Enzymes

1. Enzymes • materials, nutraceuticals, pharmaceuticals, monitoring devices, bulk chemicals, and fine chemicals • A number of enzymatic processes have already replaced conventional chemical processes. • The application of enzymes in some processes has helped to develop processes unavailable to conventional chemistry.

2. Introduction • key roles in numerous biotechnology products and processes that are commonly encountered in the form of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices • the most frequently used enzymes in biotechnology are hydrolases, which catalyze the breakdown of molecules. • Enzymes can display regio- and stereospecificity, properties that have been exploited for asymmetric synthesis and racemic resolution. • Chiral selectivity is employed to prepare enantiomerically pure pharmaceuticals, agrochemicals, chemical feedstocks, and food additives. • industrial enzyme market has expanded at a rate of about 10% annually, microbial enzymes have largely replaced the traditional plant and animal enzymes, and most of them are produced recombinantly. • DNA technology has been used to modify substrate specificity and improve stability properties of enzymes for increasing yields of enzyme-catalyzed reactions.

3. Introduction • used in metabolic engineering of cellular metabolism to increase yields of fermentation products • Enzymes are environmentally friendly; they work under moderate conditions of temperature, pH, and pressure, their catalyzed reactions rarely form wasteful side products, and the proteins themselves are biodegradable and generally pose no threat to the environment.

4. Classification They are protein catalysts, accelerate chemical reactions, the transfer of electrons, atoms, or functional groups, by several orders of magnitude while maintaining high fidelity of reaction trajectories and producing low levels of side-reaction products. may require organic (e.g., coenzymes such as flavin mononucleotide and thiamine pyrophosphate) or inorganic (e.g., Mg2þ) cofactors that, like the enzyme itself, are not consumed in the catalyzed reactions. Nomenclature Assigned longer systematic names and four-part classification numbers (EC number, ‘enzyme commission number’) by the enzyme nomenclature system maintained by the International Union of Biochemistry and Molecular Biology and the International Union of Pure and Applied Chemistry (IUPAC)

5. Classification Depending on the type of reaction catalyzed, enzymes are divided into six main classes: 1.Oxidoreductases. Transfer of electrons from one substrate molecule to another (e.g., dehydrogenases, reductases, oxidases). 2. Transferases. Transfer of functional group from one substrate molecule to another (e.g., glycosyltransferases, acetyl transferases, and aminotransferases). 3. Hydrolases. Transfer of functional group from substrate to water (e.g., glycoside hydrolases, peptidases, esterases). 4. Lyases. Elimination of functional group from substrate with the formation of double bonds. Thus, bonds are cleaved using a different principle than hydrolysis (e.g., pectatelyases break glycosidic linkages by b-elimination). 5. Isomerases. Transfer of groups from one position to another in the same molecule (e.g., glucose isomerase). 6. Ligases. Addition of function group to substrate usually coupled with ATP hydrolysis (e.g., glycine–tRNAligase).

6. Industrial Uses • Starch conversions • Production of glucose syrup • Production of high fructose corn syrup • Production of high maltose conversion syrups • Production of cyclodextrins • Production of ethanol • Lignocellulosic Biomass conversions • Cellulose conversion • Hemicellulose conversion • Lignin conversion

7. Industrial Uses Enzymes in the Production of Functional Oligosaccharides and Other Neutraceuticals Enzymes in the Modification of Fats and Oils Enzymes in the Animal Feed Industry Enzymes in the Pulp and Paper Industry Enzymes in the Fruit Juice Processing Industry Enzymes in the Meat and Fish Processing Industry Enzymes in the Dairy Industry Enzymes in Detergents Enzymes in the Leather Industry Enzymes in the Production of Bulk and Fine Chemicals Analytical Applications of Enzymes Enzyme-ReplacementTherapy

8. Starch conversions • Starch contains about 15–30% amylose and 70–85% amylopectin. • Enzymes have largely replaced the use of strong acid and high temperature to break down starchy materials. • Three types of enzymes are involved in starch bioconversion: • endo-amylase (a-amylase, EC 3.2.1.1), [Bacillus lichiniformis, Bacillus subtilis, and Bacillus amyloliquefaciens and fungisuch as Aspergillus oryzae] • exo-amylases • glucoamylaseor glucan 1,4-a-glucosidase, EC 3.2.1.3 [Endomycopsis, Aspergillus, Penicillium, Rhizopus, and Mucor]; • b-amylase, EC 3.2.1.2)[Bacillus megaterium, Bacillus cereus, Bacillus polymyxa, Thermoanaerobacterthermosulfurogenes, and Pseudomonas sp.] • debranchingenzymes (pullulanase, EC 3.2.1.41; isoamylase, EC 3.2.1.68). [Aerobacteraerogenesand isoamylase is produced by Pseudomonas amyloderamosa.]

9. In amylose these are linked  -(1, 4)-, with the ring oxygen atoms all on the same side.

10. In amylopectin about one residue in every twenty or so is also linked -(1,6)- forming branch-points.

11.  -1,4 hydrolyzes the a-1,4-glycosidic bonds in starch from the nonreducing ends, generating maltose. The enzyme is unable to bypass the a-1,6 linkages and leaves dextrins, known as b-limit dextrins Hydrolyse internal (endo) a- 1,4 but not a- 1,6 producing malto-oligosaccharides Enzymatic hydrolysis of amylose

12. cleaves glucose units from the nonreducing end of starch and it can hydrolyze both a-1,4 and a-1,6 linkages of starch, slower Enzymatic hydrolysis of amylopectin

13. Dextrins: A group of low-molecular-weight carbohydrates produced by the hydrolysis of starch. Dextrins are mixtures of linear a-(1,4)-linked D-glucose polymers starting with an a-(1,6) bond.

14. Amylases break starch into sugars All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. a-Amylases Both the salivary and pancreatic amylases are α-Amylases. They are Ca metalloenzymes,completely unable to function in the absence of calcium. They act at random locations along the starch chain hence faster than b-amylases Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. During the ripening of fruit, β-amylase breaks starch into sugar, resulting in the sweet flavor of ripe fruit. Both are present in seeds; β-amylase is present prior to germination, whereas α-amylase and proteases appear once germination has begun.

15. Enzymes for starch conversion a-amylase randomly hydrolyse a-1,4 linkages in both amylose and amylopectin to yeild mixture of glucose, maltose, maltotriose and series of a-limit dextrins. b-amylase sometimes used in place of a-amylase. They hydrolyze alternate a-1,4 linkages and yield maltose residues and b-limit dextrins Glucoamylase hydrolyses a-1,3. a-1,4 and a-1,6 linkages but is less efficient than a-amylase. Major role is to break cross links of amylopectin resulting in complete breakdown to glucose. Generally used to reduce CHO content of beers. Industrially obtained from fungus Aspergillusniger. Glucose isomeraseis used for conversion of glucose obtained after processing to fructose. Pullulanase (pullulana-1,6-glucanohydrolase) or isoamy- lase (glycogen a-1,6-glucanohydrolase) cleaves the a-1,6- linked branch points of starch and produces linear amylosaccharides of varying lengths.

16. Production of glucose syrup • Production of high fructose corn syrup • Production of high maltose conversion syrups • Production of cyclodextrins • Production of D-glucose from starch by acid hydrolysis (chemical) produces undesirable bitter sugar (gentiobiose), and the inevitable formation of salt (from subsequent neutralization with alkali) and coloring materials. • With the discovery and development of thermostablea-amylase from Bacillus licheniformis, an enzymatic process has replaced the acid hydrolysis process. • liquefaction and saccharification • Typically, glucose syrups (DE 97–98) having 96% glucose contain 2–3% disaccharides (maltose and isomaltose) and 1–2% higher saccharides. Dextrose equivalent (DE) is a measure of the amount of reducing sugars present in a sugar product, relative to glucose, expressed as a percentage on a dry substance basis (DS). an estimate of the percentage reducing sugars present in the total starch product

17. GLUCOSE ---- FURCTOSE ISOMERIZATION 2. HFCS Glucose isomerase(also known as xylose isomerase, EC 5.3.1.5) is an example of the highly successful application of enzyme biotechnology to an industrial process that has no commercially viable route through conventional chemistry. Chemical isomerization of glucose to fructose at high pH and high temperature leads to undesirable side products, some of which are colored and have off flavors. Enzyme- catalyzed isomerization (at moderate pH and temperature) does not form undesired side products. DNA TECHNOLOGY FOR INCR IN THERMAL STABILITY Streptomyces, Bacillus, Arthobacter, and Actinoplanes

18. 3. Various maltose-containing syrups are used in the brewing, baking, soft drink, canning, confectionery, and other food industries. There are three types of maltose-containing syrups: high-maltose syrup (DE 35–50, 45–60% maltose, 10–25% maltotriose, 0.5–3% glucose), extra high-maltose syrup (DE 45–60, 70–85% maltose, 8–21% maltotriose, 1.5–2% glucose), and high conversion syrup (DE 60–70, 30–47% maltose, 35–43% glucose, 8–15% maltotriose). liquefaction and saccharification, as in the production of glucose. However, in this process, the liquefaction reaction is terminated when the DE reaches about 5–10 since a low DE value increases the potential for attaining high maltose content. maltogenic amylase such as b-amylase, b-amylase with pullulanase or isoamylase, or a fungal a-amylase at pH 5.0–5.5 and 50–55oC.

19. Production of ethanol The process of making ethanol from starch involves three basic steps: (1) preparation of the glucose feedstock, (2) fermentation of glucose to ethanol, and (3) recovery of ethanol. Enzymes have major role in preparation of feedstock: Corn kernelscontain 60–70% starch

20. Wet milling process corn is steeped in acidic water solutions (corn steep liquor) and the oil, protein, and fiber fractions are successively removed as products leaving the starch fraction. Enzymatic liquefaction and saccharification of the starch fraction are then carried out for the production of glucose Glucose is fermented by the traditional yeast Saccharomyces cerevisiae to ethanol, which can be recovered by distillation.

21. Drygrindingprocess lower capital investment required in comparison to that of wet mills. In the typical dry grind process, corn is mechanically milled to a coarse flour. Oil, protein, and fiber fractions are not isolated liquefaction, enzymatic saccharification using glucoamylase fermentation using the conventional yeast are carried out simultaneously. The addition of protein-splitting enzymes (proteases) releases soluble nitrogen compounds from the fermentation mash and promotes growth of the yeast, decreasing fermentation time. The residue left after fermenting the sugars is known as distiller’s grains, which is used as animal feed.

22. Milled grain steam Gelatinized material cool a-amylase Bacillus amyloliquefaciens Liquefied material Glucoamylase Saccharified material Yeast Glucose isomerase Fermentation Fructose Alcohol Industrial production of alcohol and fructose from starch

23. Lignocellulosic Biomass conversions Various agricultural residues (straws, hulls, stems, cobs, stalks), deciduous and coniferous woods, municipal solid wastes (paper, cardboard, yard debris, wood products), waste from the pulp and paper industry, and energy crops (switchgrass, miscanthus). These materials are structurally diverse and compositions vary widely (cellulose, 35–50%; hemicellulose, 20–35%; lignin, 10–25%; proteins, oils, and ash, 3–15%). Native lignocellulosic biomass is resistant to enzymatic hydrolysis hence Pretreatment is required like steam explosion, dilute acid, concentrated acid, alkali, SO2, alkaline peroxide, ammonia fiber expansion, and organic solvents.

24. hemicellulose to simple sugars (xylose, arabinose, and other sugars) and acids (acetic, glucuronic), which are water-soluble. • insoluble residue contains cellulose and lignin. • The lignin can be extracted with solvents such as ethanol, butanol, or formic acid. • Alternatively, enzymatic hydrolysis of cellulose with lignin present produces glucose, and the residues are lignin plus any unreacted materials.

25. Cellulose (C6H10O5)n • Structural component of cell wall of green plants, many algae and fungi. Some bacteria secrete it to form biofilms. • It is the most common organic compound on earth. • 33% plant matter is cellulose. Cotton is 90% cellulose and wood is 50% cellulose. • Industrially, cellulose is obtained from wood pulp and cotton to produce cardboard and paper and derivatized to make cellophane and rayon. • Cellulose can be digested in the gut of ruminants and termites with the help of symbiotic bacteria (Trichonympha, which produces cellulases). Humans cannot digest cellulose but acts as dietary fiber and hydrophilic bulking agent for faces. • The major combustible component of non-food energy crops is cellulose, with lignin being second. • Some bacteria can convert cellulose into ethanol which can then be used as a fuel . • Cellulose is crystalline, strong, and resistant to hydrolysis, cellulose contains only anhydrous glucose residues with beta configuration.

26. Structural Unit Cellulose is a linear polymer of β-(1,4)-D-glucopyranose units in 4C1 conformation. The fully equatorial conformation of β-linked glucopyranose residues stabilizes the chair structure, minimizing its flexibility (for example, relative to the slightly more flexible α-linked glucopyranose residues in amylose).

27. Functionality • Cellulose has many uses as an anticake agent, emulsifier, stabilizer, dispersing agent, thickener, and gelling agent but these are generally subsidiary to its most important use of water holding capacity. • Water cannot penetrate crystalline cellulose but dry amorphous celluloseabsorbs water becoming soft and flexible. Some of this water is non-freezing but most is simply trapped. • Less water is bound by direct hydrogen bonding if the cellulose has high crystallinity but some fibrous cellulose products can hold on to considerable water in pores and its typically straw-like cavities; water holding ability correlating well with the amorphous (surface area effect) and void fraction (that is, the porosity). • As such water is supercoolable, this effect may protect against ice damage. Cellulose can give improved volume and texture particularly as a fat replacer in sauces and dressings but its insolubility means that all products will be cloudy.

28. Lignocellulose: structural support system for all terrestrial plants Lignocellulosics Plant biomass comprises of Lignin, hemicellulose and cellulose combined in different proportions Primary cellulosics Agricultural waste cellulosics Municipal Waste cellulosics Plant material that remain after harvesting and processing Straw, corn, rice hulls, sugarcane baggase, animal manure, timber residues Plants harvested for cellulosic content, structural use or feed Cotton, timber, hay Waste paper and discarded paper products

29. Lignin • Complex chemical compound most commonly derived from wood and an integral part of the cell walls of plants. • one of the most abundant organic polymers on Earth, superseded only by cellulose, employing 30% of non-fossil organic carbon and constituting from a quarter to a third of the dry mass of wood LIGNIN: 3D, GLOBULAR, IRREGULAR, INSOLUBLE, HIGH MW POLYMER MADE OF PHENYLPROPANE SUBUNITS NO CHAINS OF REPEATING UNITS OR BONDS THAT ARE EASILY HYDROLYSABLE In plants lignin is bonded to hemicellulose and wraps around fibres composed of cellulose Gives rigidity, resistance to mechanical stress and microbial attack Fungus: Phanerochaetechrysosporium

31. HEMICELLULOSE:SHORT CHAIN HETEROGENOUS POLYMERS CONTAN BOTH HEXOSES (C6: GLU, MAN, GAL) AND PENTOSES (C5: XYL, ARA) Difference between hemicellulose and cellulose Hemicellulose is amorphous, random and easily hydrolysed by acid or base or hemicellulases. It consists of shorter chains - 500-3000 sugar units, hemicellulose is a branched polymer. Composed of hexoses and pentoses. Cellulose is crystalline, strong and resistant to hydrolysis. Contain 7,000 - 15,000 glucose molecules per polymer and is unbranched. Composed of anhydrous glucose units joined by b-1,4. Major hemicelluloses are 1. Xylans (backbone of b-1,4 xylans with side chains to ara, glucoronic acid, arabinoglucoronic acid) present in hardwoods 2. Mannans (glucomannans, galactomannans, arabinogalactans) present in softwoods Trichoderma sp., Aspergillus sp., Fusarium sp., and Bacillus sp

32. Enzymes for cellulose hydrolysis Fungus: Trichodermareesei Cellulomonasfimi Aspergillus Endo-1,4-b-glucanse: hydrolyzes b-1,4 linkages b/w adjglumocs(cellulase, EC 3.2.1.4) Exo-1,4-b-glucanase: degrades nicked cellulose chains from non reducing ends and produced glucose, cellobiose (2 glu units) and cellotriose (3 glu units) 1,4-b-Cellobiohydrolase; type of exoglucanase removes units of 10 or more glu residues from non reducing ends (found in cellulolytic fungi) b-glucosidase or cellobiaseconverts cellobiose and cellotriose to glucose cellobiose

33. Crystalline region Amorphous region Endoglucanase Removal of oligosacc. from reducing ends Exoglucanase cellobiose cellotriose Exoglucanase Cellobiohydrolase glu Endoglucanase b-glucosidase Enzymatic biodegradation of cellulose

34. Cellulose Inhibits Cellobiose Feedback Inhibitor of cellulose hydrolysis Feedback Inhibitor of cellobiose Inhibits Glucose Decrease amount of cellobiose which prevents end product inhibition of exo and endo b-glucosidase Cloning of gene in host cell b-glucosidase not only produces glucose from cellobiose but also lowers cellobioseinhibition, allowing the cellulolytic enzymes to function more efficiently. However, like b-glucanases, most b-glucosidasesare subject to product (glucose) inhibition. Fermentation To increase rate and extent of degradation , addition of b-glucosidase enhances enzymatic utilization of cellulose