CN110305934A - A kind of method of simple and convenient measurement prolease activity - Google Patents
A kind of method of simple and convenient measurement prolease activity Download PDFInfo
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- CN110305934A CN110305934A CN201910730740.3A CN201910730740A CN110305934A CN 110305934 A CN110305934 A CN 110305934A CN 201910730740 A CN201910730740 A CN 201910730740A CN 110305934 A CN110305934 A CN 110305934A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- 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
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Abstract
The present invention provides a kind of easy, quick, efficiently measurement prolease activity method.This method is mainly using a kind of oligopeptide compounds Suc-Ala-Ala-Pro-Tyr-pNA as substrate, prepare the reaction system that the substrate solution and buffer, liquid of protease form, peptide bond in the protease hydrolytic oligopeptides between tyrosine (Tyr) and paranitroanilinum (pNA), the pNA monomer generated after peptide bond fracture is in OD410There is absorption peak at place, measures OD after reaction410Light absorption value, the enzyme activity of protease can be obtained.Process of the present invention is easy to operate, and time-consuming short, sample requirements are small, and can carry out the enzyme activity determination work of multiple samples simultaneously, can satisfy the demand of batch samples measurement work.
Description
Technical field
The invention belongs to bioengineering fields, and in particular to a kind of method of simple and convenient measurement 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 the bottom of to
The specificity of object.
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 refers to tyrosine in protease hydrolytic substrate
The ability of the peptide bond of generation.During National Standard Method measures enzyme activity, need by dilute sample, substrate hydrolysis reaction terminates anti-
It answers, reaction solution centrifuging and taking supernatant, the colour developing of forint phenol and etc., single sample measures longer and sample requirements the time required to enzyme activity
It is larger.Substrate casein used in national standard is the bisexual protein that isoelectric point is pH4.8, with Dicalcium Phosphate, phosphorus in milk
The composite form of sour tricalcium or both exists, and construction is extremely complex, up to now without completely specified molecular formula, molecular weight
About 57000-375000.Proteinase activity unit in national standard is with the casein definition of unit time consumption unit mass
's.
Summary of the invention
It is an object of the invention to be directed to the deficiency of existing National Standard Method of Determination, a kind of easy, quick, efficiently measurement is provided
The method of prolease activity.A kind of this method mainly oligopeptide compounds Suc-Ala-Ala-Pro-Tyr-pNA of utilization (abbreviation:
AAPY, molecular structural formula are as follows) it is used as substrate,
The oligopeptide compounds are synthesized, the reaction system that substrate solution and buffer, liquid of protease form, protease water are prepared
The peptide bond in the oligopeptides between tyrosine (Tyr) and paranitroanilinum (pNA) is solved, the pNA monomer generated after peptide bond fracture exists
OD410There is absorption peak at place, measures OD after hydrolysis410Light absorption value, the enzyme activity of protease can be obtained.
Further, by preparing the buffer of different pH, this method is widely used in measurement acid protease, neutral egg
The enzyme activity of white enzyme, alkali protease.
Further, enzyme activity determination method of the invention mainly includes the following steps:
1, the AAPY substrate solution of 4mM is prepared with DMSO；
2, reaction system forms: according to liquor capacity, 8 portions of buffers, 2 parts of AAPY substrate solutions, 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
For the present invention using the method for synthesis substrate A APY measurement prolease activity, process is easy to operate, and time-consuming short, sample needs
The amount of asking is small, and can carry out the enzyme activity determination work of multiple samples simultaneously, can satisfy the need of batch samples measurement work
It asks.
Detailed description of the invention
Fig. 1: the standard curve of the method for the present invention measurement prolease activity.
Fig. 2: the method for the present invention measures the comparison of protease with National Standard Method simultaneously.
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 first
Liquid 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.
The method for measuring prolease activity using AAPY in the present invention for substrate, enzyme activity are defined as follows:
1 enzyme activity unit refers to 1g enzyme powder or 1mL enzyme solution under specified conditions (40 DEG C, other is optimum condition), 1
The enzyme amount of 1 micromole substrate can be converted in minute.
Embodiment 1:AAPY substrate measures 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 AAPY substrate measurement multiple protein enzyme is utilized
1, the enzyme activity of 1398 neutral proteinases is measured using AAPY substrate
1) the AAPY substrate solution of 4mM is prepared with DMSO；
2) reaction system forms: 80 μ L phosphate buffers (pH=7.5), 20 μ L AAPY substrate solutions, 100 μ L dilution
Sample to be tested enzyme solution (3-4U/mL) afterwards；
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 AAPY substrate
1) the AAPY substrate solution of 4mM is prepared with DMSO；
2) reaction system forms: 80 μ L boraxs-sodium hydrate buffer solution (pH=10.5), 20 μ L AAPY substrate solutions,
Sample to be tested enzyme solution (3-4U/mL) after 100 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 AAPY substrate
1) the AAPY substrate solution of 4mM is prepared with DMSO；
2) reaction system forms: 80 μ L boraxs-sodium hydrate buffer solution (pH=10.5), 20 μ L AAPY substrate solutions,
Sample to be tested enzyme solution (3-4U/mL) after 100 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 AAPY measurement keratinase is utilized
1) the AAPY substrate solution of 4mM is prepared with DMSO；
2) reaction system forms: 80 μ L boraxs-sodium hydrate buffer solution (pH=10.5), 20 μ L AAPY substrate solutions,
Sample to be tested enzyme solution (3-4U/mL) after 100 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 AAPY measurement acid protease is utilized
1) the AAPY substrate solution of 4mM is prepared with DMSO；
2) reaction system forms: 80 μ L lactic acid-sodium lactate buffer (pH 2.0), 20 μ L AAPY substrate solutions, 100 μ L
Sample to be tested enzyme solution (3-4U/mL) after 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.
Above-mentioned prolease activity measurement result is as shown in the table:
Embodiment 3: 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,
Cultivate 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. termination is taken to react
Reaction solution centrifuging and taking supernatant afterwards.4. supernatant is reacted with forint phenol, 20min, and 40 DEG C.5. measuring the OD of chromogenic reaction liquid680Number
Value calculates 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 AAPY substrate solution of 4mM is prepared with DMSO；
2) reaction system forms: 80 μ L boraxs-sodium hydrate buffer solution (pH=10.5), 20 μ L AAPY substrate solutions,
Sample to be tested enzyme solution after 100 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.
Enzyme activity determination Comparative result is following (shown in Fig. 2):
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, but in the process of the present invention as the prescreening method of protease mutant, in the variation tendency of measurement result
It is upper to be consistent with National Standard Method, meet the requirement during preliminary screening.The sample size needed when National Standard Method measurement enzyme activity is larger,
Therefore it needs to obtain enough samples by shake flask fermentation.Using the method for the present invention, the sample number needed is less, can pass through 48
Orifice plate fermentation obtains sample, enormously simplifies fermentation process.It, need to be by reacting, being centrifuged, show during National Standard Method measures enzyme activity
Color and etc., error is larger, and it is cumbersome, and reaction system is larger, single sample minute is longer, needs or so hour
It completes.When measuring enzyme activity using the method for the present invention, it is only necessary to single 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), which is reacted 15 minutes, can be obtained as a result, being suitble to the measurement of batch samples
Work.
Claims (7)
1. a kind of method for measuring prolease activity, which is characterized in that the method is to utilize a kind of oligopeptide compounds Suc-
Ala-Ala-Pro-Tyr-pNA prepares the reaction system that the substrate solution and buffer, liquid of protease form, water as substrate
OD is measured after solution reaction410Light 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 AAPY substrate solution of 4mM is prepared with DMSO；
2) reaction system forms: according to liquor capacity, 8 portions of buffers, 2 parts of AAPY substrate solutions, 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 nitroaniline standard solution410Light absorption value draws standard curve, and institute is calculated according to standard curve
Measure the enzyme activity in 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 3U/mL-4U/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 pH
7.5 phosphate buffer；Or the lactic acid of pH 2.0-sodium lactate buffer；Or borax-sodium hydrate buffer solution of pH 10.5.
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 oligopeptide compounds Suc-Ala-Ala-Pro-Tyr-pNA.
7. the purposes that compound described in claim 6 is used to detect prolease activity as substrate.
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| CN111500674A (en) * | 2020-04-21 | 2020-08-07 | 华侨大学 | Method for screening aphid-resistant Cry protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108169217A (en) * | 2017-12-28 | 2018-06-15 | 阎冬 | A kind of simple and efficient proteinase activity detection method |
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| CN108169217A (en) * | 2017-12-28 | 2018-06-15 | 阎冬 | A kind of simple and efficient proteinase activity detection method |
Non-Patent Citations (2)
| Title |
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| CURRENT MICROBIOLOGY: "A new alkaline proteinase with pI 2.8 from Alkalophilic bacillus sp", 《CURRENT MICROBIOLOGY》 * |
| EVETTE S RADISKY等: "Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates", 《PROC NATL ACAD SCI U S A》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111500674A (en) * | 2020-04-21 | 2020-08-07 | 华侨大学 | Method for screening aphid-resistant Cry protein |
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Application publication date: 20191008 |