CN106636043A - Preparation method of protease - Google Patents
Preparation method of protease Download PDFInfo
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- CN106636043A CN106636043A CN201611230292.3A CN201611230292A CN106636043A CN 106636043 A CN106636043 A CN 106636043A CN 201611230292 A CN201611230292 A CN 201611230292A CN 106636043 A CN106636043 A CN 106636043A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
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Abstract
The invention relates to a preparation method of protease. A protease inhibitor is added to a culture medium constantly in a specific time period from the logarithmic phase to the stable phase during microbial growth and can inhibit self-degradation of protease effectively. Surprisingly, the protease inhibitor cannot inhibit growth of microorganisms when added to the culture medium constantly in the specific time period during microbial growth, and greatly increases the yield of protease.
Description
Technical field
The present invention relates to fermentation technical field, more particularly to a kind of method for preparing protease.
Background technology
Enzyme preparation has been widely used for the aspects such as food, medicine, light industry, chemical industry, environmental protection and the agriculture and energy.
Enzyme preparation is referred to be extracted from organic-biological or has the material of catalysis activity by the class that microorganism secretion goes out, and mainly can be passed through
The mode of submerged fermentation is using the production enzyme preparation such as bacterium, filamentous fungi and yeast.
However, during producing enzyme preparation by the method fermented, because enzyme preparation is easily degraded, less stable,
Cause yield relatively low, this point is especially true when protease is prepared by fermentation.Most of microbe-derived protease is entering
During row large-scale industrial production, because protease tends to from degraded, so being difficult to accumulate.Inhibitor can suppress albumen
Certainly the degraded of enzyme, but traditional method adds the growth that can greatly suppress fermentative microorganism after inhibitor, causes overall yield
Decline, so existing fermentation manufacturing technique cannot be applied it in the middle of lifting proteinase production.Another kind of scheme is to pass through
The method that the temperature of control sweat lifts proteinase production, but the method can not still solve protease from caused sending out of degrading
The problem that ferment yield is restricted.
To sum up, during traditional fermentable production protease, under the yield that protease causes protease from degrading
Drop is the technical problem that industry exigence is solved, and the problem becomes the bottleneck of restriction protease industrialized production.
The content of the invention
Based on this, it is necessary to provide and a kind of can solve the problem that protease from degrading so as to the yield for lifting protease prepares egg
The method of white enzyme.
A kind of method for preparing protease, comprises the steps：
Fermented and cultured microorganism in the medium, and continue to the training in the special time period of the growth of microorganism
Protease inhibitors, time of the special time period in the logarithmic phase to stationary phase of the growth of microorganism are added in foster base
In section；And
The microorganism secretion produces protease.
In one embodiment, the special time period duration is 4h～48h, and the protease inhibitors is in institute
Final concentration of 0.1g/L～the 5g/L in culture medium is stated, the speed that the protease inhibitors is added in the culture medium is
0.002g/ (Lh)～1.25g/ (Lh).
In one embodiment, the protease inhibitors is boronic acid derivatives.
In one embodiment, the protease inhibitors is selected from 4- formyl phenylboronic acids, N-2- pyrazinecarbonyl-L-
Phenylalanine-L-Leu boric acid, thiophene -3- boric acid, 3- acetamidophenyl boronic acids, N- acyl groups-peptide ylboronic acid, 4- methyl thiazoliums
Fen -2- boric acid, 2- fluoro- 5- (methoxycarbonyl group) phenyl boric acids, 5- ethylthiophene -2- boric acid, 2- fluoro- 4- (methoxycarbonyl group) phenyl boric acids,
5- bromothiophene -2- boric acid, 4- (1- piperidino methyls) phenyl boric acid, 4- (1- pyrrolidinylmethyls) phenyl boric acid, dibenzothiophenes base -
1- boric acid, 2- (methoxycarbonyl group) -4- methylphenylboronic acids, the chloro- 3- methylphenylboronic acids of 2,6- bis-, dibenzofurans -4- boric acid, 2- are different
The fluoro- 4- methylsulfonyls phenyl boric acid of propoxyl group phenyl boric acid, 2-, FURAN-2-BORONIC ACID, the fluoro- 4- methylsulfonyls phenyl boric acids of 3-, 1-Boc-7- methoxies
Base indoles -2- boric acid, furans -3- boric acid, 4- (1- piperidinylsulfonyls) phenyl boric acid, 3- acetamidobenzeneboronic acids, 3- methoxyl groups
Thiophene -2- boric acid, 2- methyl -4- methoxyphenylboronic acids, 5- methyl -3- pyridine boronic acids, 5- n-propyl thiophene -2- boric acid, 10- benzene
Base -9- anthracene boric acid, 3 bromo thiophene boric acid, 5- methyl -6- fluoro-3-pyridine boric acid, 3 bromo thiophene -4- boric acid, the fluoro- 3- pyrroles of 2,6- bis-
The chloro- 4- methoxycarbonyl groups phenyl boric acid of pyridine boric acid, 4- formyl phenylboronic acids, 3-, phenyl boric acid, 2- chloro-4-methoxy phenyl boric acids, 5- second
Base FURAN-2-BORONIC ACID, 3- methyl -4- chlorophenylboronic acids, hexichol are for boric acid, 4- (Cyclopropylsulfamoyl base) phenyl boric acids and 5- methyl oxygen
At least one in FURAN-2-BORONIC ACID.
In one embodiment, it is additionally included in the special time period of the growth of microorganism and continues to the culture medium
Middle addition albumen enzyme stabilizers.
In one embodiment, at least one of the albumen enzyme stabilizers in magnesium ion and calcium ion.
In one embodiment, the special time period duration is 4h～48h, the magnesium ion and the calcium from
At least one final concentration of 0.005mol/L～0.1mol/L in the culture medium in son.
In one embodiment, the microorganism is Bacillus subtillis, in the medium withered grass described in fermented and cultured
Start to add 4- formyl phenylboronic acids, the 4- formyl phenylboronic acids in the culture medium after bacillus 35h～40h
Into the culture medium, the 4- formyl phenylboronic acids are in the culture medium for lasting addition within the time period of 5h～20h
In final concentration of 0.2g/L～0.6g/L, the speed that the 4- formyl phenylboronic acids are added in the culture medium is
0.01g/ (Lh)～0.12g/ (Lh).
In one embodiment, it is additionally included in the special time period of the growth of microorganism and continues to the culture medium
Middle addition albumen enzyme stabilizers, at least one of the albumen enzyme stabilizers in magnesium ion and calcium ion, the magnesium ion
With at least one final concentration of 0.0125mol/L～0.025mol/L in the culture medium in the calcium ion.
In one embodiment, the microorganism is Pichia pastoris, in the medium Pichia pastoris described in fermented and cultured
Start after 30h～40h in the culture medium add 4- formyl phenylboronic acids, the 4- formyl phenylboronic acids 5h～
Lasting addition is into the culture medium in the time period of 20h, end of the 4- formyl phenylboronic acids in the culture medium
Concentration is 0.2g/L～0.6g/L, and the speed that the 4- formyl phenylboronic acids are added in the culture medium is 0.01g/ (L
H)～0.12g/ (Lh).
The above-mentioned method for preparing protease, in the special time period of the logarithmic phase to stationary phase of growth of microorganism, continues
Add protease inhibitors in culture medium, protease inhibitors can effectively protease inhibition from degrading.And inventor
Have been surprisingly found that protease inhibitors is continuously added into culture medium in the special time period of growth of microorganism, fermentation can't be suppressed
The growth of microorganism, greatly lifts the yield of protease.
In addition, continuing to add albumen enzyme stabilizers, protease in culture medium in the special time period of growth of microorganism
Inhibitor coordinates with albumen enzyme stabilizers, can further lift the yield of protease.
Description of the drawings
Fig. 1 is the flow chart of the method for preparing protease of an embodiment；
Fig. 2 is the growth curve schematic diagram of Bacillus subtillis SCK6 in embodiment 1；
Fig. 3 is the growth curve schematic diagram of Pichia pastoris GS115 in embodiment 1.
Specific embodiment
It is understandable to enable the above objects, features and advantages of the present invention to become apparent from, it is below in conjunction with the accompanying drawings and concrete real
Apply example to be described in detail the specific embodiment of the present invention.Elaborate many details in the following description in order to
Fully understand the present invention.But the present invention can be implemented with being much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is not embodied as by following public
Restriction.
Fig. 1 is referred to, the method for preparing protease of an embodiment comprises the steps S110～S120.
S110, in the medium fermented and cultured microorganism, and continue to culture in the special time period of growth of microorganism
Protease inhibitors is added in base, in time period of the special time period in the logarithmic phase to stationary phase of growth of microorganism.
Specifically, the present invention refers to the conventional training of any suitable growth of microorganism for the culture medium of fermented and cultured microorganism
Foster base, including minimal medium and complex medium.The acquisition of culture medium can be by buying, or in reference from commercial supplier
State's General Microbiological Culture collection (China General Microbiological Culture Collection
Center, CGMCC), China typical culture collection center (China Centre for Type Culture
Collection, CCTCC), American type culture collection (American Type Culture Collection,
ATCC), German microorganism and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen
Und Zellkulturen, DSMZ) etc. mechanism announce preparation method, using methods known in the art prepare.
The method for preparing protease of the present invention can be used in the production of the protease industrial fermentation of any scale, example
Such as, can be used for scale and reach 10L, 100L, 1000L, the production of the Zymolysis Equipment of 10000L, even 100000L.Fermented and cultured
Technique can adopt batch, repeat the fermentation process of batch, fed-batch and continuous feeding.In one embodiment, micro- life
The method that thing uses Batch fermentation using the fermentation of bacillus subtilis.In another embodiment, microorganism adopts
The method that the fermentation of Pichia pastoris uses fed-batch fermentation.The conditions such as temperature, the pH of fermented and cultured are also dependent on concrete
The characteristic of the microorganism of culture is selected, and fermented and cultured can also include microbe inoculation, one-level Amplification Culture, two grades of expansions
The operation such as culture, the method operation by culture microorganism, therefore not to repeat here.
Specifically, microorganism is also referred to as protease production strain, and generation protease can be secreted after fermentable culture.This
Microorganism in bright includes bacterium and fungi.Bacterioprotein enzyme-producing bacteria is mainly bacillus, and the example of bacillus is included
But it is not limited to bacillus subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus
Licheniformis), Brevibacillus brevis (Brevibacillus brevis), bacillus cereus (Bacillus
Cereus), bacillus thuringiensis (Bacillus thuringiensis), bacillus megaterium (Bacillus
Megaterium), bacillus mycoides (Bacillus mycoides).Researcher can obtain from culture collection mechanism
Above-mentioned bacterial strains, such as China General Microbiological DSMZ (China General Microbiological Culture
Collection Center, CGMCC), China typical culture collection center (China Centre for Type Culture
Collection, CCTCC), American type culture collection (American Type Culture Collection,
ATCC), German microorganism and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen
und Zellkulturen,DSMZ)。
Mycoprotein enzyme-producing bacteria is mainly yeast, and the example of yeast includes but be not limited to torulopsis
(Torulopsis) cell, kluyveromyces (Kluyveromyces) cell, Hansenula polymorpha (Hansenula
PoIymorpha) cell, Candida (Candida) cell, fission yeast (Schizosaccharomyces) cell are red with complete
Yeast (Pichia) cell.Researcher can obtain above-mentioned bacterial strains, such as China General Microbiological from culture collection mechanism
DSMZ (China General Microbiological Culture Collection Center, CGMCC),
China typical culture collection center (China Centre for Type Culture Collection, CCTCC), U.S.'s allusion quotation
Type culture collection (American Type Culture Collection, ATCC), German microorganism and cell culture
Thing collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ).
Specifically, according to growth of microorganism speed and specific growth rate during fermented and cultured, by the growth feelings of microorganism
Condition is divided into the different stages.By detecting that microorganism biomass under Incubation Condition can be somebody's turn to do over time
Under the conditions of the microorganism growth curve.Growth curve can react four periods of growth of microorganism, period of delay, logarithmic phase,
The concrete time that stationary phase and decline phase are answered, the rule that cell viability changes over time can be reflected.It should be noted that
Different types of microorganism, different condition of culture and training strategy can all make growth curve change.Determine sweat
The biomass of middle microorganism can pass through light absorption value of the detection zymotic fluid at 600nm wavelength, i.e. OD600Value；Can also pass through
The weight in wet base of cell in detection zymotic fluid.In one embodiment, microorganism is using the fermentation detection of bacillus subtilis
OD600Value.In another embodiment, microorganism using Pichia pastoris fermentation detection be wet cell weight value.
Specifically, the logarithmic phase of growth referred to according to growth of microorganism speed and specific growth rate in sweat, will be micro-
Biological growing state is divided into the different stages, wherein being referred to as when growth of microorganism reaches the specific growth rate maximum stage
Exponential phase.
Specifically, the stationary phase of growth refer to according to growth of microorganism speed and specific growth rate in sweat, will be micro-
Biological growing state is divided into the different stages, wherein the rank when microorganism growth speed and death rate in dynamic equilibrium
Section is referred to as stationary phase.
In present embodiment, the growth curve of the microorganism cultivated under certain condition can be determined by preliminary experiment, it is determined that
The concrete time point that the period of delay of growth of microorganism, logarithmic phase, stationary phase and decline phase are answered, so that it is determined that being initially added into albumen
The time point of enzyme inhibitor and the duration of addition.Specifically, continue to training in the special time period of growth of microorganism
During the operation of protease inhibitors is added in foster base, time of the special time period in the logarithmic phase to stationary phase of growth of microorganism
In section.Moment point to the microorganism for starting logarithmic phase growth from microorganism terminates arbitrarily cutting between the moment point of stable growth
Take the time period continues in culture medium to add protease inhibitors as special time period.More specifically, special time period is in
In the time period of the early stage in the latter stage of the logarithmic phase of growth of microorganism to stationary phase.
In one embodiment, logarithmic phase of the time that special time period starts in microbial growth curve.Another
In one embodiment, stationary phase of the time that special time period starts in microbial growth curve.
In one embodiment, special time period duration is 4h～48h, and protease inhibitors is in the medium
Final concentration of 0.1g/L～5g/L, add in culture medium the protease inhibitors speed be 0.002g/ (Lh)～
1.25g/(L·h).Specifically, protease inhibitors can be configured to solution shape, protease inhibitors is within the specific time period
Uniformly it is added drop-wise in culture medium.In another embodiment, the special time period duration is 5h～45h.At another
In embodiment, the special time period duration is 10h～36h.In another embodiment, the special time period duration
For 15h～24h.Protease inhibitors is configured to solution shape can be uniformly added drop-wise in culture medium.The inventors discovered that micro-
Biological exponential phase is continuously added into protease inhibitors to growth period is stablized, and effectively protease inhibition can degrade certainly, phase
Than can substantially obtain higher proteinase production in disposable addition protease inhibitors of same time, and will not suppress micro-
Biological growth.
Specifically, protease inhibitors is to refer to reversible with proteinase activity central area or irreversibly combined, and is made
Proteinase activity declines or disappears, but will not generally cause the material of protein denaturation.In present embodiment, protease inhibitors
For boronic acid derivatives, boronic acid derivatives can reduce the vigor of protease by way of Competitive assays.Boron atom is in element week
Phase table belongs to second round, and organic boronic shows as under normal operation sp2 hydridization forms, when acting on hydrone, hydrone
The lone pair electrons of middle oxygen atom discharge a proton by into the unoccupied orbital of boron, and organic boronic is also changed into sp3 hydridization from sp2
Form.In general, the different phenylboric acids for replacing, its acidity is changed greatly with the difference of substituent, and pKa value scope is 4.5
～8.8.Under physiological ph conditions, organic boric acid compounds are easily changed into the anion form of sp3 hydridization, this structure from sp2 hydridization
It is quite similar with the tetrahedron transition state of substrate for enzymatic activity, therefore organic boron acid compounds can be used as enzyme inhibitor application.
From 1970, boric acid is found to have the characteristic of inhibitory enzyme activity, and since then scientists have designed and synthesized a large amount of such chemical combination
Thing, wherein it is hydrolase inhibitor (such as protease inhibitors) and proteasome inhibitor to study more.It is of the present invention
Protease inhibitors is selected from 4- formyl phenylboronic acids, bortezomib, thiophene -3-
Boric acid, 3- acetamidophenyl boronic acids, N- acyl groups-peptide ylboronic acid, 4- methylthiophene -2- boric acid, 2- fluoro- 5- (methoxycarbonyl group) benzene
Boric acid, 5- ethylthiophene -2- boric acid, 2- fluoro- 4- (methoxycarbonyl group) phenyl boric acids, 5- bromothiophene -2- boric acid, 4- (1- piperidyl methyls
Base) phenyl boric acid, 4- (1- pyrrolidinylmethyls) phenyl boric acid, dibenzothiophenes base -1- boric acid, 2- (methoxycarbonyl group) -4- methylbenzenes
The chloro- 3- methylphenylboronic acids of boric acid, 2,6- bis-, dibenzofurans -4- boric acid, 2- isopropoxy phenyl boric acids, the fluoro- 4- methylsulfonyls benzene of 2-
The fluoro- 4- methylsulfonyls phenyl boric acid of boric acid, FURAN-2-BORONIC ACID, 3-, 1-Boc-7- methoxy-Indole -2- boric acid, furans -3- boric acid, 4-
(1- piperidinylsulfonyls) phenyl boric acid, 3- acetamidobenzeneboronic acids, 3- methoxythiophene -2- boric acid, 2- methyl -4- methoxybenzenes
Boric acid, 5- methyl -3- pyridine boronic acids, 5- n-propyl thiophene -2- boric acid, 10- phenyl -9- anthracene boric acid, 3 bromo thiophene boric acid, 5- first
Base -6- fluoro-3-pyridine boric acid, 3 bromo thiophene -4- boric acid, the fluoro- 3- pyridine boronic acids of 2,6- bis-, 4- formyl phenylboronic acids, 3- are chloro-
4- methoxycarbonyl group phenyl boric acids, phenyl boric acid, 2- chloro-4-methoxy phenyl boric acids, 5- ethyl furan -2- boric acid, 3- methyl -4- chlorobenzene boron
Acid, hexichol are at least one in boric acid, 4- (Cyclopropylsulfamoyl base) phenyl boric acids and 5- methyl oxygen FURAN-2-BORONIC ACIDs.
In one embodiment, protease inhibitors final concentration of 0.1g/L～5g/L in the medium.
In another embodiment, protease inhibitors final concentration of 0.2g/L～2.5g/L in the medium.
In another embodiment, protease inhibitors final concentration of 0.4g/L～1.25g/L in the medium.
In another embodiment, protease inhibitors final concentration of 0.2g/L～0.6g/L in the medium.
Specifically, calculate by the final concentration according to protease inhibitors in the medium, and the time being continuously added into
Go out the addition speed of corresponding protease inhibitors.
In one embodiment, microorganism is bacillus, and protease inhibitors is 4- formyl phenylboronic acids, albumen
Enzyme inhibitor final concentration of 0.2g/L～0.6g/L in the medium.4- formyl phenylboronic acids can improve bacillus protein
The yield of enzyme.It is described improve protease yield for example may refer to add protease inhibitors after scheme proteinase production and
It is added without the scheme proteinase production difference 5% or more of protease inhibitors.
In one embodiment, it is additionally included in the special time period of growth of microorganism and continues to add in the culture medium
Enter albumen enzyme stabilizers.The composition that albumen enzyme stabilizers and protease inhibitors are cooperatively formed can be more significantly lifting egg
The yield of white enzyme.
Specifically, albumen enzyme stabilizers are referred to is beneficial to the material that protease maintains normal configuration, and albumen enzyme stabilizers can
At least one in magnesium ion and calcium ion.The source of calcium ion for example can be CaSO4、CaCl2、Ca(H2PO4)2、
CaHPO4、Ca(HCO3)2、CaCO3With Ca (NO3)2In at least one.The source of magnesium ion for example can be MgSO4、MgCl2、
Mg(H2PO4)2、MgHPO4、Mg(HCO3)2、MgCO3And Mg (NO3)2In at least one.
Specifically, at least one final concentration of 0.005mol/L in the medium in magnesium ion and calcium ion～
0.1mol/L.For example, at least one final concentration of 0.075mol/L in the medium in magnesium ion and calcium ion～
0.05mol/L.Or for example, at least one final concentration of 0.01mol/L in the medium in magnesium ion and calcium ion～
0.025mol/L。
In one embodiment, magnesium ion can be individually added into as albumen enzyme stabilizers, magnesium ion is in the medium
Final concentration of 0.005mol/L～0.1mol/L.
In another embodiment, calcium ion can be individually added into as albumen enzyme stabilizers, calcium ion is in culture medium
In final concentration of 0.005mol/L～0.1mol/L.
In another embodiment, while adding magnesium ion and calcium ion as albumen enzyme stabilizers, magnesium ion is in training
Final concentration of 0.005mol/L～0.1mol/L in foster base, calcium ion final concentration of 0.005mol/L in the medium～
0.1mol/L。
Specifically, protease inhibitors and albumen enzyme stabilizers are added into culture medium, or difference after can mixing
In adding culture medium.The inventors discovered that when producing protease using fermentable, using addition albumen enzyme level
On the premise of agent, when adding calcium salt and/or magnesium salts, the yield of protease can be further improved.Specifically, the raising egg
The yield of white enzyme for example can be referred to do not adopt as the proteinase production of stabilizer and aforementioned stable using calcium salt or magnesium salts
The proteinase production difference 5% or more of agent.
In one embodiment, the composition that protease inhibitors and albumen enzyme stabilizers are formed is added to culture medium
In, the addition of composition is 1g/L～100g/L.Composition includes the protease that weight percent content is 0.1%～10%
Stabilizer and weight percent content are 0.01%～10% protease inhibitors.
In one embodiment, microorganism is Bacillus subtillis, in the medium fermented and cultured Bacillus subtillis
Start to add 4- formyl phenylboronic acids, time of the 4- formyl phenylboronic acids in 5h～20h in culture medium after 35h～40h
Lasting addition is into culture medium in section.4- formyl phenylboronic acids final concentration of 0.2g/L～0.6g/L in the medium.
The speed that 4- formyl phenylboronic acids are added in culture medium is 0.01g/ (Lh)～0.12g/ (Lh).Fermented and cultured withered grass
After bacillus 35h～40h, Bacillus subtillis is in logarithmic phase latter stage, now starts to add protease suppression in culture medium
Preparation, and lasting addition within the time period of 5h～20h is into culture medium, 4- formyl phenylboronic acids can protease inhibition
From degraded.And inventors be surprised to learn that lasting in the 5h～20h time periods after culture Bacillus subtillis 35h～40h
By protease inhibitors add into culture medium, the growth of Bacillus subtillis can't be suppressed, so as to greatly lift egg
The yield of white enzyme.
More specifically, addition albumen enzyme stabilizers, albumen after fermented and cultured Bacillus subtillis 35h～40h are additionally included in
At least one of the enzyme stabilizers in the magnesium ion and calcium ion, in magnesium ion and calcium ion it is at least one in the medium
Final concentration of 0.0125mol/L～0.025mol/L.4- formyl phenylboronic acids coordinate with magnesium ion and/or calcium ion, can
Further the degraded certainly of protease inhibition, lifts the yield of protease.
In another embodiment, microorganism is Pichia pastoris, in the medium fermented and cultured Pichia pastoris 30h～
Start to add 4- formyl phenylboronic acids, 4- formyl phenylboronic acids to hold within the time period of 5h～20h in culture medium after 40h
Continuous addition into culture medium, 4- formyl phenylboronic acids final concentration of 0.2g/L～0.6g/L in the medium.Fermentation training
After foster Pichia pastoris 30h～40h, incipient stage of the Pichia pastoris in stationary phase growth, now start to be added in culture medium
Protease inhibitors, and lasting addition within the time period of 5h～20h, into culture medium, 4- formyl phenylboronic acids can suppress
Certainly the degraded of protease.And inventors be surprised to learn that and held in the 5h～20h time periods after culture Pichia pastoris 30h～40h
It is continuous by protease inhibitors add into culture medium, the growth of Pichia pastoris can't be suppressed, so as to greatly lift egg
The yield of white enzyme.
The microorganism secretion of fermented and cultured produces protease in S120, S110.
Protease is also referred to as protease, proteolytic enzyme, peptase or peptidohydrolase, is that a class can be with aminosal
Or the general name of the enzyme of peptide chain.It is divided into two kinds of endopeptidase and peptide ending enzyme according to its mode for hydrolyzing.According to the name side of NC-IUBMB
Method, protease belongs to any enzyme in EC3.4 enzymes race, different based on their catalyst mechanism altogether containing 13 subspecies, is divided into
Serine protease (S), cysteine proteinase (C), asparagus fern protease (A), metalloproteinases (M), and it is unknown or not yet
The protease (U) of classification, was found hydroxyproline enzyme (G) and serine/threonine protein enzyme (T) again later, see in detail
Handbook of Proteolytic Enaymes,Neil D.Rawlings and Guy Salvesen,Acadimic
Press,2013.For the origin of the protease of application present invention production is not limited, as long as can be produced by microorganism secretion
Raw albumen enzyme.Thus, the term protease not only includes the neutral protease from organism, also has including other
There are any mutant and fragment or artificial synthesized protease of proteinase activity.
Specifically, zymotic fluid is obtained after the microorganism of fermented and cultured, microorganism secretion produces protease in zymotic fluid.This
Embodiment also includes the step of reclaiming to protease, and involved in the present invention is on one side the recovery of protease, i.e.,
For the downstream processes that ferment.For example can be by flocculant (including chemical floc and biological flocculant) pretreated fermentation
Liquid；The cell in fermentation and remaining nutritional substrate are removed by filtering or being centrifuged；By evaporating or being concentrated by ultrafiltration enzyme preparation, plus
Entering stabilising system preserves protease.
After obtaining protease, also include determining prolease activity.The present invention can be using any assay method to reclaiming
Protease carry out the measure of vigor, for example can be determined using one or more substrate, the substrate includes protease specificity
Related peptide bond, the temperature of assay method and pH value are using the temperature and pH value for being adapted to the protease.By the albumen to reclaiming
Enzyme carries out the yield that the measure of vigor can determine fermentation, so as to evaluate the yield etc. of the method for preparing protease.
The above-mentioned method for preparing protease in the special time period of the logarithmic phase to stationary phase of growth of microorganism, continue to
Add protease inhibitors in culture medium, protease inhibitors can effectively protease inhibition from degrading.And invention people's will
Outer discovery protease inhibitors is continuously added into culture medium in the special time period of growth of microorganism, and fermentation can't be suppressed micro-
Biological growth, greatly lifts the yield of protease.The above-mentioned method for preparing protease can effectively solve the problem that protease from drop
Solution causes the technical problem that the yield of protease declines, and realizes the large batch of industrialized production of protease.
In addition, continuing to add albumen enzyme stabilizers, protease in culture medium in the special time period of growth of microorganism
Inhibitor coordinates with albumen enzyme stabilizers, can further lift the yield of protease.
It is below specific embodiment.
Except as otherwise noted, preparing the environment of the experiment at 25 DEG C or so such as solution and test activity is carried out.
The vigor of microorganism secretion generation protease is determined by following methods in following examples.
1st, solution is prepared：
(1) preparation of Tris buffer solutions：Prepare Tris of the specification for 0.1M/L；The CaCl of 0.01mol/L2；0.005%
TritonX-100, pH are 8.6 buffer solution.Specially weigh the Tris base of 12.1g and be dissolved in 800mL distilled water,
The CaCl of 1.1g is added in solution2It is well mixed with the TritonX-100 of 50mg, then pH to 8.6 is adjusted with the HCl of 6mol/L
(commercially available concentrated hydrochloric acid concentration is generally 12mol/L, uses after dilution), with distilled water 1000mL is settled to, as far as possible now with the current.
(2) preparation of substrate solution is stored：Prepare synthetic peptide N-Succinyl-Ala-Ala- of the specification for 20mg/mL
Pro-Phe-p-nitroanilide (SIGMA S7388), with DMSO as solvent.180mg substrates are added in the DMSO of 9mL,
Mixed dissolution, is fitted into centrifuge tube and is wrapped up with masking foil, is placed at shady and cool lucifuge, is not run out abandoning after one month.
(3) work substrate solution：Prepare synthetic peptide N-Succinyl-Ala-Ala-Pro-Phe-p- of the specification for 1mg/mL
Nitroanilide, will store substrate and dilute 20 times, as needed matching while using with Tris buffer solutions, and it is unspent unnecessary to abandon
Substrate.
(4) configuration (including control group) of liquid of protease：The liquid of protease first step to be measured is diluted into about 250 times, then
Second step dilute about 125 times, for the enzyme activity that testing protein enzyme liquid can first survey final dilution, if with compare gap compared with
Greatly, then determine the first step dilution obtain enzyme liquid.Specially accurately weigh 0.1 ± 0.05g of liquid of protease (being accurate to 0.1mg) in
In 5mL centrifuge tubes, then with the Tris buffer solutions of 0.1M to 25g, stir 5 minutes, then accurately weigh 0.2 ± 0.05g deposits
Liquid stirs 5min in another 50mL centrifuge tube, then with the Tris buffer solutions of 0.1M to 25g.
2nd, using ELIASA high throughput assay prolease activity：
Single reaction system cumulative volume is 200 μ L, and each 180 μ L liquid of protease to be measured is added separately into 96 orifice plates
In, 96 orifice plates that will be equipped with sample are positioned over the sample bin of ELIASA, and program setting automatically opens up cabin to be incubated 5min at 37 DEG C
Door, now using the volley of rifle fire by 20 μ L work substrates, in being added to each hole equipped with sample, quick concussion 20s mixes system,
By the speed (OD for determining the paranitroanilinum for discharging405) obtain reaction dynamics data.The enzyme to be measured for finally drawing it is anti-
Answer speed to carry out ratio calculation with enzyme reaction speed is compareed, calculate enzyme activity.
Embodiment 1
Bacillus subtillis SCK6 fermenting and producings prepare protease
1st, culture medium is prepared：
(1), primary inclined plane seed culture medium (100mL)：Agar powder 1.5g；Glucose 5g；Corn starch 1g；Dusty yeast
1.5g；Soybean protein 0.75g；(NH4)2SO41.5g；MgSO4·7H20 0.4g；K2HPO40.2g；MnSO40.001g；
FeSO40.001g；CaCl20.2g；Benefit is filled with water to 100mL, and 8mL is dispensed in every test tube after heating, micro- after high pressure steam sterilization to incline
It is tiltedly stand-by after cooled and solidified.
(2), secondary liquid seed culture medium (100mL)：Glucose 5g；Corn starch 1g；Dusty yeast 1.5g；Soybean protein
0.75g；(NH4)2SO41.5g；MgSO4·7H20 0.4g；K2HPO40.2g；MnSO40.001g；FeSO40.001g；
CaCl20.2g；It is stand-by after high pressure steam sterilization.
(3), three-level liquid seed culture medium：It is identical with secondary liquid seed culture medium.
(4), fermentation medium (1L)：Cornstarch 100g；Pearling cone meal 40g；Soy hydrolysate 30g；Soybean protein 10g；
MgSO4·7H20 4g；Na2HPO45g；MnSO40.01g；FeSO40.01g, adds 1g AMSs to boil and stir after preparation
Mix to starch liquefacation, being filled in fermentation tank carries out in-situ sterilization.
2nd, inoculated and cultured
(1), primary inclined plane seed is cultivated：
The bacterial classification SCK6 of -80 DEG C of preservations of thawed on ice, picks a ring and is spread evenly across on slant medium with oese, puts
In 37 DEG C of incubators, 16h is cultivated.
(2), secondary liquid seed is cultivated：
The thalline grown on slant medium is scraped with oese, tapers of the 1000mL equipped with liquid seed culture medium is accessed
In bottle, liquid amount is 100mL, is placed in rotation concussion shaking table, and rotating speed 200rpm, 37 DEG C of temperature, culture 12h starts to determine cell life
Long concentration, cultivates to OD600Seed is harvested during=0.7-0.9.
(3), three-level liquid seeds are cultivated
Secondary seed is accessed in conical flasks of 5 2000mL equipped with liquid seed culture medium with 10% inoculum concentration, dress
Liquid measure is 200mL, is placed in rotation concussion shaking table, and rotating speed 200rpm, 37 DEG C of temperature starts to determine cell life respectively after culture 12h
Long concentration, cultivates to OD600Seed is harvested during=0.7-0.9.
(4), Bacillus subtillis SCK6 fermented and cultureds secretion produces protease
Three-level seed is accessed into 10L fermentation tanks with 6% inoculum concentration, liquid amount is 7L, and throughput is controlled to 0.8Nm3/ h,
Defoamer is added and is controlled to automatically, and tank body pressure remains 0.02MPa, and fermentation temperature is 35 DEG C, and incubation time continues 60h, often
2h collects sample 10mL by probe tube.
Dissolved oxygen level is controlled：It is set as automatically controlling, scope is 25%～45%, is adjusted by the change of mixing speed
Section.
Mixing speed：It is set to be linked with dissolved oxygen, adjustable range is 100rpm～900rpm.
According to Bacillus subtillis SCK6, with this understanding biomass over time, draws its growth curve such as Fig. 2
It is shown, the time corresponding to period of delay, logarithmic phase, stationary phase and decline phase in its fermenting and producing is obtained, determine therefrom that fermentation is opened
10h, 20h, 35h and 40h are respectively as the time started for adding protease inhibitors, protease inhibitors in the present embodiment after beginning
For 4- formyl phenylboronic acids.And while record yield when being added without protease inhibitors.
The tactful as shown in table 1 below of protease inhibitors 4- formyl phenylboronic acids, each strategy is added to carry out one batch
Secondary fermentation of bacillus production protease experiment, compare the highest enzyme activity that obtains under Different Strategies and its be added without albumen
The ratio of the enzyme activity of the fermentation protein enzyme of enzyme inhibitor：
Table 1：Different protease inhibitors addition strategies with without proteinase production contrast
Wherein, * zymotic fluids=FB (Fermentation broth), add the time started represent culture microorganism more than when young
When after time point, what the duration represented in special time period continuous uniform adds albumen enzyme level in culture medium
Agent.Final concentration (g/L) represents 4- formyl phenylboronic acids final concentration in the medium.Yield is by above-mentioned prolease activity
Method is determined.Enzyme activity ratio is the yield of the yield divided by the 0th batch of corresponding batch.
The fermenting and producing experimental result of 1～36 batch shows in table 1, batch 22,23,24,25,26,27,31,32,33,
34th, 35 and 36 high enzyme yield is obtained, and is significantly higher than the control batch 0 for not adding protease inhibitors, illustrated in bacillus
After SCK6 is when logarithm production period later stage and stationary phase being fermented and cultured 35h and 40h, continues 5h～20h and add albumen enzyme level
When agent is to final concentration of 0.2g/L～0.6g/L, the yield of proteinase can be obviously improved.
Respectively in table 1 1～36 batch fermenting and producing experiment gather fermentation time be 0h, 2h, 4h, 6h, 8h, 10h,
The zymotic fluid of 12h, 14h, 16h, 18h, 20h, 22h, 24h, 30h, 35h, 40h, 60h, 80h and 100h determines OD600Value, and
Draw growth curve, obtain batch 22,23,24,25,26,27,31,32,33,34,35 and 36 growth curve results with figure
2 is consistent, illustrates the addition 4- formyls in culture medium of the continuous uniform in the special time period of Bacillus subtillis SCK6 growths
Base phenylboric acid will not produce impact to the growth of Bacillus subtillis SCK6.
Embodiment 2
Pichia pastoris GS115 fermenting and producing prepares protease
1st, culture medium is prepared：
(1), general trace element (PTM1) liquid storage (100mL) of Pichia pastoris：FeSO4·7H2O, 6.5g；ZnCl2, 2.0g；
CuSO4·5H2O, 0.6g；MnSO4H2O, 0.3g；CoCl2, 0.05g；Biotin, 0.03g；Na2MoO4·2H2O, 0.02g；
NaI, 0.008g；H3BO3, 0.002g；Dense H2SO4(98%, m/v), 0.5mL.
(2), general trace element (PTM4) liquid storage (100mL) of Pichia pastoris：FeSO4·7H2O, 2.2g；ZnCl2, 0.7g；
MnSO4·H2O, 0.3g；CuSO4·5H2O, 0.2g；CoCl2, 0.05g；CaSO4·2H2O, 0.05g；Biotin, 0.02g；
Na2MoO4·2H2O, 0.02g；NaI, 0.008g；H3BO3, 0.002g；Above material is prepared after being uniformly dissolved, and is carefully added into dense
H2SO4(98%, m/v), 0.1mL.
(3), YPD culture mediums (90mL)：Yeast extract 1g, peptone 2g.
The sterile-filtered solution thereof for adding 10mL to contain 2g glucose after high pressure steam sterilization.
(4), FM22 culture mediums (100mL)：KH2PO4, 4.3g；(NH4)2SO4, 0.5g；CaSO4·2H2O, 0.1g；K2SO4,
1.5g；MgSO4·7H2O, 1.2g；The PTM4 liquid storages of glycerine, 4g and 0.25mL
(5), fermentation tank glycerine supplemented medium (100mL)：The general micro- liquid storage of glycerine 50g, PTM1 Pichia pastoris
1.2mL。
(6), fermentation tank formaldehyde supplemented medium (100mL)：The general trace element storage of methyl alcohol 98.8mL, PTM1 Pichia pastoris
Liquid 1.2mL.
2nd, inoculated and cultured
(1) activation of bacterial classification is produced：The preservation Pichia pastoris GS115 protease production strain inoculation that ultralow temperature magnetic bead is preserved
Activate to OD in the YPD culture mediums containing 26 μ g/mL bleomycin resistances of 10mL600=3.5,
(2) seed is cultivated：Fresh activation culture liquid is accessed in 375mL seed culture medium YPD with 3% amount, is placed in
29 DEG C of culture 20-24h detection OD of horizontal shaking table600Stop after 4-5 culture to reaching.
(3) proteinase
Using FM22 culture mediums as fermentation medium, initial liquid amount is 3.75L in 10L fermentation tanks, after in-situ sterilization,
The seed culture fluid 375mL for harvesting is accessed into fermentation tank, dissolved oxygen is controlled in 15-45% in sweat, and throughput is 1.0Nm3/
H, mixing speed setting is automatically adjusted and arranged with dissolved oxygen and links, and excursion is 100-900rpm, the temperature in thalli growth stage
Degree is set as 28 DEG C, and temperature is set as 26 DEG C into after induction period, and it is 5.5 to keep pH constant, and alkali is only needed under normal circumstances
Property solution adjust, mend alkali using being automatically added to ammoniacal liquor, froth breaking is arranged using being automatically added to defoamer, when detection carbon source exhausts, plus
Enter 400mL fermentation tank glycerine supplemented mediums.
When fermentation tank dissolved oxygen shows and begins to ramp up to more than 15%, formaldehyde supplemented medium is added, addition is to make to send out
Fermentation tank final concentration is about 0.5% (v/v), then starts with the speed stream of 6mL/h～14mL/h plus formaldehyde supplemented medium 8h；This
Afterwards the addition speed of formaldehyde supplemented medium is controlled according to the change of dissolved oxygen, dissolved oxygen is maintained between 15%～45%, generally,
The speed that stream adds is 3mL/L/h～10mL/L/h, until fermentation ends, need periodically sampling in sweat, determines yeast
The content of biomass, fermented supernatant fluid prolease activity and protein.
According to Pichia pastoris, with this understanding biomass over time, draws its growth curve as shown in figure 3, obtaining
Time in its fermenting and producing corresponding to period of delay, logarithmic phase, stationary phase and decline phase, it is determined that fermentation start rear 10h, 20h,
30h and 40h respectively adds the time started of protease inhibitors, and protease inhibitors is 4- Fonnylphenyls in the present embodiment
Boric acid.And while record yield when being added without protease inhibitors.
The strategy for adding protease inhibitors 4- formyl phenylboronic acids is shown in table 2 below, and each strategy carries out one batch
Secondary Pichia pastoris proteinase fermenting experiment, compare Different Strategies under obtain highest enzyme activity and its be added without
The ratio of the enzyme activity of the fermentation protein enzyme of protease inhibitors：
Table 2 it is different protease inhibitors addition strategy with without proteinase production contrast
* zymotic fluid=FB (Fermentation broth), after adding the time started to represent culture microorganism how many hours
Time point, what the duration represented in special time period continuous uniform adds protease inhibitors in culture medium.Eventually
Concentration (g/L) represents 4- formyl phenylboronic acids final concentration in the medium.The method that yield passes through above-mentioned prolease activity
Determine.Enzyme activity ratio is the yield of the yield divided by the 0th batch of corresponding batch.
The fermenting and producing experimental result of 1～36 batch shows in table 2, batch 22,23,24,25,26,27,31,32,33,
34th, 35 and 36 high enzyme yield is obtained, and is significantly higher than the control batch 0 for not adding protease inhibitors, illustrated in Pichia pastoris
GS115 logarithm production period later stages and stationary phase ferment when starting 30h and 40h, continue 5h～20h and add protease inhibitors extremely
During final concentration of 0.2g/L～0.6g/L, the yield of proteinase can be lifted.
In table 2 1～36 batch fermenting and producing experiment gather fermentation time be 0h, 2h, 4h, 6h, 8h, 10h, 12h,
The zymotic fluid of 14h, 16h, 18h, 20h, 22h, 24h, 30h, 40h, 60h, 80h, 100h, 120h and 140h determines wet cell weight
Value, and growth curve is drawn, obtain batch 22,23,24,25,26,27,31,32,33,34,35 and 36 growth curve results
It is consistent with Fig. 3, illustrate the addition 4- first in culture medium of the continuous uniform in the special time period of Pichia pastoris GS115 growth
Aminosulfonylphenyl boric acid will not produce impact to the growth of Pichia pastoris GS115.
Embodiment 3
Protease inhibitors is added to lift the yield of protease
Production bacterial strain and fermentation conditions used by fermentation of bacillus is in the same manner as in Example 1.
Protease inhibitors addition species is shown in Table 3, and each protease inhibitors carries out the fermenting experiment of a batch, adds
Plus strategy is carried out after 40h for fermentation, start to add protease inhibitors in zymotic fluid, continue each protease inhibitors of 20h
Ultimate density is 0.4g/L, corresponding fermenting enzyme yield result and with the fermenting experiment yield of enzyme for being not added with protease inhibitors
The ratio result of (control) is shown in table 3 below
Table 3 adds protease inhibitors and contrasts with the proteinase production without protease inhibitors
The result of table 3 shows, the albumen enzyme level shown in addition table 3 is started when fermentation of bacillus subtilis carries out 40h
Agent, continues at the uniform velocity to add 20h, each protease inhibitors final concentration is reached after 0.4g/L, can lift the yield of protease.
Embodiment 4
Add protease inhibitors and protease stabiliser compositions that the yield of protease is produced to fermentation of bacillus
Affect
Production bacterial strain and fermentation conditions used by fermentation of bacillus is in the same manner as in Example 1.
Composition addition strategy is carried out after 40h for fermentation, starts the composition for adding protease inhibitors and stabilizer, is held
It is continuous at the uniform velocity to add 20h, the ultimate density of each composition and it is shown in table 4 below with corresponding fermenting enzyme yield result：
Table 4 add protease inhibitors and stabiliser compositions with without proteinase production contrast
The composition energy of addition protease inhibitors, calcium salt and/or magnesium salts is can be seen that from the result shown with upper table 4
The yield of bacillus SCK6 fermentation protein enzymes is enough significantly improved, and only adds the impact of calcium salt and/or magnesium salts to the yield of enzyme
It is not notable.
Embodiment 5
Add the product of protease inhibitors and protease stabiliser compositions to Pichia pastoris GS115 proteinase
The impact of amount
Pichia pastoris fermentation production bacterial strain used and fermentation conditions are in the same manner as in Example 2.
Composition addition strategy is carried out after 30h for fermentation, starts the composition for adding protease inhibitors and stabilizer, is held
Continuous at the uniform velocity 20h, the ultimate density of each composition and is shown in down with corresponding fermenting enzyme yield result
Table 5：Addition protease inhibitors and stabiliser compositions with without proteinase production contrast
The composition energy of addition protease inhibitors, calcium salt and/or magnesium salts is can be seen that from the result shown with upper table 5
The yield of Pichia pastoris GS115 fermentation protein enzyme is enough significantly improved, experimental result equally shows only adds calcium salt and/or magnesium salts pair
The amount of Pichia pastoris producing enzyme affects not notable.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but and
Can not therefore be construed as limiting the scope of the patent.It should be pointed out that for one of ordinary skill in the art comes
Say, without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. a kind of method for preparing protease, it is characterised in that comprise the steps：
Fermented and cultured microorganism in the medium, and continue to the culture medium in the special time period of the growth of microorganism
Middle addition protease inhibitors, time period of the special time period in the logarithmic phase to stationary phase of the growth of microorganism
It is interior；And
The microorganism secretion produces protease.
2. the method for preparing protease according to claim 1, it is characterised in that the special time period duration is
4h～48h, final concentration of 0.1g/L～5g/L of the protease inhibitors in the culture medium, add in the culture medium
The speed for entering the protease inhibitors is 0.002g/ (Lh)～1.25g/ (Lh).
3. the method for preparing protease according to claim 1, it is characterised in that the protease inhibitors spreads out for boric acid
It is biological.
4. the method for preparing protease according to claim 1 or 3, it is characterised in that the protease inhibitors is selected from
4- formyl phenylboronic acids, bortezomib, thiophene -3- boric acid, 3- acetylaminos
Phenylboric acid, N- acyl groups-peptide ylboronic acid, 4- methylthiophene -2- boric acid, 2- fluoro- 5- (methoxycarbonyl group) phenyl boric acids, 5- ethyl thiophenes
Fen -2- boric acid, 2- fluoro- 4- (methoxycarbonyl group) phenyl boric acids, 5- bromothiophene -2- boric acid, 4- (1- piperidino methyls) phenyl boric acid, 4-
(1- pyrrolidinylmethyls) phenyl boric acid, dibenzothiophenes base -1- boric acid, 2- (methoxycarbonyl group) -4- methylphenylboronic acids, 2,6- bis-
Chloro- 3- methylphenylboronic acids, dibenzofurans -4- boric acid, 2- isopropoxy phenyl boric acids, the fluoro- 4- methylsulfonyls phenyl boric acids of 2-, furans -
The fluoro- 4- methylsulfonyls phenyl boric acid of 2- boric acid, 3-, 1-Boc-7- methoxy-Indole -2- boric acid, furans -3- boric acid, 4- (1- piperidyls
Sulfonyl) phenyl boric acid, 3- acetamidobenzeneboronic acids, 3- methoxythiophene -2- boric acid, 2- methyl -4- methoxyphenylboronic acids, 5- first
Base -3- pyridine boronic acids, 5- n-propyl thiophene -2- boric acid, 10- phenyl -9- anthracene boric acid, 3 bromo thiophene boric acid, the fluoro- 3- of 5- methyl -6-
Pyridine boronic acid, 3 bromo thiophene -4- boric acid, the fluoro- 3- pyridine boronic acids of 2,6- bis-, 4- formyl phenylboronic acids, the chloro- 4- methoxycarbonyl groups of 3-
Phenyl boric acid, phenyl boric acid, 2- chloro-4-methoxy phenyl boric acids, 5- ethyl furan -2- boric acid, 3- methyl -4- chlorophenylboronic acids, hexichol generation
At least one in boric acid, 4- (Cyclopropylsulfamoyl base) phenyl boric acids and 5- methyl oxygen FURAN-2-BORONIC ACIDs.
5. the method for preparing protease according to claim 1, it is characterised in that be additionally included in the growth of microorganism
Continue to add albumen enzyme stabilizers in the culture medium in special time period.
6. the method for preparing protease according to claim 5, it is characterised in that the albumen enzyme stabilizers selected from magnesium from
At least one in son and calcium ion.
7. the method for preparing protease according to claim 6, it is characterised in that the special time period duration is
4h～48h, at least one final concentration of 0.005mol/L in the culture medium in the magnesium ion and the calcium ion
～0.1mol/L.
8. the method for preparing protease according to claim 1, it is characterised in that the microorganism is Ko subtilis bar
Bacterium, starts to add 4- formoxyls in the culture medium in the medium after Bacillus subtillis 35h～40h described in fermented and cultured
Phenylboric acid, the lasting addition within the time period of 5h～20h of the 4- formyl phenylboronic acids is described into the culture medium
Final concentration of 0.2g/L～0.6g/L of the 4- formyl phenylboronic acids in the culture medium, adds described in the culture medium
The speed of 4- formyl phenylboronic acids is 0.01g/ (Lh)～0.12g/ (Lh).
9. the method for preparing protease according to claim 8, it is characterised in that be additionally included in fermented and cultured in culture medium
Start to add albumen enzyme stabilizers, the albumen enzyme stabilizers in the culture medium after the Bacillus subtillis 35h～40h
At least one in magnesium ion and calcium ion, at least one in the magnesium ion and the calcium ion is in the culture medium
In final concentration of 0.0125mol/L～0.025mol/L.
10. the method for preparing protease according to claim 1, it is characterised in that the microorganism is Pichia pastoris,
Start to add 4- formyl phenylboronic acids in the culture medium after Pichia pastoris 30h～40h described in fermented and cultured in culture medium,
The lasting addition within the time period of 5h～20h of the 4- formyl phenylboronic acids into the culture medium, the 4- formoxyls
Final concentration of 0.2g/L～0.6g/L of the phenylboric acid in the culture medium, the 4- formoxyls are added in the culture medium
The speed of phenylboric acid is 0.01g/ (Lh)～0.12g/ (Lh).
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