Everything you need to know about enzyme technology. Some of the most frequently asked questions are as follows:-
Q.1. How have new strains of yeast been possible by recombinant DNA technology?
Ans: The new strains of yeasts which show amylolytic activity have been prepared using recombinant DNA technology. To achieve this objective glucoamylase genes from Aspergillus, β-1, 3/1, 4- glucanase genes from Bacillus subtilis and cellulase (endo- β -1, 4 glucanase) and β-galactosidase genes from Trichoderma have been transferred and expressed in brewer’s yeast.
Q.2. Name the organisms and the enzymes involved in brewing and wine making, cheese-making and production of vinegar.
Ans: These are presented in Table 41.1.
Q.3. Name the mould used to produce citric acid. Why Mn2+ and Fe3+ are used in low concentration.
Ans: The growth conditions of Aspergillus Niger are arranged to stimulate the overproduction of citric acid. The growth medium used has high concentration of sugars and low concentration of Mn2+ Fe3+. The low concentration of Mn2+ is kept because Mn2+ deficiency represses all the TCA cycle enzymes except citrate syntheses permitting citric acid accumulation. The citrate is an allosteric inhibitor of phosphofructokinase. Mn2+ deficiency also lead to accumulation of NH4+ ions which relieves citrate inhibition.
Q.4. Give the use of microorganisms in production of organic chemicals.
Ans: A large number of microorganisms are involved in production of organic chemicals. Some of these are listed in Table. 41.2.
Q.5. Which method is generally used for isolation of intracellular enzymes?
Ans: It is high pressure homogenization method, generally used.
Q.6. Name the enzymes used in industry to metabolize carbohydrates.
Ans: These are amylase, exo-1, 4 -α-D-glucosidase (amyloglucosidase), cellulase, polygalacturonase, β-D-fructofuranosidase, glucose oxidase, xylose isomerase generally called glucose isomerase.
Q.7. Which enzymes are used to metabolize proteins in the industry?
Ans: These are papain, chymosin (rennin), trypsin, chymotrypsin, fungal proteases and bacterial proteases.
Q.8. On what activity aeration or CO2 production depend in manufacture of bread?
Ans: It depends on adequate a- and 0- amylase activity.
Q.9. Why is chymosin and not trypsin (a proteolytic enzyme) used in cheese-making?
Ans: Chymosin (rennin) obtained largely from calf stomachs is an aspartic acid rotease leads to clotting of milk which involves the cleavage of a single peptide bond in k-casein between Phe 105 and Met 106, releasing the acidic c-terminal peptide.
The release of c-terminal peptide is followed by Ca2+ induced aggregation of the modified micelles to form a gel. The micelles are an aggregate of amphipathic molecules in water with the nonpolar portions in the interior and the polar portions at the exterior surface exposed to water. On the other hand proteolytic enzymes like trypsin also result clotting of casein (milk protein) but further degrade casein. If it happens in cheese-making it leads to impart undesirable flavours.
Q.10. What are the substitutes of chymosin obtained from calf stomachs with the ever increasing number of vegetarians?
Ans: The fungal aspartate proteases from Mucor pucillus, Rhizomucor miehei and Cryphonectria parasitica serve as substitute for chymosin but they do not possess good clotting proteolysis ratio. However, recombinant DNA chymosin in which the chymosin gene has been closed into expression systems such as Aspergillus oryzae and Kluyveromyces lactis have also been used in cheese-making since 1994.
Q.11. Which enzymes are used in tenderization of meat?
Ans: The toughness in meat is mainly due to collagen and elastin, and also due to actomysin. Lower- quality cuts of meat are just as nutritious as prime cuts. Therefore, to make maximum use of carcass (dead body of animal for cutting up as meat.
The carcass meat is contrasted from tinned or corned meat) efforts have been made to tenderize the quality cuts. Proteases are used both for the purpose of tenderization as well as marination. Traditionally, enzyme papain is used but bromelain trypsin, chymotrypsin and microbial proteases from the mould Aspergillus are also used for the purpose.
Q.12. Which are the enzymes used as sweetening agents?
Ans: Two important enzymes used for the process are exo-1, 4 – α -D- glucosidase obtained from the fungus Aspergillus Niger, and xylose isomerase from Streptomyces spp. The xylose isomerase is also called glucose isomerase since it also isomerize glucose.
Q.13. Give the role of enzymes in classification of beverages.
Ans: Turbidity in beers, wines and fruit juices is undesirable because turbit products are not accepted by the customer. The fruit juices are more easily prepared from pulp if the viscosity is not very high. In case of beer the turbidity called as haze may be formed on cooling because of a complex between tannin and protein. It can be removed by adding small amounts of proteases often papain.
But a very careful balance has to be maintained because proteins because of their foaming properties account for the formation c f head or the froth on the top. The proteolysis not only remoes the chilled haze but good head also. Therefore, the head needs to be protected. In case of wines and fruit juices the turbidity is because of starch or pectin which can be removed by addition of amylases or polygalacturonases.
Q.14. Give the two main uses of immobilized enzymes.
Ans: Two main uses of immobilized enzymes are:
1. Preparative – as large scale isomerization of glucose and production of L-amino acids racemic mixtures.
2. Analytical – as use of immobilized glucose oxidase.
Q.15. How enzyme works as sweetener in production of syrups from corn starch?
Ans: It is well known that fructose is more satisfactory than glucose as it is sweeter and does not crystalize so readily in concentrated solutions. The overall process comprises of two enzyme catalyzed steps as below:
Starch → exo-1, 4-α-D- glucosidase, glucose → xylose isomerase, fructose
Immobilized whole cells used in amino acid inter-conversions:
Q.16. How immobilized whole cells may be used for amino acid inter-conversions? Discuss.
Ans: Not only single enzymes rather whole cells also can be immobilized by trapping the cells in polymerizing acrylamide and N, N’ – methylenebisacrylamide around the cells. Undamaged or complete cells of Escherichia coli and Pseudomonas putida having suitable enzymes, have been used for the production of aspartate, phenylalanine tryptophane, senna, urocanic acid 6 – aminopenicillanic acid by entrapment technique.
Q.17. What is acrylamide? Discuss its enzymatic synthesis.
Ans: Acrylamide is used as a starting material for the production of a large number of polymers, as an ingredient in coagulators and as soil conditioner. Acrylamide can be made by reacting acrylonitrile and water in the presence of appropriate inorganic catalysts like copper but this leads to undesirable side reactions. On the other hand nitriles are found in plants, bacteria and fungi, e.g., in antibiotics like toyocamycin and treponemycin and some microorganisms are capable of using nitrile both as carbon and nitrogen source.
Aliphatic nitriles in this way are degraded in two steps:
1. The first step is catalysed by nitrile hydratase
2. The second is catalysed by an amidase
While Rhodococcus rhodochrous is grown on the optimum culture medium to induce nitrile hydratase, the latter stands for more than 50% of total soluble protein. The purification of enzyme is not necessary rather simply to entrap the organisms in a polyacrylamide gel. Thus it is one of the major products made using immobilized enzymes.
Q.18. Give one of the most common biological recognition mechanism or use of biosensors in clinical diagnosis.
Ans: More than half of the existing applications of biosensors are the devices to measure blood glucose.