Chapter 5 Microbial Biotechnology……

1. BTEC3301 Chapter 5Microbial Biotechnology……

2. Microbial Biotechnology MICROORGANISMS AS TOOLS INDUSTRIAL USE OF ENZYMES • Several thermostable enzymes, like the Taq polymerase have been identified and widely used in PCR and other reactions. • Cellulase is obtained from E.coli and degrades cellulose, a polysaccharide in plant cells.

3. Microbial Biotechnology MICROORGANISMS AS TOOLS INDUSTRIAL USE OF ENZYMES • The denim jean is treated with cellulase, from fungi Trichoderma reesei and Aspergillus niger, to give the faded look and texture. • The protease subtilisin, from Bacillus subtilis, forms component of Laundry detergent to remove and degrade protein stains.

4. Microbial Biotechnology MICROORGANISMS AS TOOLS INDUSTRIAL USE OF ENZYMES • Enzymes can rightly be called the catalytic machinery of living systems. • Enzymes are responsible for fermentation of sugar to ethanol by yeasts, a reaction that forms the bases of beer and wine manufacturing. • Enzymes oxidize ethanol to acetic acid. This reaction has been used in vinegar production for thousands of years.

5. Microbial Biotechnology MICROORGANISMS AS TOOLS INDUSTRIAL USE OF ENZYMES • Similar microbial enzyme reactions of acid forming bacteria and yeasts are responsible for aroma forming activities in bread making. • Presently more than 2000 different enzymes have been isolated and characterized. • More than 75% of industrial enzymes are hydrolases. • 40% of all enzyme sales are Protein-degrading enzymes .

6. Microbial Biotechnology MICROORGANISMS AS TOOLS Enzyme Production by Microbial Fermentation • Extracellular enzymes are secreted outside the cell makes the recovery and purification process much simpler compared to production of intracellular enzymes.

7. Microbial Biotechnology MICROORGANISMS AS TOOLS Enzyme Production by Microbial Fermentation • Intracellular enzymes must be purified from thousands of different cell proteins and other components. • The organism producing the enzymes should have a GRAS-status, which means that it is Generally Regarded as Safe. This is especially important when the enzyme produced by the organism is used in food processes.

8. Microbial Biotechnology MICROORGANISMS AS TOOLS Enzyme Production by Microbial Fermentation • The organism should be able to produce high amount of the desired enzyme in a reasonable time frame. • The industrial strains typically produce over 50-g/l extracellular enzyme proteins. • Most of the industrial enzymes are produced by a relatively few microbial hosts like Aspergillus and Trichoderma fungi, Streptomyces and Bacillus.

9. Microbial Biotechnology MICROORGANISMS AS TOOLS Enzyme Production by Microbial Fermentation • Most of the industrially used microorganisms have been genetically modified to overproduce the desired activity and not to produce undesired side activities.

10. Microbial Biotechnology MICROORGANISMS AS TOOLS Protein engineering • Often enzymes do not have the desired properties for an industrial application. E.g extreme thermo stability or overproduction of the enzyme.

11. Microbial Biotechnology MICROORGANISMS AS TOOLS Protein Engineering • Protein engineering is used to improve commercially available enzyme to be a better industrial catalyst. • Several enzymes have already been engineered to function better in industrial processes. These include proteinases, lipases, cellulases and few amylases

12. Microbial Biotechnology MICROORGANISMS AS TOOLS Protein Engineering • Xylanase from fungus Trichoderma sps. is a good example of an industrial enzyme, used in pulp and paper industry and needs to be stable in high temperature. • Xylanases is a good example of engineered enzyme from Trichoderma. Its xylanase has been purified and crystallized. By designed mutagenesis its thermal stability has been increased about 2000 times at 70oC and its pH-optimum shifted towards alkaline region by one pH-unit.

13. Microbial Biotechnology MICROORGANISMS AS TOOLS Enzyme Technology • Enzyme technology involves how to use enzymes. • The simplest way to use enzymes is to add them into a process stream where they catalyse the desired reaction and are gradually inactivated during the process.

14. Microbial Biotechnology MICROORGANISMS AS TOOLS Enzyme Technology • Example liquefaction of starch with amylases, bleaching of cellulose pulp with xylanases or use of enzymes in animal feed. • An alternative way to use enzymes is to immobilize them so that they can be reused. • One method of immobilization is to use ultrafiltration membranes in the reactor system. The large enzyme molecules cannot pass the membrane but the small molecular reaction products can. Therefore enzymes are retained in a reaction system and the products leave the system continuously.

15. Reading Assignment for Quiz:Detergent, Food & BeveragesAnimalfeed,personal care etc slide 15-40 Large scale enzyme applications Detergents • Detergents were the first large scale application for microbial enzymes. • Bacterial proteinases are still the most important detergent enzymes. Some products have been genetically engineered to be more stable in the hostile environment of washing machines with several different chemicals present.

16. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applicationsDetergents • Lipid degrading enzymes, lipase, were used in powder and liquid detergents to decompose fats. • Lipase is produced in large scale by Aspergillus oryzae host after cloning the Humicola gene into this organism. • Amylases are used in detergents to remove starch based stains.

17. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applicationsDetergents • Cellulases have been part of detergents since early 90s. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose. • In textile cellulases remove cellulose microfibrils, which are formed during washing. • Alkaline cellulases are produced by Bacillus strains and neutral and acidic cellulases by Trichoderma and Humicola fungi.

18. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity • Yogurt, cheese, chocolate, butter, pickles, sauerkraut, soy sauce, food supplements (such as vitamins and amino acids), food thickeners (produced from microbial polysaccharides), alcohol (beer, whiskeys, wines) and silage for animals are all products of microbial activity.

19. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity • The industrial microbiologist/ biotechnologist may be involved in producing concentrated microbial inocula for fermentations or the maintenance of fermentation systems utilized in production facilities. • The use of starch degrading enzymes, amylase, was the first large-scale application of microbial enzymes in food industry.

20. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity • Enzymes have many applications in drink industry. The use of chymosin in cheese making to coagulate milk protein. • Another enzyme used in milk industry is beta-galactosidase or lactase, which splits milk-sugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers.

21. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity • Enzymes are used also in fruit juice manufacturing. Pectins are substances in fruit lamella and cell walls. The cell wall contains also hemicelluloses and cellulose. Pectinase, xylanase and cellulase improve the liberation of the juice from the pulp. • Pectinases and amylases are used in juice clarification.

22. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity • Brewing is an enzymatic process. Malting is a process, which increases the enzyme levels in the grain. In the mashing process the enzymes, amylase, are liberated and they hydrolyse (Break down) the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide. • Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the yield.

23. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity • Brewing is an enzymatic process. Malting is a process, which increases the enzyme levels in the grain. In the mashing process the enzymes, amylase, are liberated and they hydrolyse (Break down) the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide. • Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the yield.

24. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages cured or improved by microbial activity • Production of coffee, tea, cocoa, summer sausage, vanilla, cheese, olives and tobacco all require microbial activity.

25. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Food flavoring agents and preservatives • Organic acids such as citric, malic and ascorbic acids and monosodium glutamate are microbial products commonly used in foods.

26. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods • Mushrooms, truffles and some red and green algae are consumed directly. Yeasts are used as food supplements for humans and animals.

27. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Oil recovery/mining • Oil recovery may be facilitated by the development of unique bacteria which produce a surfactant that forces trapped oil out of rocks. • Extraction of minerals from low-grade ores is enhanced by some bacteria (microbial leaching).

28. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Waste and Wastewater Management • Isolating or developing microbial strains capable of degrading and detoxifying hydrocarbon and halogenated hydrocarbon waste (for example organophosphates, acetylcholinesterase inhibitors) of industrial, agricultural or military origin is essential in waste management.

29. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Textiles • The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology. • Starch has for a long time been used as a protective glue of fibers in weaving of fabrics. This is called sizing.

30. MICROORGANISMS AS TOOLS Large scale enzyme applications Textiles • Enzymes are used to remove the starch in a process called desizing. Amylases are used in this process since they do not harm the textile fibers. • Laccase – a polyphenol oxidase from fungi is used to degrade lignin the aromatic polymer found in all plant materials.

31. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Animal Feed • The net effect of enzyme usage in feed has been increased animal weight. • The first commercial success was addition of beta-glucanase into barley based feed diets. Barley contains beta-glucan, which causes high viscosity in the chicken gut.

32. MICROORGANISMS AS TOOLS Large scale enzyme applications Animal Feed • Xylanase, from Trichoderma, are added to wheat-based broiler feed and are nowadays routinely used in feed formulations and animals gain weight. • Enzymes have become an important aspect of animal feed industry. In addition to poultry, enzymes are used in pig feeds and turkey feeds.

33. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Baking • Alpha-amylases have been most widely studied in connection with improved bread quality and increased shelf life. • Both fungal and bacterial amylases are used in bread making and excess may lead to sticky dough.

34. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Pulp and Paper • The major application is the use of xylanases in pulp bleaching for paper.

35. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Leather • Leather industry uses proteolytic and lipolytic enzymes in leather processing. • Enzymes are used to remove animal skin, hair, and any unwanted parts.

36. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Leather • The used enzymes are typically alkaline bacterial proteases. • Lipases are used in this phase or in bating phase to specifically remove grease.

37. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Enzymes in Personal Care products Personal care products are a relatively new area for enzymes and the amounts used are small but worth to mention as a future growth area.

38. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Enzymes in Personal Care products • One application is contact lens cleaning. Proteinase and lipase containing enzyme solutions are used for this purpose. Hydrogen peroxide is used in disinfections of contact lenses. The residual hydrogen peroxide after disinfections can be removed by a heme containing catalase enzyme, which degrades hydrogen peroxide.

39. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Enzymes in Personal Care products • Some toothpaste contains glucoamylase and glucose oxidase. • Dentures can be cleaned with protein degrading enzyme solutions. • Enzymes are also used for applications in skin and hair care products.

40. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Enzymes in DNA-technology • DNA-technology has revolutionized both traditional biotechnology and opened totally new fields for scientific study. • Recombinant DNA-technology allows one to produce new enzymes in traditional overproducing and safe organisms.

41. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Enzymes in DNA-technology • Protein engineering is used to modify and improve existing enzymes. • DNA is basically a long chain of deoxyribose sugars linked together by phosphodiester bonds. Organic bases, adenine, thymine, guanine and cytosine are linked to the sugars and form the alphabet of genes. The specific order of the organic bases in the chain constitutes the genetic language.

42. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Enzymes in DNA-technology • Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process. E.g.:

43. Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Enzymes in DNA-technology • Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process. E.g.: • Restriction enzymes recognise specific DNA sequences and cut the chain at these recognition sites. • DNA modifying enzymes synthesize nucleic acids, degrade them, join pieces together and remove parts of the DNA. 3. DNA-polymerases synthesize new DNA-chains. Many of them need a model template, which they copy. 4. Ligases join adjacent nucleotides together.

44. Microbial Biotechnology MICROORGANISMS AS TOOLS Therapeutic Proteins by Gene Transfer • Recombinant DNA technology led to the rapid development and production of Therapeutic protein. • There are many proteins essential to good health that some people cannot produce because of genetic defects.

45. Microbial Biotechnology MICROORGANISMS AS TOOLS Therapeutic Proteins by Gene Transfer • These proteins include various blood-clotting factors causing hemophilia, insulin (resulting in diabetes), growth hormone (resulting in lack of proper growth), and other proteins, the administration of which corrects pathological conditions or results in other therapeutic benefits. • Plasmids are used to transfer human genes to bacterial cells.

46. Microbial Biotechnology MICROORGANISMS AS TOOLS Therapeutic Proteins by Gene Transfer • If the gene inserted into the plasmid of bacteria is the human gene for insulin, for example, the bacteria into which this gene is inserted produces human insulin. • Bacteria as such do not produce insulin, but the recombinant bacterial cells do produce insulin, it was an outstanding example of microbial biotechnology.

47. Courtesy © John J. Cardamone, Jr. Insertion of a DNA section into a plasmid

48. Microbial Biotechnology MICROORGANISMS AS TOOLS Therapeutic Proteins by Gene Transfer cDNA: • Human genes composed of coding and non- coding sequences. The copy of the coding sequences is called cDNA. • The synthesis of the insulin cDNA will allow the production of a functional insulin molecule.

49. Transfer of the Insulin gene into a plasmid vector (schematic)

50. Microbial Biotechnology MICROORGANISMS AS TOOLS Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism): • Insulin was first synthesized in 1979 in E. coli cells through the use of recombinant DNA techniques. • Insulin is produced by beta cells in pancreas in humans.