Production of Enzymes

fermentation for the production of enzymes

1. MONTICA SAWANT 13FET1007 INDUSTRIAL PRODUCTION OF microbial ENZYMES

2. Introduction Enzymes have been used ever since mankind discovered ways to process food. Food processing steps like milk acidification, milk clotting, alcohol fermentation and soy bean fermentation are enzyme-mediated processes carried out by microorganisms. However, it was not until the late 19th century that purified enzymes were used for food processing. History : 1901: Eduard Bucher won the Nobel prize in biochemistry for proving the existence of enzymes. Approx 20 years before, Christian Hansen invented and commercialized a process to purify rennet from calf stomach which revolutionized the diary industry. Marks Beginning of global food enzyme industry. Global sales US $ 3.3 billion in 2010 Market leaders: Novozymes (Denmark), Danisco (now DuPont), DSM (Netherlands)

3. METABOLIC PROCESS METABOLIC PROCESS Substrates, Physical Parameters Biomass, By products Enzyme 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 maximum REGULATORY MECHANISMS: Simple Induction Repression Complex Global regulation

4. PROS VS CONS

5. BiotechnoLOgical process of enzyme production

6. Enzymes sold on the basis of activity FUNGI ( Aspergillus & Trichoderma ) YEASTS ( Saccharomyces, Hansenula , Kluyveromyces ) BACTERIA ( E.coli , Bacillus) SPECIES SELECTION

7. METABOLIC ENGINEERING Construction of production strain is a long process involving: classical mutagenesis, screening for improved production & targeted deletion of unwanted genes Strain improvement through classical selection Chemical Agents and UV radiation have been used to quickly find more useful variants Loss of regulatory Function results in enhanced enzyme production Strain improvement through extra copies of gene or interest, removal of inhibitory regulation and enhancement of positive regulation, increasing turnover rate of limiting step in synthesis, increase the rate of mRNA production by constructing plasmids with desired genes Gene shuffling and targeted evolution Today almost all production strains are GM Chymosin (calf rennet( production Strain by . A niger var. awamori

8. 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.hr qe = specific enzyme syn rate units/ g.hr Cx =biomass conc in g/l u= specific growth rate KS=saturation in g/l A)Growth coupled synthsis B) Saturated synthesis C) Saturated synthesis with CR D)Repressed Synthesis If qe strong function of u; maintenance of high u is important; oxygen and HT limiting: USE CONTINUOUS CULTURE METHOD If enzyme produced as secondary metabolite use RECYLCING REACTOR, Decrease u If complex relationship exists: FED-BATCH process to control growth rate

9. PROPAGATION AND PROCESS CONTROL PROPAGATION

10. FERMENTATION 1) Solid Surface Fermentation Media: W heat bran, whose high content of nutrients includes minerals and salts Applications: production of amylase, protease, and lipase from Aspergillus and Mucor species, as well as for pectinase and cellulase from Aspergillus and Penicillium species. TRAY PROCESS: substrate is spread in a thin layer in incubation rooms DRUM PROCESS: horizontal rotating drums After cultivation of fungi with spores, the mycelia are extracted with water or salt solution in countercurrent mode, and the concentrated enzyme solution is precipitated. DISADVANTAGES: Handling costs and control of infection, temperature, humidity, and aeration

11. 2) SUBMERGED CULTURE METHOD (CSTR) Less risk of infection and offer reduced handling costs and higher yields Mechanical Stirred reactors in BATCH OR FED-BATCH MODE Capacity: 10,000-100,000 l for 30-150 hours Equipment and techniques are most often adapted from antibiotic fermentations. Continuous mode not preferred because of risk of enzyme inactivation of media sterilization (successfully used for Glucose isomerase ) The formation of enzyme and many secondary metabolites is often subject to catabolite repression by high concentrations of glucose. 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. Addition of surface-active agents may lead to increased excretion of extracelluar enzymes. MIXING IS MOST IMPORTANT PARAMETER AFFECTING OXYGEN AND NUTRIENT DISTRIBUTION. Rushton turbines(radial flow impellers) have replaced traditional ones for mycelial fermentations, Optimal scale for production process depends on on technical and strain specific considerations, but also on the balance between economy and risk assessment.

12. 1) Inoculate seed fermenter 2)Batch process to propagate biomass Type of sugar depends on genetic set up of fungus Maltose 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) Fomulation 9)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. A wamori )

13. CHYMOSIN PRODUCTION USING e.coli (Pfizer, 1990) T his 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

14. COMAPRISON OF THE DIFFERENT FERMENTATION PROCESSES

15. ENZYME FORMULATION Enzymes are sold as stabilized liquid concentrates or as particulate solids WHY FORMULATION? P rimary 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 odor HOW 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. PACKAGING Careful 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.

16. 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

17. Different formulations depending on applications

18. Because of generally 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, recycling is generally not possible because of the presence of metabolites. Solid organ remains and mycelium, which are used as animal feed, can be separated. The latter must be carefully 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 chemically or thermally inactivated before disposal to ensure that no live organisms escape into the environment. WASTE DISPOSAL

19. APPLICATIONS