CN110283880A - A kind of new method measuring prolease activity - Google Patents
A kind of new method measuring prolease activity Download PDFInfo
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- CN110283880A CN110283880A CN201910730238.2A CN201910730238A CN110283880A CN 110283880 A CN110283880 A CN 110283880A CN 201910730238 A CN201910730238 A CN 201910730238A CN 110283880 A CN110283880 A CN 110283880A
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- VBEGHXKAFSLLGE-UHFFFAOYSA-N n-phenylnitramide Chemical compound [O-][N+](=O)NC1=CC=CC=C1 VBEGHXKAFSLLGE-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
Abstract
The present invention provides a kind of new method for measuring prolease activity, can more comprehensively, more accurately evaluate the practical hydrolysis ability of protease.This method is mainly using a kind of composition being made of 20 kinds of oligopeptide compounds as substrate, and the oligopeptide compounds have general formula Suc-Ala-Ala-Pro-X-pNA, and wherein X represents any one of 20 kinds of common amino acids；20 kinds of oligopeptide compounds are synthesized, prepare the reaction system that mixed substrates solution and buffer, liquid of protease form, the pNA monomer generated after the peptide bond between protease hydrolytic amino acid X and paranitroanilinum pNA is in OD410There is absorption peak at place, by being used as substrate after mixing 20 kinds of oligopeptides, measures OD after protease hydrolytic mixed substrates410Value, measure OD after reaction410Light absorption value, the maximum enzyme activity of protease can be obtained.
Description
Technical field
The invention belongs to bioengineering fields, specifically relate to a kind of new method for measuring prolease activity.
Background technique
In numerous enzyme preparations, protease occupies critically important status due to its extensive use in the industry.Albumen
Enzyme can be hydrolyzed to amino acid, peptide, peptone with the peptide bond in catalytic proteins macromolecular, so also referred to as peptase.Due to albumen
The hydrolysis that enzyme has, and it is widely used in the industries such as leather industry, detergent, medicine, food and feed processing.It is washing
A kind of protease that subtilisin is used in agent, greatly improves washing effect.
Protease is one of most important and the most abundant enzyme system of biological industry, and the source of protease is wider, is widely distributed in
In pluck, microorganism and plant stem-leaf, fruit, wherein the protease of bacterial origin occupies world market nearly 20%.From plant
The protease that object extracts is mainly neutral proteinase, wherein representative is papain, keratinase, bromelain
Enzyme and ficin.The source of current protease includes plant origin, and animal origin is microbe-derived.Wherein, micro- life
The protease in object source occupies 60% or more of global protease sales volume.
Protease is divided into different types according to different mode classifications, and some is divided with activated centre, or with effect
Mode divides, also some is divided with optimal pH, is academicly all divided with activated centre.By activated centre protease can be divided into
Lower four classes: (1) serine protease, (2) cysteine proteinase, (3) aspartic protease, (4) metalloproteinases.Silk
The member of serine protease family is widely present in bacterium, animal pancreas, in mould.Serine protease may be used also with its specificity
It is divided into trypsase type, four seed type of chymotrypsin type, staphylococcus type and slime bacteria.The optimum pH of serine protease exists
9.5-10.5 is alkali protease, but has individual serine proteases to belong to neutral proteinase.Their active site all contains
Ser, His, Asp, and catalyst mechanism having the same.The difference of they and substrate-binding site also determines respectively to substrate
Specificity.
Protease hydrolytic peptide bond generates amino acid monomer, and the principle of National Standard Method measurement proteinase activity power is to utilize albumen
The phenolic group on tyrosine that enzyme hydrolysis casein generates can react with forint phenol in alkaline environment and generate blue precipitate, this
Blue precipitate is in OD680There is the principle of absorption peak, measures the ability for the peptide bond that tyrosine is formed in protease hydrolytic substrate.Foundation
The principle of National Standard Method measurement prolease activity is it is found that the enzyme activity of National Standard Method measurement is only for junket ammonia in protease hydrolytic substrate
The ability for the peptide bond that acid generates, and actually protease can hydrolyze all categories Amino acid profile peptide bond, therefore national standard
Method measurement enzyme activity it is practical be protease whole hydrolysising peptide key ability subset.During National Standard Method measures enzyme activity, need
To pass through dilute sample, substrate hydrolysis reaction is terminated and is reacted, reaction solution centrifuging and taking supernatant, the colour developing of forint phenol and etc., single sample
Longer and sample requirements are larger the time required to product measure enzyme activity.Substrate casein used in national standard is that isoelectric point is pH4.8
Bisexual protein, exist in milk with the composite form of Dicalcium Phosphate, tricalcium phosphate or both, construct it is extremely complex,
Up to now without completely specified molecular formula, molecular weight is about 57000-375000.
Summary of the invention
It is an object of the invention to the deficiencies for prolease activity measuring method in the prior art, provide a kind of measurement egg
The new method of white enzyme activity can more comprehensively, more accurately evaluate the practical hydrolysis ability of protease.
Technical solution of the present invention is summarized as follows: the method for the present invention is mainly to be made of using a kind of 20 kinds of oligopeptide compounds
For composition as substrate, the oligopeptide compounds have general formula Suc-Ala-Ala-Pro-X-pNA (abbreviation: AAPX, molecular structure
Formula is as follows), wherein X represents any one of 20 kinds of common amino acids；Protease hydrolytic whole can be measured using this method
The enzyme activity of peptide bond.
20 kinds of oligopeptide compounds are synthesized, the reaction system that mixed substrates solution and buffer, liquid of protease form, egg are prepared
The pNA monomer generated after peptide bond between white enzyme hydrolysis amino acid X and paranitroanilinum pNA is in OD410There is absorption peak at place, hydrolysis
OD is measured after reaction410Light absorption value, the maximum enzyme activity of protease can be obtained.
Further, by configuring the buffer of different pH, this method is widely used in measurement acid protease, neutral egg
The maximum enzyme activity of white enzyme, alkali protease.
Further, enzyme activity determination method of the invention mainly includes the following steps:
1, the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；
2, reaction system forms: according to liquor capacity, 8 portions of buffers, 2 parts of mixed substrates solution, albumen after 10 parts of dilutions
Enzyme solution；
3, it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4, with the concentration and OD of nitroaniline standard solution410Light absorption value draws standard curve, is calculated according to standard curve
To the enzyme activity in measured sample enzyme solution.
Preferably, after the dilution enzyme activity of liquid of protease in 3U/mL-4U/mL.
Preferably, the phosphate buffer (being suitable for measurement neutral proteinase) that the buffer is pH 7.5；Or pH
2.0 lactic acid-sodium lactate buffer (is suitable for measurement acid protease)；Or borax-sodium hydrate buffer solution of pH 10.5
(being suitable for measurement alkali protease).
The utility model has the advantages that
1, the present invention can be commented comprehensively using the enzyme activity of the mixed substrates measurement protease containing 20 kinds of amino acid peptide bonds
Maximum effectiveness during valence protease hydrolytic, true hydrolysis of the enzyme activity index measured closer to protease in practical applications
Ability.
2, the method for the present invention operating procedure is simple, and speed is fast, and time-consuming short, sample requirements are small, can carry out simultaneously multiple
The enzyme activity determination of sample works, and can satisfy the demand of batch samples measurement work.
Detailed description of the invention:
Fig. 1: the standard curve of the method for the present invention measurement prolease activity.
Specific embodiment:
The method of the present invention is described below by specific embodiment.Unless stated otherwise, the technology hand used in the present invention
Duan Junwei method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from
Under the premise of spirit and scope of the present invention, to the various changes or change of material component and dosage progress in these embodiments
Also belong to protection scope of the present invention.
Partial buffer solution used is formulated as follows in the embodiment of the present invention:
Phosphate buffer (pH 7.5): weighing disodium hydrogen phosphate 6.02g and sodium dihydrogen phosphate 0.5g, is dissolved in water and fixed
It measures and corrects using pH meter after holding to 1000mL.
Lactic acid-sodium lactate buffer (pH 2.0): solution A: weighing lactic acid (60%-85%) 12.0g, is dissolved in water and fixed
Hold to 1000mL, second liquid: weighs sodium lactate (50%-70%) 18.0g, be dissolved in water and be settled to 1000mL.It prepares: taking solution A
10mL adds second liquid 2mL, mixes, and forms 0.05mol/L acidity after one times of dilution and rushes solution, pH meter is recycled to measure and correct.
Borax-sodium hydrate buffer solution (pH 10.5): solution A: weighing Boratex (borax) 10.0g, is dissolved in water and fixed
Hold to 500mL, second liquid: weighing sodium hydroxide 2.0g, is dissolved in water and is settled to 500mL.Preparation: solution A 500mL, second liquid are taken
400mL is mixed, and is measured and is corrected using pH meter after being diluted with water to 1000mL.
20 kinds of common amino acids refer to glycine, alanine, valine, leucine, isoleucine, phenylalanine, dried meat ammonia
Acid, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, paddy
The amino acid of this 20 kinds composition human body proteins of propylhomoserin, lysine, arginine and histidine.
Measuring method of the present invention is defined as follows enzyme-activity unit: 1 enzyme activity unit refers to 1g enzyme powder or 1mL enzyme solution
Under specified conditions (40 DEG C, other is optimum condition), the enzyme amount of 1 micromole substrate can be converted in 1 minute.
Embodiment 1: mixed substrates measure the drafting of the standard curve of proteinase activity
1) p-nitrophenyl amine aqueous solution of various concentration is prepared with DMSO.
2) reaction system forms: 80 μ L phosphate buffers (pH=7.5), 20 μ L p-nitrophenyl amine aqueous solutions, 100 μ L phosphoric acid
Salt buffer (pH=7.5).
3) using 0 pipe without paranitroanilinum as blank, in OD410When measure absorbance respectively.
4) using absorbance A as ordinate, the final concentration C of paranitroanilinum is abscissa, and drawing standard curve, (curve should lead to
Zero crossing).The concentration of the paranitroanilinum when absorbance is 1, as absorbance constant K are calculated according to mapping.Standard
Curve is as shown in Figure 1.
Embodiment 2: the enzyme activity of Oligopeptide Compositions measurement multiple protein enzyme is utilized
1, the enzyme activity of 1398 neutral proteinases is measured using Oligopeptide Compositions
1) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；
2) reaction system forms: 80 μ L phosphate buffers (pH=7.5), 20 μ L mixed substrates solution, after 100 μ L dilution
Sample to be tested enzyme solution (3-4U/mL)；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzyme activity in enzyme solution to be measured is calculated, as a result with a fractional representation.
2, the enzyme activity of the alkali protease in Bacillus clausii source is measured using Oligopeptide Compositions
1) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；
2) reaction system forms: 80 μ L boraxs-sodium hydrate buffer solution (pH=10.5), 20 μ L mixed substrates solution, and 100
Sample to be tested enzyme solution (3-4U/mL) after μ L dilution；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzymatic activity in enzyme solutions is calculated, as a result with a fractional representation.
3, the enzyme activity of 2709 alkali proteases in bacillus licheniformis source is measured using Oligopeptide Compositions
1) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；
2) reaction system forms: 80 μ L boraxs-sodium hydrate buffer solution (pH=10.5), 20 μ L mixed substrates solution, and 100
Sample to be tested enzyme solution (3-4U/mL) after μ L dilution；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzymatic activity in enzyme solutions is calculated, as a result with a fractional representation.
4, the enzyme activity of Oligopeptide Compositions measurement keratinase is utilized
1) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；
2) reaction system forms: 80 μ L boraxs-sodium hydrate buffer solution (pH=10.5), 20 μ L mixed substrates solution, and 100
Sample to be tested enzyme solution (3-4U/mL) after μ L dilution；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzymatic activity in enzyme solutions is calculated, as a result with a fractional representation.
5, the enzyme activity of acid protease is determined using Oligopeptide Compositions
1) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；
2) reaction system forms: 80 μ L lactic acid-sodium lactate buffer (pH 2.0), 20 μ L mixed substrates solution, 100 μ L are dilute
Release rear sample to be tested enzyme solution (3-4U/mL)；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzymatic activity in enzyme solutions is calculated, as a result with a fractional representation.
Above-mentioned prolease activity measurement result is as shown in the table:
Embodiment 3: trypsase vitality test
Trypsase is the more significant protease of a species specificity, the peptide bond formed for arginine (R) and lysine (K)
There is higher cutting efficiency, reacted respectively with 20 kinds of oligopeptide compounds using the enzyme, can verify and reflect egg with Oligopeptide Compositions
The feasibility of white zymolyte cleavage specificity.
It is reacted respectively using mixed substrates and 20 kinds of oligopeptide compounds with trypsase, concrete operations are as follows:
1) prepare the AAPX substrate solution of 4mM respectively with DMSO (X represents any one of 20 kinds of common amino acids)；Reaction
System composition: 80 μ L phosphate buffers (pH=7.5), 20 μ L AAPX substrate solutions, enzyme solution (3-4U/ after 100 μ L dilution
mL)；
2) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；Reaction system composition: 80 μ L phosphate buffers (pH=7.5), 20 μ L mixed substrates solution, enzyme solution after 100 μ L dilution
(3-4U/mL)；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzyme activity in enzyme solution to be measured is calculated, as a result with a fractional representation.
Experimental result is as follows:
| Substrate kind | AAPK | AAPI | AAPH | AAPG | AAPF | AAPE | AAPD |
| Enzyme activity (U/mL) | 67.1 | 0.3 | 0.7 | 0.9 | 4.4 | 0.2 | 0 |
| Substrate kind | AAPC | AAPW | AAPV | AAPT | AAPS | AAPR | AAPQ |
| Enzyme activity (U/mL) | 0.6 | 0.4 | 0 | 0 | 0 | 33.1 | 0 |
| Substrate kind | AAPP | AAPN | AAPM | AAPY | AAPA | AAPL | Mixed substrates |
| Enzyme activity (U/mL) | 0 | 0.5 | 1.5 | 4.5 | 0.2 | 1.6 | 79.2 |
The results show that trypsase has higher enzyme-activity data after reacting respectively with AAPK, AAPR, side proves such bottom
Specificity when object can be used for measuring the enzyme activity of protease and show protease cutting peptide bonds.It include 20 indsoles in mixed substrates
Object, in 20 in the presence of substrate whole, trypsase can still be directed to AAPK, and AAPR has higher cleavage specificity,
But remaining substrate of the cutting of meeting small frequency.The enzyme-activity data that the slightly above most suitable substrate of enzyme-activity data that measurement obtains measures.
Embodiment 4: basic protein enzyme activity determination
Peptide bond in alkali protease hydrolyzable protein also has hydrolysis amido bond, ester bond and transesterification and turns peptide
Function.Respectively using mixed substrates and 20 kinds of oligopeptide compounds and basic protein enzyme reaction, enzyme-activity data is measured.Concrete operations step
It is rapid as follows:
1) prepare the AAPX substrate solution of 4mM respectively with DMSO (X represents any one of 20 kinds of common amino acids)；Reaction
System composition: 80 μ L phosphate buffers (pH=7.5), 20 μ L AAPX substrate solutions, enzyme solution (3-4U/ after 100 μ L dilution
mL)；
2) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；Reaction system composition: 80 μ L phosphate buffers (pH=7.5), 20 μ L mixed substrates solution, enzyme solution after 100 μ L dilution
(3-4U/mL)；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzyme activity in enzyme solution to be measured is calculated, as a result with a fractional representation.
Experimental result is as follows:
| Substrate kind | AAPK | AAAPI | AAPH | AAPG | AAPF | AAPE | AAPD |
| Enzyme activity (U/mL) | 6.1 | 0.6 | 7.5 | 1.2 | 75.1 | 0.9 | 75.1 |
| Substrate kind | AAPC | AAPW | AAPV | AAPT | AAPS | AAPR | AAPQ |
| Enzyme activity (U/mL) | 2.5 | 5.1 | 3.6 | 0.6 | 0.3 | 0.3 | 0.8 |
| Substrate kind | AAPP | AAPN | AAPM | AAPY | AAPA | AAPL | Mixed substrates |
| Enzyme activity (U/mL) | 7.1 | 7.1 | 19.4 | 25.2 | 3.4 | 17.5 | 126.3 |
Experimental result is shown: the specificity used in experiment when alkali protease cutting peptide bonds is not strong, not for 20 kinds
Peptide bond in same substrate has cutting power, and there are tendentiousness.Alkali protease cuts the peptide bond that phenylalanine is formed
Efficiency highest, the efficiency for the peptide bond that cutting tyrosine is formed illustrate AAPY inferior to the efficiency for the peptide bond that cutting phenylalanine is formed
It may not be the most suitable substrate of alkali protease.National Standard Method measures the principle of prolease activity, is to utilize protease hydrolytic junket
The phenolic group on tyrosine that albumen generates, which can be reacted in alkaline environment with forint phenol, generates blue precipitate, by detecting this indigo plant
Color is deposited in the absorption peak at OD680, to obtain prolease activity.Principle according to National Standard Method measurement prolease activity can
Know, the prolease activity characterized with National Standard Method, be actually only the ability of protease cutting tyrosine peptide key, but fails to reflect
Protease cuts the ability of other amino acid peptide bonds, i.e. the hydrolysis vigor of protease whole out.And the method for the present invention is by containing
The composition of 20 kinds of amino acid peptide bonds can more comprehensively, thoroughly reflect the hydrolysis vigor of protease as hydrolysis substrate.
Experimental result shows that the enzyme-activity data of the alkali protease of mixed substrates measurement is higher than the basic protein of all single substrate measurements
The enzyme-activity data of enzyme.
Embodiment 5: multiple basic protein enzyme samples are measured simultaneously using National Standard Method and the method for the present invention
The alkaline protease gene and its variant that Bacillus clausii source is expressed in bacillus subtilis, obtain more
A bacterial strain, fermentation measures the enzyme-activity data of these bacterial strains in this experiment.The protease gene in Bacillus clausii source is compiled
Number are as follows: CP019985.1.It chooses original strain and 5 mutant strains carries out the experiment.The naming rule of mutant strain: using " original
The amino acid of amino acid position replacement " indicates mutant strain, amino acid residue using generally acknowledging one-letter symbol form in nomenclature,
Such as G283A, indicate that the 283rd amino acid is substituted for alanine (A), the volume of position by the glycine (G) of parent protease
Number correspond to by NCBI number CP019985.1 the encoded protease of gene order amino acid sequence number.
Sample preparation in National Standard Method: 1. connect bacterium to plate, 37 DEG C of culture 12h from glycerol tube.2. choosing single colonie access
LB culture medium test tube, cultivates 12h by 37 DEG C, 220rpm.3.250mLLB shake flask fermentation, 2% inoculum concentration, 37 DEG C, 220rpm, training
Support 48h.4. enzyme solution sample is made in fermentation liquid centrifuging and taking supernatant.
National Standard Method: sample by gradient dilution is diluted to suitable multiple by 1., and (every milliliter of enzyme activity of sample is in 10- after dilution
15U).2. sample and prepared tyrosine substrate reactions after dilution are terminated reaction ten minutes later.3. after taking termination to react
Reaction solution centrifuging and taking supernatant.4. supernatant is reacted with forint phenol, 20min, and 40 DEG C.5. measuring the OD of chromogenic reaction liquid680Numerical value,
Calculate enzyme activity.Concrete operation step is shown in national standard GB/T 23527-2009.
Enzyme activity defines in National Standard Method: 1g solid enzyme powder (or 1mL liquid enzymes), under certain temperature and pH value condition, 1min
Caseinhydrolysate generates 1 μ g tyrosine, and as 1 enzyme activity unit is indicated with U/g (U/mL).
The sample preparation of the method for the present invention: 1. connect bacterium to plate, 37 DEG C of culture 12h from glycerol tube.2. choosing single colonie to connect
Enter LB culture medium test tube, 37 DEG C, 220rpm, cultivates 12h.3. it is fermented using 48 orifice plates, 2% inoculum concentration, 37 DEG C, 750rpm, training
Support 48h.4. fermentation liquid centrifuging and taking supernatant obtains enzyme solution sample.
The method of the present invention measures the enzyme activity of alkali protease and its mutant, and steps are as follows:
1) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is
4mM；
2) reaction system forms: 80 μ L lactic acid-sodium lactate buffer (pH 2.0), 20 μ L mixed substrates solution, 100 μ L are dilute
Release rear sample to be tested enzyme solution (3-4U/mL)；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) enzymatic activity in enzyme solutions is calculated, as a result with a fractional representation.
Enzyme activity determination result is as follows:
National Standard Method of Determination is different from definition of the method for the present invention for enzyme-activity unit, and the data of acquisition cannot be directly used to
Compare enzyme activity height.The method of the present invention can measure the maximum capacity of protease hydrolytic peptide bond, more complete compared to National Standard Method
Face and accurate.It is larger that National Standard Method measures the sample size needed when enzyme activity, it is therefore desirable to enough samples are obtained by shake flask fermentation.
Using the method for the present invention, the sample size needed is less, can be fermented by 48 orifice plates and obtain sample, enormously simplified and fermented
Journey.During National Standard Method measures enzyme activity, it need to be centrifuged by reaction, colour developing and etc., error is larger, and it is cumbersome, and react
System is larger, and single sample minute is longer, and a hour is needed to complete.When measuring enzyme activity using the method for the present invention, it is only necessary to one
Step reaction, and reaction system is smaller, can be completed in 96 orifice plates, volley of rifle fire batch measurement enzyme activity can be used, primary (a batch) is anti-
Answering 15 minutes can be obtained as a result, being suitble to the measurement work of batch samples.
Claims (7)
1. a kind of method for measuring prolease activity, which is characterized in that the method is using one kind by 20 kinds of oligopeptide compounds
The composition of composition has general formula Suc-Ala-Ala-Pro-X-pNA as substrate, the oligopeptide compounds, and wherein X represents 20
Any one of kind common amino acid；The reaction system that mixed substrates solution and buffer, liquid of protease form is prepared, hydrolysis is anti-
Should after measure OD410Light absorption value, the enzyme activity of protease can be obtained.
2. a kind of method for measuring prolease activity as described in claim 1, which is characterized in that the method mainly includes as follows
Step:
1) the mixed substrates solution that 20 kinds of oligopeptide compounds are prepared with DMSO, wherein the concentration of every kind of oligopeptide compounds is 4mM；
2) reaction system forms: according to liquor capacity, 8 portions of buffers, 2 parts of mixed substrates solution, liquid of protease after 10 parts of dilutions；
3) it is uniformly mixed, 40 DEG C of standings react 10min, measure OD410When light absorption value；
4) with the concentration and OD of paranitroanilinum standard solution410Light absorption value draws standard curve, is calculated according to standard curve
Enzyme activity in measured sample enzyme solution.
3. a kind of method for measuring prolease activity as claimed in claim 1 or 2, which is characterized in that protease after the dilution
The enzyme activity of liquid is in 3 U/mL-4 U/mL.
4. a kind of method for measuring prolease activity as claimed in claim 1 or 2, which is characterized in that the buffer is pH7.5
Phosphate buffer；Or the lactic acid of pH2.0-sodium lactate buffer；Or borax-sodium hydrate buffer solution of pH10.5.
5. as claimed in claim 1 or 2 it is a kind of measure prolease activity method, which is characterized in that the protease include but
It is not limited to neutral proteinase, acid protease or alkali protease.
6. a kind of composition, the composition is by 20 kinds of oligopeptide compounds with general formula Suc-Ala-Ala-Pro-X-pNA
Composition, wherein X represents any one of 20 kinds of common amino acids.
7. the purposes that composition described in claim 6 is used to detect prolease activity as substrate.
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| US11655464B2 (en) | 2020-12-21 | 2023-05-23 | Tianjin University Of Science And Technology | Alkaline protease mutant, and gene, engineered strain, preparation method and application thereof |
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