Enzyme Engineering Exam Questions and Answers

                                                 

                  

Enzyme Engineering Test Questions (A)

1. Explanation of nouns (3 points for each question, 30 points in total)

 1. Enzyme engineering: also known as enzyme technology, is a technology for the large-scale production and application of enzyme preparations.

2. Suicide substrate: After the substrate is catalyzed by the enzyme, its potential reactive group is exposed, and then acts on the enzyme to become an irreversible inhibitor of the enzyme. This substrate is called a suicide substrate.

3. Allosteric enzymes: After the regulator binds to the regulatory center of the enzyme molecule, it causes a change in the conformation of the enzyme molecule, thereby changing the affinity of the catalytic center to the substrate. This effect is called the allosteric effect. allosteric enzyme

4. Induced enzymes: Some enzymes are not synthesized or rarely synthesized under normal circumstances, and will be synthesized in large quantities when an inducer is added. Such enzymes are called induced enzymes

1. Mol catalytic activity: Indicates the number of molecules converted by each active center in an enzyme molecule per unit time

6. Ion exchange chromatography: using ion exchangers as carriers, these carriers have a certain charge under certain conditions. When molecules with opposite charges pass through, they will be adsorbed by the carrier due to electrostatic attraction. This separation method is called ion exchange. chromatography.

7. Immobilized enzyme: By physical or chemical methods, the enzyme is bound to a water-insoluble carrier, or the enzyme is bound to a certain space, restricting the free flow of enzyme molecules, but enabling the enzyme to play a catalytic role

8. Modified enzymes: enzymes formed by covalently linking enzymes and some reagents in vitro with certain chemical methods

9. Non-aqueous enzymology: Usually, the catalytic effect of enzymes is carried out in the water phase. The subject of studying the catalytic mechanism of enzymes in the organic phase is non-aqueous enzymology.

10 Simulated enzymes: Non-protein molecules with catalytic functions that are simpler than enzymes and synthesized by organic chemical synthesis methods are called simulated enzymes.

Two fill-in-the-blank questions (1 point for each blank, 30 points in total)

1. There are two factors that determine the catalytic activity of enzymes, one is   the molecular structure of the enzyme   , and the other is    the reaction conditions.

2. The most commonly used method for finding Km is   the double reciprocal graphing method   .

3. The kinetic mechanism of multi-substrate enzymatic reaction can be divided into two categories, one is   sequence mechanism     and the other is  ping-pong mechanism.

4. Reversible inhibition can be divided into   competitive , anti-competitive , non-competitive and mixed;

5. For the bacteria that produce enzymes, we must consider the following conditions, one is to see if it is a  pathogenic bacteria    , the other is to be able to use cheap raw materials, the fermentation cycle   is short   , and the enzyme production    is high . The third is that the bacteria are not easy to      Fourth       , it is best to select strains that can produce   extracellular     enzymes, which is conducive to the separation and purification of enzymes, and the recovery rate is high.

6. The assay method of enzyme activity can be      terminated   reaction method and    continuous       reaction method.

7. There are four types of enzyme preparations, namely   liquid    enzyme preparations,    solid    enzyme preparations,   pure enzyme preparations and    immobilized  enzyme preparations.

8. Usually, enzyme immobilization methods include   adsorption method , embedding   method,  cross-linking  method,

  covalent bonding method

  9. The in vitro modification of enzyme molecules includes surface   modification and    internal   modification of enzymes .

10. The two types of mimic enzymes are  semi-synthetic enzymes and  total synthetases   .

11. The preparation methods of abzyme include  copy    method and   introduction    method.

Three questions and answers (10 points each, 40 points in total)

1. What are the advantages and disadvantages of immobilized enzymes compared with free enzymes?

Solution: Advantages (1) It is easy to separate the immobilized enzyme from the substrate and the product, and there is no enzyme residue in the product solution, which simplifies the purification process

(2) It can be used continuously for a long time (3) The reaction process can be strictly controlled, which is conducive to process automation (4) The stability of the enzyme is improved

(5) More suitable for multi-enzyme reactions

(6) Enzyme use efficiency is high, yield is high, and cost is low

shortcoming

1. Loss of enzyme activity upon immobilization
2. More suitable for water-soluble substrates
3. Not suitable for multi-enzyme reactions compared to intact cells.  

1. Write down three methods for separating and purifying enzyme proteins, and briefly describe their principles.

Solution: Method: dialysis and ultrafiltration, centrifugal separation, gel filtration, salting out, isoelectric point precipitation, co-precipitation, adsorption chromatography, electrophoresis affinity chromatography, thermal denaturation, acid-base denaturation, surface denaturation, etc. (principle omitted)

1. Why is the production of enzyme preparations mainly based on microorganisms?

Solution: (1) There are many types of microorganisms, rich in enzymes, and the strains are easy to mutate, and the strains are diverse

(2) Microorganisms grow and multiply quickly, and enzymes are easy to extract, especially extracellular enzymes

(3) Wide range of sources and cheap prices

1. Microorganisms are easy to obtain and the growth cycle is short
2. Microcomputer technology can be used to control the fermentation production of enzymes, which can be continuous, automated, and economically beneficial
3. Molecular biology technology mainly based on genetic engineering can be used to select and transform strains, increase enzyme production rate and develop new enzyme species

4 The following is some data measured by someone on the enzyme, based on which the maximum reaction speed and Michaelis constant of the enzyme can be obtained

[S](mol/L)	V0(form/min)
0.5´10-6	28
4.0´10-6	40
1.0´10-5	70
2.0´10-5	95
4.0´10-5	112
1.0´10-4	128
2.0´10-4	139
1.0´10-2	140

Solution: The maximum reaction speed is 140 , Km: 1.0´10-5

Enzyme Engineering Test Questions (B)

a term explained

1 Abzyme: It is a kind of immunoglobulin with catalytic effect, which belongs to chemical artificial enzyme

2 Enzyme reactor: It is a device that uses biochemical principles to enable enzymes to complete the catalytic action. It provides a suitable place and optimal reaction conditions for enzymatic reactions to maximize the conversion of substrates into substances.

3 Simulated enzymes: Non-protein molecules with catalytic functions that are simpler than enzymes and synthesized by organic chemical synthesis methods are called simulated enzymes.

4 Substrate inhibition: In an enzymatic reaction, a high substrate concentration reduces the reaction rate.

5. Stable pH: The enzyme is stable within a certain pH range, and beyond this limit, it is volatile and inactivated. Such a pH range is the stable pH of the enzyme

6 Enzyme production kinetics: mainly study the rate of cell enzyme production and the influence of various factors on the enzyme production rate, including macroscopic enzyme production kinetics and microscopic enzyme production kinetics.

7. Gel filtration: also known as molecular exclusion chromatography, molecular sieve chromatography, molecular sieves are filled in the chromatography column, the sample to be purified is added and then rinsed with an appropriate buffer. After the molecules in the sample pass through the chromatography column at a certain distance, Chromatographic methods that elute sequentially according to molecular size and are separated from each other.

8. Immobilized enzyme: By physical or chemical methods, the enzyme is bound to a water-insoluble carrier, or the enzyme is bound to a certain space, which restricts the free flow of enzyme molecules, but enables the enzyme to play a catalytic role.

9 Non-aqueous enzymology: Usually, the catalytic effect of enzymes is carried out in the water phase, and the subject of studying the catalytic mechanism of enzymes in the organic phase is non-aqueous enzymology

10 Liquid fermentation method: the method of breeding microorganisms and producing enzymes using liquid culture medium as raw material, and is divided into liquid surface fermentation method and liquid submerged fermentation method according to different ventilation methods.

Two fill-in-the-blank questions (1 point for each blank, 30 points in total)

1. When the Km value increases, the inhibitor is a   competitive  inhibitor; if the Km remains unchanged, the inhibitor is a  non-competitive inhibitor ; if the Km decreases, the inhibitor is  an anti-competitive   inhibitor.

2. The methods generally used for strain culture include    solid    culture method and    liquid    culture method.

3. The quality of the strain is the main factor affecting the enzyme production fermentation. In addition, the fermentation conditions also have a great impact on the enzyme production of the strain. The fermentation conditions generally include   temperature    ,   PH     ,     oxygen    ,   stirring    ,    humidity      and     foam     .

   4. Control conditions     ,    genetic control      ,     and other methods      can break the restriction of enzyme synthesis regulation mechanism .

5. The mode of enzyme biosynthesis is divided into   synchronous synthesis type ,   continuous synthesis type   ,   intermediate synthesis type     and   lagging synthesis type    .

6. The purification methods established according to the differences in the stability of enzymes and proteins include    heat denaturation    ,   acid-base denaturation    and  surface   denaturation

7. Usually, enzyme immobilization methods include    cross-linking    method , embedding    method,   adsorption method and   covalent binding     method

   8. The in vitro modification of enzyme molecules includes surface       modification and     internal     modification of enzymes .

9. The important difference between enzymes and antibodies is that enzymes can bind and stabilize  the filter state   of chemical reactions , thereby reducing  the energy barrier   of substrate molecules , while the antigen bound by antibodies is only a  ground   state molecule, so it has no catalytic ability

Three questions and answers (10 points each, 40 points in total)

1. In production practice, what are the requirements for enzyme-producing bacteria?

Generally, the following conditions must be met:

1. 1. It should not be a pathogenic bacteria, and it is best to have nothing to do with pathogenic bacteria in terms of phylogeny
   2. Can use cheap raw materials, short fermentation cycle, high enzyme production
   3. Bacteria are not easy to mutate and degenerate, and are not easy to infect phages
   4. It is best to use bacteria that produce extracellular enzymes, which is conducive to the separation and purification of enzymes and has a high recovery rate

In the application of food and pharmaceutical industries, safety issues are more important

1. What factors should be considered when chemically modifying an enzyme?

Solution: (1) The nature of the modified enzyme, including the stability of the enzyme, the condition of the active center of the enzyme, the nature and reactivity of the side chain group

(2) Conditions of the modification reaction, including pH and ionic strength, modification reaction time and temperature, ratio of enzyme to modifier in the reaction system, etc.

1. List the functional groups on the enzyme protein that can be combined with the carrier when the enzyme is immobilized by covalent bonding

Solution: (1) The α-amino group at the N-terminal of the enzyme protein or the Σ-amino group of lysine

(2) The carboxyl group of the C-terminal of the enzyme protein and the β carboxyl group of aspartic acid or the γ carboxyl group of glutamic acid

(3) sulfhydryl group of cysteine ​​1 point

1. Hydroxyl on Serine Chroine Threonine
2. The phenyl rings on phenylalanine and tyrosine
3. imidazolyl on histidine

Indolyl on tryptophan

1. After the primary extract of an enzyme is purified once, the following data are obtained by measurement, and the specific activity, recovery rate and purification multiple are calculated.

	Volume (ml)	Vitality unit (u/ml)	Protein nitrogen (mg/ml)
primary extract	120	200	2.5
ammonium sulfate precipitation	5	810	1.5

Solution: (1) Initial total energy: 200´120＝24000 (unit)

(2) Initial specific activity: 200¸2.5=80 (unit/mg protein nitrogen)

(3) Total activity after purification 810´5＝4050 (unit) 2

(4) Purified specific activity 810¸1.5=540 (unit/mg protein nitrogen)  

(5) Yield (percentage yield): 4050¸24000 = 17%   

(6) Purification multiple: 540¸80=6.75