar and Singh, 2003; Singh, 1987; Singh et al., 2007) and enzymology

(Bhatnagar and Singh, 2004; Singh, 2007; Singh and Sinha, 1982b; Sinha and Singh, 1980;

Sinha et al., 1981).

Each microbe has its own special conditions for the maximum enzyme production (Kumar

and Takagi, 1999). The general rules for the optimization of microbial protease production

are affected by various physical factors, which include pH, cultivation temperature, shaking

condition, nutrients, incubation period and aeration. These factors are important to promote

the production of proteases (Rahman et al., 2005).

Although extensive work has been reported on the proteases and other extracellular enzymes

of thermophiles, the research work carried out and reported on production of extracellular

proteases in T. vulgaris (wild-type 1227 and its biochemical mutant strains 1261, 1278, 1279

and 1286) is rather very limited. This situation provided the necessary motivation to explore

the issue thoroughly and undertake the present investigation with a view to establish optimal

conditions for the production of extracellular protease from these strains. The objectives of

the present investigation are:

1. To screen the wild-type strain (1227) of Thermoactinomyces vulgaris for the production

of extracellular protease with respect to substrate utilization.

2. To optimise the culture conditions for production of extracellular protease in T. vulgaris

(1227) in terms of incubation period, pH and temperature.

3. To study the effect of various carbon sources on the production of protease in T.

vulgaris (1227).

4. To study the effect of various metal ions on the production of proteases in T. vulgaris

(1227).

5. To compare the production of extracellular protease in wild-type (1227) and the mutant

strains (i.e. 1261, 1278, 1279, 1286) of T. vulgaris under static and shake culture

conditions.

1

INTRODUCTION TO THERMOACTINOMYCES VULGARIS AND

PROTEASES

The genus Thermoactinomyces was proposed by Tsiklinsky (1899) with the single species

Thermoactinomyces vulgaris. Other Thermoactinomyces species, which have been described

are: T. sacchari (Lacey, 1971), T. peptonophilus (Nonomura and Ohara, 1971), T. candidus

(Kurup et al., 1975), T. intermedius (Kurup et al., 1980), T. thalpophilus (Lacey and Cross,

1989) and T. putidus (Lacey and Cross, 1989). T. dichotomicus was proposed for ‘Actinobifida

dichotomica’ (Krasil’nikov and Agre, 1964; Cross and Goodfellow, 1973). More recently, T.

candidus was reclassified as a synonym of T. vulgaris, while T. thalpophilus as a synonym of T.

sacchari on the basis of DNA-DNA relatedness data (Yoon et al., 2000). Thermoactinomyces

species were long recognized as actinomycetes because of their morphological characteristics,

forming aerial and substrate mycelia. On the basis of endospore formation, DNA G+C

content and phylogenetic data, the genus Thermoactinomyces has now been placed within

the family Bacillaceae, not the order Actinomycetales (Lacey and Cross, 1989; Yoon and

Park, 2000). Thermoactinomyces species are aerobic, gram positive and thermophilic, with

the exception of one mesophilic species, T. peptonophilis (Nonomura and Ohara, 1971).

The genus Thermoactinomyces is defined and characterized by the presence of an aerial

mycelium and by the formation of single spores on both aerial and vegetative mycelia

(Henssen, 1957; Tsiklinsky, 1899; Waksman and Corke, 1953). Globose and often ridged

spores of T. vulgaris grows in size upto 0.5-1.2 micron at its optimum growth temperature

of 50◦C. Its cell wall thickness is about 22 nm, while plasma membrane thickness is around

10 nm.

Inhalation of the spores can produce pulmonary fibrosis, which can have sudden symptoms

and progress rapidly to death. It is also known as Farmer’s Lung Disease, especially when

it results from exposure to moldy hay. In urban areas, mold growth on air conditioners has

been tied to the disease.

Usaite et al. (1980) studied methods of storage (as soil cultures, in peptone corn agar, and

in the freeze-dried state) of the thermotolerant actinomycete T. vulgaris RA II-4, a protease

producer. The best method of storage with high level of proteolytic activity was found to be

freeze-drying. The optimal temperature for the storage of the freeze-dried culture was 4◦C.

The physiological behaviour of T. vulgaris was analysed by Kretschmer et al. (1982), during

2

fermentation. The consumption of nutrients and oxygen as well as the rates of macromolecular

and protease synthesis, during 38 h were also measured by them. The morphological and

ultrastructural studies of the mycelia showed that the mycelia grew exponentially for about

5 h. After a short lag and a second slower growth phase, growth continued about linearly

until the end, as was indicated by a constant rate of incorporation of labelled thymidine.

However, a portion of hyphae, up to 45%, underwent lysis. According to the changing ratio

of growing and lysing material, the formation of the protease started at the transition to the

slow growth phase and continued linearly. The ability to produce protease was attributed to

a mycelium being formed after the shift down caused by limitation of supply of utilizable

nitrogen compounds. The arrest of protease production after 10 h was correlated to a drastic

decrease of the respiratory activity of the mycelia, probably caused by exhaustion of easily

utilizable carbohydrates.

2.1 Taxonomy of Thermoactinomyces vulgaris

Thermoactinomyces is a group of typically thermophilic bacterium, found in composts, soil and

heated environments. The microbe forms substratum and aerial mycelium with spores which

is a fungus-like feature and favors the placement of this genus in actinomycetes. However, the

resistance patterns of endospores and 16S rRNA oligonucleotide sequence data indicates its

placement in bacteria (Stackebrandt and Woese, 1981). Further, Thermoctinomyces vulgaris

is phylogenetically related to the genus Bacillus, based on 5S rRNA sequences (Park et al.,

1993).

The wall composition and the chemicals present in endospores like menaquinones (Collins et

al., 1982; Tseng et al., 1990) and dipicolinic acid (Cross and Johnson, 1971; Cross et al., 1968;

Lacey and Vinci, 1971) alongwith low mol % G+C content (Lacey and Cross, 1989) suggest

that the genus Thermoactinomyces should be placed in the family Bacillaceae, irrespective

of the actinomycete-like morphology (Goodfellow and Cross, 1984). The developmental

stages of sporulation in T. vulgaris like formation of spore protoplast, cortex formation, coat

formation and growth within the sporangium appears to be similar to those reported for

Bacillaceae (Ensign, 1978; Kalakoutskii et al., 1969; Becker et al., 1965).

Recently, phylogenetic inference, basedon16SrDNAsequence, showedthatThermoactinomyces

species is related to the family Bacillaceae (Yoon and Park, 2000). Thus, the accepted

taxonomic position of the cellular organism, T. vulgaris can be described as follows:

3

Kingdom: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Thermoactinomycetaceae

Genus: Thermoactinomyces

Species: vulgaris

Specific descriptor: vulgaris-Tsiklinsky 1899 (Approved Lists 1980)

Scientific name: Thermoactinomyces vulgaris Tsiklinsky 1899 (Approved Lists 1980)

Other members (species) of the genus Thermoactinomyces are:

T. candidus, T. glaucus, T. intermedius, T. lanuginosus, T. monospora , T. monosporus, T.

thalpophilus and T. viridis.

2.2 Importance of Thermoactinomyces vulgaris

T. vulgaris secretes enzymes at higher temperature, which are economically useful like

lipases (Elwan et al., 1978) , proteases (Roberts et al., 1977) and amylases (Allam et al.,

1975). Thermitase is an extracellular enzyme isolated from this organism. This enzyme is a

protease with temperature optimum of 60◦C, which lie above the optimal growth temperature

(50-52◦C) of this organism (Zhao and Frances Arnold, 1999). This shows that thermophilic

enzymes are more stable and tolerate extreme conditions beyond the optimum temperature

limit to ensure survival. This is an ecological adaptation of the organism to the extreme

environments for its survival. Enzymes of T. vulgaris are industrially important because

they are thermostable and can survive adverse conditions. Specially, thermostable amylases,

proteases and lipases produced by this genus have attracted much attention.

Among the Thermoactinomyces genus, T. vulgaris is an excellent example of natural

thermophilic transformation system. Transformation in this organism was first of all reported

by Hopwood and Wright, (1971) and later it was described by them in detail alongwith the

description of standard transformation conditions (Hopwood and Wright, 1972). T. vulgaris

is very well known for the inherent transformability in the presence of extracellular DNA and

for the thermostability of the cellular machinery. Recently Ca2+- dependence and inhibition

of transformation by trifluoperazine and chlorpromazine in T. vulgaris has been reported by

Bhatnagar and Singh (2003, 2004).

4

2.3 Introduction to Proteases

Figure 2.1: Protease catalysis of peptide bonds (proteolysis) (Gençkal, 2004)

Proteases (EC 3.4.21-24 and 99; peptidyl-peptide hydrolases) are a group of enzymes (also

known as peptidases, proteinases or proteolytic enzymes) that hydrolyse (break down) a

variety of proteins via the addition of water across peptide bonds (i.e., bonds that join

two adjacent amino acids to form a polypeptide) and catalyse peptide synthesis in organic

solvents and in solvents with low water content (Beg et al., 2003; Sookkheo et al., 2000).

Proteolysis: The process of breaking down protein with the help of proteases as shown in

Figure 2.1 is called proteolysis. It is a catabolic process in which proteins are digested

(broken down) directly or intramolecular digestion takes place. It is the process of hydrolysis

of peptide bonds to break down polypeptide chain into smaller amino acid sequences. The

products of proteolysis are protein and peptide fragments, and free amino acids.

Called by many as molecular knives i.e. biochemical version of Swiss army knives, proteases

are hydrolytic enzymes that cut long sequences of amino acids into fragments, a process that is

essential for the synthesis of all proteins, controlling their size, composition, shape, turnover,

and ultimate destruction and thus regulate most physiological processes (Barrett et al., 2003;

Vandeputte and Gros, 2002). They are vitally important in the life cycle. Different types of

5

proteases have different action mechanisms and biological processes, as explained in section

2.6. Regulating most physiological processes by controlling the activation, synthesis, and

turnover of proteins, proteases play pivotal regulatory roles in conception, birth, digestion,

growth, maturation, aging, and even death of all organisms (Howard and Yang, 2000).

Owing to their important regulatory roles in the life cycle, proteases have become important

targets for medical research and drug development (Cudic and Fields, 2009; Hooper, 2002;

Southan, 2001). The actions of proteases can be extremely selective, with each protease

being responsible for splitting very specific sequences of amino acids under a preferred set

of environmental conditions (Hedstrom, 2002).

Proteases are divided into four major groups according to the character of their catalytic

active site and conditions of action: serine proteinases, cysteine (thiol) proteinases, aspartic

proteinases, and metalloproteinases. Attachment of a protease to a certain group depends

on the structure of catalytic site and the amino acid (as one of the constituents) essential

for its activity.

Proteases are used throughout by an organism for various metabolic processes. Acid proteases

secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin

and chymotrypsin) enable us to digest the protein in food; proteases present in blood serum

(thrombin, plasmin, Hageman factor, etc.) play important role in blood clotting, as well

as lysis of the clots, and the correct action of the immune system. Other proteases are

present in leukocytes (elastase, cathepsin G) and play several different roles in metabolic

control. Proteases determine the lifetime of other proteins, playing important physiological

roles like hormones, antibodies, or other enzymes – this is one of the fastest "switching on"

and "switching off" regulatory mechanisms in the physiology of an organism. By complex

cooperative action, the proteases may proceed as cascade reactions, which result in rapid

and efficient amplification of an organism’s response to a physiological signal.

Proteases represent one of the largest group of industrial enzymes and account for about

60% of the total worldwide sale of enzymes (Rao et al., 1998). Their application in the

leather industry for dehairing and bating of hides to substitute currently used toxic chemicals

is a relatively new development and has conferred added biotechnological importance to

them (Rao et al., 1998). The vast diversity of proteases, in contrast to the specificity of

their action, has attracted worldwide attention in attempts to exploit their physiological and

biotechnological applications (Fox and Bjamason, 1991; Poldermans, 1990).

6

2.4 Different Sources of Proteases and Significance of Microbial Proteases

Proteases are found in all forms of organisms regardless of kingdom, as exemplified in

table 2.1. Some examples include the plant proteases, papain of papaya and bromalein

of pineapple, trypsin, chymotrypsin, renin and pepsin of the animal and human digestive

proteases. Proteases of bacteria, fungi and viruses are increasingly studied due to their

importance and subsequent application in industry and biotechnology. Examples of these

include application of bacterial neutral and alkaline proteases from the Bacillus sp. in

fermentation and detergent industry (Noomen et al., 2009) and acid protease of Aspergillus

sp. in food industry namely, the production of cheese (Sumantha et al., 2006). The detailed

applications of microbial proteases have been discussed in section 2.8.

In general, microbial proteases are extracellular in nature and are directly secreted into the

fermentation broth by the producer, thus simplifying downstream processing of the enzyme

as compared to proteases obtained from plants and animals (Gupta et al., 2002a). Microbial

proteases, especially from Bacillus sp. have traditionally held the predominant share of the

industrial enzyme market of the worldwide enzyme sales with major application in detergent

formulations (Beg et al., 2003).

Usually, all bacteria produce multiple enzymes, but Bacillus species are specific producers

of extracellular enzymes like proteases, lipases and amylases. For example, Tambekar et al.

(2009) found that 27 Bacillus strains, isolated from soil of saline belt of Purna river basin of

Vidarbha region of Maharashtra state, showed multiple enzyme activity by producing protease

(41%), amylase (33%) and lipase (26%). All 3 enzymes were produced by 9 isolates and

showed multienzyme production capacity. Shah et al. (2007) isolated 25 strains of Bacillus,

out of which 8 were able to produce all 3 enzymes. Limpon and Kalita (2007) also reported

that out of 24 isolated strains of Bacillus sp., 21 produced protease, 19 produced amylase,

15 produced lipase and 9 showed cellulase activity.

The inability of the plant and animal proteases to meet current world demands has led to

an increased interest in microbial proteases. Microorganisms represent an excellent source

of enzymes owing to their broad biochemical diversity and their susceptibility to genetic

manipulation (Rao et al., 1998; Gupta et al., 2002a). Proteases from microbial sources are

preferred to the enzymes from plant and animal sources, since they possess almost all the

characteristics desired for their biotechnological applications. Commercial application of

7

Tabl

e2.

1:Pr

otea

seso

urce

s(R

aoet

al.,

1998

)

Sour

ces

ofPr

otea

ses

Prot

ease

Ext

ract

edD

escr

iptio

n

Plan

tsPa

pain

Ext

ract

edfr

omth

ela

tex

ofC

aric

apa

paya

frui

ts,

activ

ebe

twee

npH

5.0

and

9.0

and

isst

able

upto

80or

90°C

inth

epr

esen

ceof

subs

trat

es,

exte

nsiv

ely

used

inin

dust

ryfo

rth

epr

epar

atio

nof

high

lyso

lubl

ean

dfla

vore

dpr

otei

nhy

drol

ysat

es.

Bro

mel

ain

Prep

ared

from

the

stem

and

juic

eof

pine

appl

es,

char

acte

rize

das

acy

stei

nepr

otea

se,

activ

efr

ompH

5.0

to9.

0an

dits

inac

tivat

ion

tem

pera

ture

is70

°C.

Ani

mal

sTr

ypsi

nA

seri

nepr

otea

sean

dm

ain

inte

stin

aldi

gest

ive

enzy

me

resp

onsi

ble

for

the

hydr

olys

isof

food

prot

eins

,us

edin

the

prep

arat

ion

ofba

cter

ial

med

iaan

din

som

esp

ecia

lized

med

ical

appl

icat

ions

.

Chy

mot

ryps

inFo

und

inan

imal

panc

reat

icex

trac

t,us

edex

tens

ivel

yin

the

deal

lerg

eniz

ing

ofm

ilkpr

otei

nhy

drol

ysat

es,

stor

edin

the

panc

reas

inth

efo

rmof

apr

ecur

sor,

chym

otry

psin

ogen

,an

dis

activ

ated

bytr

ypsi

nin

am

ultis

tep

proc

ess.

Peps

inA

cidi

cpr

otea

se(p

H1.

0-2.

0)fo

und

inth

est

omac

hsof

alm

ost

all

vert

ebra

tes,

cata

lyze

sth

ehy

drol

ysis

ofpe

ptid

ebo

nds

betw

een

two

hydr

opho

bic

amin

oac

ids.

Ren

nin

Peps

in-l

ike

prot

ease

,pr

oduc

edas

anin

activ

epr

ecur

sor,

pror

enni

n,in

the

stom

achs

ofal

lnu

rsin

gm

amm

als,

conv

erte

dto

activ

ere

nnin

byth

eac

tion

ofpe

psin

orby

itsau

toca

taly

sis,

used

exte

nsiv

ely

inth

eda

iry

indu

stry

topr

oduc

ea

stab

lecu

rdw

ithgo

odfla

vor.

Mic

robe

sB

acte

rial

neut

ral

prot

ease

s(n

eutr

ase)

Act

ive

ina

narr

owpH

rang

e(p

H5.

0to

8.0)

and

have

rela

tivel

ylo

wth

erm

otol

eran

ce,

Som

eof

the

neut

ral

prot

ease

sbe

long

toth

em

etal

lopr

otea

sety

pean

dre

quir

edi

vale

ntm

etal

ions

for

thei

rac

tivity

,w

hile

othe

rsar

ese

rine

prot

eina

ses,

whi

char

eno

taf

fect

edby

chel

atin

gag

ents

.B

acte

rial

alka

line

prot

ease

sC

hara

cter

ized

byhi

ghac

tivity

atal

kalin

epH

,e.

g.,

pH10

.0,

and

broa

dsu

bstr

ate

spec

ifici

ty,

The

irop

timal

tem

pera

ture

isar

ound

60°C

,us

edex

tens

ivel

yin

the

dete

rgen

tin

dust

ry

Fung

alpr

otea

ses

(aci

d,al

kalin

ean

dne

utra

lpr

otea

ses)

Fung

alac

idpr

otea

ses

(pH

2.5-

6.0)

used

inch

eese

mak

ing

indu

stry

.Fu

ngal

neut

ral

prot

ease

s(p

H7.

0)ar

em

etal

lopr

otea

ses

and

supp

lem

ent

the

actio

nof

plan

t,an

imal

,an

dba

cter

ial

prot

ease

sin

redu

cing

the

bitte

rnes

sof

food

prot

ein

hydr

olys

ates

.Fu

ngal

alka

line

prot

ease

sar

eus

edin

food

prot

ein

mod

ifica

tion

Vir

alpr

otea

ses

Seri

ne,

aspa

rtic

,an

dcy

stei

nepe

ptid

ases

are

foun

din

vari

ous

viru

ses.

All

ofth

evi

rus-

enco

ded

pept

idas

esar

een

dope

ptid

ases

,th

ere

are

nom

etal

lope

ptid

ases

.

8

microbial proteases (predominantly extracellular) is attractive due to the relative ease of large

scale production as compared to proteases from plants and animals (Ward et al., 2009).

Although protease production is an inherent property of all organisms, only those microbes

that produce a substantial amount of extracellular protease have been exploited commercially.

Microbes serve as a preferred source of these enzymes because of their rapid growth, the

limited space required for their cultivation, and the ease with which they can be genetically

manipulated to generate new enzymes with altered properties that are desirable for their

various applications. General features and classification of proteases, their mechanism of

action, physiological functions of proteases, protease engineering, industrial production of

proteases, industrial applications of proteases and use of proteases in organic synthesis have

been recently reviewed by Ward et al. (2009). The industrial production of most microbial

proteases is further enhanced by the advancement in fermentation technologies.

2.5 Classification of Proteases

Investigations concerned with the localization of proteolytically active enzymes in the cells

of T. vulgaris showed that the enzymes are present in a dissolved form in the cellular extract

(75%) as well as linked to the solid cell components (25%) (Schalinatus et al., 1983a). The

ratio of the activities of the soluble periplasmic fraction to the soluble cytoplasmic fraction

to the insoluble cytoplasmic membrane fraction was found to be 2:1:1. They measured the

formation of proteins during the cultivation by detecting the activity in the cellular extract.

As soon as enough of biomass was present (weighable) in the medium, the protease activity

can be detected in the cellular extract, although a correlation between the formation of

biomass and enzymes was not existent.

The characterization and comparison of biochemical properties of the intracellular proteases

(IP) of the cellular extract of T. vulgaris with the corresponding extracellular proteases

(EP) was done by Schalinatus et al. (1983b). The storage stability, the temperature and

pH behaviour (optimum, stability) of both of the proteases were found to be identical.

However, differences were detected between IP and EP after the action of several effectors

and different substrates. After a column chromatographic separation of the IP of the cellular

extract, it was found to be composed of at least 3 proteases, two of them were serine

proteases, which could be inhibited unspecifically by p-chloromercuribenzoate. The purified

intracellular proteases were found to be very unstable.

9

According to the Nomenclature Committee of the International Union of Biochemistry and

Molecular Biology, proteases are classified in subgroup 4 of group 3 (hydrolases) (Rao et

al., 1998). The three major criteria currently used for the classification of peptidases are:

1. The reaction catalysed,

2. The chemical nature of the catalytic site and

3. The evolutionary relationship, as revealed by the structure (Rao et al., 1998).

With reference to the classification by the reactions they catalyse, as shown in table 2.2,

protease may be divided into two subclasses of peptide hydrolases (class 3.4) depending on

the location of the enzymatic action, either exopeptidase or endopeptidase. Exopeptidases

cleave peptide bonds at the amino terminus (aminopeptidase) or the carboxy terminus

(carboxypeptidases) of a peptide substrate. Endopeptidases, on the other hand, cleave peptide

bonds internally, away from either termini of the protein substrate. Thus, on their mode of

attack proteases are classified into two broad groups:

1. Endoproteases (EC Number 3.4.21-24 and 3.4.99 for unknown): These are proteases

which attack peptide bonds on the interior of the protein chain. The hydrolysis products

are usually smaller polypeptides and peptides. Therefore, most endoproteins will not

produce a great deal of free amino acids as end products.

2. Exoprotease (EC Number 3.4.11-3.4.19): These are proteases which can cleave off

single amino acids from either end of the protein chain. The exopeptidases thus

act only near the ends of polypeptide chains. Based on their site of action at the

N or C terminus, they are classified as aminopeptidases (EC number 3.4.11-3.4.14)

and carboxypeptidases (EC number 3.4.15-3.4.19), respectively (Rao et al., 1998).

Eventually under right conditions, a protein can be reduced down to single amino acid.

Based on the functional group present at the active site, endopeptidases are further classified

into six prominent groups:

1. Serine proteases (EC 3.4.21)

2. Aspartic proteases (EC 3.4.23)

3. Cysteine proteases (EC 3.4.22)

4. Metalloproteases (EC 3.4.24)

5. Threonine protease (EC 3.4.25) and

6. Glutamic acid protease

10

Tabl

e2.

2:C

lass

ifica

tion

ofpr

otea

ses

(Kum

aret

al.,

2008

)

Seri

nePr

otea

ses

(Try

psin

,C

hem

otry

psin

,Su

btili

sin)

Seri

nere

sidu

epr

esen

tat

cata

lytic

cent

er,

hist

idin

ean

das

part

icre

sidu

esal

sopr

esen

tE

C3.

4.21

Cys

tein

epr

otea

ses

(Pap

ain,

Cat

heps

inB

)C

yste

ine

grou

p(-

SH)

pres

ent

atca

taly

ticce

nter

EC

3.4.

22

End

opep

tidas

esA

spar

ticPr

otea

ses

(pep

sin,

chym

osin

)A

spar

ticac

idre

sidu

epr

esen

tat

cata

lytic

cent

erE

C3.

4.23

Met

allo

prot

ease

s(t

herm

olys

in,

carb

oxyp

eptid

ase

A)

Glu

tam

atic

acid

resi

due

pres

ent

atca

taly

ticce

nter

.R

equi

reZ

n2+,

Cu2+

,M

g2+to

hydr

olys

epe

ptid

ebo

ndE

C3.

4.24

Thr

eoni

neE

ndop

eptid

ases

Thr

eoni

nere

sidu

epr

esen

tat

cata

lytic

cent

erE

C3.

4.25

Prot

ease

sE

ndop

eptid

odas

esof

unkn

own

cata

lytic

mec

hani

sm

Rea

ctio

nm

echa

nism

has

not

been

com

plet

ely

eluc

idat

edE

C3.

4.99

Am

inop

eptid

ases

EC

3.4.

11Pe

ptid

ylam

ino-

Aci

dH

ydro

lase

sor

Acy

lam

ino-

Aci

dH

ydro

lase

sE

C3.

4.12

Am

inop

eptid

ases

Dip

eptid

ase

EC

3.4.

13E

xope

ptid

ases

Dip

eptid

ylpe

ptid

ase

and

Trip

eptid

ylpe

ptid

ase

EC

3.4.

14Pe

ptid

yl-d

ipep

tidas

esE

C3.

4.15

Seri

neca

rbox

ypep

tidas

esE

C3.

4.16

Car

boxy

pept

idas

esC

yste

ine

carb

oxyp

eptid

ases

EC

3.4.

18M

etal

lo-c

arbo

xype

ptid

ases

EC

3.4.

17O

meg

aPe

ptid

ases

Hyd

roly

seam

ino

acid

resi

due

subs

titut

edei

ther

from

Nte

rmin

al(3

.4.1

4)or

from

Cte

rmin

al(3

.4.1

5)E

C3.

4.19

11

Some parameters like molar mass, optimum pH and temperature etc. have been given in table

2.3. Serine-type peptidases have an active centre serine involved in the catalytic process,

the cysteine-type peptidases have a cysteine residue in the active centre, the aspartic-type

endopeptidases depend on two aspartic acid residues for their catalytic activity, and the

metallopeptidases use a metal ion (commonly zinc) in the catalytic mechanism. Some

properties of these proteases have been summarised in table 2.3. A number of endopeptidases

cannot yet be assigned to any of the sub-subclasses EC 3.4.21-24 and these form a separate

sub-subclass EC 3.4.99 of the enzyme list. However, aspartic proteases are rare for bacteria

and to date, none have been reported for bacterial pathogens. Metalloproteases, on the other

hand, seem to be a common feature in most bacterial pathogens.

The threonine and glutamic-acid proteases were not described until 1995 and 2004, respectively

(Sims et al., 2004; Baird et al., 2006). The mechanism used to cleave a peptide bond involves

making an amino acid residue that has the cysteine and threonine (peptidases) or a water

molecule (aspartic acid, metallo- and glutamic acid peptidases) nucleophilic so that it can

attack the peptide carboxyl group. One way to make a nucleophile is by a catalytic triad,

where a histidine residue is used to activate serine, cysteine, or threonine as a nucleophile.

Threonine proteases are proteases that have threonine, bonded at the active site. It is

responsible for functioning of proteasome, the large protein-degrading apparatus. Threonine

proteases have a conserved N-terminal threonine at each active site. Pre-proteins, which

are catalytic beta subunits, are activated when the N-terminus is cleaved off. This makes

threonine the N-terminal residue. Threonine proteases are activated by primary amines. The

mechanism for the threonine protease was described first in 1995 (Seemuller et al., 1995;

Baird et al., 2006). The mechanism showed the cleaving of a peptide bond, which made

an amino acid residue (usually serine, threonine, or cysteine) or a water molecule become

a good nucleophile that could perform a nucleophilic attack on the carboxyl group of the

peptide. The amino acid residue (in this case threonine) is usually activated by a histidine

residue.

Glutamic acid proteases were described after 2004. These proteases require water for their

catalytic activity. Glutamic acid proteases are involved in nucleophilic reactions (Sims et

al., 2004).

Aminopeptidases act at a free N terminus of the polypeptide chain and liberate a single

amino acid residue, a dipeptide, or a tripeptide. They are known to remove the N-terminal

12

Tabl

e2.

3:C

lass

ifica

tion

ofpr

otea

ses

base

don

func

tiona

lgr

oup

pres

ent

atac

tive

site

and

thei

rpr

oper

ties.

(Sum

anth

aet

al.,

2006

)

Prot

ease

EC

No.

Mol

arm

ass

rang

e/k

Da

pHop

timum

Tem

pera

ture

optim

um/◦

C

Met

alio

nre

quir

e-m

ent(

s)

Act

ive

site

amin

oac

id(s

)M

ajor

Inhi

bito

rsM

ajor

Sour

ces

Seri

nePr

otea

se3.

4.21

18-3

56.

0-11

.050

-70

Ca2+

Seri

ne,

hist

idin

ean

das

part

e

PMSF

,D

IFP,

ED

TA,

soyb

ean

tryp

sin

inhi

bito

r,ph

osph

ate

buff

er,

indo

le,

phen

ol,

tria

min

oac

etic

acid

Bac

illus

,A

sper

gillu

s,A

nim

altis

sue

(gut

),Tr

itira

chiu

mal

bum

(the

rmos

tabl

e)

Cys

tein

eor

thio

lpr

otea

se3.

4.22

34-3

52.

0-3.

040

-55

-A

spar

tate

orcy

stei

neIn

doac

etam

ide,

p-C

MB

Asp

ergi

llus,

stem

ofpi

napp

le(A

nana

sco

mor

us),

late

xof

figtr

ee(F

icus

sp.),

papa

ya(C

aric

apa

paya

),St

rept

ococ

us,

Clo

stri

dium

Asp

artic

orca

rbox

ylpr

otea

se

3.4.

2330

-45

3.0-

5.0

40-5

5C

a2+A

spar

tate

orcy

stei

nePe

psta

tin

Asp

ergi

llus,

Muc

or,

End

othi

a,R

hizo

pus,

Peni

cilli

um,

Neu

rosp

ora,

Ani

mal

tissu

e(s

tom

ach)

Met

allo

-pr

otea

se3.

4.24

19-3

75.

0-7.

065

-85

Zn2+

Ca2+

Phen

y-al

anin

eor

leuc

ine

Che

latin

gag

ents

such

asE

DTA

,E

GTA

Bac

illus

,A

sper

gillu

s,P

seud

omon

as,

Peni

cilli

um,

Stre

ptom

yces

13

methionine that may be found in heterologously expressed proteins but not in many naturally

occurring mature proteins. Aminopeptidases occur in a wide variety of microbial species

including bacteria and fungi. In general, aminopeptidases are intracellular enzymes, but there

has been a single report on an extracellular peptidase produced by A. oryzae (Rao et al.,

1998).

Carboxypeptidases act at C terminals of the polypeptide chain and liberate a single amino

acid or a dipeptide. Carboxypeptidases can be divided into three major groups, serine

carboxypeptidases, metallo carboxypeptidases, and cysteine carboxypeptidases, based on the

nature of the amino acid residue at the active site of the enzymes (Rao et al., 1998). Omega

carboxypeptidases have been recently described by Mayas et al. (2008).

Proteases are also classified into different families and clans, depending on their amino acid

sequences and evolutionary relationships. Within each of the broad groups, peptidases have

been classified into families of related peptidases (Barrett et al., 2003). For example, within

the serine peptidases, families are labeled Sx, where S denotes the serine catalytic type and

the x denotes the number of the family, for example S1 (chymotrypsins). An up-to-date

classification of peptidases into families is found in the MEROPS database.

Proteases are also classified by the optimal pH, in which they are active as:

1. Acid proteases (pH < 7.0)

2. Neutral proteases (pH ≈ 7.0)

3. Basic/alkaline proteases (pH 7.5-12.0)

Automated methods that can identify proteases and their types both timely and effectively

based on the protein sequence information alone have been developed (Shen and Chou,

2009).

Acidic Proteases

These are found in animal cells, moulds and yeasts, but seldom in bacteria. Rennins from

calf stomach and pepsin of humans are the well known examples of acid proteases, catalysing

hydrolysis of protein around pH 2.0-4.0. Some of the fungi also produce acid proteases,

which are rennin-like and used mainly in cheese production. Acid proteases are also used in

the preparation of digestive syrup, soy protein digestion during sauce preparation, hydrolyzing

the gluten from wheat dough used for preparing biscuits in bakery making them crispy. Silver

from the film roll is recovered by digesting the gelatin by acid proteases. Alcaligens, Bacillus,

14

Corynebacterium, Lactobacillus, Pseudomonas, Serratia, Streptococcus and Streptomyces are

the bacteria, and Aspergillus, Candida, Coriolus, Endothia, Endomophthora, Irpex, Mucor,

Penicillium¸Rhizopus, Sclerotium and Torulopsis are the some of the fungi producing rennin

like proteases, which find applications in cheese processing (Sumantha et al., 2006).

The microbial rennin-like proteases have been derived from Mucor miehei (Escobar and

Barnett, 1993; Fernandez-Lahore et al., 1999). M. pusillus, Endothia parasitica, M. hiemalis,

M. racemosus and M. bacilliformis are other species of Mucor that present protease activity

of commercial value (Fernandez-Lahore et al., 1998, 1997). Pepsin-like acid proteases are

derived from Aspergillus sp. (Tremacoldi et al., 2004) and Rhizopus sp. (Kumar et al.,

2005). Calf rennet, composing of 88-94% chymosin and 6-12% pepsin, is extracted from

the fourth stomach of the unweaned calf (Burgess and Shaw, 1983). Many of them contain

aspartate as the active amino acid and their specificity is defined by the presence of aromatic

or bulky side chains at both sides of the cleaving bond. The carbohydrate content of these

enzymes confers heat stability to these biocatalysts (Tsujita and Endo, 1978). Production of

acid proteases from thermophilic Penicillium sp. has also been reported (Hashimoto et al.,

1973).

Neutral Proteases

Neutral proteases are obtained from plants, e.g., papain (from Carica papaya), bromelain (from

Ananas comorus) and ficin (from Ficus sp.), which are cysteine proteases. Neutral proteases

are produced by bacteria (Clostridium histolyticum, Streptococcus sp., Bacillus subtilis, B.

cereus, B. megaterium, B. stearothermophilus, B. thuringiensis, B. pumilus, B. polymyxa,

B. licheniformis, B. amyloliquefaciens, Pseudomonas aeruginosa, Streptomyces griseus) and

fungi (Aspergillus oryzae, A. sojae, Penicillium sp., Pericularia oryzae) (Sumantha et al.,

2006). These microbial neutral proteases are either cysteine or metalloproteases. Neutral

metalloproteases have specificity towards peptide linkages that contain hydrophobic amino

acids to the amino side. The neutral proteases are unstable and require calcium, sodium

and chloride ions for their stability. Not only the pH range for these proteases is small,

but they also get inactivated at elevated temperatures. Commercial fungal neutral proteases

are used in baking, food processing, protein modification and in leather, animal feeds

and pharmaceutical industries. A neutral protease, isolated from Bacillus sp. 158, having

optimum pH at 7.0 and optimum temperature at 30◦C has been effectively used to remove

protein deposit from contact lenses, indicating its potential to increase in transmittance of

15

lenses (Pawar et al., 2009).

Alkaline Proteases

Alkaline proteases are very important industrial enzymes. They are used in the manufacture

of detergents, food, pharmaceuticals and leather, in the production of protein hydrolysate,

bioprocessing of used X-ray films for silver recovery and for waste processing (Gupta et al.,

2002a). Proteases to be used as detergent additive should be stable and active in the presence

of surfactants, bleaching agents, bleach activators, fillers, fabric softeners and various other

formulation of a typical detergent. In textile industry, proteases may also be used to remove

the stiff and dull gum layer of sericine from the raw silk fibre to achieve improved luster

and softness. Protease treatment also modifies the surface of wool and silk fibers to provide

unique finishes. The alkaline proteases also have potential application in removal of gelatin

from the used photographic films vis-a-vis recovery of silver from them. To develop an

economically feasible technology, research efforts are mainly focused on: (i) improvement

in the yields of alkaline proteases; and (ii) optimization of the fermentation medium and

production conditions in industrial scale. However, it is known that 40% of the production

cost of industrial enzymes in large scale is estimated to be accounted for the cost of the

growth medium (El Enshasy et al., 2008). Concerning this fact, the use of cost-effective

growth medium for the production of alkaline protease is especially important.

Among bacteria, Bacillus strains are active producers of extracellular alkaline proteases (Rao

et al., 1998). Currently, large portions of commercially available alkaline proteases are

derived from these Bacillus strains. The alkaline serine protease from alkaliphilic Bacillus

sp. B18 has optimal pH 12.0-13.0 and temperature 85◦C (Fujiwara et al., 1993). The

optimum pH and temperature for trypsin like thermostable serine alkaline protease from

thermophilic and alkaliphilic Bacillus sp. JB-99 has been reported to be at pH 11.0 and

70-80◦C, respectively; and this protease retained 78% activity even after 1 h heat treatment

at 80◦C (Johnvesly and Naik, 2001).

Alkaline protease from Bacillus licheniformis NCIM-2042 has been reported to be produced

optimally at pH 9.5, temperature 30◦C, incubation period 72 h, agitation 200 rpm and airflow

rate 3 vvm (volume of air per volume of media per minute)(Potumarthi et al., 2007). It has

been reported that B. subtilis, B. amyloliquefaciens and B. megaterium produce proteases

optimally at pH 10.0, while B. licheniformis shows optimal protease production at pH 9.0

16

and 11.0 (Boominadhan et al., 2009). Asokan and Jayanthi (2010) found that optimum

protease production in Bacillus licheniformis and Bacillus coagulans was at pH 10.0 and at

temperature 30◦C, and hence the proteases of these bacteria are quite suitable for detergent

applications.

Viability of commercial production of alkaline protease, having pH optimum at pH 8.0-9.0

and temperature optimum at 37-45◦C by a thermophilic strain of Bacillus subtilis DM-04

under solid-state fermentation, using potato peel (a kitchen waste) and the notorious weed,

Imperata cylindrica grass (commonly known as Ulukher in Assam) in an optimal ratio of 1:1

(w/w) as cheap source of carbon and nirogen, has been recently demonstrated (Mukherjee

et al., 2008). This protease retained 67% of its original activity post-heating at 60◦C for

15 minutes and exhibited a significant stability and compatibility with most of the tested

commercial laundry detergents, demonstrating its feasibility for inclusion in laundry detergent

formulation.

A significant improvement (14-fold) in the production of protease by the Bacillus mojavensis

A 21 strain was accomplished, using cheap carbon and nitrogen substrates (i.e. hulled grain

of wheat and sardinella peptone), which may result in a significant reduction in the cost of

medium constituents for industrial production of alkaline proteases (Haddar et al., 2010).

An alkaline protease (thermitase) was also purified from T. vulgaris by means of isoelectrical

focussing in the flat-bed procedure, using granulated gel (Frommel et al., 1978). A highly

purified protease with a uniform N-terminal end group was isolated by isoelectrical focussing

in a single step. The enzyme had an isoelectric point (IP) at pH 9.0 and a molecular weight

of 37,400 Da. The enzyme consisted of a polypeptide chain with arginine as the N-terminal,

and tyrosine as the C-terminal end groups. In addition to an essential serine residue, it had

a -SH group, which is hardly accessible in the native enzyme.

2.6 Mechanism of Catalysis

Study of the structure-function relationships in protease-catalyzed hydrolysis of the peptide

bond has advanced our understanding of enzyme catalysis and specificity. Three-dimensional

structures have been solved for each mechanistic class of protease, and these studies have been

extended using site-directed mutagenesis to characterize the amino acid residues required for

enzyme function (Phillips and Fletterick, 1992). The catalytic mechanisms have been well

established for serine proteases (Fig. 2.2), aspartic proteases (Fig. 2.3), cysteine proteases

17

(Fig. 2.4) and metalloproteases (Fig. 2.5) by Raih et al. (2005).

Figure 2.2: Catalytic mechanism of serine proteases (Raih et al., 2005)

18

Figure 2.3: Catalytic mechanism of aspartic proteases (Raih et al., 2005)

19

Figure 2.4: Catalytic mechanism of cysteine proteases (Raih et al., 2005)

20

Figure 2.5: Catalytic mechanism of metalloproteases (Raih et al., 2005)

21

2.7 Thermophilic Proteases and Thermitase

Thermophilic enzymes are potentially applicable in a wide range of industrial processes,

particularly and mainly due to their denaturant tolerance and extraordinary operational stability

at high temperatures. Such enzymes are used in chemical, food, pharmaceutical, paper,

textile and other industries (Haki and Rakshit, 2003; Turner et al., 2007; Zamost et al., 1991).

A thermostable neutral protease from Bacillus BT1 was found to be optimally active at 82°C

and 50% of the enzyme activity was retained after incubation at 76°C for 30 minutes (Vecrek

and Kyslik, 1995). Another thermophilic neutral protease from thermophilic Bacillus strain

HS08 was found to be stable during the 1 h incubation at 50◦C (Guangrong et al., 2006).

The optimal pH and optimal temperature for the activity of this protease were at pH 7.5

and 65◦C, respectively.

A thermophilic extracellular serine alkaline protease from Bacillus stearothermophilus AP-4

was found to be optimally active at pH 9.0 and 55◦C (Dhandapani and Vijayaragavan, 1994).

The optimum pH, agitation rate and temperature for protease production by Bacillus sp.

(No.1) were found to be at pH 8.0, 150 rpm and 50°C, respectively, wherein the protease

production coincided with the late exponential or early stationary phase of bacterial growth

and corresponded with the sporulation of this bacterium (Razak et al., 1997).

A thermophilic alkaline protease from alkalophilic Bacillus pumilus MK6-5 was found to

be optimally active at pH 11.5 and temperature of 50-60◦C (Kumar, 2002). The serine

and metalloproteases produced by Streptomyces sp. 594 in submerged (SF) and solid-state

fermentation (SSF), using feather meal, an industrial poultry residue (keratinous waste) and

corn steep liquor (corn processing by-product), were found to be active over a wide range

of pH (5.0-10.0) and high temperatures (55-90◦C) (De Azeredo et al., 2006a,b).

A keratinolytic metalloprotease from Bacillus subtilis MTCC (9102) was found to be optimally

active at pH 6.0 and remained stable upto 70◦C, thereby having promising and potential

applications in the bioconversion of keratinous wastes and eco-friendly dehairing in the

leather industry (Balaji et al., 2008).

Behnke et al. (1978a) purified a microbial protease "thermitase" from the submerged cultivation

of T. vulgaris. After treating the culture filtrate with ethanol or Na2SO4, the precipitate was

vacuum dried and purified by column chromatography on Sephadex G-75, DEAE-Cellulose

and Sephadex G-50. The proteolytically active fractions were in each case pooled, freeze

22

dried and tested for protein components and protease activity by gel electrophoresis. The

isolated protease was further characterised, after passing through the third column. It was

found that the specific activity of the enzyme was increased 4.5-fold and isoenzyme pattern

showed three protease bands. The freeze dried fraction contained 85% protein and 4%

carbohydrates (glucose as single monomer component after acid hydrolysis). Its molecular

weight was found to be 11,000 Da.

“Thermitase” (EC 3.4.21.14), a thermostable extracellular serine protease from T. vulgaris,

binds one calcium ion with a dissociation constant of about 10−4M at 25◦C and pH 7.5 to 3.5

(Frommel and Hohne, 1981). It was shown by experiments with a calcium-selective electrode

that two calcium ions were bound more tightly to the enzyme. The single most weakly bound

calcium ion caused a slight quenching of the protein fluorescence emission, accompanied by

a stabilization against thermal denaturation or autolysis and an increase of esterolytic activity

by 10% (approximately). The tightly bound calcium ions had only a slight influence on

activity, thermal denaturation and autolytic degradation. The activation parameters of thermal

denaturation indicated that “thermitase” belongs to the class of thermostable enzymes with

a high intrinsic stability.

The substrate specificity and properties of partially purified thermitase from T. vulgaris were

studied by Behnke et al. (1978b). Among the protein substrates tested, urea denaturated

hemoglobin was split best, followed by gelatin, casein, field bean protein, serum albumin

and gluten. The weakest rate of hydrolysis was observed with elastin. In contrast to

acetyl-(L-ala)3-methylester, a substrate for elastase, was split best from all the esters tested.

Only 8% of the activity could be found with the chymotrypsin substrates, acetyl-L-tyr-ethylester

and acetyl-L-phe-ethylester and 1% of the above activity with the trypsin substrates, tosyl-L-

arg-methylester and benzoyl-L-arg-methylester. The fatty acid esters and the p-nitroanilides

were hydrolyzed much more slowly. The pH optimum of thermitase was found in the

alkaline range at pH 9.0. There were only small differences between the individual high

and low molecular weight substrates. The temperature optimum was between 60 and 75◦C

for esters and p-nitroanilides as substrates and at 90◦C for casein. The enzyme was quickly

inactivated above 70◦C (Schalinatus et al., 1979). The cleavage specificity of thermitase from

T. vulgaris was determined by the cleavability of insulin β -chain, casein and haemoglobin

by this enzyme as compared to other proteases (i.e. trypsin, chymotrypsin, proteases from B.

megaterium and cytophages) (Schalinatus et al., 1979). Thermitase, unspecifically hydrolysed

23

all the substrates (i.e. insulin β -chain, casein and haemoglobin) efficiently.

Thermitase, the main component of the proteases in the culture medium of T. vulgaris,

was degraded by autolysis (increase of liberated amino groups) and inactivated at high

temperature, at alkaline pH and in the absence of substrates (Behnke et al., 1982). Complete

disappearance of the thermitase band on polyacrylamide gel electrophoresis was observed

after heating the enzyme at 85◦C for 5 minutes. The quantitative comparison of autolysis and

heat inactivation as well as the kinetics of reversible inhibition of the enzyme by HgCl2, at

different temperatures showed that above 60◦C, thermal denaturation of the enzyme protein

contributes to thermitase inactivation. It was also found that Ca2+-ions (20 mM) had a

stabilizing effect against both autolysis and thermal denaturation (inactivation) of thermitase

(Behnke et al., 1982).

An anionic protease component, which frequently contaminates preparations of routinely

isolated cationic protease (i.e. thermitase), was isolated and purified by rechromatography on

controlled pore glass (CPG-10) from T. vulgaris (Kleine and Kettmann, 1982). The purified

anionic enzyme shared several properties with thermitase such as size, sensitivity against

phenylmethanesulfonylfluoride and Hg2+, UV-spectral, immunological and pH behavior. On

the other hand, the isoelectric point (at pH 6.5), temperature dependence (more heat stable)

and enzymatic activity (less active) of anionic protease differed significantly from thermitase.

At pH 6.0-8.0 and temperature 4-25◦C, anionic protease was hydrolysed completely by

thermitase. Like other protein substrates, anionic protease simultaneously acts as a stabilizer

for thermitase. In contrast to thermitase, the anionic protease partially changed during

long-term storage at 4◦C and at pH 6.0 to a cationic protein species endowed with proteolytic

activity.

Some structural and catalytic properties of purified thermitase were studied by Kleine

(1982). The crystal data and the high elastinolytic action showed the possible relationship of

thermitase and pancreatic elastase. The enzyme showed maximal stability between pH 6.0

to 7.5 and maximal activity between pH 7.5 to 9.5. The larger the substrate, the higher was

its optimum temperature (60◦C for esterolysis and 85◦C for proteinolysis). The stability of

thermitase significantly improved by acetates and chlorides at 1M concentration. Besides its

high hydrolytic action on soluble proteins, thermitase was capable for efficient degradation

of the insoluble proteins such as elastin and collagen.

The modification of the serine and histidine residues in the active centre of thermitase

24

with diisopropylfluorophosphate (DFP) and L-1-tosylamide-2-phenylethyl chloromethylketone

(TPCK) and of the only -SH group of the enzyme, with Hg-compounds resulted in the loss

of activity against hydrolysis of 4-nitrophenylacetate (Hansen et al., 1982). It was shown

that modification of cysteine prevented reaction of serine and histidine in the active centre

of the enzyme with DFP and TPCK, respectively, while Hg2+ and CF3Hg binding to the

-SH group after modification of essential amino acid residues in the active centre is retained.

Ca2+ ions were found to have a stabilizing effect on all the modified products of thermitase,

as well as on the native enzyme. Simultaneous modification of the cysteine and serine led

to an increase in thermostability of thermitase, whilst double modification at the cysteine

and histidine caused destabilization of the enzyme (Hansen et al., 1982).

The dried proteolytic enzyme “Thermitase” was prepared from the cultural filtrate of T. vulgaris

(Leuchtenberger et al., 1983). The procedure comprised of concentration, sedimentation,

ethanol, sodium sulfate precipitation and drying in laboratory and semitechnical scales. A

sedimentation procedure using caoline or an aqueous solution of CaCl2 and Na2HPO4 or

filtration was elaborated by them to eliminate the lipid components in the concentrated

solution. The method was convenient to get good yields of the thermostable protease.

The influence of chloromethyl ketones and methyl ketones of N-acylated peptides on the

thermal denaturation of thermitase in the presence and the absence of calcium ions was

investigated by Schreier et al. (1984). The chloromethyl ketone derivatives were found to

react irreversibly with the enzyme, whereas the corresponding methyl ketones were found to

be reversible inhibitors. The irreversible inhibition of thermitase caused a marked stabilization

against thermal denaturation. On the other hand, the enzyme stability was not influenced

by the binding of reversible inhibitors. The Ca2+interaction (bridge formation) in the active

site region of thermitase played an important role for its thermal stability.

Dieter and Kleine (1984) tested seven microbial peptide inhibitors (i.e. chymostatin, antipain,

elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon) for their efficiency to inhibit

thermitase. Chymostatin and antipain were found to be the most effective inhibitors, with Ki

values of 7×10−8M and 2×10−7M, respectively. Except for leupeptin, all inhibitors resisted

hydrolysis by thermitase. Leupeptin, however, was cleaved by thermitase between the two

leucyl residues. The covalent immobilization of thermitase increased its thermostability

(Mozhav, 1990). At 68◦C, the catalytic activity of the immobilized enzyme was more than

10 times greater than that of the native thermitase.

25

A serine protease was isolated from the cultural filtrate of T. vulgaris strain INMI-4a by a

procedure using affinity chromatography on bacitracin-Sepharose, ion-exchange separation

on aminosilochrome and gel-filtration on Sephadex G-25 (Stepanov et al., 1980). A 194-fold

purification and the 55% yield of the enzyme was achieved by this procedure. The

molecular weight of the enzyme, as determined by polyacrylamide gel electrophoresis in

the presence of Na-SDS as well as by gel-filtration on Sephadex G-75 was 28,000 Da.

The isoelectric point was at pH 8.0-9.0. The pH optimum for the hydrolysis of peptide

substrate (Z-L-Ala-LAla-L-leu-pNA) was at pH 8.2. The enzyme was stable at pH 7.0-9.0.

The temperature optimum of the proteolytic activity was at 55◦C; however, the enzyme was

stable after heating for 1h at 37◦C (Stepanov et al., 1980). This enzyme was completely

inactivated by the serine protease specific inhibitors like phenylmethylsulphofluoride and

the protein inhibitor IT-AjT from Actinomyces and p-chloromercuribenzoate. The enzyme

showed lytic activity against the cells of E. coli, Micrococcus lysodeicticus and of the yeast.

Akparov et al. (2007) showed that site-directed mutagenesis in the active site of T. vulgaris

carboxypeptidase T(CpT), which is capable of hydrolyzing both hydrophobic and positively

charged substrates. Grishin et al. (2008) also studied the influence of residues at positions

260 and 262 on a broad substrate specificity of T. vulgaris carboxypeptidase T(CpT) by

mean of site-directed mutagenesis.

2.8 Industrial Applications of Microbial Proteases

Proteases constitute one of the most important groups of industrial enzymes, accounting for

at least 25% of the total global enzyme production sales. Proteases are by far the most

important group of enzymes produced commercially and are used in the detergent, protein,

brewing, meat, photographic, leather and dairy industries. Fibrous proteins, such as horn,

feather, nails and hair, are abundantly available in nature as wastes, but these can be converted

to useful biomass, protein concentrate or amino acids using proteases derived from certain

microorganisms. (Gupta et al., 2002a,b; Kumar and Takagi, 1999; Rao et al., 1998; Ward

et al., 2009).

2.8.1 Detergents

Enzymes have long been of interest to the detergent industry for their ability to aid the

removal of proteinaceous stains and to deliver unique benefits that cannot otherwise be

obtained with conventional detergent technologies (Gupta et al., 2002a,b).

26

Table 2.5: Commercial Bacillus sp. alkaline proteases used in detergent formulations (Sumantha etal., 2006)

Tradenames

Source organism OptimumpH

Optimumtemperature

(ºC)

Manufacturer

Alcalase B. licheniformis 8.0-9.0 60 Novo NordiskSavinase Alkaliphilic Bacillus sp. 9.0-11.0 55 Novo NordiskEsperase Alkaliphilic Bacillus sp. 9.0-11.0 60 Novo NordiskMaxacal Alkaliphilic Bacillus sp. 11.0 60 Gist-BrocadesMaxatase Alkaliphilic Bacillus sp. 9.5-10.0 60 Gist-BrocadesOpticlean Alkaliphilic Bacillus sp. 10.0-11.0 50-60 Solvay EnzymesOptimase Alkaliphilic Bacillus sp. 9.0-10.0 60-65 Solvay EnzymesProtosol Alkaliphilic Bacillus sp. 10.0 50 Advanced

BiochemicalsWuxi Alkaliphilic Bacillus sp. 10.0-11.0 40-50 Wuxi Synder

Bioproducts

The ideal detergent protease should possess broad substrate specificity to facilitate the removal

of a large variety of stains due to food, blood, and other body secretions. Activity and stability

at high pH, temperature and compatibility with other chelating and oxidizing agents added

to the detergents are among the major prerequisites for the use of proteases in detergents.

The key parameter for the best performance of a protease in a detergent is its pI (Isoelectric

point). It is known that a protease is most suitable for this application if its pI coincides

with the pH of the detergent solution.

The major use of detergent-compatible alkaline proteases is in laundry detergent formulations.

Detergents available in the international market such as Dynamo, Eraplus (Procter & Gamble),

Tide (Colgate Palmolive), contain proteolytic enzymes, the majority of which are produced

by members of the genus Bacillus (Anwar and Saleemuddin, 1998). Esperase and Savinase T

(Novo Industry), produced by alkalophilic Bacillus sp., are two commercial preparations with

very high isoelectric points (pI 11); hence, they can withstand higher pH ranges (Maurer,

2004). Some more industrial alkaline proteases have been listed in table 2.5.

Purification and biochemical characterization of a detergent-stable serine alkaline protease

named SAPB, which has a very important catalytic efficiency and stability at alkaline pH,

high temperature and in the presence of detergent additives is amply demonstrated (Jaouadi

et al., 2008).

Alkaline protease from Bacillus circulans has been purified and characterized in detail for

27

its robustness and its eco-friendly application potential in leather processing and detergent

industries because it has revealed stain removal property and dehairing activity for animal

hide without chemical assistance and without hydrolyzing fibrous proteins (Subba Rao et

al., 2009).

Wash performance analysis revealed that the crude enzyme of alkalophilic Bacillus licheniformis

NH1 strain, which contains at least five major extracellular proteases and a unique amylase,

as showed by zymography technique, could effectively remove a variety of stains, such

as blood, chocolate and barbecue sauce and as such can be used as a detergent additive

(Noomen et al., 2009). The latest status of application of microbial proteases as laundry

detergent additive has been described by Kumar et al. (2008).

2.8.2 Leather Industry

Leather processing involves several steps such as soaking, dehairing, bating, and tanning.

The major building blocks of skin and hair are proteinaceous. Proteases are used for selective

hydrolysis of noncollagenous constituents of the skin and for removal of nonfibrillar proteins

such as albumins and globulins. The purpose of soaking is to swell the hide. Microbial

alkaline proteases are used to ensure faster absorption of water and to reduce the time required

for soaking. The use of non ionic and, to some extent, anionic surfactants is compatible with

the use of enzymes. Alkaline proteases with hydrated lime and sodium chloride are used for

dehairing (Sivasubramanian et al., 2008), resulting in a significant reduction in the amount

of waste water generated. Pancreatic trypsin is used in combination with other Bacillus and

Aspergillus proteases for bating (Arunachalam and Saritha, 2009; Choudhary et al., 2004;

Hamid and Ikram, 2008; Palanisamy et al., 2004). The selection of the enzyme depends on

its specificity for matrix proteins such as elastin and keratin, and the amount of enzyme

needed depends on the type of leather (soft or hard) to be produced. Increased usage of

enzymes for dehairing and bating not only prevents pollution problems but also is effective

in saving energy. Novo Nordisk manufactures three different proteases, Aquaderm, NUE,

and Pyrase, for use in soaking, dehairing, and bating, respectively.

Zambare et al. (2007) had demonstrated that Bacillus cereus produced extracellular protease,

when grown on a medium containing starch, wheat bran and soya flour. The ammonium

sulphate precipitated (ASP) enzyme was applied for dehairing of buffalo hide.

28

2.8.3 Dairy and Food Industry

In cheesemaking, the primary function of proteases is to hydrolyze the specific peptide bond

(the Phe105-Met106 bond) to generate para-k-casein and macropeptides (Rao et al., 1998).

Chymosin is preferred due to its high specificity for casein, which is responsible for its excellent

performance in cheesemaking. The proteases produced by GRAS (genetically regarded as

safe)-cleared microbes such as Mucor michei, Bacillus subtilis, and Endothia parasitica are

gradually replacing chymosin in cheesemaking (Sumantha et al., 2006). Chymosin produced

through recombinant DNA technology has been successful in dairy industry (Hicks et al.,

1988; Justesen et al., 2009).

Cow milk also contains whey proteins such as lactalbumin and lactoglobulin. The denaturing

of these whey proteins, using proteases, results in a creamier yogurt product. Destruction

of whey proteins is also essential for cheese production (Gupta et al., 2002a; Kumar and

Takagi, 1999; Sumantha et al., 2006; Ward et al., 2009).

The most significant property of acidic proteases is the ability to coagulate proteins, as is

evidenced by their widespread application in the dairy industry for their ability to coagulate

milk protein (casein) to form curds from which cheese is prepared after the removal of whey.

By virtue of this property, microbial acidic proteases have largely replaced the calf enzyme

(rennet), facilitating the expansion of the cheese manufacture industry whose development

was hurdled by animal rights issues. A protease from Pseudomonas fluorescens R098 has

been reported to hydrolyse the peptides found in cheese, which are responsible for the bitter

taste, and thus finds application as a debittering agent (Sumantha et al., 2006).

The enzymes used in the food industry include Alcalase, Netrase, Esperase, Protamex,

and Novozym FM (Sumantha et al., 2006). These enzymes are commercially marketed

by Novozymes, Denmark.These bacterial proteases are used for improving the functional,

nutritional and flavor properties of proteins. Netrase is a bacterial protease which is used

in alcohol production for improving yeast growth (Sumantha et al., 2006). In baking, it is

used to degrade proteins in flour for biscuits, crackers and cookies. In brewing, it is used

for extracting sufficient proteins from malt and barley and for obtaining the desired level of

nitrogen nutrients. It is also involved in lactose reduction and flavor modification in dairy

applications.

Acid protease from Aspergillus saitoi, aspergillopepsin I is commercially marketed as Molsin

29

F by Kikkoman Corp., Japan (Sumantha et al., 2006). The enzyme is useful for the

production of seasoning materials from the foods containing various proteins, the degradation

of the turbidity complex resulting from protein in fruit juices and alcoholic liquors, and

the improvement of quality of protein-rich foods. Flavourzyme is a fungal complex of

exopeptidases and endoproteases derived from Aspergillus oryzae used for extensive hydrolysis

of proteins. Kojizyme is a similar complex, which finds application in the fermentation of

soy sauce. These enzymes are also products of Novozymes, Denmark.

2.8.4 Baking Industry

Wheat flour is a major component of baking processes. It contains an insoluble protein called

gluten, which determines the properties of the bakery doughs. Endo- and exoproteinases from

Aspergillus oryzae have been used to modify wheat gluten by limited proteolysis. Enzymatic

treatment of the dough facilitates its handling and machining and permits the production of

a wider range of products. The addition of proteases reduces the mixing time and results

in increased loaf volumes. Bacterial proteases are used to improve the extensibility and

strength of the dough (Moodie, 2001).

2.8.5 Manufacture of Soy Products

The alkaline and neutral proteases of fungal origin play an important role in the processing

of soy sauce. Proteolytic modification of soy proteins helps to improve their functional

properties. Treatment of soy proteins with alcalase at pH 8.0 results in soluble hydrolysates

with high solubility, good protein yield, and low bitterness. The hydrolysate is used in

protein-fortified soft drinks and in the formulation of feeds.

Positive results to produce effective antioxidant hydrolysates from soy proteins have been

reported in a recent investigation on two commercial microbial proteases viz. neutral protease

from Bacillus subtilis (NP) and alkaline protease from Bacillus licheniformis (AP) have been

very recently reported by Zhang et al. (2010).

2.8.6 Debittering of Protein Hydrolysates

The bitterness of protein hydrolysates is proportional to the number of hydrophobic amino

acids in the hydrolysate. The presence of a proline residue in the center of the peptide

also contributes to the bitterness. The peptidases that can cleave hydrophobic amino acids

and proline are valuable in debittering protein hydrolysates. Aminopeptidases from lactic

30

acid bacteria are available under the trade name Debitrase. Carboxypeptidase A has a high

specificity for hydrophobic amino acids and hence has a great potential for debittering. A

careful combination of an endoprotease for the primary hydrolysis and an aminopeptidase

for the secondary hydrolysis is required for the production of a functional hydrolysate with

reduced bitterness (Rao et al., 1998).

2.8.7 Synthesis of Aspartame

An immobilized preparation of thermolysin from Bacillus thermoprotyolyticus is used for

the enzymatic synthesis of aspartame, a non calorific artificial sweetener (Rao et al., 1998).

Toya Soda (Japan) and DSM (Dutch State Mines Co. based at The Netherlands) are the

major industrial producers of aspartame.

2.8.8 Pharmaceutical Industry

The wide diversity and specificity of proteases have a great advantage in developing effective

therapeutic agents. Oral administration of proteases from Aspergillus oryzae (i.e. Luizym

and Nortase) has been used as a digestive aid to correct certain lytic enzyme deficiency

syndromes. Clostridial collagenase or subtilisin is used in combination with broad-spectrum

antibiotics in the treatment of burns and wounds. An asparginase isolated from E. coli

is used to eliminate aspargine from the bloodstream in the various forms of lymphocytic

leukemia. Alkaline protease from Conidiobolus coronatus was found to be able to replace

trypsin in animal cell cultures (Rao et al., 1998).

Taken together, proteases represent a group of diverse and relatively unexplored agents for

use in insect pest management. A key issue before broad application of such proteases for

pest control relates to target specificity. For reduced risk associated with any insect control

technology, insect specificity is highly desirable. The use of proteases employed in plant

defense against herbivory holds particular promise for future development of insect resistant

transgenic plants. Greater understanding of the biology of virulence factors in the genomics

and transcriptomics era may facilitate identification of candidate proteases for use in pest

management (Harrison and Bonning, 2010).

2.8.9 Photographic Industry

Alkaline proteases are very important in the bioprocessing of used X-ray or photographic

films for silver recovery. These waste films contain 1.5-2.0% silver by weight in their gelatin

31

layer, which can be used to recover silver for different purposes. As the silver is bound

to gelatin, it is possible to extract silver from the protein layer by proteolytic treatments

(Shankar et al., 2010). Enzymatic hydrolysis of gelatin helps in extracting silver, as well as

the polyester film base can be recycled. Alkaline protease from Bacillus subtilis decomposed

the gelatin layer within 30 minutes at 50-60°C and released the silver (Fujiwara et al.,

1989; Ishikawa et al., 1993). Ishikawa et al. (1993) have demonstrated the use of alkaline

protease of Bacillus sp. B21-2 for the enzymatic hydrolysis of gelatin layers of X-ray films

to release silver particles. The alkaline proteases of Bacillus sp. B18 (Fujiwara et al., 1991)

and Bacillus coagulans PB-77 (Gajju et al., 1996) were also competent in decomposing the

gelatinous coating on used X-ray films from which the silver could be recovered.

2.8.10 Silk Degumming

As waste solution from the silk degumming process contains high nitrogen levels, waste water

must be treated prior to discharge. Pilanee et al. (2008), tested the silk degumming waste

solution as a nutrient substrate for microbial growth and protease production by Bacillus

licheniformis TISTR 1010 and Aspergillus flavus TISTR 3130, TISTR 3366, TISTR 3135 and

TISTR 3041. All strains were preliminarily screened for their protease activity by growing

on casein-agar plates with B. licheniformis TISTR 1010 being chosen as the best producer

of protease. Cultivation in a silk degumming solution as the nutrient source demonstrated

that the highest protease activity was achieved at an optimum pH of 10.0 for 36 h. Among

the culture media used, the specific activity of released protease was best with a medium

containing 6% protein from the silk degumming waste, 1% malt extract, 1% polypeptone

and 1% Na2CO3. They demonstrated the use of silk degumming waste as a nitrogen source

for microorganism growth and protease production and as such suggested an alternative

way to convert wastes into more valuable and marketable products. Optimization of silk

degumming protease production from Bacillus subtilis C4 has been recently reported by ?,

wherein the optimised conditions are reported to be quite suitable for industrial production

of silk degumming protease.

2.8.11 Alkaline Protease: a Tool to Clean Environment

Alkaline proteases are being extensively used in detergents, food processing and leather

industry. Production of these enzymes using low cost substrates would reduce the cost of

production (Gupta et al., 2002a).

32

Extracellular enzymes are produced during a fermentation process and possess the ability

to break down bonds within organic compounds and/or catalyze their transformation into

less toxic and more biodegradable forms. Unlike many microbes, enzymes remain effective

in a wide range of pH and temperature ranges, particularly if they are immobilized on

some carrier, and they can degrade a wide variety of compounds. Alkaline proteases have

been demonstrated of being able to reduce pathogen counts, reduce the solids content, and

increase deflocculation in sludge. Currently, high production costs inhibit the widespread

use of extracellular enzymes for remediation, but bench studies and field studies have shown

enzymatic treatment to be feasible options for bioremediation (Timothy and Jeffrey, 2006).

Alkaline serine proteases are the most important group of enzymes exploited commercially.

Alkaline proteases are one of the most important groups of enzymes, used in various

industrial products/processes as detergents, pharmaceuticals, leather, meat tenderizers, protein

hydrolyzates, food products and even in the waste processing (Anwar and Saleemuddin,

1998; Gupta et al., 2002a).

During the last few years many enzymes have been isolated from alkalophilic/ thermophilic

organisms which are capable of surviving in extremes of pH and temperature and are also

stable against a wide variety of denaturants such as urea and detergents. Subtle alterations

in the amino acid sequences, using techniques such as site-directed mutagenesis, could

be beneficial in producing enzymes with enhanced stability in soluble form for industrial

applications (Gupta et al., 2002a).

2.8.12 Medical Uses of Proteases

Neutral protease (dispase) is a bacterial enzyme produced by Bacillus polymyxa that

hydrolyses N-terminal peptide bonds of non-polar amino acid residues and is classified as

an aminopeptidase. This enzyme is marketed by many companies such as Invitrogen Corp.,

USA, BD Biosciences, USA, Worthington Biochemical Corp., USA, etc. Its mild proteolytic

action makes the enzyme especially useful for the isolation of primary and secondary cells

(subcultivation), since it maintains cell membrane integrity (Sumantha et al., 2006). Dispase

is also frequently used as a secondary enzyme in conjunction with collagenase and/or other

proteases in many primary cell isolation and tissue dissociation applications. It dissociates

fibroblast-like cells more efficiently than epithelial-like cells, so it has also been used for

differential isolation and culture applications. Other advantages are its non-mammalian

33

(bacterial) source and its ability to be inhibited by EDTA. Collagenase, which hydrolyses

native collagen, has been used for debridement of dermal ulcers and burns and also finds

application in the lysis of diseased invertebral disks.

Urokinase has been used for the treatment of clotting disorders (Sumantha et al., 2006).

Proteases of Aspergillus find application as digestive aids in gastro-intestinal disorders such

as dyspepsia (Sumantha et al., 2006).

Brinase, a plasmin-like acid protease, hydrolyses fibrin and fibrinogen. This is applied on

patients on chronic haemodialysis with clotted arteriovenous cannulae. Several minutes of

brinase treatment restores vessel function. But the enzyme exhibits toxic side effects and is

also inhibited by serum inhibitors.

Clear-Lens Pro, also marketed by Novozymes, Denmark, is used in contact lens cleaning

formulations to remove protein-based deposits and protein films from contact lenses. This

protease produced by submerged fermentation of a Bacillus, hydrolyses the protein in the

deposits and films, making them readily dissolvable and dispersible in the cleaning liquid.

This enzymic formulation is available both as a liquid preparation and as a microgranulate

(Sumantha et al., 2006).

A review of the status of human protease research and prospects for future protease-targeted

drugs has been reported by Turk B. (2006) with reference to some key examples where

protease drugs have succeeded or failed.

Proteases have found by now a wide range of applications in various industries such as food,

pharmaceutical, cosmetic, etc. and have been widely commercialised by various companies

throughout the world. Though, the production of these enzymes has improved significantly

by the utilization of hyper-producing strains of fungi and bacteria and genetically modified

microbes as well, efforts are still being done to find newer sources of enzymes, better

production techniques and novel applications of these enzymes in unexplored fields. Hence,

proteases from thermophilic microorganisms are being exploited extensively to be used

industrially, since they can withstand high pH and are highly thermostable.

2.9 General Consideration

Several proteases were reported to be produced by different strains of T. vulgaris (Behnke et

al., 1978a,b; Desai and Dhala, 1969; Kleine and Rothe, 1977; Stepanov et al., 1980; Taufel

34

et al., 1979), showing their optimal proteolytic activity at 55◦C (Demidyuk et al., 1997;

Leuchtenberger et al., 1979). The substrate specificity and properties of partially purified

thermitase from T. vulgaris were studied by Behnke et al. (1978b). Among the protein

substrates tested, urea denaturated hemoglobin was hydrolysed best, followed by gelatin,

casein, field bean protein, serum albumin and gluten. The most intense splitting effect on a

variety of substrates, insulin β -chain, casein and haemoglobin, was exerted by thermostable

protease (thermitase, EC 3.4.21.14), suggesting the unspecificity of this enzyme (Schalinatus

et al., 1979, 1983b).

Thermitase’ is an alkaline serine protease of T. vulgaris, which is an extracellular endopeptidase

that is reportedly well suited for use in the food industry (Frommel et al., 1978; Frommel and

Hohne, 1981; Kleine, 1982; Kleine and Kettmann, 1982). It exhibited its temperature optimum

between 60◦C and 75◦C for esters and p-nitroanilides as substrates and at 90◦C for casein.

The enzyme was quickly inactivated at temperatures above 70◦C (Behnke et al., 1978a). This

enzyme binds calcium ions, which influence thermal stability (Frommel and Hohne, 1981).

Serine protease was found to be capable for efficient degradation of the insoluble proteins,

elastin and collagen (Kleine, 1982). The influence of chloromethyl ketones and methyl ketones

of N-acylated peptides on the thermal denaturation of thermitase was found to be affected

by Ca2+ (Schreier et al., 1984). The substrate specificity of a thermostable serine protease

from T. vulgaris, with several oligo- and polypeptide substrates was investigated by Bromme

and Kleine (1984). The nucleotide sequence of Thermoactinomyces sp. 27a containing the

metalloproteinase gene was determined (Zabolotskaya et al., 2004). A thermostable neutral

protease from Thermoactinomyces thalpophilus has been purified (Obido and Obi, 1988).

The kinetic parameters (Km and kcat) and the proteolytic coefficients for thermitase were

studied by Rothe et al. (1982).

In our laboratory also lot of work has been carried on the catalytic properties of proteases

isolated from T. vulgaris. The extracellular protease, a thermophilic metalloenzyme, exhibits

its optimal catalytic activity at 65◦C, and 60-65◦C in the absence and in the presence of

an activatory divalent cation (Mn2+), respectively. For its high-temperature catalysis, Mn2+

increased both slope and Arrhenius energy of activation (EA), suggesting thereby that this

divalent cation enhanced the rate of enzyme catalysis at the expense of EA. Mn2+ increased

Vmax of the enzyme without changing the Km for the substrate. The thermal inactivation

profiles of protease suggested that, although this enzyme was thermophilic in nature, it was

35

found to be thermolabile, as it was not completely stable even at the growth temperature of

the organism (i.e., at 50◦C of pretreatment). This was further supported by the observation

that, in the absence of protein synthesis (i.e., in the presence of chloramphenicol added in

the culture medium at 8 h of growth), a decrease in its specific activity was observed as

compared to control at subsequent hours of growth. Thus, T. vulgaris possesses thermolabile

protease, which is constantly replenished as a result of their rapid resynthesis or rapid

turnover. The extracellular protease of T. vulgaris showed, in all, four isoenzymes (ProI,

ProII, ProIII and ProIV), which exhibited their differential expression in the wild-type and

auxotrophic mutant strains of this thermophilic bacterium (Kedia, 2009).

36

MATERIALS AND METHODS

3.1 Microorganism Used and Culture Conditions

Figure 3.1: Sporulating aerial mycelium of wild-type (strain 1227) of T. vulgaris observedunder scanning electron microscope (X 4000).

A wild-type (strain 1227) of Thermoactinomyces vulgaris (Fig. 3.1) and its mutant derivatives

(strains 1261, 1278, 1279 and 1286) were used in the present investigation. These strains

were kindly supplied by Professor D. A. Hopwood, John Innes Institute, Norwich, U.K.

These strains were grown on the media described by Hopwood and Wright (1972) with

certain modifications as described by Singh (1980). The basic composition of the medium

per litre was: NaNO3 – 2 g ; KCl – 0.5 g ; MgSO4 – 0.5 g ; Sodium β -glycerophosphate

(C3H7O6PNa2) – 0.5 g ; FeSO4 – 0.01g ; Sucrose – 30 g ; Agar – 15 g. 6.0 g of casein

hydrolysate was added to the minimal medium, supplemented with the growth factors required

by the wild-type strain (1227) auxotrophic mutant strains of T. vulgaris described in table

3.1 and made upto 1 litre with distilled water. The pH of the medium was adjusted to 6.8.

Hopwood’s medium (30 ml each) was taken in a 250 ml conical flask and autoclaved at 15

lb/inch2 for 15 minutes.

37

Table 3.1: Growth factor requirements of different strains of Thermoactinomyces vulgaris.

Strains Characteristics Growth factor(s) requirement

1227 nic+thi+ura+strs Not required (prototroph)

1261 nic−thi−strr Nicotinamide (1µg/ml), thiamine (1µg/ml)

1278 thi−ura−strr Thiamine (1µg/ml), uracil (10µg/ml)

1279 nic−ura−strr Nicotinamide (1µg/ml), uracil (10µg/ml)

1286 thi−strs Thiamine (1µg/ml)

Streptomycin sulphate powder (Ambistryns) was added to the growth medium at a concentration

of (25 µg/ml) in the case of streptomycin-resistant strains (strr). All the cultures were

incubated at 50-52◦C for 24 hours.

Normal saline (8.7 g NaCl dissolved in 1000 ml of distilled water) was used to suspend

the spores. The suspension was shaken on a vortex mixer to break the chains and filtered

through sterilized double layered cheese cloth to get rid of mycelial debris. Spore density was

determined with the help of haemocytometer. Approximately 1×109 spores were inoculated

in 30 ml of minimal liquid medium contained in a 250 ml conical flask. The incubation

was done in a rotatory shaker at an optimum temperature of 50◦C at 150 revolutions per

minute. After optimum incubation, the culture medium was centrifuged at 10,000 rpm for

30 minutes. The supernatant was collected and used to check and quantify extracellular

protease activity.

3.2 Assay for Protease Activity in T. vulgaris Strains

The protease activity test in the cultures of both wild-type and mutant strains of T. vulgaris

were determined, through plate assays and spectrophotometrically by the method of Folin

and Ciocalteu (1929).

38

Enzyme solution (1 ml)

+

1% casein solution (2 ml) in 50 mM potassium phosphate buffer (pH 7.5)

↓Mixed by swirling and incubated at 50◦C for 30 minutes

↓Trichloroacetic acid (TCA) reagent (3 ml of 20% TCA) added and kept for 10 minutes at

room temperature

↓Centrifuged at 10,000 rpm for 15 minutes

↓Supernatant (1 ml) used as test filtrate

↓Sodium carbonate solution (2.5 ml of 0.5 M) was added

↓Folin and Ciocalteu’s phenol reagent (1 ml of 5 times diluted) was added for development

of colour

↓Absorbance read at 660 nm

3.3 Screening of Thermoactinomyces vulgaris (1227) for the Production of Extracellular

Protease with respect to Substrate Utilization

Protease activity was determined, using 1% of casein, Bovine serum albumin (BSA), gelatin

and skimmed milk, as a substrate in Hopwood’s medium (Hopwood and Wright, 1972).

Modified sterilized Hopwood’s agar medium was poured on sterile Petri-plates and allowed

to solidify at room temperature. It was then inoculated with wild-type (1227) strain of T.

vulgaris and incubated at 50-52◦C for 24 and 48 h. The formation of a halo zone surrounding

the colony was considered to be a positive test for protease production.

39

3.4 Effect of Incubation Period on the Production of Extracellular Protease

The effect of incubation period on the production of extracellular proteases in T. vulgaris

(wild-type strain, 1227) was studied by assaying the enzyme as described in section 3.2 at

different incubation periods (i.e., 4, 8, 12, 16, 20, 24, 28 and 32 hours).

3.5 Effect of pH on the Production of Extracellular Protease

The effect of pH on the production of extracellular proteases in T. vulgaris (wild-type strain,

1227) was studied by assaying the enzyme after 16 hours of incubation at varying range of

pH (i.e., at pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0).

3.6 Effect of Temperature on the Production of Extracellular Protease

The effect of temperature on the production of extracellular proteases in T. vulgaris (wild-type

strain, 1227) was studied by assaying the enzyme at pH 7.0 after 16 hours of incubation

period in the culture medium at varying temperatures (i.e., 40, 45, 50, 55, 60 and 65◦C).

3.7 Effect of Carbon Sources on the Production of Extracellular Protease

The effect of different carbon source on the production of extracellular proteases in T.

vulgaris (wild-type, 1227) was studied by assaying the enzyme after 16 hours of incubation,

at temperature of 50◦C, pH 7.0 and 1% (w/w) of various carbon sources (i.e., Dextrose,

Starch, Sucrose, Lactose, Trisodium Citrate and Citric Acid).

3.8 Effect of Metal Ions on the Production of Extracellular Protease

The effect of different metal ions on the production of extracellular proteases in T. vulgaris

(wild-type strain, 1227) was determined by the addition of each of the corresponding ion at

a final concentration of 10 mM to the culture medium, and the assay was performed under

standard conditions. The metal ions used were:

Ca2+ , Mn2+, Mg2+, Fe2+, Cu2+, Na2+ and Zn2+.

3.9 Comparison of Static and Shake Culture Conditions for the Production of Protease

in Five Strains of T. vulgaris

Protease production in static and shake (150 rpm) cultures was determined by using T.

vulgaris wild-type (1227) and its mutant strains (i.e., 1261, 1278, 1279 and 1286). The

culture media in both conditions were incubated at temperature 50◦C and pH 7.0 for 16 h.

The assays were performed under standard conditions.

40

RESULTS AND DISCUSSION

Generally, proteases produced from microorganisms are constitutive or partially inducible

in nature (Beg et al., 2002; Kalisz, 1988). Most of the Bacillus sp. produce extracellular

proteases during post exponential and stationary phases (Razak et al., 1997). Extracellular

protease production in microorganisms is influenced by media components e.g. variation

in C/N ratio, presence of some easily metabolizable sugars, such as glucose (Beg et al.,

2002), and metal ions (Varela et al., 1997). Protease synthesis is also affected by rapidly

metabolizable nitrogen sources, such as amino acids in the medium. Beside these, several

other physical factors, such as aeration, inoculum density, pH, temperature and incubation

period also affect the protease production (Hameed et al., 1999; Puri et al., 2002). Keeping

these factors in mind, the production of extracellular proteases in the wild-type strain (1227)

of Thermoactinomyces vulgaris was optimised in the present study.

4.1 Screening of T. vulgaris (strain 1227) for the Production of Extracellular Protease

with respect to Substrate Utilization

Based on the zone of hydrolysis, screening for protease producing ability of T. vulgaris

was tested in the Hopwood’s medium containing different substrates such as casein, BSA,

gelatin and skimmed milk (Fig. 4.1). It was observed that protease of T. vulgaris (1227)

used casein most efficiently as a substrate, followed by skimmed milk. BSA and gelatin

were not used as a substrate by proteases of T. vulgaris, as no zone of hydrolysis was

observed. Secreening of protease activity in Bacillus licheniformis RP1 by Sellami-kamoun

et al. (2008) was done using skimmed milk as substrate, but enzyme assay for its activity

was done using casein as a substrate. Similar results were reported in Thermoactinomyces

sp. E79, where casein was used as a substrate (Lee et al., 1996).

41

Figure 4.1: Culture plates showing control (A1 & A2) and zone of hydrolysis of proteaseproduced by T. vulgaris (1227) using 1% of casein (B1 & B2), skimmed milk (C1 & C2),BSA (D1 & D2) and gelatin (E1 & E2) as substrate after 24 h (A1, B1, C1, D1, E1) and48 h (A2, B2, C2, D2, E2) of growth at 50◦C.

4.2 Standard Curve of Tyrosine

Figure 4.2: Standard curve of tyrosine

4.3 Effect of Incubation Period on the Production of Extracellular Protease

Protease production was determined at different incubation periods. Maximum protease

production was obtained at 16 h of incubation in T. vulgaris (Fig. 4.3). Any prolongation

in incubation period decreased enzyme production.

Incubation period plays a substantial role in the maximum protease production. The optimum

incubation time for protease production in T. vulgaris was found to be 16 h at 150 rpm

and at 50◦C. Similar observations have been reported in Thermoactinomyces sp. E79, which

showed maximum protease production after 16 h at 250 rpm and at 50◦C (Lee et al., 1996);

whereas Bacillus sp. SSR1 gave maximum protease production after 18 h of incubation

at 150 rpm and at 40◦C (Singh et al., 2001a). Results of present study indicated that

production of protease was dependent on the bacterial cell growth. The cells of T. vulgaris

42

started multiplication within 5 h of incubation and reached to the maximum growth in 9 h

(Singh, 1980) under shake culture conditions. The mycelial growth remained high upto 9 h

and thereafter, mycelia disintegrated. It is clear from Figure 4.3 that the maximum enzyme

production was obtained during post exponential and stationary phase i.e. after 16 h of

incubation and thereafter, bacterial growth stopped and sporulation started due to depletion

of carbon source and other nutrients.

Figure 4.3: Protease production by T. vulgaris at varying incubation periods.

The proteases are known to be associated with the onset of stationary phase, which is a

transition state from vegetative growth to sporulation stage in spore-formers. Therefore,

protease production is often related to the sporulation stage in many bacilli such as Bacillus

subtilis (O′Hara and Hageman, 1990) and Bacillus licheniformis (Hanlon and Hodges, 1981).

On the contrary, a few reports also suggested that sporulation and protease production,

although co-occuring were not related, as spore deficient strains of Bacillus licheniformis were

43

not protease deficient (Fleming et al., 1995). It is also established that protease production

and sporulation are two independent events in stationary phase (Khan, 2000).

4.4 Effect of pH on the Production of Extracellular Protease

The effect of pH on the production of crude protease enzyme was investigated; and it was

observed that the protease production was found to be maximum at pH 7.0. The production

decreased significantly at pH above 7.5 and pH below 6.5 (Fig. 4.4).

Figure 4.4: Protease production by T. vulgaris at varying pH values.

Similar to our study, Hameed et al. (1999) optimised the production of protease at pH 7.0 in

Bacillus sp. K2. Similar results have also been reported in Bacillus mojavensis (Beg et al.,

2002), Bacillus sp. RGR-14 (Oberoi et al., 2001; Puri et al., 2002) and Bacillus licheniformis

44

ATCC 21415 (Mabrouk et al., 1999). The other prokaryotes like Thermoactinomyces sp.

E79 (Lee et al., 1996) and Streptomyces NCIB 10070 (Yeoman and Edwards, 1997) also

exhibited maximum protease production at pH 7.0. As T. vulgaris grows optimally at pH

7.0, so we can correlate the protease production with growth. At high pH values (i.e. above

pH 7.5), the inactivation of protease occurs probably due to the accumulation of metabolites.

Because of the optimal protease activity at pH 7.5, this enzyme in T. vulgaris is classified

as neutral protease, which might play an important role in enzyme industry (Kedia, 2009).

4.5 Effect of Temperature on the Production of Extracellular Protease

Figure 4.5: Protease production by T. vulgaris at varying temperatures.

The effect of critical physical parameter, temperature was investigated on the production of

proteases in T. vulgaris (strain 1227) at varying temperatures. The optimal growth temperature

45

was found to be 50◦C for protease production, but considerable enzyme production was

observed at 45◦C (Fig. 4.5).

My findings are similar to those obtained for maximum protease production (at 50◦C) in

Thermoactinomyces sp. E79 (Lee et al., 1996) and Bacillus mojavensis (Beg et al., 2002).

Whereas, in case of Bacillus subtilis PE-11, the optimum temperature was found to be 60◦C

(Pastor et al., 2001). Subsequently, 37 and 30◦C were reported to be the best temperatures

for protease production in certain bacilli (Gupta et al., 2002b). Recently Boominadhan et

al. (2009) have reported 50◦C to be the temperature optimum for protease production in B.

subtilis, B. amyloliquefaciens, B. megaterium and B. licheniformis.

As T. vulgaris also grows optimally at 50◦C, so I have good reason to correlate its growth

with protease production at this temperature. Thermostable protease from T. vulgaris can

withstand temperature upto 65◦C (Kedia, 2009). Therefore, this enzyme can be exploited

for diverse industrial applications.

4.6 Effect of Carbon Sources on the Production of Extracellular Protease

Different carbon sources (1% w/v) each of sucrose, starch, lactose, dextrose, trisodium

citrate and citric acid were employed in the study to determine growth and production of

extracellular protease in T. vulgaris (strain 1227). The basal production medium had sucrose

as a carbon source for protease production. When sucrose was replaced by various other

carbon sources, starch was found to be an effective carbon source, followed by dextrose

for protease production (Fig. 4.6). T. vulgaris utilized all these carbon sources for growth,

except trisodium citrate and citric acid.

In the present study, the best carbon source for extracellular protease production and growth

was starch. Although there was a good growth in case of dextrose, lactose and sucrose, but

the protease production was comparatively less (Fig. 4.6).

Starch as best carbon source in protease production has also been reported in Thermoactinomyces

sp. E79 (Lee et al., 1996), Bacillus sp. RGR-14 (Oberoi et al., 2001; Puri et al., 2002), B.

licheniformis and B. coagulans (Asokan and Jayanthi, 2010). In thermophilic Bacillus sp.

strain SMIA-2, starch was the best carbon source, followed by trisodium citrate, citric acid

and sucrose in the protease production (Nascimento and Martins, 2004). In some Bacillus

species such as B. subtilis (Boominadhan et al., 2009), B. mojavensis (Beg et al., 2002),

Bacillus sp. P-2 (Kaur et al., 2001), B. sphaericus (Singh et al., 2001b) and Bacillus sp.

46

IE-3 (Purva et al., 1998), glucose was found to be the best carbon source for protease

production. On the other hand, lactose in Bacillus brevis MTCC B0016 (Banerjee et al.,

1999) and Bacillus sp. SSR-1 (Singh et al., 2001a), and sucrose in Serratia marcescens

ATCC 25419 (Romero et al., 2001), were reported as a good carbon sources for protease

production. In B. amyloliquefaciens and B. megaterium, fructose had been reported as the

best carbon source for protease production (Boominadhan et al., 2009). Hence carbon source

plays an important role in growth as well as protease production.

Figure 4.6: Effect of different carbon sources (1% each) on protease production by T.vulgaris.

47

4.7 Effect of Metal Ions on the Production of Extracellular Protease

The effect of different metal ions (10 mM each) on the production of extracellular protease in

T. vulgaris (strain 1227) was investigated. Cu2+ appeared to significantly (2.5 fold) enhance

the protease production in T. vulgaris (Fig. 4.7).

Figure 4.7: Effect of metal ions on the production of proteases by T. vulgaris.

The supplementation of the culture medium with a solution of metal traces improved

substantially the protease production in T. vulgaris (Fig. 4.7), thereby indicating the

requirements of some metal ions for protease production by this thermophilic bacterium. This

can be supported by earlier findings of metal ions enhancing the activity of proteases (Janssen

et al., 1994). Ferrero et al. (1996) reported the use of trisodium citrate alongwith MgSO4,

CaCl2, MnSO4 and ZnSO4 for increased protease production by Bacillus licheniformis MIR

29. Nascimento and Martins (2004) observed that protease produced by Bacillus sp. was

enhanced by Mn2+and Ca2+. Ca2+ is known to play a major role in enzyme stabilization

48

by increasing the activity and thermal stability of alkaline proteases at higher temperature

(Kumar, 2002; Lee et al., 1996). Other metal ions, such as Ba2+, Mn2+, Mg2+, Co2+,

Zn2+ and Fe3+are also used for stabilising proteases (Johnvesly and Naik, 2001). Metal

ions Mg2+, Fe3+ and Cu2+, enhancing the activity of alkaline protease (pH 10.0) from

the fungus Microsporum canis (JNU-FGC#503) has been recently reported by Gupta et al.

(2010). These metal ions protect the enzyme against thermal denaturation and play a vital

role in maintaining the active conformation of the enzyme at higher temperatures.

4.8 Comparison of Static and Shake Culture Conditions for the Production of Protease

in Different Strains of T. vulgaris

Figure 4.8: Protease production under static and shake culture conditions in different strainsof T. vulgaris.

49

A comparative analysis of protease production was done under static and shake culture

conditions (50◦C, pH 7.0 and 16 h of incubation) in the wild-type strain 1227 and four

auxotrophic mutant strains (i.e., 1261, 1278, 1279 and 1286.

The protease production was more in the shake culture as compared to static culture conditions

in wild-type strain1227 of this bacterium and its mutant strains (1261, 1278, 1279 and 1286)

(Fig. 4.8). This might be due to the fact that under static conditions, the quantity of dissolved

O2 in the culture broth is smaller and inhibits aerobic Thermoactinomyces. (Singh, 1980).

As shown in Figure 4.8, the mutant strains 1278, 1279 and 1286 of T. vulgaris exhibited

a huge difference in protease activities under static and shake culture conditions. These

three strains thus appear to be efficient candidates for industrial production of protease by

employing shake cultures. And, out of all these three strains, strain 1286 was found to

be the best candidate for extracellular protease production, and hence it need to be further

investigated in detail.

50

CONCLUDING REMARKS

The proteases are essential for all life forms, including prokaryotes, fungi, animals and

plants. Microbial proteases are the most important hydrolytic enzymes. Microorganisms

have a large array of proteases, which can be divided into two categories: intracellular and

extracellular proteases. Intracellular proteases are important for various cellular and metabolic

processes, whereas extracellular proteases are important for the hydrolysis of proteins in

cell free environments. These extracellular proteases have been commercially exploited in

industries. Microorganisms account for a two third share of commercial protease production

in the world (Gupta et al., 2002b).

Thermostable proteases from thermophiles such as T. vulgaris are advantageous in some

applications, as higher processing temperature can be employed, resulting in faster reaction

rates, increase in the solubility of non gaseous reactants and products and reduced incidence of

microbial contamination by mesophilic organisms. Therefore, extracellular proteases secreted

by thermophilic bacteria are increasingly useful in a range of commercial applications (Singh

et al., 2001a). Bacilli mostly produce two groups of proteases i.e. alkaline and neutral

proteases (Rao et al., 1998).

The proteases from T. vulgaris are maximally active near neutral pH and hence they are

neutral proteases. Neutral proteases have relatively low thermotolerance (Barrett, 1994; Kedia,

2009). This property is advantageous for controlling their activity during the production of

food hydrolysates with a low degree of hydrolysis. Bacterial neutral proteases generate less

bitterness in hydrolysed food proteins than animal proteases, and hence are valuable for use

in the food industry (Rao et al., 1998). It has been reported that some neutral proteases

belong to the metalloprotease (E.C. 3.4.24.4) type and require divalent metal ions for their

activity (Brenner, 1998). In order to assess the utility of the bacterial proteases for industrial

use, it is desirable to have a search for new proteases with novel properties from as many

different sources as possible. That is why, the aim of present study was to optimise the

conditions for maximum production of extracellular protease from T. vulgaris.

In the present study, various parameters such as incubation time, pH, temperature, metal ions

and carbon source are optimised for maximum production of protease in T. vulgaris (1227).

Since T. vulgaris could be a potential source of thermophilic neutral protease production,

therefore these parameters and various other aspects related to these parameters need further

51

investigations.

The striking outcomes of the present investigation are:

1. Proteases of T. vulgaris (1227) used casein most efficiently as a substrate, followed

by skimmed milk; while BSA and gelatin were not used as a substrate by protease of

T. vulgaris.

2. Protease production in T. vulgaris (1227) was found to be maximum at pH 7.0, and

the production decreased significantly at pH above 7.5 and pH below 6.5.

3. The optimum growth temperature was found to be 50◦C for protease production in T.

vulgaris (1227), but considerable enzyme production was observed at 45◦C.

4. Incubation period of 16 h was found to be optimum for maximum protease production

in T. vulgaris (1227).

5. Starch is the best carbon source for protease production in T. vulgaris (1227).

6. Among various metal ions tested, Cu2+ appeared to significantly (2.5 fold) enhance

the protease production in T. vulgaris (1227).

7. As compared to wild-type strain 1227, mutant strains (1278, 1279 and 1286) were

more efficient in protease production; and among all the mutant strains tested, strain

1286 was most efficient under shake culture conditions.

The contention, that wild-type strains of most organisms are the best sources of industrial

enzymes, does not hold true, as in some cases (such as this), the mutants could be more

useful than the wild-type strain.

52

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CHAPTER 1

INTRODUCTION

CHAPTER 2

INTRODUCTION TO

THERMOACTINOMYCES VULGARIS

AND PROTEASES

CHAPTER 3

MATERIALS AND METHODS

CHAPTER 4

RESULTS AND DISCUSSION

CHAPTER 5

CONCLUDING REMARKS

REFERENCES