A biochemical question 5 on enzyme purification

Total active unit Total protein = enzyme specific activity (u mg).

The most efficient step, i.e., the one that increases the most than viability for the first time, is affinity chromatography.

The least efficient step is the one where the enzyme activity is most lost, i.e., pH precipitation.

The results in the table are not indicative of absolute protein purity. Other methods to evaluate purity include: SDS-PAGE electrophoresis (whether the band is single), chromatography (whether the peak shape is smooth and complete), etc.

a.Specific activity of the enzyme solution.

This is the total activity of the total protein corresponding to each purification step.

Units are all U mg

b. 4c. 3

d.Yes, there is no increase in activity at step 6 and can be assessed using SDS-PAGE.

Chapter 3 Enzymes.

First, the group of enzymes is scattered.

Simple enzymes: Enzymes that are made up of only amino acid residues.

Conjugating enzymes: enzyme proteins: determine the specificity of the reaction;

Cofactors: determine the type and nature of the reaction; It can be metal ions or small molecule organic compounds.

It can be divided into coenzymes: it binds loosely to enzyme proteins and can be removed by dialysis or ultrafiltration.

Prosthetic group: tightly bound to enzyme proteins, cannot be removed by dialysis or ultrafiltration.

The complex formed by the combination of enzyme proteins and cofactors is called holoenzyme, and only holoenzyme has a catalytic effect.

Vitamins involved in the composition of coenzymes.

The group that transfers the coenzyme or prosthetic group of the vitamin contained.

Hydrogen atom NAD + NADP + Niacin amide (vitamin PP).

FMN FAD Vitamin B2

Aldehyde TPP vitamin B1

Acyl CoA lipoic acid pantothenic acid, lipoic acid.

Alkyl cobalamins, coenzymes, vitamin B12

Carbon dioxide, biotin, biotin.

Amino pyridoxal phosphate Pyridoxal (vitamin B6).

Methyl, equal one-carbon unit tetrahydrofolate folic acid.

Second, the active center of the enzyme.

The active center of the enzyme is composed of the essential groups of the enzyme, which are close to the specific spatial structure in the spatial position, and can specifically bind to the substrate and convert the substrate into a product. For conjugate enzymes, cofactors are involved in the composition of the enzyme's center of activity. However, there are some essential groups that do not participate in the composition of the active center.

3. Enzyme reaction kinetics.

The speed of the enzymatic reaction depends on substrate concentration, enzyme concentration, pH, temperature, agonists and inhibitors, etc.

1. Substrate concentration.

1) When the substrate concentration is low, the reaction rate increases with the increase of substrate concentration, increases the substrate concentration, slows down the reaction speed, further increases the substrate concentration, and the reaction speed no longer accelerates with the increase of substrate concentration, reaching the reaction speed, at this time, the active center of the enzyme is saturated by the substrate.

2) Michaelis equation.

v＝vmax［s］／km＋［s］

a.The Michaelis constant km value is equal to the substrate concentration at half the speed of the enzymatic reaction.

The smaller the value, the greater the affinity of the enzyme for the substrate.

The value is one of the characteristic constants of the enzyme, which is only related to the structure of the enzyme, the substrate catalyzed by the enzyme and the reaction environment such as temperature, pH, and ionic strength, and has nothing to do with the concentration of the enzyme.

is the reaction rate when the enzyme is completely saturated with the substrate and is proportional to the enzyme concentration.

2. Enzyme concentration.

In the enzyme reaction system, when the substrate concentration greatly exceeds the enzyme concentration, so that the enzyme is saturated with the substrate, the reaction speed is proportional to the concentration of the enzyme.

3. Temperature Temperature has a dual effect on the speed of enzymatic reactions. On the one hand, increasing the temperature can speed up the enzymatic reaction, and at the same time, increase the denaturation of the enzyme. The ambient temperature at which the enzymatic reaction is fastest is called the optimal temperature for the enzymatic reaction.

Although the activity of the enzyme decreases with the decrease of temperature, the low temperature generally does not destroy the enzyme.

The optimal temperature of an enzyme is not a characteristic constant of the enzyme, it is related to the time when the reaction takes place.

Summary. 2. In the process of biochemical reactions, some enzymes have the activity of a variety of enzymes (such as topoisomerase, which can be both hydrolyzed and linked.

Topoisomerases are capable of both hydrolyzing and linking phosphodiester bonds to DNA molecules.

2. In the process of biochemical reactions, some enzymes have the activity of a variety of enzymes (such as topoisomerase, which can be both hydrolyzed and linked.

2. In the process of biochemical reactions, some enzymes have a variety of enzymes (such as topoisomerase can be hydrolyzed and connected topoisomerase can hydrolyze DNA molecules, and can be linked to phosphodiester bonds).

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When purifying enzyme preparations, the main indicators of enzyme purity are: Liyin () aProtein concentration.

b.Enzyme amount. c.Enzyme activity.

d.The specific activity of the enzyme limb front carries the god.

e.Physicochemical properties of enzymes.

Correct answer: d

Answer]: The enzyme activity determination during purification should be considered as:

1) In order to quickly know the purification results with dismantling, the determination method is required to be fast and simple, and the accuracy is relatively high to a certain extent, and even 5% and 10% errors are allowed, so spectrophotometry, electrical determination and other determinations are commonly used.

2) Holoenzymes may lose cofactors in the process of separation and purification, so it is sometimes necessary to add corresponding substances to the reaction system.

3) Because substances that affect or interfere with the reaction and determination of enzymes are introduced during the purification process, it is sometimes necessary to perform dialysis or add chelating agents before viability.

9.When enzyme-catalyzed hydrolysis to obtain secondary glycosides, the most suitable temperature and time is bond only ()bFerment for 4-8 hours.

d.Ferment for 24 hours.

Correct touch the source answer laugh: ad