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

[00:00:05]Welcome everyone. Let me present myself Priyanka Vidyad Kandipatil, assistant professor, department of microbiology.

[00:00:12]In the previous lecture we were explore the microbial production of ami enzyme.

[00:00:18]So today we will explore the enzyme proteasis. Our main focus is on extraction, purification and industrial applications of that particular protease enzyme. So before going further, we should know the basics of protease enzyme. So this particular protease enzyme refers to the group of enzyme belonging to the class of hydrolases whose catalytic function is to hydraize peptide bonds of that particular proteins.

[00:00:53]The protease enzyme is also known as a proteolytic enzyme. That means this particular enzyme is responsible to digest proteins also known as proteinasis.

[00:01:08]So this proteus enzyme are ubiquitous in nature and it is widely used industrial enzyme with lots of applications including the physiological as well as commercial fields. Moving forward, let's discuss the sources of protease enzyme including plant proteasis, animal proteasis, microbial proteasis having bacterial as well as fungal proteasis and the last source that is viral proteasis.

[00:01:45]So first that is plant proteasis including pepin. So pepin basically it is the proteolytic enzyme extracted from the papaya fruit. That's why that particular enzyme is known as pepin and this particular enzyme is responsible to digest the proteins. Next is bromelain.

[00:02:08]So the bromelain that particular proteolytic enzyme is extracted from the pineapple fruit. Next one keratinise. So keratin is this particular enzyme which is a specialized proteolytic enzyme responsible to digest keratin protein which is a tough fibrous protein abundant in hair, nails, feathers as well as horns of that particular animals.

[00:02:39]Next, carnine and melain. These three enzymes which are the plant proteasis are the specialized serene proteasis.

[00:02:54]So here the first that is victin it is the highly theostable serene proteasis which basically extracted from the victi tree. Next one is carnine which is also the theostable serin proteus milline. This particular serene proteus is extracted from the euphoria millie the milky latex of that particular plant parts and the last one that is fine. So this ficin is also the proteolytic enzyme which is extracted from the fig fruit.

[00:03:37]Next one that is animal proteasis including pancreatic trine, kimotriine, pepsine and the last one that is rein.

[00:03:48]So the pancreatic trine, kimotriin and pepsine are the most commonly used proteolytic enzyme which is responsible to digest proteins. So here the pancreatic trinotin present in the pancreas basically in small intestine and the pepsin that particular enzyme present in stomach.

[00:04:17]The rein this particular animal proteus produced by the kidney and its main function is to control the blood pressure then also control the body secretions body fluid levels present in that particular human body as well as the electrolyte level.

[00:04:40]Next protease that is microbial proteas including bacterial as well as fungal proteasis.

[00:04:48]The bacterial proteasis work under the pH range that is 5 to 8 and temperature around 60° and the fungal proteases which produce acid neutral as well as alkaline proteases.

[00:05:08]And this fungal proteasis are active over a wide pH range that is 4 to 11.

[00:05:17]And it exhibit broad substrate specificity.

[00:05:22]And the last source of proteas that is viral proteus.

[00:05:29]So next that is types of proteases. So depending on their site of action proteases are classified into two main types that is exopeptidase and endopeptidase. Exopeptidase this particular proteeptide bonds proximal to amino or caroxil ends of that particular enzyme and it acts on the terminal peptide bonds and second one that is endopeptidase. So this particular peptidase is responsible to cleave the peptide bonds and it basically acts within the polyeptide chain.

[00:06:11]Second, based on the functional group which are present at the active site of that particular enzyme, the proteasis are classified into four main types that is serene proteasis, aspartic proteasis, cyine thol proteasis and the fourth type that is metalloproteasis.

[00:06:32]So seren proteasis are also known as alkaline proteasis characterized by its serene group or the presence of serene group at the active site of that particular enzyme. Okay. So serene it is the basic amino acid that's why that particular proteus is known as alkaline proteasis.

[00:07:12]This particular serene proteuses contain three residues at their active site including serene histine and aspartate.

[00:07:23]So that's why that particular structure is known as catalytic triad because the three residues present at their active site that is first one is serene itself second one is histine and the third one which is the aspartic acid. Right.

[00:07:57]Second type that is aspartic proteasis.

[00:08:00]This particular proteasis also known as acidic proteasis because the aspartic acid it is the acidic amino acid. That's why it is called as acidic proteasis.

[00:08:14]So the aspartic proteasis which are endopeptides acts within the polyeptide chains. So here this particular proteasis contain two highly conserved aspartic residues at their active site. That's why it is also known as catalytic di because two highly conserved catalytic aspartic residues are present in their active site which is also responsible for the catalytic activity.

[00:08:54]Third one that is cyine theol proteasis.

[00:08:58]So cyine thol proteasis occur in both procariots as well as ukariots.

[00:09:06]So 20 families of cysteine theol proteasis have been recognized and the cyine thol proteases are also contain two catalytic subunits including cyine and histine. So it is also known as catalytic diode. And the last type that is metalloproteases which contain the metal ions at their active site which function for the catalyzis for the hydraysis. So this is all about the types of proteasis. Moving forward, the production of a bacterial proteasis involve four main steps. First one that is selection of microorganism.

[00:09:59]Second one that is formulation of medium. Third production process and the fourth one recovery and purification of that particular enzyme. So we see one by one. So the first that is selection of microorganisms.

[00:10:16]Here the microorganisms prefer which produce the maximum amount of proteus enzyme in less time. So for this strain improvement we can use different types of mutagens including physical mutagens chemical as well as biological mutagens.

[00:10:35]Along with that for this trend development we can use the horizontal gene transfer techniques that is conjugation, transduction and transformation.

[00:10:46]So here we use the microorganisms for example basilus lycheniformis basilus amloqueifacience and the third that is basilus stereothermopilus.

[00:11:01]So this three basilus spaces responsible to produce proteus enzyme in high amount. Second step that is a formulation of medium. So medium used to contain the ground barley as a carbon source. So here ground barley as a carbon source.

[00:11:32]Next the starch level is limited in that particular medium. And here the protein hydrolysis and sodium glutamate which act as the nitrogen source.

[00:11:58]Next step that is production process. So the production process during the fermentation the insoluble proteins are firstly hydrayed by boiling with dilute acids as well as giving the enzyatic treatments. So by using various types of enzymes or by using dilute acids we can hydrayze that insoluble protein present in that particular fermentation medium.

[00:12:32]Next, this particular medium have some optimum conditions including the pH range that is 6 to 7, neutral pH range, temperature is about 37° C and the production period is 3 to 5 days. And the last step that is recovery and purification of that particular enzyme. So the fermentation broth is filtered and then centrifuged to remove the cell debris, certain impurities and the solid substances present in that particular media.

[00:13:13]Next there is the removal of nucleic acids by adding polycans for example polyamines, steptoycine and polyethile amine. So this particular polycans are responsible to precipitate that particular nucleic acid.

[00:13:29]Next precipitation by using ammonium sulfate and ammonium hydroxide.

[00:13:36]Next we go for the purification by using the chromatography techniques.

[00:13:44]Most commonly used chromatography techniques for the purification of that particular enzyme that is gel filtration chromatography and ion exchange chromatography along with we can also use dialysis for the purification purpose.

[00:14:00]Next crystallization.

[00:14:02]So this particular precipitate is crystallized at 4° C and then drying. So drying is very important step because there is the removal of moisture content water contain present in that particular enzyme to avoid the contamination and by using film evaporator as well as freeze dryer.

[00:14:29]Moving forward next we see the production of fungal proteasis.

[00:14:37]Again there are four steps we see one by one. So the first step that is selection of microorganisms.

[00:14:46]So here the main producing organisms are asperulus ori aspergilus niger aspergilus soji. Next aspulus venti mucer pucilus and mucer mei. Along with that mucer dilemma and amyomicis roxy.

[00:15:07]So this particular microorganisms are responsible to produce the fungal proteasis.

[00:15:14]Next step that is a formulation of a medium. So here the fungus is grown on wheat brand. So here we use the solid substrate that is wheat brand for the growth of certain microorganisms.

[00:15:34]Next here the fermentation conditions similar to those of the amias production. So for the fungus pH range is about 4 to six that is acidic pH range is required and the temperature is about 30 to 40° at this sporulation.

[00:15:57]So it is the process of releasing or producing the reproductive cells that particular reproductive cells are known as spores. So at this corulation stage this proteolytic enzyme are present in the medium. So here in the preparation of inoculum medium there are some trace elements used for the growth of that particular microorganisms including zinc sulfate, magnes sulfate, copper sulfate, cobalt sulfate etc. So during the production of that particular fungal proteases we can use two medium. One medium is for the inoculum preparation and one medium for the production. So the inoculum preparation media contain quinine hydrayzeed as a nitrogen source. Then soya proteust, yeast extract, soluble starch, de manitol, trace elements and FeSO4.

[00:17:07]For the production media, so this particular media composition contain soya bean, milk, glucose, NO3, skim milk, KH2P4 and MgSO4.

[00:17:24]Next step that is a production process.

[00:17:27]So the proteasis from aspergilus ori and aspergilus soji. So this particular microorganisms produced by solid substrate fermentation. So here the semi-olid culture this particular culture is produced with agar of concentration of 5% or less than 5%. And it has the soft custard like consistency. Here we use for the growth of aspergilus orasas as well as the aspergilus suji that is a solid substrates including rice brand as well as moisten wheat brand.

[00:18:13]So the mixture of enzyme containing acid aspartil proteasis neutral metalloproteesis and the serene proteasis.

[00:18:24]So this particular proteus responsible to produce certain acidic neutral as well as alkaline proteasis proteasis from aspergilus or by using submerged fermentation process. So here we use the liquid media instead of solid substrate along with lots of nutrients in it including the cheap source of carbon, nitrogen, amino acids, trace elements and the little amount of salts.

[00:18:59]The aspilus are closely related spaces with the black spores often produce the acid proteasis. Here the optimum pH range of that particular production media that is 4 to 5 that is acidic pH range we maintain during the production of that particular proteus enzyme.

[00:19:23]The last step that is recovery and purification of enzymes.

[00:19:29]Same first that particular fermentation broth is filtered then centrifuged then there is the remole of nucleic acids then precipitation by using ammonium sulfate then purification by using different chromatography techniques crystallization and the last step that is drying. But here in the recovery and purification of that particular fungal proteasis include after the growth the harvest can be dried at 50° C or less than 50° C then it ground to liberate the proteasis.

[00:20:11]Proteases can also be extracted by water followed by the addition of alcohol for the precipitation.

[00:20:21]And here the drying is occur by using the film evaporator at 55° C.

[00:20:31]The last part that is the applications of that particular proteus enzyme. So it is widely used industrial enzymes with lots of applications in both physiological as well as commercial field. So the first application that is detergent industry.

[00:20:49]So this protease enzyme is used to cleaning the contact lenses also responsible to remove the stains which are present in the cloths. So these stains include the food stain, blood, grass as well as the body secretions.

[00:21:08]Next application that is leather industry. So the selective hydrarolyis of non collagenous constituents of skin and for the rem of nonfibbrillar proteins.

[00:21:23]Third application in food industry. So first industry that is dairy industry in the manufacturer of cheese.

[00:21:32]Next is baking industry to modify wheat gluten by limited proteolyis that is the breakdown or the destation of that particular protein.

[00:21:45]Next it is also used for the soya sauce production.

[00:21:51]Then meat tenderization.

[00:21:53]So here this particular protease enzyme responsible to improve the tenderness of that particular meat. So the tenderness is basically reduce the toughness of that particular meat. Next it is also responsible to synthesize the aspartame.

[00:22:14]So aspartame is nothing but the nonspecific artificial sweetener.

[00:22:20]And next application in pharmaceutical industries to correct the litic enzyme deficiency syndromes also responsible to cure the inflammation in that particular body parts also reduce the pain and the last application that is therapeutics to diagnosis or it is responsible for the dissation purpose.

[00:22:48]So this is all about the microbial enzyme production of GTS enzyme. Thank you so much.