Production of enzymes ppt

Production of secondary metabolites : enzymes which involves the upstream technological process
Introduction
History
Process involved
Contribution of different micro-organisms
Flowchart
Example: Methods Production of Amyalse in industrial view

1. Subject: UPSTREAM PROCESSING TECHNOLOGY Topic - Secondary metabolite production: Production of enzymes

2. What are enzymes Enzymes are proteins and are the biocatalysts synthesized by living cells They classified into Oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases etc… Enzymes have been used ever since mankind discovered ways to process food

3. History The first enzyme produced industrially was a fungal amylase in 1896, in United States. It was used as a pharmaceutical agent to cure digestive disorders. In 1901 Eduard Bucher won the Nobel prize in biochemistry for proving the existence of enzymes A German scientist (Otto Rohm) demonstrated in 1905 that extracts from animal organs (pancreases from pig and cow) could be used as the source of enzymes-proteases, for leather softening.

4. BIOTECHNOLOGICAL PROCESS OF ENZYME PRODUCTION 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

5. Different organisms contribution in the production of enzymes a)Fungi – 60% b)Bacteria – 24% c)Yeast – 4% d)Streptomyces – 2% e)Higher animals – 6% f)Higher plants – 4%

6. FLOW CHART

7. Regulation of enzyme productions A maximal production of microbial enzymes can be achieved by optimizing the fermentation conditions Fermentation Surface cultures ( solid-substrate cultures) Submerged cultures ( liquid cultures)

8. Solid state fermentation Solid state fermentation has been defined as “the fermentation process occurring in the absence or near absence of free water utilizing the solid substrate”. It is a bio-molecule manufacturing process used in the food, pharmaceutical, cosmetic, fuel and textile industries. These bio-molecules are mostly metabolites generated by microorganisms grown on a solid support selected for this purpose. This technology for the culture of microorganisms is an alternative to liquid or submerged fermentation, used predominantly for industrial purposes

9. Benefits of SSF Simple and cost effective Less effluent release, reduce pollution Aeration is easy Resembles the natural habitat of some fungi and bacteria SSF utilizes solid substrate, thus nutrient rich waste materials can be easily recycled as substrate Substrate are used very slowly and steadily so the same substrate can be used for longer fermentation period SSF is best suited for fermentation techniques involving fungi and microorganism that require less moisture content

10. Submerged fermentation / liquid fermentation Submerged fermentation is the techniques of cultivation of microorganism in liquid broth which breaks down the nutrient to release the desired bio-active compound into solution. In this method, selected microorganism are grown in closed vessels containing a broth rich in nutrients and high concentration of oxygen. In SMF substrate are utilized quite rapidly hence need to be constantly replaced or supplemented with nutrients Bacteria that requires high moisture content or high water activity are best suited for submerged fermentation.

11. Substrates Submerged fermentation (SMF) Solid state fermentation (SSF) Soluble sugar Wheat bran Molasses Rice and wheat straw Liquid media Fruit and vegetable waste Fruit and vegetable juices Paper pulp Sewage / waste water Bagasses

12. Enzyme Formulation 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, to avoid the precipitation or haze formation, minimizing formation of sensitizing dust or aerosols and improving color and odor

13. Different formulations depending on applications 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

14. PACKAGING One should use tight bottles PACKAGING and stoppers to prevent access to moisture and 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

15. Solid state fermentation

16. Microorganism, culture conditions, and substrates The fungal strain A. oryzae is obtained from the Microbial Type Culture Collection The strain is maintained on yeast extract,malt extract ,agar and Czapek Dox agar, respectively. Potato–dextrose agar is also used for the growth and maintenance of the cultures. The cultures are grown at 30 °C for seven days and then stored at 4 °C. The powdered edible oil cakes, namely groundnut, coconut, and sesame oil cakes are selected as substrates The substrates are powdered and sieved using standard sieves to eliminate the foreign materials and stored in aseptic conditions

17. Substrates

18. Inoculum preparation and solid-state fermentation Spores of 7-day-old fungal cultures are scrapped using an inoculation loop and aseptically transferred to sterile distilled water containing 0.1% Tween-80(polysorbate 80). Exactly 1 ml of spore suspension is used as inoculum for the entire fermentation. About 5 g of dry oil cake is taken into a 250-mL flask, containing 2 ml of mineral salts solution , containing 2 g of potassium di-hydrogen phosphate , 5 g of ammonium nitrate , 1 g of sodium chloride and 1 g of magnesium di-hydrogen sulfate in a liter of distilled water to adjust the required moisture level All the contents are mixed, autoclaved at 121 °C for 20 min, and cooled. Spore suspensions are inoculated on the sterile solid substrate and incubated in a solid-state fermenter maintained at 37 °C.

19. Development of a pilot-scale solid state fermenter 600L SSF is used for optimization A fan is fixed at the center of the chamber to maintain the temperature. A relative humidity (RH) sensor is placed inside the chamber .A compressor and water supply are connected to spray water inside the chamber whenever RH decreases Under optimum conditions, the microorganisms or cells are able to perform their desired functions. Temperature and RH inside the chamber is monitored and controlled using sensors and controllers The bioreactor has four trays, an RH sensor, a temperature controller, and a control system networked together. The pH electrode is also set and working in the range of 0 to 13 can be used. When pH change exceeded the set range, 0.1 N HCl or 0.1 N NaOH can be added based on the measured value

20. Enzyme extraction A known quantity of fermented substrate is mixed with double distilled water along with 0.1% Tween 80. The contents are shaken in a rotary shaker and then centrifuged at 7000 rpm at 4 °C for 10 min The reaction mixture consist of 1.25 ml of 1% soluble starch, 0.25 ml of 0.1 M acetate buffer (pH 5.0), 0.25 ml of distilled water, and 0.25 ml of crude enzyme extract. After 10-min incubation at 50°C, the liberated reducing sugars (glucose equivalents) is estimated using the dinitrosalicylic acid (DNS) method. The intensity of color develops and measured at 540 nm One unit (IU) of α-amylase is defined as the amount of enzyme releasing one μmol glucose equivalent per minute

21. Enzyme purification The crude enzyme is saturated up to 50% using ammonium sulfate and incubated at 4 °C overnight for precipitation of proteins. The sample is centrifuged for 15 min (7000 rpm at 4 °C) in a refrigerated centrifuge The supernatant-containing residues can be discarded and the precipitate is used as an enzyme source

22. Reference Optimization and scale-up of α- amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes M. Balakrishnan1*, G. Jeevarathinam1 , S. Kiran Santhosh Kumar1 , Iniyakumar Muniraj2 and Sivakumar Uthandi2*

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