MONTICA SAWANT13FET1007

INDUSTRIAL PRODUCTION OF

MICROBIAL ENZYMES

INTRODUCTION Enzymes have been used ever s ince mankind d iscovered ways to process food. Food processing steps l ike mi lk ac id ification, mi lk c lotting, a lcohol fermentation and soy bean fermentation are enzyme-mediated processes carr ied out by microorganisms. However, i t was not until the late 19th century that pur ified enzymes were used for food processing.

History : 1901: Eduard Bucher won the Nobel pr ize in b iochemistry for proving the ex istence of enzymes. Approx 20 years before, Chr istian Hansen invented and commerc ia l i zed a process to pur i fy rennet f rom cal f stomach which revolutionized the d iary industry. Marks Beginning of g lobal food enzyme industry.

Global sa les US$ 3.3 b i l l ion in 2010 Market leaders: Novozymes (Denmark) ,

Danisco (now DuPont) , DSM (Nether lands)

METABOLIC PROCESS

METABOLIC PROCESS

Substrates,Physical Parameters

Biomass,By productsEnzyme Synthesis,

Transcription,Translation,

Post translational processing

Metabolic Conversions

INPUT PARAMETERS

OUTPUT PARAMETERS

STRUCTURAL COMPONENTS

Regulatory Mechanism

The Regulatory Mechanism dictates the rate of enzyme synthesis from 0 to maximumREGULATORY MECHANISMS:• Simple Induction• Repression• Complex Global

regulation

The existence of different types of enzyme activityThe rapidity and stability of production through the inexpensive, reproducible, and safe microbial fermentation routeIncreased yield by genetic engineering. Stable under wide range of pH and temperatureBetter control over specificity (stereo specificity)Milder reaction conditionsEconomic feasibility

AllergenicExpensiveUnstableNeed for co-factors or co-enzymesLegal factors (GM enzymes limited acceptability)

ADVA

NTA

GES

DISADVANTAGES

PROS VS CONS

1• Screening• Choosing an appropriate micro-organism for the desired enzyme

2• Modification• Possible application of genetic engineering to improve the microbial strain

3• Laboratory Scale Pilot• To determine the optimum conditions for growth of micro-organism

4• Pilot Plant• Small scale fermenter to clarify optimum conditions

5• Industrial Scale Fermenter

BIOTECHNOLOGICAL PROCESS OF ENZYME PRODUCTION

Enzymes sold on the basis of activity

Species selection

High yield

Short fermentation period

Utilizes inexpensiv

e substrate

Reproducibility

GRAS

Safe and non-toxic

Extracellular

enzymes

Cost effective

FUN GI (A sperg i l lus & Tr ichode rma)

YEA STS (Saccharomyces , Hans enula , K luy veromyces)

BAC TER IA (E . co l i , Bac i l lus )

SPECIES SELECTION

METABOLIC ENGINEERING

C o n s t r u c t i o n o f p r o d u c t i o n s t r a i n i s a l o n g p r o c e s s i nv o lv i n g : c l a s s i c a l m u t a g e n e s i s , s c r e e n i n g f o r i m p r ove d p r o d u c t i o n & t a r g e te d d e l e t i o n o f u nw a n t e d g e n e sS t r a i n i m p r o v e m e n t t h r o u g h c l a s s i c a l s e l e c t i o nC h e m i c a l A g e n t s a n d U V r a d i a t i o n h ave b e e n u s e d t o q u i c k ly f i n d m o r e u s e f u l va r i a n t sL o s s o f r e g u l a t o r y F u n c t i o n r e s u l t s i n e n h a n c e d e n z y m e p r o d u c t i o nS t r a i n i m p r o v e m e n t t h r o u g h e x t r a c o p i e s o f g e n e o r i n te re s t , r e m o va l o f i n h i b i t o r y r e g u l a t i o n a n d e n h a n c e m e n t o f p o s i t ive re g u l a t i o n , i n c re a s i n g t u r n ove r ra t e o f l i m i t i n g s t e p i n s y n t h e s i s , i n c r e a s e t h e ra te o f m R N A p r o d u c t i o n by c o n s t r u c t i n g p l a s m i d s w i t h d e s i r e d g e n e sG e n e s h u f f l i n g a n d t a r g e t e d e vo l u t i o nTo d ay a l m o s t a l l p r o d u c t i o n s t r a i n s a r e G M

Wild type overproducing fungal glucoamylase strain

selected

Major protease removed, expression plasmid inserted into

genome

Only 300 mg/l of active chymosin

Extensive mutagenesis and screening approach

by regulating glucoamylase promoter and subjecting to CCR

Reducing CCR by screening for

deoxyglucose mutantProduction yields : 1g/l

Chymosin(calf rennet( production Strain by. A niger var. awamori

OPTIMIZATION

Optimization means finding highest specific synthesis rate (qe in units/g of biomass per h) for a given amount of biomass. As, enzyme synthesis depends on primary metabolism, conditions favoring enzyme synthesis favor growth. Thus, we need to determine, conditions favoring growth and the relationship between growth rate and enzyme synthesis rate.

Re= enzyme synthesis rate in units/lit.hrqe= specific enzyme syn rate units/g.hrCx=biomass conc in g/lu= specific growth rateKS=saturation in g/l

A)Growth coupled synthsis B) Saturated synthesisC) Saturated synthesis with CR D)Repressed Synthesis

1) If qe strong function of u; maintenance of high u is important; oxygen and HT limiting: USE CONTINUOUS CULTURE METHOD

2) If enzyme produced as secondary metabolite use RECYLCING REACTOR, Decrease u3) If complex relationship exists: FED-BATCH process to control growth rate

PROPAGATION AND PROCESS CONTROL

PROPAGATION

FERMENTATION

o 1) So l id Surface Fermentationo Media : Wheat bran, w hose h igh content of

nutr ients inc ludes minera ls and s a l tso Appl ications: production of amylase , pro tease ,

and l ipase f rom As perg i l lus and Mucor spec ies , as wel l as fo r pectinas e and ce l lu las e f rom As perg i l lus and Pe nic i l l i um spec ies .

o TRAY PROCESS: substrate i s spread in a th in layer in incubatio n roo ms

o DRUM PROCESS: ho r izonta l rotating drumso After cu ltivatio n o f fung i w i th spo res , the mycel ia

are extracted w ith water or sa l t so lution in co untercurrent mo de, and the co ncentrated enzyme so lution i s prec ip i tated.

o DISA DVANTAGES: Handl ing co sts and co ntro l o f infectio n, temperature , humidi ty, and aeratio n

2) SUBMERGED CULTURE METHOD (CSTR)

o Less risk of infection and offer reduced handling costs and higher yieldso Mechanical Stirred reactors in BATCH OR FED-BATCH MODE o Capacity: 10,000-100,000 l for 30-150 hourso Equipment and techniques are most often adapted from antibiotic

fermentations. o Continuous mode not preferred because of risk of enzyme inactivation

of media sterilization (successfully used for Glucose isomerase) o The formation of enzyme and many secondary metabolites is often

subject to catabolite repression by high concentrations of glucose. o In addition to the influences of nutrient medium and size and age of

inoculum, operational parameters such as pH, aeration, and agitation must be taken into account to optimize the production of enzymes.

o Addition of surface-active agents may lead to increased excretion of extracelluar enzymes.

o MIXING IS MOST IMPORTANT PARAMETER AFFECTING OXYGEN AND NUTRIENT DISTRIBUTION. Rushton turbines(radial flow impellers) have replaced traditional ones for mycelial fermentations,

o Optimal scale for production process depends on on technical and strain specific considerations, but also on the balance between economy and risk assessment.

1) Inoculate seed fermenter

2)Batch process to propagate biomass

Type of sugar depends on genetic set up of fungusMaltose and maltodextrins preferred substrate for Chymosin production (Glucoamylose promoter)

3) Fed batch process

4)After about 1 week, fermentation stopped by acid. At pH 2, biomass inactivated

8)Fomulation9)Packaging

6) Downstream processing: Filter aid to separate biomass and enzyme(liq)

5)Holding time of several hours

7)Chromatography (EBA) for turbid liquids, alternatively, spray drying or UF

Chymosin production by A. niger var. Awamori)

CHYMOSIN PRODUCTION USING e.coli (Pfizer, 1990)

This product was the first GM-

derived food enzyme on the

market. The production

organism is an E. coli K-12

strain having the prochymosin

gene under the control of the

trp promoter in pBR322-

derived vector system.

C-limiting feeding strategy to gain biomass and product Conc, and inhibit acetic acid accumulation at unlimited growth conditions

Chymosin accumulates in Ibs,Cells distrupted by homogenization, IB collected by centrifugation

After washing at pH 2, the inclusion bodies are dissolved by addition of urea (7–9 M) and the pH is increased to 10. After the renatura- tion step with NaCl and Na3(PO4) buffers, the pH is stepwise adjusted to 5.5. Chymosin can be purified and concentrated by anion exchange chromatography.

Renaturate protein aggregates, refold and convert to active chymosin

FUN

GI+Extracellular secretion+Easy downstream processing+High recovery Yields-Long process time (4-6 days)-High energy consumption-Higher risk of contamintaion-Challenges in upscaling and reproducibility-Diffculties in morphology and rheology

Bact

eria+fast and uncomplicated

growth+mineral salts can be used to improve reproducibility+less than half the time of fungal cultivation+Cost saving in energy consumption & expensive fermentation capacity-Intracellular secretion-Complex downstream processing-Large amount of chemicals pose a major waste problem

GM

K. lactis and several Bacillus expression hosts combine the advantages of fungal systems (ie. high production levels and secretion in the external medium) with some of the advantages of E. coli-based systems like fast growth. The most commonly used host systems in industry at this time are Aspergillus and Bacillus species.

COMAPRISON OF THE DIFFERENT FERMENTATION PROCESSES

ENZYME FORMULATION Enzymes are sold as stabilized liquid concentrates or as particulate solids

WHY FORMULATION?Primary task of formulation is to minimize losses in enzymatic activity during transport, storage and use. Secondary purposes include, prevention of microbial contamination, avoidance of precipitation or haze formation, minimizing formation of sensitizing dust or aerosols and improving color and odorHOW IT WORKS?By preventing denaturation, catalytic-site deactivation and proteolysis i.e. PREVENT UNFOLDING by altering the protein’s environment so as to induce a compact protein structure. There are several ways to accomplish this.

PACKAGINGCareful selection of packaging materials. One should us tight bottles and stoppers to prevent access to moisture and s should not release any traces of heavy metals or other enzyme-inactivating substances into the enzyme solution or suspension. In some cases, enzymes must be protected from light and packaged in brown glass bottles.

FUNCTION METHOD

Inducing compact protein structure

Preferential exclusion of water from protein surface by adding sugars, polyols and lyotropic salts

Combat active site inactivation

Sufficient levels of any required cofactors, reversible inhibitors & exclusion of oxidizing or reactive species in formulation

Overall enzyme purity and quality

Choice of raw materials and enzyme recovery process

Removal or impurities, problems related to color, odor & precipitation

Downstream operations like diafiltration, adsorption, chromatography, crystallization, extraction

Prevention of physical precipitation

Formulating near pI of enzyme with hydrophilic solvents like glycerol or PPG

Prevention of salting out Moderate levels of solvating salts are aded

Addressing Microbial contamination

Combination of filtration, acidification, minimization of free water, biocides with limits

FORMULATION TECHNIQUES

T-granulates: physical strength and minimum dust. High shear

granulation and coating techniques. Detergent industry.

BG/SG granulates: smaller particle size, easy incorporation into

flour, safety. Spray drying & Fluidized bed drying. Bakery

industry.

Micro granulates: Fluidized bed Drying for finer particle size distribution and

safety (Non-dusting) in food industry

CT-Granulates: (coated-Tough) for heat sensitive enzymes to prevent

denaturation. Feed Industry.

Immobilized Enzyme: High productivity at low cost. Enzyme immobilized on a carrier or in a matrix, enhancing stability and preventing leakage into substrate during

application. Starch, Oil & fat industry.

Liquid Formulations: liquid product formulated and stabilized with

polyols like glycerol, sorbitol, MPG, sugar, salts to decrease water activity

Different formulations depending on applications

Because of general ly low concentration of enzyme in the starting material, the volume of material that must be processed is large and substantial amounts of waste accumulate.

The spent fermentation medium can still contain large amounts of unused nutrients. However, recycl ing is general ly not possible because of the presence of metabolites.

Solid organ remains and mycel ium, which are used as animal feed, can be separated. The latter must be careful ly checked for undesired metabolites, eg) antibiotics before being fed to animals

In rDNA techniques, the need to maintain absolute containment is of great concern. Waste must be chemical ly or thermally inactivated before disposal to ensure that no l ive organisms escape into the environment.

WASTE DISPOSAL

APPLICATIONS