Enzyme technology

In the field of biotechnology there are many industrial applications that result in biotech products that we use everyday at home. Some of these are food science applications that utilize enzymes to produce or make improvements in the quality of different foods. In the dairy industry, some enzymes are required for the production of cheeses, yogurt and other dairy products, while others are used in a more specialized fashion to improve texture or flavour.

Isolation and Purification of Enzymes
Enzymes are unstable molecules with a definite physico chemical organization. Even a slight change in this organization reduces the activity of enzyme and sometimes the enzyme is totally inactivated.
Therefore, the enzymes have to be isolated under controlled conditions of pH, ionic strength and temperature. Since they are proteinaceous in nature, standard extraction and purification procedures for enzymes are the same as those used for proteins except that the activity of the enzyme is assayed at each of the following four steps of extraction and purification.
Purification of Enzymes - Enzyme purification involves three steps, electrophoresis. These three techniques described in the following text
1.Dialysis
2.Chromatography.

1. Enzyme Technology Presented by – Sumer Pankaj Class – Msc . EST (Sem. 3) Roll no. – 16EST41 Institute of Science and Technology for Advanced Studies and Research Affiliated to Sardar Patel University Recognized under section 2(f) and 12 (B) of UGC act 1956 Mota Bazaar, Vallabh Vidyanagar, Anand , Gujarat 38812058 PG Department of Environmental Science and Technology

2. Content Background Products of Enzyme Technology Structure of an enzyme Mechanism of an Enzyme Production of Enzymes Properties of Enzyme Sources of Enzyme Classification of Enzyme Isolation of enzymes Uses of Enzymes Application of enzymes in various industries Use of Nanoparticles in Enzyme Technology Enzyme immobilization Single cess enzyme nanoparticle Enhancement of Enzyme activity and thermos ability Enzyme Metallography A case study on Bioluminescence of Fireflies

3. Background Enzyme - A biological catalyst that promotes & speeds up a chemical reaction without itself being altered in the process, they fulfil their role by binding specific substrates at their active sites. Enzyme technology is concerned with the application of enzymes as tools of industry, agriculture and medicine . The value of using enzymes over inorganic catalysts in the technological field is due to their efficiency, selectivity and specificity. Enzymes are able to operate at wide range of temperatures, atmospheric pressure and within normal pH ranges – all of which create energy savings for industry. Enzymes are biodegradable and, unlike many inorganic catalysts, cause less damage to the environment . Catabolism Anabolism Metabolism

4. Primary metabolites During cell growth the nutrients of the substrate are converted to cell mass. The chemical compounds produced in this process are called “primary metabolites”. „ The cell mass itself consists mainly of proteins, but a number of primary waste products are also formed, for instance carbon dioxide, lactic acid, ethanol, etc. Primary metabolites are produced in parallel with the cell mass.

5. Secondary metabolites „The formation of secondary metabolites is not directly related to cell growth. They are the side products of bacterial life. In nature, they are produced in low concentration, but through laboratory mutation and selection, cells can be optimized to overproduce these metabolites. Many antibiotics and vitamins are secondary metabolites. The formation of secondary metabolites is not directly proportional to primary metabolism and cell growth.

6. Micro-organisms have been used for thousands of years for making products such as wine, beer, vinegar, soy sauce, bread and cheese. Products of Enzyme Technology NOTE - The micro-organisms (such as yeast) are really used as a source of enzymes during the manufacture of these products of biotechnology. Many industrial processes now make use of pure sources of enzymes, i.e. the enzymes have been ISOLATED from the micro-organisms before use Protease found in papaya as meat tenderizer

7. Structure of an enzyme NOTE :- All enzymes are proteins but all proteins are not enzymes .

8. Mechanism 0f Enzymes - Lock and Key hypothesis Enzymes possess specifically shaped active sites for reacting with one specific substrate thereby generating pure products free from unwanted by-products.

9. MODIFICATION – possible application of genetic engineering to improve the microbial strain LABORATORY SCALE PILOT – to determine the optimum conditions for growth of the Micro-organism PILOT PLANT – small scale fermenter to clarify optimum operating conditions SCREENING – choosing an appropriate micro-organism for the desired enzyme INDUSTRIAL SCALE FERMENTATION The Biotechnological Process of Enzyme Production

10. Pectinase is obtained from the fungus Aspergillus niger Aspergillus niger produces pectinase as an extracellular enzyme Commercial Enzyme Production - An Example

11. Control ripening. Cause food spoilage (rotting). Responsible for changes in flavor, color, texture and nutritional properties. Can be inactivated by heat to extend storage stability of foods. Control oxidation and spoilage ( bioconservation ) Increase nutritive values ( phytase , proteases etc.) Used for fermentation purposes in foods. Can be extracted and purified to a high degree. Properties of enzymes

12. Sources of enzymes There are three major sources of enzymes : Plants (  4%) ( papain , bromilain ) Animals (  8%) ( renet ) Microorganisms (>80%) (yeast, fungi and bacteria)

13. VEGETABLE COAGULANTS Vegetable coagulants are plant extracts which contain a mixture of  proteolytic enzymes and contribute  unique characteristics to cheese. Vegetable coagulants are used primarily in niche products where specific flavours and consistency are desired, or in remote regions where other coagulants are unavailable . Extracts of the Cardo flower ( Cynara cardunculus ) used in Spain and Portugal for the production of some DOP ( Denominación de Origen Protegida ) cheeses, these cheeses are often very soft or semi-liquid and may be spoonable. The flavour profile is strong, with some bitterness.  Extracts of the  Calotropis procera plant used in Ghana for the production of Wara or Wagashi cheese, having a  hard texture and high melting point suitable for grilling. Due to high production costs, low yield and development of off- flavors , vegetable coagulants are not used in industrial cheese production.

14. Enzymes Extracted from animals Lipase :- Lipolytic enzymes extracted from the throat of cows, sheep or goats which enhance cheese flavour. These are very expensive Produced from stomach of cows, enzyme chymosin (rennin) obtained either from calf stomach or more recently from a microorganism . Cut casein proteins in milk into smaller pieces solidify milk Rennet

15. Taq polymerase Thermo stable enzyme essential for PCR reactions Isolated from hot-spring dwelling species Thermus aquaticus ENZYMES PRODUCED BY MICROORGANISMS

16. Invertase Derived from Saccharomyces cerevisiae Also known as sucrase Enzyme digests sucrose into glucose and fructose Used to create candies with a soft center Most commonly used to make chocolate covered cherries

17. Enzyme Production/Isolation Methods

18. Production And Isolation of Enzymes

19. Uses of enzymes Analytical Applications of Enzymes The Animal Feed Industry The Meat and Fish Processing Industry The Dairy Industry The Leather Industry CIP and cleaning of microfilters – Detergents The modification of Fats and Oils The Pulp and Paper Industry The Fruit Juice Processing Industry The Production of Bulk and Fine Chemicals Enzyme-Replacement Therapy

20. Industrial Use of Enzymes Enzyme Source Action in food Food application Papain Latex of unripe papaya fruit protein hydrolysis Meat tenderisation Bromelain Pineapple juice and stem Muscle and connective tissue protein hydrolysis Meat tenderisation Ficin Fig fruit latex Muscle and connective tissue protein hydrolysis As bromelain & papain but not widely used due to cost Chymosin (rennet) Calf abomasum Kappa casein hydrolysis Cheese making Pepsin Bovine abomasum casein hydrolysis in cheese Help for rennet action

21. Lysozyme Hen egg white Hydrolysis of bacterial cell wall polysaccharides Prevention of late blowing defects in cheese by spore-forming bacteria Lactoperoxidase Cheese whey: bovine colostrum Oxidation of thiocyanate ion to bactericidal Hypothiocyanate Cold sterilisation of milk Aminopeptidase Lcictococcus lactix Axpergillux spp. Rhizopux oryzae Releases free amino acids from N-terminus of proteins and peptides Releases free amino acids from N-terminus of proteins and peptides Lipase/ esterase Gullet of goat&lamb : calf abomasum : pig pancreas Triglyceride (fat) hvdrolvsis Flavour enhancement in cheese products:

22. Use of enzymes in Food industry

23. Fruit & Vegetable Enzymes break down specific components within fruit & vegetables such as pectin, starch, proteins and cellulose which results in increased yields, shortening of processing time and improving sensory characteristics.   Some examples: Pectinases and Cellulases are used to break down cell walls in fruit and vegetables, resulting in improved extraction and increase in yield. They can also be used to decrease the viscosity of purees or nectars, and to provide ‘cloud stability’ and texture in juices.

24. Brewing & Alcohol Production Serial no. Enzyme Purpose/Function 1. Cellulases , beta- glucanases , alpha amylases, proteases, maltogenic amylases For liquefaction, clarification and to supplement malt enzymes 2. Amyloglucosidase Conversion of starch to sugar

25. Baking industries/Bakery Serial no. Enzyme Purpose/Function 1. Alpha-amylases Breakdown of starch, maltose production 2. Amyloglycosidases Saccharification 3. Maltogen amylase ( Novamyl ) Delays process by which bread becomes stale 4. Protease Breakdown of proteins 5. Pentosanase Breakdown of pentosan , leading to reduced gluten production 6. Glucose oxidase Stability of dough

26. Some other Products Sr. no. Industry Enzyme Purpose/Function 1. Wine & fruit Juice Pectinase Glucose oxidase Increase of yield and juice clarification Oxygen removal 2. Meat Protease Meat tenderising 3. Protein Proteass , trypsin, aminopeptidases Breakdown of various components 4. Starch Alpha amylase, glucoamylases , hemicellulases , amylases, Modification and conversion ( eg to dextrose or high fructose syrups) 5. Insulin Inulinases Production of fructose syrup

27. Some Enzymes and their functions Alpha-amylase:   Converts starch to dextrins in producing corn syrup. Solubilizes carbohydrates found in barley and other cereals used in brewing. Glucoamylase :   Conversion of dextrins to glucose in the production of corn syrup. Conversion of residual dextrins to fermentable sugar in brewing for the production of "light" beer. Beta- glucanase :   Breakdown of glucans in malt and and other materials to aid in filtration after mashing in brewing. Lipase:   Enhancing flavor development and shortening the time for cheese ripening. Production of specialty fats with improved qualities. Production of enzyme-modified cheese/butter from cheese curd or butterfat . Papain :  Used as meat tenderizer. Used in brewing to prevent chill-haze formation by digesting proteins that otherwise react with tannins to form insoluble colloids . Chymosin :  Curdling of milk by breaking down kappa-caseins in cheese making. Microbial proteases:  Processing of raw plant and animal protein. Production of fish meals, meat extracts, texturized proteins, and meat extenders . Pectinase:  Treatment of fruit pulp to facilitate juice extraction and for clarification and filtration of fruit juice.

28. Continue… Lactase:   Additive for dairy products for individuals lacking lactase. Breakdown of lactose in whey products for manufacturing polyactide . Acetolactate decarboxylase:   Reduction of maturation time in wine making by converting acetolactate to acetoin . Glucose oxidase:   Conversion of glucose to gluconic acid to prevent Maillard reaction in products caused by high heat used in dehydration . Cellulase :   Conversion of cellulose waste to fermentable feedstock for ethanol or single-cell protein production. Degradation of cell walls of grains, allowing better extraction of cell contents and release of nutrients.

29. Advancements in the field of Enzyme Technology – Use of nanoparticles

30. Nanotechnology is the study of manipulating matter on an atomic scale. Nanotechnology refers to the constructing and engineering of the functional systems at very micro level or we can say at atomic level . A Nanometer is one billionth of a meter, roughly the width of three or four atoms. The average human hair is about 25,000 nanometers wide. Use of Nanoparticles in Enzyme Technology The first ever concept was presented in 1959 by the famous professor of physics Dr. Richard P.Feynman . Invention of the  scanning tunneling microscope   in 1981 and the discovery of fullerene (C60) in 1985 lead to the emergence of nanotechnology. The term “Nano-technology" had been coined by Norio Taniguchi in 1974

31. ENZYME IMMOBILISATION Application of nanomaterials as novel supporting materials for enzyme immobilisation has generated incredible interest in the biotechnology community. These robust nanostructured forms, such as nanoparticles , nanofibres , nanotubes , nanoporous , nanosheets , and nanocomposites , possess a high surface area to volume ratios that can cause a high enzyme loading and facilitate reaction kinetics, thus improving biocatalytic efficiency for industrial applications. The current status of versatile nanomaterial support for biofuel production employing cellulases and lipases is described in details nanomaterials will become an integral part of sustainable bioenergy production .

32. How nanoparticles helps enzyme in increasing the catalytic activity ???

33. SINGLE ENZYME NANOPARTICLES SEN (single enzyme nanoparticles ) Enzyme lead short and brutal lives ,to increase the enzymes longevity and versatility, a a team at department of Energy’s Pacific Northwest , National Laboratory in Richlad caged single enzyme to create a new class of catalysts called SENs The nanostructure protects the catalyst, allowing it to remain active for several months Kim and Grate , working in te W.R Wiley Environmental molecular sciences laboratory modified a common protein splitting enzyme called alpha chymotrypsin

34. ENHANCEMENT OF ENZYME ACTIVITY AND THERMOSTABILITY Study on Impaired Pectate Lyase from Attenuated Macrophomina phaseolina  in Presence of Hydroxyapatite Nanoparticle Hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting thermostability ) but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL) with low native state activity. The purified enzyme showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP.

35. EnzMet (Enzyme Metallography ) EnzMet (Enzyme Metallography) is  a new biological labeling and staining method  developed at Nanoprobes .  It uses a targeted enzymatic probe with a novel metallographic substrate to provide a quantum leap in staining clarity over conventional chromogenic and fluorescent substrates . EnzMet ™ has proven highly sensitive both for  in situ hybridization (ISH) , where it readily  visualizes endogenous copies of single genes , and  immunohistochemistry (IHC) detection .

36. NANOTECHNOLOGY TO HARNESS THE NATURAL LIGHT PRODUCED BY FIREFLIES By designing a way to chemically attach genetically manipulated luciferase enzymes directly to the surface of nanorods , scientists at Syracuse University found a new way to harness the natural light produced by fireflies. Fireflies produce light through a chemical reaction between luciferin and it’s counterpart, the enzyme luciferase . In Maye’s laboratory, the enzyme is attached to the nanorod’s surface; luciferin , which is added later, serves as the fuel. A Case Study

37. Continue…. The energy that is released when the fuel and the enzyme interact is transferred to the nanorods , causing them to glow. The process is called Bioluminescence Resonance Energy Transfer (BRET). The nanorods are composed of an outer shell of cadmium sulfide and an inner core of cadmium seleneide . 

38. Refrences http://www.azonano.com/article.aspx?ArticleID=736 http://omicsonline.org/2153-0777/2153-0777-2-e114.pdf http://www.nanoprobes.com/products/EnzMet-SISH-enzyme-metallography-for-ISH-and-IHC.html http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0063567 http://www.che.udel.edu/research_groups/wilfred/Current%2013%20final.pdf www.journals.elseveir.com/biological-control/ http://microbewiki.kenyon,edu.index.php/Biocontrol www.sciencedirect.com/science/article/pii/0261219491900385 J. Srilakshmi , ;J. Madhavi , Lavanya S and Ammani (2014) Commercial Potential of Fungal Protease: Past, Present and Future Prospects , Journal of Pharmaceutical, Chemical and Biological Sciences ,2(4)p.218-234. Sumantha (2006).Food-Grade Proteases, journal of Food Technology & Biotechnology. 44 (2), p.211–220 Riddhi Sawant and Saraswathy Nagendran (2014),protease: an enzyme with multiple industrial application, www.wjpps.com .

39. Thank you…