Girld Iind Typing Plant of Experiment

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Isolation, production and

Characterization of alpha-

amylase from Bacillus

amyloliquefaciens by UV

treatment.

A

Project Report

Submitted to

Gyani Inder in!h Institue

of

"rofessional tudies

Dehradun


2/57

By

Neeta Lohani

(B.Sc. Biotechnology)


3/57

Acknowledgement

ir!t o" all my heart"elt !alutation to the "eet o"

Almighty #od $ho be!to$ed upon me the !trength %eal a!

a re!ult o" $hich thi! arduou! ta!& ha'e been completed.

hrough thi! project * $ould li&e to e+pre!! my gratitude

to tho!e $ho ha'e ungrudgingly and pain!ta&ingly !pared

both time and energy "or me. ,y all prai!e goe! to

almighty $ho!e merci"ulne!! and bene'olence! gi"ted the

e'er caring parent! $ho!e prayer! and !o"tne!! ge!turema&e thi! $or& to reach it! "ruit"ul de!tination.

* con!ider it a! a great pri'ilege to ha'e e!teemed Dr.

Ra'indra -umar Re!earch Scienti!t lonegen

Biotechnology P't. Ltd. Noida a! my project ad'i!or. * ta&e

thi! opportunity to e+pre!! my !incere gratitude to him

$ho through con!tant ad'ice and con!tructi'e critici!mnouri!hed my intere!t in the !ubject and pro'ided a "ree

and plea!ant atmo!phere to $or& again!t all odd

!ituation!.

* o$n a hea'y debt o" gratitude to ,r. Amit Pandey

a! my Project incharge and ,!. -aranjot -aur #hai (Project

#uide) lonegen biotechnology P't. Ltd. Noida (/.P) are!cholar! a compendium o0 &indne!! and ma!ter in their

pro"e!!ion. * am proud o" getting increa!ing in!piration

throughout the cour!e o" my $or&. 1ithout their !upport it

$ould ha'e been impo!!ible "or me to pre!ent thi! project.

* am great"ul to ,r. ,anoj -umar 2erma Project

manager "or their guidance to$ard! thi! project $or& * am


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too than&"ul to Dr. Ajit Sri'a!ta'a Director "or hi! &ind

!upport or it.

* am highly indebted to my belo'ed parent! "or their

prayer! la!ting lo'e con!tant !upport and continued

encouragement rendered to me throughout the period o"

my !tudie! and li"e $ithout $hich my !tudy $ould not

ha'e been a reality.

La!t but not the lea!t * e+pre!! my !incere than&! to all

the 'olunteer! and !ubject! "or the 'aluable co3operation

in thi! project $or&.

Neeta Lohani


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Date March 07, 2010

CERTIFICATE

This is to certify that Ms. Neeta Lohani student of B.Sc. biotechnology of Gyani Inder

Singh Institute of professional Studies , Garhwal Uniersity, !ehradun has undergone

pro"ect entitled #Isolation, $roduction and %haracteri&ation of 'lpha( a)ylase fro)

Bacillus a)yloli*uefaciens by U+ treat)ent with us for a period of Three )onths.

The data shall not be published anywhere in any without the per)ission of !irector

cloneGen Biotechnology $t. Ltd-

Ms. Neeta Lohani is a sincere and diligen student. e wish her eery success in het

career.

Ravinder Kumar, Ph. D. A.K.Srivatava

Sr. /esearch Scientist $ro"ect Manager%lonegen Biotechnology $t. Ltd. %loneGen Biotechnology $t. Ltd

N0I!' N0I!'.


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Index

S.No. Contents Page No.

1. Index of Tables

2. Index of Graphs and figures3. General Instructions

4. Safet Instructions

!. Instru"entation

#. $bbre%iations

&. Introduction

'. (e%ie) of *iterature

+. ,aterial and "ethods

1-. Plan of xperi"ent 11. Procedure

Isolation

,edia

Gra" Staining

/ioche"ical test

Confir"ator test

Production

Characteri0ation

n0"e assa

Protein Isolation

SS

/radford assa

N$ isolation

$.G. PC(.

12. bser%ations and (esults

13. Conclusion

14. (eference


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INST(,NT$TIN

1 .IGIT$* IG5ING /$*$NC

Ma1i). /ange 234 g) 5 )in. range is 4.42 g). Turn on the balance 5 wait till

war)ing co)pleted 6war)ing light in off )ode7 5 Stable light is on then you can

weigh you stuff, "ust after weighing clean the pan, )achine table 5 put all the stuff to

their appropriate position in the Lab. 'lways use weighing paper to weigh your stuff,

Neer use news paper, alu)inu) foil or other loose sheets.

2 .$N$*6TIC$* IG5ING /$*$NC

Ma1i)u) range 34.4g). 5 )ini)u) range is 4.4442 g). e use this for +ery

)inute a)ount of stuff. Instru)ent should be turn on only for short ti)e during

weighing. 'lways use weighing paper to weigh you stuff.


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3. ,$GNTIC STI(((

It can be sued to get 8o)ogenous solution for preparation of culture )edia-

)olecular biology reagents. 9or continuous sha:ing-)i1ing it is ery helpful

instru)ent. Solidified culture )edia can also be )elted by using hot $late.

4. 7(T8 ,I8(

+orte1 )i1er is ery helpful instru)ent in )olecular biology lab for )i1ing of the

co)ponent, especially for test tubes and centrifuge tubes.


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!. p5 ,T(

To opti)i&e ph- physiological p8 of any solution, reagent, p8 )eter is helpful. Before

5 after use, glass electrode should be wiped and cleaned properly as any spot- pla*ue

on electrode )ay affect your result.

#. $TC*$7

'utoclae is used for sterili&ation of culture )edia, )olecular biology reagents.

Before using the instru)ent, one )ust chec: the water leel, )aterial to be sterili&ed,

:nob etc. sterili&ation )ust be )ini)u) of continuous 2; )inutes, ti)e )ay increase

depending upon the content olu)e of culture )edia etc.


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&. 5T $I( 7N

8ot 'ir oen is used to sterili&e glass wares 6li:e bea:er, conical flas:, test tubes,

$etri plates etc.7 - steel wares. 9or sterili&ation, te)perature should be 2 hrs. It can be used for dry heat treat)ent aboe nor)al

incubation ti)e i.e. =?4%.

'. *$,IN$( $I( 9*

9irst of all turn on blower for =4 )in. then turn off blower 5 close the door, turn on

U+ light for 2; )in. 'gain turn off U+ light, turn on blower and white light. %lean

carefully the wor:ing area and all the unnecessary ite)s, and only re*uired ite)

should be :ept in L'9. Before starting our e1peri)ent, wipe your hands with surface

disinfectants. Li:e ?4@ ethanol or sprit or =4@ acetic acid.


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+. /$CT(I*GIC$* INC/$T(

Bacteriological incubator proides us a continuous te)perature for growth 5

cultiation of different )icro organis)s. 0pti)u) growth te)perature for bacteria is

=;4% to A;4% 5 or fungi is 3;4% to =44%.

1-. S5$:( INC/$T(

e will use sha:er incubator for cultures in li*uid )edia i.e. broth culture. e can

opti)i&e growing te)p. by digital Te)p. regulator and speed by speedo)eter.

Sha:ing is necessary for proper growth of )icrobes in li*uid )edia. Before opening

the door, )a:e sure sha:er is off and inbuilt blower is on.


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11. $T( /$T5

9or thawing fro&en che)ical and presered cultures. It is necessary to :eep the) in

bacteriological water bath for 24(=4 )in. It can also be used to war)ing che)icals 5

reagent for te)p. range of =?4% to 2444%.

12. CNT(I9G ,$C5IN

Separation of Suspended particles in a solutions can be done y this instru)ent,

it acts on the principle of centrifuge force. !uring putting the sa)ple in %entrifuge

)achine it, should be properly balanced )eans no. of sa)ple should be een innu)ber.

ach centrifuge tube )ust contain e*ual a)ount and type of solution.


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13. $G$(S G* *CT(P5(SIS

lectronics of !N' of /N' can be done by 'G'/0S GL L%T/0$80/SIS

UNIT. %oncentration of 'garose will be fro) 4.?@ to 2;@ depending upon your

sa)ple.

14. SS P$G

lectrophoresis of protein is done by S!S $'G acryl a)ide solution with Tris %l,

S!S TM! 5 '$S is )i1ed in desired ratio 5 is pored in between two glass plates

for poly)eri&ation. $oly)eri&ation of resoling- separating Gel should be done first

followed by Sta:ing gel. 'fter loading 5 electrophoresis, gel is soa:ed staining

solution, again destain for obsering bands of protein using white light

Transillu)inator.


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1!. 7 T($NSI**,IN$T(

9or obsering bands of nucleic acid U+ T/'NSLLUMIN'T0/ are used for

isuali&ation, florescent dye 6thidiu) Bro)ide7 is used wither in gel or in sa)ple

depending upon nature of the sa)ple. By using the intensity :nob one can increase

the intensity of U+ light to obsering lighter 5 dar:er bands, depending upon the

sa)ple a)ount.

1#. 5IT *IG5T T($NSI**,IN$T(

9or obsering bands of proteins hite Light T/'NSLLUMIN'T0/ is used. 9or

isuali&ation, dye 6%oo)assie Brilliant blue /(3;47 is used, after staining and de

staining the gel.


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1&. SPCT(,T(

%alculating of concentration, absorbency at particular nano)eter of waelength

trans)itiity of any suspension- solution is do)e by S$%T/0MT/. Before

putting the sa)ple into the instru)ent, clean the cuets and should be :ept in proper

way. %ontrol and )onitoring of the instru)ent is done by an attached $% in which its

software is installed.

1'. PC(

Instru)ents )ust be connected with down(step as shown is figure in any slight

deiation in oltage )ay charge your result. $%/ reaction )i1ture is prepared 34@ in

e1tra re(distributed in separate $%/ tubes. $rogra) is set for particular e1peri)ent

according to your re*uire)ent and allowed to co)plete the cycles. 'fter $%/

'garose gel lectrophoresis is done in 2.4@ 'garose.


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1+. ,IC(PPTTS

e hae different range of )icropipettes 5 can be used according to our re*uire)ent.

'ppropriate olu)e to be ta:en is set by the :nob of the pipette and again set it )a1.

'fter use. Use sa)e )icro tips for sa)e solution and separate tips for separate

solution. Micro pipette should be in ertical position during pipetting.


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'BB/+'TI0NS

C5,IC$*S; ($GNTS

'$S '))oniu) per sulphate

BS' Boine seru) albu)in

%BB %oo)assie brilliant blue

!N' !eo1yribonucleic acid

!- !istilled ater

!NS =, ;(dinitro salicylic acid

dNT$s !eo1yribonucleotide phosphates

!T' thylene !ia)ine Tetra 'cetic acid

tBr thydiu) Bro)ide

Na%l Sodiu) %hloride

N'M Nutrient 'gar Mediu)

NB Nutrient Broth$BS $hosphate Buffer Syste)

S!S Sodiu) !odicyl Sulphate

T%' Tri chloro acetic acid

TM! N,NC, N Dtetra )ethylethylene dia)ine

T Tris !T'

T' Tris !T' 'cetic acid


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!"ITS# APPARAT!S

KD Kilo Dalton

Mins Minutes

ml Milli litres

O.D. Optical Density

Rpm Revolutions per minute

UV Ultra Violet

g Microgram

A.G.E. Agarose Gel Electrophoresis

PAGE Poly Acryl amie Gel Electrophotesis

P!R Polymerase !hain Reaction


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INT(CTIN

Before proceeding with the description of our ai)s and ob"ecties of the

e1peri)ents. I would li:e to briefly introduce first the Da)ylase and the need to

wor: on the). 'lpha ')ylases are e1tracellular endo en&y)es. It has been deried

fro) seeral fungi, yeasts, bacteria and actino)ycetes the )ost abundantly used

bacterial (a)ylases were deried fro) /acillus a"loli


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M*+ecu+ar -eiht/;3,444(;A,444 F! 6%o&&one et al. 2?47.

C*m*iti*n/ The en&y)e is a glycoprotein. Its single polypeptide chain of about

A?; residues has two S8 groups and four disulfide bridges and contains a tightly

bound %a3H. It e1ists in two for)s 6I and II7 which hae identical en&y)atic

properties, differing only in electrophoretic )obility. ' binding site for %l has been

reported which effects a confor)ational change that enhances actiity.

'timum &/?.4.

Seciicit/ 'lpha(a)ylase cataly&es the hydrolysis of internal alpha(2,A(glucan

lin:s in polysaccharides containing = or )ore alpha(2, A(lin:ed !(glucose units

yielding a )i1ture of )altose and glucose.

Inhi3it*r/ Urea and other a)ide reagents

Activat*r/ %l is essential.

Sta3i+i4er/ %alciu) and chloride ions are necessary for stability.

2%C(ASSIFICATI'"

'n am+ae is an en&y)e that brea:s starch down into sugar. ')ylase can be

classified into three groups 'lpha(a)ylase, Beta( a)ylase and Ga))a(a)ylase.

56AM)(ASE

The alpha a)ylase is calciu) )etalloen&y)e, co)pletely unable to function in the

absence of calciu). By acting at rando) location along the starch chain, alpha

a)ylase brea:s down long chain carbohydrates ulti)ately yielding )altotriose 5

)altose fro) a)ylase or )altose. Because it can act any where on the substrate 'lpha

a)ylase trends to be a faster acting than B(a)ylase. In ani)als it is a )a"or digestie

en&y)e. In hu)an physiology two type of a)ylase found one is saliary a)ylase and

pancreatic a)ylase, both ate (a)ylase.

6AM)(ASE

(')ylase is an other for) of a)ylase. It is also synthesi&ed by Bacteria, 9ungi and

$lants. or:ing fro) the non reducing end, J(a)ylase catalyse the hydrolysis of the

second ( 2, A D glycosidic bond, cleaing off two glucose units 6Maltose7 at a ti)e.

!uring the ripening the fruits, (a)ylase brea:s the starch into sugar. and (

a)ylase both are present in the seeds. (a)ylase present prior to ger)ination where

as ( a)ylase appears once ger)ination has begun. Many )icrobes also produce


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a)ylase to degrade the e1tra cellular starch. 'ni)als tissue do not contain (a)ylase,

although it )ay be present in )icroorganis).

6 AM)(ASEIt is also called the Glucon 2,A(( glucosidase, glucoa)ylase In addition to cleaing

the last(2,A(glucosidic lin:age at the non reducing end of a)ylase and a)ylopectin,

yielding glucose,(a)ylase can cleae (2,> glycosidic lin:age.

$8% APP(ICATI'"S 'F A6AM)(ASE

$a% Te9ti+e6 The use of en&y)e in te1tile industries is one of the )ost rapidly

growing fields in industrial en&y)ology. Starch has for a long ti)e been used as a

protectie gie of fibers in weaing fabrics. This is called si&ing. n&y)es are used to

re)oe the starch in a process called desi&ing. ')ylases are used in this process since

they do not har) the te1tile fibers.

$3% :a;in6 'lpha(a)ylase has been widely studied in connect with i)proed bread

*uality and increase shelf life. Both fungal and bacterial a)ylases are used. 0er

dosage )ay lead to stic:y dough to the added a)ount needs to be carefully controlled.

$c% Deterent6 ')ylases are used in detergents to re)oe starch based stains. They

hydrolyses gelatini&ed starch, which tends to stic: on te1tile fibers and bind other

stain co)ponents.

$d% :re-in6 In the )ashing process the en&y)e are liberated and )altose. 'lpha(

a)ylase can be used to help the hydrolysis, filtration and storage.

$e% Feed6 Intensie study to used en&y)es in ani)al feed stared in early


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RE


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sufficiently low substrate concentration, where transglycosylation and condensation

can be ignored. This )ethod was applied to ealuate the sub site affinities of Ta:e(

a)ylase '. Based on a reaction sche)e which inoles hydrolysis, transglycosylation

and condensation, the ti)e courses of the for)ation of arious products were

si)ulated, using the /unge(Futta(Gill )ethod. Good agree)ent with the

e1peri)ental results was obtained.

Growth of "acillus amyloli#ue$aciens TN 24> p'T; and synthesis of

plas)id(encoded protein 6alpha ( a)ylase7 are inestigated in batch, continuous, and

fed(batch cultures using a defined )ediu) containing glucose and-or starch as the

carbohydrate source. The batch culture studies reeal that reduced aailability of

arginine ha)pers growth of reco)binant cells 6which lac: an arginine synthesis gene

but pro)otes production of alpha(a)ylase and substitution of glucose by starch as the

carbohydrate source leads to slower growth of reco)binant cells and increased

production of alpha( a)ylase per unit cells )ass. /etention of reco)binant cells oer

prolonged periods in continuous cultures is not possible without continues application

of antibiotic selection pressure owing to segregational plas)id instability. 9ed(batch

e1peri)ents with constant olu)etric feed are de)onstrate that alpha( a)ylase

production is enhanced at lower feed concentration of starch 6ole carbohydrate

source7 and lower olu)etric feed rate. Such slow addition of starch is howeer not

conducie for growth of reco)binant cells. The e1pression of the ther)ostable alpha(

a)ylase gene carried on the reco)binant plas)id p'T; 6deried fro) a plas)id

isolated fro) a ther)ophilic bacteriu)7 is pro)oted at higher te)perature, while

growth of reco)binant cells is depressed. In all batch and fed(batch e1peri)ents,

production of alpha( a)ylase is obsered to be inersely related to growth of

reco)binant cells. The efficacy of two(stage bioreactor operations, with growth of

reco)binant cells being pro)oted in the first stage and alpha Da)ylase production in

the second stage, in attaining increased bul: alpha Da)ylase actiity is de)onstrated.

The idea for the gene cloning techni*ue first arose in Noe)ber 2?3 at a

scientific )eeting 8onolulu. %ohen who had been studying plas)ids was intrigued by

boyerCs presentation on bacterial en&y)es which cut at specific sites in the !N'

)olecule. 0n a late eening e1cursion to a delicatessen in ai:i:i, the two scientific

tal:ed about a collaboration co)bining their areas of scientific e1pertise.

Molecular cloning refers to the procedure of isolating a defined !N'

se*uence and obtaining )ultiple copies to it in io. %loning is fre*uently e)ployed


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to a)plify !N' frag)ents containing genes, but it can be used to a)plify any !N'.

It is utili&ed in a wide array of biological e1peri)ents and practical application such

as large scale protein production, occasionally, the ter) cloning is )isleadingly used

to refer to the identification of the chro)oso)al location of a gene associated with a

particular phenotype of interest, such as in positional cloning. In practice, locali&ation

of the gene to a chro)oso)e or geno)ic region does not necessarily enable one to

isolate or a)plify the releant geno)ic se*uence. In essence, in order to a)plify any

!N' se*uence in a liing organis), that se*uence )ust be lin:ed to an origin of

replication, a se*uence ele)ent capable of directing the propagation of itself and any

lin:ed se*uence. In practice, howeer, a nu)ber of other features are desired and a

ariety of speciali&ed cloning ector e1ist that allow protein e1pression, tagging

single stranded /N' and !N' production and a host of other )anipulations.

%loning of any !N' frag)ent essential inoles four steps frag)entation,

legation, transfect ion and screening-selection. 'lthough these steps are inariable

a)ong cloning procedure a nu)ber of alternatie routes can be selection. 'lthough

these steps are inariable a)ong cloning procedure a nu)ber of alternatie routes can

be selected, these are su))ari&ed as a Ocloning strategyC.

Initially, the !N' of interest needs to be isolated to proide !N' seg)ents of

suitable si&e. Subse*uently, a legation procedure is used where the a)plified frag)ent

is inserted into a ector. The ector 6which is fre*uently circular7 is line raised using

restriction en&y)es, and incubated with the frag)ent of interest under appropriate

condition with en&y)es called !N' lipase. 9ollowing legation the ector with the

insert of interest of interest is transected into cells. ' nu)ber of alternatie techni*ues

are aailable, such as che)ical sensiti&ation of cells, electro oration and ballistics.

9inally, the transected cells are cultured. 's the afore)entioned procedures are of

particularly low efficiency, there is a need to identify the cells that hae been

successfully transected with the ector construct containing the desired insertion

se*uence in the re*uired orientation. Modern cloning ectors include selectable

antibiotic resistance )ar:ers, which allow only cells in which the ector has been

transected, to grow. 'dditionally, the cloning ectors )ay contain color selection

)ar:ers which proide blue-white screening 6(factor co)ple)entation7 on P(gal

)ediu). Neertheless, these selection steps do not absolutely guarantee that the !N'

insert is presently in the cells obtained. 9urther inestigation of the resulting colonies


25/57

is re*uired to confir) that cloning was successful. This )ay be acco)plished by

)eans of $%/, restriction frag)ent analysis and-or Molecular cloning refers to the

procedure of isolating a defined !N' se*uence and obtaining )ultiple copies of it in

io. %loning is fre*uently e)ployed to a)plify !N' frag)ents containing genes,

but it can be used to a)plify any !N' se*uence such as pro)oters, non(coding

se*uences and rando)ly frag)ented !N'. It is utili&ed in a wide array of biological

e1peri)ents and practical applications such as large scale protein production.

0ccasionally, the ter) cloning is )isleadingly used to refer to the identification of the

gene to a chro)oso)e or geno)ic region does not necessarily enable one to isolate or

a)plify the releant geno)ic se*uence.

In essence in order to a)plify any !N' se*uence in a liing organis), that

Se*uence )ust be lin:ed to an origin of replication, a se*uence ele)ent capable of

direction the propagation of itself and any lin:ed se*uence. In practice, howeer, a

nu)ber of other features are desired and a ariety of speciali&ed cloning ectors e1ist

that allow protein e1pression, tagging, single stranded /N' and !N' production and

a host of other )anipulations.

%loning of any !N' frag)ent essentially inoles four steps frag)entation, legation,

transfect ion, and screening selection. 'lthough these steps are inariable a)ong

cloning procedures a nu)ber of alternatie routes can be selected, these are

su))ari&ed as a Ocloning strategyC.

Initially, the !N' of interest needs to be isolated to proide a !N' seg)ent of

suitable si&e. Subse*uently, a legation procedure is used where the a)plified frag)ent

is inserted into a ector. The ector 6which is fre*uently circular7 is linearised using

restriction en&y)es, and Incubated with the frag)ent if interest under appropriate

conditions with an en&y)es called !N' ligase. 9ollowing ligation the ector with the

insert of interest is transfected into cells. ' nu)ber of alternatie techni*ues are

aailable, such as che)ical sensitiation of cells, electroporation and biolistics.

9inally, the transfected cells are cultured. 's the afore)entioned procedures are of

particularly low efficiency, there is a need to identify the cells that hae been

successfully transfected with the ector construct containing the desired insertion

se*uence in the re*uired orientation. Modern cloning ectors include selectable

antibiotics resistance )ar:ers, which allow only cells in which the ector has been

transfected, to grow. 'dditionally, the cloning ectors )ay contain color selection

)ar:ers which proide blue-white screening 6a factor co)ple)entation7 on P(gal


26/57

Mediu). neertheless, these Selection steps do not absolutely that the !N' insert is

present in the cells obtained 9urther inestigation of the resulting colonies is re*uired

to confir) that cloning was successful. This )ay be acco)plished be )eans of $%/,

restriction frag)ent analysis and-or !N' se*uencing.

The ector itself is generally a !N' se*uence that consists of an insert

6transgene7 and a larger se*uence that seres of the #bac:bone of the ector. The

purpose of a ector to transfer genetic infor)ation to another cell is typically ti

isolate, )ultiply, or e1press the insert in the target cell. +ectors called e1pression

ectors e1pression constructs7 specifically are for the e1pression of the transgene in

the target cell, and generally hae a pro)otors se*uence that dries e1pression of the

transgene. Si)pler ectors called transcription ectors are only capable of being

transcribed but not translated they can be replicated in a target cell but not e1pressed,

unli:e e1pression ectors. Transcription ectors are used to a)plify their insert.

To study genes in the laboratory, it is necessary to hae )any copies on hand

use as sa)ple of different e1peri)ents. Such e1peri)ents include southern or norther

blots, in which genes labeled with radioactie or florescent che)icals are used as

probes for detecting specific genes that )ay be present in co)ple1 )i1ture of !N'.

%loned genes also )a:e it easier to study the protins they encode. Because the genetic

code of bacteria is identical to that of eu:aryotes, a cloned ani)al or plant gene that

has been introduce in to a bacteriu) can often direct the bacteriu) to produce its

protrin product, which can then be purified and used for bioche)ical e1peri)entation.

%loned genes can also be used for !N' se*uencing which is the !eter)ination of the

precise order of all the base pairs in the gene. 'll of these applications re*uire )any

copies of the !N' )olecule that is being studied.

Bacilli are :nown to secrete a nu)ber of en&y)es into the e1tracellular )ediu). 0ne

of the )a"or classes of these e1tracellular )ediu). 0ne of the )a"or classes of these

e1tracellular en&y)es are a)ylases. Both alpha(a)ylases and beta(a)ylases

hydroly&e alpha %I%(0(%A bonds. 8ow eer, alpha(a)ylases and beta(a)ylases are

distinguished by different en&y)atic action patterns obsered in the hydrolysis of

starch.

!uring the early stages of starch digestion, alpha(a)ylases split starch essentially at

rando) . The products of the hydrolysis are alpha()altose, alpha(glucose, and alpha(

li)it de1trins. The alpha(a)ylases are endoen&y)es that can bypass the alpha(2,>


27/57

branch points of a)ylopectin. Their action results in a rapid decrease in iscosity

6li*uefaction7 and loss of iodine(binding color 6de1trinogenic capacity7.

In contrast, beta(a)ylases are e1oen&y)es that re)oe )altose stepwise fro) the

nonreducing end of starch. The products of this hydrolysis are beta()altose, beta(li)it

de1trins, and ery s)all a)ounts of beta(glucose 6for)ed by the degradation of

a)ylase )olecules containing an odd nu)ber of sugar residues7.Beta(a)ylases do not

hydroly&e a)ylopectin internally to the alpha(2,>, branch points. !uring the early

stages of starch hydrolysis, beta(a)ylases generate relatiely large a)ounts of

)altose, as co)pared with the a)ounts of de1trin frag)ents liberated. That is, beta(

a)ylases are saccharifying en&y)es.

It should, howeer, be reali&ed that alpha(a)ylases are #partially saccharifying and

that beta(a)ylases are #partially li*uefying and de1trinogenic. Indeed, Bacillus was

once thought to produce two Types of alpha a)ylases, one li*uefying and the other

saccharifying. Based upon !N' hybridi&ation results, the organis) secreting en&y)e

has been rena)ed". amyloli#ue$aciens.

MATERIA(S A"D MET&'DS

$1% MICR':I'('>)/6

Chemica+# Item C*man :rand

Nutrient 'gar-Broth 8igh Media

peptone( ;.4 g

Na%l( ;.4 g

Beef 1tract( =.4g

'gar( 2;.4 g

!-( 2444 )l

Borer Borosil

'utoclae, 8ot air oen, Incubator Qarco8ori&ontal La)inar air flow cha)ber Qarco

!ry and %lean conical flac: Borosil

$etri $lates, Test Tube Borosil

Micro Tips, Micropipette Tarsons 5 Gilsons

Measuring %ylinder Borosil

$2% SDS PA>E/6

Chemica+ # Item C*man :rand

/esoling Gels-Stac:ing Gel

A4@ 'cryla)ide Biorad2 M Tris 8%l p8


28/57

24@ S!S Biorad

2@$ero1y !isulphate Biorad

TM! Biorad

2 M Tris 8%l p8 >.< Biorad

'$S Merc:

%oo)assie Brilliant Blue 6/(3;47 Sig)aS!S Gel lectrophoretic Unit Biorad

Micropipettes 5 Tips Gilson 5 Tarsons

!ry 5 %lean Test Tubes Borosil

$8% D"A IS'(ATI'" A"D A.>.E/6


Te Buffer 6Saline7 Ultrapure

24@ S!S Biorad

$roteinase : Bangalore Genie$henol chlorofor) )i1ture Spec:pure

!T' Ultra pure

Sodiu) acetate and Isopropanol Ultrapure

?4@ thanol RualigenCs

'garose Gel lectrophoretic Unit Biorad

'garose Gel(4.?@ Ultrapure

2P T'(Tris

(!T'

(Glacial 'cetic 'cid

spec:pure

spec:pure

spec:pure

tBr Biorad

/unning Buffer( 2P T' Spec:pure

>P Loading !ye(Pylene cyanol

(glycerol

(Bro)ophenol Blue

Nitrogen

$?% PR'TEI" ASSA) $:RADF'RD ASSA)%/6


Bradford /eagent ( Sig)a%oo)assie Blue G( 3;4

thanol ;4)l RualigenCs

$hosphoric 'cid 6


29/57


Micropipette, Tips Tarsons 5 Gilson

$% PCR/6

%he)icals-Ite)s %o)pany Brand

$%/ Unit

/eaction Mi1ture( Ta* $oly)erase

en&y)e

Bangalore Genei

( Ta* buffer

( dNT$s

( $ri)er6IB/%(?7

( !istilled ater$%/ Tubes

$@% E")ME ASSA)

Chemica+#Item C*man :rand

Spectrophoto)eter Unit Biorad


Micropipette, Tips Tarsons 5 Gilson

2@ Starch

!NS(!NS $owder$otassiu) Sodiu) Tartarate Ultra pure

sodiu) 8ydro1ide

!istilled ater

$BS( Sodiu) 8ydrogen 0rtho $hosphate Spec:pure

!isodiu) 8ydrogen 0rtho $hosphate

Sodiu) %hloride

ater Bath Unit


30/57

P(A" 'F EBPERIME"T

IS'(ATI'"/6

Isolation of". amyloli#ue$aciensfro) soil by

eria+ di+uti*n meth*d

Sreadin

6%ountable colonies occur in 24(A and 24(; dilutions7

Strea;in

6subculturing7

!ifferent isolated colonies were then chec:ed through

>ram Stainin

Gra) $ositie colonies were then sub"ected to

:i*chemica+ tet $Cata+ae Tet%

%onfir)atory test $Citrate Tet%was perfor)ed on

%atalade $ositie %olony

%itrate Negatie colony was then Strea:ed to

$roduce Pure Cu+ture


31/57

PR'D!CTI'" /6

Ta:en $ure %ulture

IN0%UL'T in Nutrient Broth

IN%UB'T for 3A(=> hours

ach plate is then treated under U+ continuously

Petri +ate n*. Time * !< Treatment

2. 4 )ins 6control7

3. = )ins.

=. A )ins.

A. ; )ins.

;. > )ins.

>. ? )ins

Incubate each plate at =?o% for A< hours

$repare > Nutrient Broth for > culture

Incubate at =?o% in sha:er for 3A hours


32/57

C&ARACTERIATI'" /6

Ta:e each culture and perfor) the

9ollowing techni*ues

Pr*tein

I*+ati*n

En4me Aa

6Ti)e para)eter

%onsidered7

D"A I*+ati*n

:rad*rd Aa Partia+

Puriicati*n

'garose Gel

lectrophoresis

PCR

'garose Gel

lectrophoresis

SDS 6 PA>E


33/57

PR'CED!RE

1% I*+ati*n */. a"loli


34/57

$lates were :ept for incubation for =(A days at =?o%

'fter this, nu)ber of colonies obsered on plates 6which show different

growing pattern 5 )orphology7, was pic:ed up 5 purified by trea;in on different

solidified agar plates.

$lates were then :ept for incubation for 3A(A< hours at =?o%

2. Media rearati*n

"utrient Aar Media $"AM%

S. "*. Inredient uantit

2. Beef(e1tract =.4g)

3. $eptone ;.4g)

=. Na%2 ;.4g)

A. 'gar 2;.4g)

;. !istilled ater 2444)l

6p8(>.


35/57

p8 was set to >. )ins.

>. ? )ins

*ncu)ate each petriplate at 02o! $or 5 hours


39/57

Prepare 1 +" $rom a)ove 1 cultures

Incubate at =?o% in sha:er incubator for 3A(=> hours

6These cultures are further used for their characteri&ation7


40/57

@% Characteri4ati*n * the !< treated/. a"loli


41/57

9or crude en0e"e "ethod

%a&e 6 ml o$ ". amyloli#ue$aciens culture

!entri$uge at 3444 rpm $or 3 mins.

%a&e supernatant >crude en0"e?

7%his is per$orme (ith each UV treate culture separately8

9ere en:yme activity is chec&e (ith time parameter.


42/57

1% En4me activit * the c*ntr*+ cu+ture.

S. "*.


43/57

8% En4me activit * the ? min. !< treated cu+ture.

S. "*.


44/57

;% En4me activit * the @ min. !< treated cu+ture.

S. "*.


45/57

>rah h*-in the ma9imum en4me activit in 8 min. !< treated

cu+ture.


46/57

$3% PR'TEI" IS'(ATI'"

Ta:e 2)l of B. a)yloli*uefaciens culture

%entrifuge at 23444 rp) for 2;)ins.

Ta:e the supernatant

%entrifuge at 23444 rp) for 2;)ins

Ta:e the supernatant $crude r*tein%6This is perfor)ed with each U+ treated culture separately7

8ere en&y)e actiity is chec:ed with ti)e para)eter

$A% :rad*rd Aa

6$rotein esti)ation7


47/57

SDS6PA>E

Princi+e / It is the Separation of protein, according to their charges as well as

)olecular weights. $roteins are a)photeric in nature.

$olyacryla)ide gels hae a s)all range of separation, but ery high resoling power.

Pr*cedure /

>e+ rearati*n

S. "*. C*m*nent * e+ 10m+ * earatin e+ m+ * tac;in e+

2. A4@ 'cryl a)ide gel =.2)l 4.;;)l

3. 24@ S!S 244l ;4l

=. Tris cl 3.; )l 6p8 .. 24@ '$S 244 )l A;4 l

Runnin 3uer G IB SDS D 4.3;M Tris cl

( 4.; M Glycine

( 2@ S!S

Stainin *+uti*n G 4.3@ %.B.B.dye 6%oo)assie Blue /(3;47 in )ethanol

ater glacial acetic acid 6A;A;247

Detainin *+uti*n G Methanol ater Glacial acetic acid 63;>;247

Sam+e / De H 3 2


48/57

Catin, +*adin runnin Gprepare separating gel

%ast it ?4@ in casting plate

Then fill it with !- to aoid bubble for)ation

's the gel solidifies, re)oe the water and add stac:ing

Gel and put co)b and allow it to solidify

Set the plate in the S!S electrophoretic unit and fill it with /unning buffer

Load the sa)ple and )ar:er and run at ?4 + for = hours

Transfer the gel into staining solution for oernight

Transfer the gel fro) staining to destaining solution for oer night

0bsere the gel under white light


49/57

:RADF'RD ASSA)

Prearati*n * :rad*rd reaent $:.R.%

Ta:e 3.; )l of %.B.B. !ye 6G(3;47

!issoled in ;)l of =. 3;4 3;4 23.; 3 4.24

?. =44 344 2;.4 3 4.3=3


50/57

Standard :SA c*nc. rah

Pr*tein Etimati*n * the am+e and it rah

S.

"*.

Suernatant

' !?l of T buffer

( =4 l of 24@ S!S

( = l of proteinase :

Incubate at =?o% for 2 hour

'dd 4.;)l of e*ual olu)e of phenol chlorofor) )i1ture

'nd )i1 gently

%entrifuge at 23444 rp) for 2;)ins

Ta:e top a*ueous layer and add 2-24tholu)e of sod. 'cetate

H 4.>(2 ol. of ice cold isopropanol

Incubate at 4% for half an hour-oernight

%entrifuge at 23444 rp) for 24)ins.

!iscard supernatant and to the pellet add 344l of ?4@ethanol

'nd )i1 gently

%entrifuge at 23444 rp) for 24)ins

!iscard supernatant and air dry the pellet

!issole pellet in T buffer 6!N' sa)ple7 and perfor) A.>.E.


52/57

A>AR'SE >E( E(ECTR'P&'RESIS $A.>.E.%

It is the separation of charge particles by )ass charge ratio.

$rocedure

Ta:e 4.=;g of 'garose 5 dissole in ;4)l 2 1 T'

8eat it in oen for =4(A4 second

%ool it to roo) te)p. and add 3l of tBr

Set the co)b and pour it in casting plate

's it solidifies re)oe the co)b and place it in the lectrophoresis unit and fill it with

2P T' running buffer

Load the sa)ple with > P loading dye in A2 ratio

/un at ?4+ for 2 8ours

0bsere in U+ Transillu)inator


53/57

P'()MERASE C&AI" REACTI'" $PCR%

This is for !N' a)plification.

Prearati*n * reacti*n mi9ture /

6 !N' sa)ple D 3l

6 Ta* poly)erase en&y)e D 4.; l

( Ta* Buffer D 3 l

( dNT$s D 3l

( $ri)er 6IB/%(?7 3l

( Ma:e up final ol. 34l with distilled water

Pr*cedure /

$repare reaction )i1ture for each sa)ple in $%/ tubes

$ut the) in $%/ and start the progra)

Pr*ram *++*- /

2. Initiation at Ao% for ; )ins.

3. !enaturation at Ao% for 2)in.

=. 'nnealing at =>(A3o% for 34(A4 seconds.

A. 1tension at ?3o% for 2)in.

;. Go to step D == ti)es.

>. 9inal e1tension at ?3o% for ; )ins.

?. 8old at Ao%

To chec: the a)plification of the sa)ple perfor)

'.G.. with 2@ 'garose


54/57

/S(7$TIN $N (S*TS

'ccording to the result basis, we got a result that shows after = )inutes U+ treat)ent

'lpha ')ylase production will be )ore.

n&y)e actiity in = )inutes U+ treated plate is )a1i)u) in en&y)e assay.

'lso in Bradford 'ssay, it shows that = )inutes U+ treated plate is haing )ole

protein content as co)pared to other U+ treated plates.

'fter !N' Isolation, it is the = )inutes U+ treated plate that show better bands whichget a)plified after $%/.


55/57

CNC*SIN

The aboe result can be concluded as follows

9inally, it has been concluded that the U+ treat)ent was able to induce so)e

)utations which resulted in increased 'lpha ')ylase production.

The )ost effectie U+ treat)ent was proed to be of = )inutes as it yielded the )ost

stable en&y)e.


56/57

(9(NCS

Nagara"an !/, /a"agopala) G/a"agopala), G Frishan %, depart)ent of

Biotechnology, India Institute of Technology Madras, %hennai >444=>, India.

$urification and !%%24=, characteri&ation of a )altooligosaccharide D for)ing

alpha(a)ylase fro) a new Bacillus subtilis, 'ppl Microbiol Biotechnology. 344> !ec

?= 6=7 ;2(?. pub 344> Kul 2.

hi , Song K, 0uyang 9. National Fey Laboratory of Bioche)ical ngineering,

Institute of $rocess ngineering, %hinese 'cade)y of Sciences, Bei"ing 2444=(, pub 344A No 2


57/57

cellular alpha D a)ylase fro) Bacillus subtilis 'P34, $rotein 1pr $urif. 344; Kun

A2 637 3A(;A. $MI! >AA22>3 $ubMed D Inde1ed for M!LIN.

!as F, !oley /, Mu:her"ee 'F, !epart)ent of Molecular Biology and

Biotechnology, Te&pur Uniersity, Te&pur ?

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Girld Iind Typing Plant of Experiment

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