Microbial Production of Amylase | By Miss Priyanka Kande-Patil | RSML

[00:00:05]Welcome everyone. Let me present myself Priyanka Vidadar Gandhi Patil, assistant professor, department of microbiology.

[00:00:12]So today we will explore the microbial enzyme production and our main focus on the extraction, purification and industrial application of amias enzyme.

[00:00:25]started with basic introduction of microbial enzyme. Microbial enzymes are the biological catalyst for the biochemical reaction leading to microbial growth and respiration as well as the formation of fermentation product or the desired product. So we know about the biocatalyst.

[00:00:49]Biocatalyst are nothing but the biological catalyst which are the chemical substances responsible to accelerate the chemical reaction within the living organism.

[00:01:03]Biocatalyst they are highly specific in nature. Each biocatalyst target specific reactant or substrate within the living organisms and they are fitting together like lock and key.

[00:01:22]They are reusable that means we use repeatedly over time.

[00:01:29]This particular bar catalyst are sensitive to certain environmental conditions.

[00:01:35]So we know that all enzymes are proteins but all proteins are not enzymes. So enzymes are proteins that's why they work under based optimum conditions for example normal body temperature and pH.

[00:01:55]If environment become too acidic or too hot, so this particular proteins lose their shape and stop working simply they undergo dennaturation process. So this is the basic introduction of microbial enzyme. So by using various types of microorganisms and by utilizing different biotechnology tools we can develop or we can form the desired microbial enzymes in less time or at a laboratory level. So moving forward the microorganisms that produce enzymes.

[00:02:39]So the first category that is bacteria and actinois.

[00:02:44]So this particular category include azuspilum.

[00:02:57]Next baselison, riseobium and stptomicesis.

[00:03:25]Next category that is fungi east and mold. So this particular category include aspergilus niger, aspergilus oris, penicyium notatum, penicyium chryogenum, tririccoderma and the most common east that is saccharomyis cervici. And the last category that is algae which includes microalgae and chlorella.

[00:03:57]So this various types of microorganisms are responsible to produce microbial enzymes including amias protease and lipase. Moving forward.

[00:04:11]So what is the culture media? Culture media it is the source of nutrition during the production of enzymes for the microorganisms.

[00:04:22]So they can grow very well. Culture medium is nothing but the ideal medium which contain cheap source of carbon, nitrogen, amino acids, stress elements and little amount of salts within it. So this particular culture medium is responsible to support the growth of microorganisms at the certain laboratory conditions to obtain a maximum amount of microbial enzymes.

[00:04:53]So two types of cultures we can use in this enzyme production process.

[00:05:00]The first culture that is semi-olid culture and second one that is submerged culture. So the semiolid culture it is prepared with agar at concentration of 0.5% or less than 0.5%.

[00:05:16]It has soft custard like consistency. So here we use a semi-olid substrates for example moisten wheat and rice brand with lots of nutrients in it and which is responsible for the growth of microorganisms and this particular microorganisms give the maximum amount of enzymes.

[00:05:41]The second culture that is submerged culture. So here instead of semi-olid substrate we can use liquid media with lots of nutrients for the growth of microorganisms.

[00:05:54]So moving forward let's discuss about the types of amias.

[00:06:01]There are three types of amias. The first type that is alpha amias which is the endoenzyme. So what is the endoenzyme? It is also known as intracellular enzyme. So this particular enzyme function or secrete inside the cell that's why it is called as endoenzyme.

[00:06:23]So this particular alphaami responsible to cleave or break the internal alpha 14 glycoidic linkage.

[00:06:35]It is also known as alpha 14 dlucan glucano hydrolase.

[00:06:42]So this particular alphaase act as the calcium metallo enzyme. The calcium ions strictly required for the enzyme catalytic activity, thermal stability and structural integrity of that particular enzyme structure and this particular type of ami work under the pH range that is 6.7 to 7 approximately the neutral pH range is required for the growth of that particular alphaami enzyme.

[00:07:18]or the production of that particular alphaase enzyme and this particular alphaami is responsible for the breakdown of starch into two units.

[00:07:31]First that is amyos and second one is amilopectin.

[00:07:35]So amyos and amilopectin this two are the primary polyaccharides.

[00:07:41]The structure of amyos include linear unbranched chain and amyopectin include highly branched chain. This particular amyos responsible to cleave or break alpha 14 glycoidic linkage and amopectin this particular unit responsible to cleave both alpha 14 and alpha 16 glycoidic linkage.

[00:08:09]The amyos is 20 to 30% proportion of that particular amyos present in starch and typically 70 to 80% proportion of amylopectine present in starch. So amyos have slower digestive capacity and the amyopectin has rapid digestive capacity.

[00:08:35]Next type that is betailease which is the exoenzyme. Exoenzyme which is also known as extracellular enzyme. This particular type of ami work or secreted or function outside the cell. That's why it is called as exoenzyme.

[00:08:55]So it is also known as alpha 14 dlucane maltohydrolase.

[00:09:01]Why it is called as maltohydrolase?

[00:09:04]Because the non-reducing end producing maltos that's why it is called as malto hydrolase. This particular betamase enzyme synthesized by using different types of microorganisms then fungi and plants.

[00:09:22]So this betaamiase during the ripening of fruit this beta amiioise is responsible to break the starch into maltos sugar and this maltos sugar give the sweet flavor to that particular ripe fruit that is the function of beta amias enzyme. This particular type of amias work under the pH range that is 4 to 5 that is acidic pH range. The last type that is gamma amias which is also known as glucoamase.

[00:09:59]Why it is called as gluccomi? Because this particular gamma amias is responsible to break the amylo and amyopectine into glucose. That's why it is called as glucami.

[00:10:13]So this particular gamma amias is responsible to break down both alpha 14 as well as alpha 16 glycoidic linkage and it is also known as alpha 14 gluccoidase. So this is all about the classes of amias enzyme. Moving forward the microbial producers of amiasis. It includes bacteria fungi and yeast bacteria for example basilus subtilus basilus lycheniformis and basilus amyo liquifacians. So basically basilus amyo liquifacian it is the primary industrial producer with a high thermal stability.

[00:11:02]Next category that is fungi which includes aspergilus niger aspergilus ori penicylium notatum penicylium chryogenum and risopus species with excellent for the acid stable amiases. So these all are acid stable amiases. The third category that is used is the most commonest that is saccharis cervicy and the candida spaces which are responsible to produce the amiasis enzyme.

[00:11:34]So why this organisms prefer to produce the amus enzyme because they are have generally recognized as a safe status.

[00:11:45]They are high extracellular enzyme secretions, well characterized genetics, stable fermentation profiles and they have high thermo stability. That's why we prefer this microorganisms for the amiasis production.

[00:12:04]Moving forward stepbystep amias production. So here the first step that is strength selection. Strength selection is nothing but the selection of microorganisms. So here microorganisms preferred which produce maximum amount of enzymes in less time.

[00:12:22]So for this strength development here we can use different types of mutagens.

[00:12:27]strain development or strain improvement. It is the process where we modify the genetic makeup of that particular new industrial microorganisms to enhance the desired properties of that particular microorganism.

[00:12:44]So for this 10 development we can use horizontal gene transfer techniques including conjugation, transformation and transduction. Also we use classical methods including UV light and chemical mutagenesis.

[00:13:00]So in this process we can expose the cell to the UV light and certain specific chemical agents that basically change their genetic makeup.

[00:13:14]Also we use the targeted approach technique that is crisper cast 9.

[00:13:24]So crisper castine system it basically clustered regularly interspaced short palendromic ripets with associated with cast 9 protein. So this particular system is responsible to find, delete and replace the faulty section of DNA and it is responsible to cure the genetic disorders as well as enhance the crop yield and recover the illness for example cancer. So this strain improvement techniques we use to develop the strength which give the maximum amount of enzyme.

[00:14:09]Next step that is inoculum preparation.

[00:14:12]So the pure culture of selected microorganism is maintained on the nutrient agar slants as well as starch agar slants and they are particularly transferred to the liquid medium as the inoculum.

[00:14:28]Next step that is media formulation or the preparation of fermentation media.

[00:14:35]So this particular medium contain soluble starch, pepone, yeast extract, mineral salts including KH2 P4, then magnesium sulfate, calcium chloride, Na2HP4, potassium chloride and magnesium chloride.

[00:15:00]We maintain pH range that is 6.8. Eight.

[00:15:05]Some key nutrient sources also used in the media composition including carbon sources including maltos, sucrosse and glucose. Nitrogen sources including ammonium sulfate, sodium hydrogen phosphate and ammonium chloride. And some common substrates are used here that is molasses, corn constip liquid, pepone, east extract, malt extract, soya meal and vi.

[00:15:36]Moving forward next step that is the fermentation process. Here we use two fermentation technique. First is submerged fermentation and second one is solid state fermentation. So in submerged fermentation technique microorganisms grown in liquid medium containing starch pepone and east extract. So this particular submerged fermentation technique is used to produce the bacterial amias enzyme.

[00:16:10]Next solid state fermentation. So here we use the solid substrates like wheat bran, rice brand, casava. So these solid substrates are used to grow the microorganisms and this particular fermentation technique used to produce the fungal amiis enzyme. So here some optimum conditions are required. So for the bacteria temperature is maintained 40 to 45° or 55°C pH that is 6 to 7 and for the fungi temperature is maintained 30 to 40° pH range that is acidic 4.5 to6. So here the aerobic condition is maintained.

[00:16:59]This whole procedure is uh occurred in the presence of oxygen and we required the production time that is 3 to 5 days.

[00:17:10]So within 3 to 5 days we obtain a desired product that is amias.

[00:17:16]Moving forward let's discuss about the recovery and purification of ami enzyme. So here the ferment fermentation broth is filtered and centrifuged.

[00:17:31]So this particular process that is filtration and centrifugation is responsible to remove cell debris certain impurities and solid substrates which are present in that particular fermentation broth. That particular process is known as clarification.

[00:17:50]So next there is the removal of nucleic acids by adding polycatans includes streptoycine polyamines and polyeththile amines that particular polycatons are responsible to precipitate that nucleic acid.

[00:18:12]Next step that is precipitation by using ammonium sulfate and ammonium hydroxide.

[00:18:20]This particular chemicals are responsible to precipitate amias and this precipitate is collected by using filtration technique. We use here filters.

[00:18:32]Next the purification.

[00:18:34]So we purify that particular desired product by using the chromatography techniques including gel filtration and ion exchange chromatography. Along with that we use dialysis and lastly the crystallization and drying. The precipitate is crystallized at 4° C and then this precipitate is dried by using film evaporator and freeze dryer. So drying is required to remove the moisture content present in that particular desired product to avoid the contamination. So this is all about the production steps.

[00:19:19]Last part that is application of amileas enzyme. We know that amiles enzyme is widely used industrial enzyme in food fermentation technique textile detergent industry, medicine, pharmaceuticals etc. So first we say food and beverage industry. So here am enzyme is used in baking industry. So particularly amias is responsible to break the starch into fermentable sugars and this particular fermentable sugar is responsible to improve or to handle the texture of that particular bread.

[00:20:08]Next that is breathing and distilling industry. So in this particular industry the amias enzyme is used in beer and whiskey production and it is converted into cereal starch into the malttos sugar. Also it is converted into glucose for the east fermentation process.

[00:20:33]Then it is also used in fruit and juice industry for the clarification purpose that basically increases the sugar content to enhance the flavor of that particular fruit or juices.

[00:20:49]Next application in textile and detergent industry. In textile it is used in desizing of fabrics and in detergent industry.

[00:21:01]The ami enzyme is responsible to remove the starchbased stance. Food stance basically include rice, gravy, chocolate and custard from the cloths. Next application that is paper and pulp industry. So amus enzyme used for the starch modification in paper coating then improve the strength of that particular paper then smoothness and the print quality of that particular paper. Next application in pharmaceuticals and medicine. So amias enzyme act as the digestive aid.

[00:21:45]Basically the amias enzyme play important role to diagnose the pancreatitis.

[00:21:53]So pancreatitis this particular disease is nothing but the inflammation occur in pancreas.

[00:22:02]Next application bofail industry amias enzyme play key role in bioeththanol production and dairy industry. So amias enzyme is used to produce the lactosefree and low viscosity dairy products to avoid the lactose intolerance in humans.

[00:22:26]And the last application that is waste management. By using various types of microorganisms and by utilizing different biotechnology tools we can easily develop or we can easily produce amias enzyme in laboratory level in less time. So this is all about the applications of amiasis.

[00:22:49]So thank you so much. This is all about the microbial production of amias.