CN106754840B - The curdled milk enzyme mutant that a kind of enzyme activity and thermal stability improve - Google Patents
The curdled milk enzyme mutant that a kind of enzyme activity and thermal stability improve Download PDFInfo
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- CN106754840B CN106754840B CN201611195945.9A CN201611195945A CN106754840B CN 106754840 B CN106754840 B CN 106754840B CN 201611195945 A CN201611195945 A CN 201611195945A CN 106754840 B CN106754840 B CN 106754840B
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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
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- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6478—Aspartic endopeptidases (3.4.23)
- C12N9/6483—Chymosin (3.4.23.4), i.e. rennin
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/23—Aspartic endopeptidases (3.4.23)
- C12Y304/23004—Chymosin (3.4.23.4), i.e. rennin
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Abstract
The invention discloses the curdled milk enzyme mutants that a kind of enzyme activity and thermal stability improve, and belong to technical field of enzyme engineering.The present invention replaces the 75th leucine of renin with glutamine, the 222nd methionine is replaced with threonine, and double mutation building curdled milk enzyme mutants are carried out to the amino acid in above-mentioned site, by mutant in Pichia pastoris GS115 heterogenous expression, curdled milk enzyme activity is carried out to the crude enzyme liquid of expression, protease activity, heat resistance detection.The crude enzyme liquid curdled milk enzyme activity of double-mutant Leu75Gln-Met222Thr reaches 28.0SU/mL, and C/P value reaches 11.7, and the curdled milk enzyme activity and C/P value compared to starting strain P-cMCE improve 155.6% and 195.0%.The thermal stability of 45 DEG C of constant temperature half an hour reduces 13.6%.
Description
Technical field
The present invention relates to the curdled milk enzyme mutants that a kind of enzyme activity and thermal stability improve, and belong to technical field of enzyme engineering.
Background technique
Renin is one kind most early in the aspartic protease found in the calf stomach not weaned, can exclusively be cut
Peptide bond in cream between κ-casein Phe105-Met106, destroying casein micelles makes milk condensing, the curdled milk energy of renin
Power and proteolytic activity become the key enzyme that texture and flavour are formed in cheesemaking, are widely used in
The production of cheese and Yoghourt.
Enhance the curdled milk ability of renin, reducing the hydrolysis ability to albumen and reducing heat resistance is always renin egg
The popular direction of white transformation.Chitpinityol etc. (1998) carries out threonine to asparagus fern to 77 sites using site-directed mutagenesis technique
The change of propylhomoserin causes muton relatively recombination wild type curdled milk enzyme activity to be declined；So far, for calf chymosin etc.
The research of the fungies renin such as animal rennet and Mucor pusillus is more, domestic for the research of bacterial origin renin rite-directed mutagenesis temporary
It does not find.
Summary of the invention
The first purpose of the invention is to provide a kind of curdled milk enzyme mutant, amino acid sequence such as NO.1~3 SEQ ID
It is shown.
A second object of the present invention is to provide the genes for encoding the curdled milk enzyme mutant.
Third object of the present invention is to provide the genetic engineering bacterium for expressing the curdled milk enzyme mutant, the genetic engineerings
Bacterium is host with Pichia pastoris using pPIC9K-cMCE as carrier, expression NO.1~3 SEQ ID it is any shown in curdled milk enzyme mutant
Body.
In one embodiment of the invention, the genetic engineering bacterium is host with Pichia pastoris GS115.
Fourth object of the present invention is to provide the method for constructing the genetic engineering bacterium, and the method is by pPIC9K-
CMCE is connect with the gene for encoding the curdled milk enzyme mutant, is converted into Pichia pastoris.
In one embodiment of the invention, the construction method of the pPIC9K-cMCE is disclosed in number of patent application and is
201611157248.4 application for a patent for invention in；The Pichia pastoris is Pichia pastoris GS115.
Fifth object of the present invention is to provide a kind of methods for improving renin enzyme activity, and being will be shown in SEQ ID NO.4
The 75th leucine of renin sport glutamine and obtain mutant shown in SEQ ID NO.1, or by the 222nd egg ammonia
Acid replaces with threonine and obtains mutant shown in SEQ ID NO.2.
In one embodiment of the invention, the method is bright by renin the 75th shown in SEQ ID NO.4
Histidine mutations are glutamine, and the 222nd methionine is replaced with threonine and obtains mutant shown in SEQ ID NO.3.
It is that the genetic engineering bacterium is seeded to hair sixth object of the present invention is to provide a kind of method of producing lab ferment
In ferment culture medium, 25~30 DEG C of 24~96h of culture.
In one embodiment of the invention, the fermentation efficiently induces Pichia pastoris extracellular expression by stream plus methanol
Renin.
The present invention also provides the curdled milk enzyme mutant food, biology, field of medicaments application.
In one embodiment of the invention, the application is specially to prepare the dairy products such as cheese.
The utility model has the advantages that (1) present invention uses for the first time in bacillus amyloliquefaciens source rennet-based because of upper carry out rite-directed mutagenesis
The one-step method that PCR is mediated, carries out single-site mutant and double mutations, whole process simple and clear, success rate reach 90% with
On.(2) Leu75Gln and Met222Thr double mutations effect is best, and crude enzyme liquid curdled milk enzyme activity, C/P value improves 155.6%
With 195.0%, thermal stability is also reduced, and reduces 13.6% compared to starting strain.Improve the quotient of such renin
Industry value, has widened commercial field, and the industrial curdled milk enzyme product to obtain excellent has established important foundation.
Detailed description of the invention
Fig. 1 is mutated the verifying of recombinant plasmid double digestion；Wherein, M, 15000bp Marker；0, starting strain P-Cmce；1~6
Respectively correspond recombinant plasmid pPIC9K-cMCE-Leu75Gln, pPIC9K-cMCE-Val81Asp, pPIC9K-cMCE-
Met222Thr, pPIC9K-cMCE-Leu75Gln-Val81Asp, pPIC9K-cMCE-Leu75Gln-Met222Thrp, and
PIC9K-cMCEVal81Asp-Met222Thr；
Fig. 2 is the SDS-PAGA figure for being mutated transformant shaking flask culture 96h crude enzyme liquid；Wherein, M, Marker；0, starting strain
P-Cmce；1~6, respectively correspond recombinant plasmid pPIC9K-cMCE-Leu75Gln, pPIC9K-cMCE-Val81Asp, pPIC9K-
CMCE-Met222Thr, pPIC9K-cMCE-Leu75Gln-Val81Asp, pPIC9K-cMCE-Leu75Gln-Met222Thrp,
And PIC9K-cMCEVal81Asp-Met222Thr；
Fig. 3 is mutant compared with the thermal stability of control.
Specific embodiment
1) condenser water level measures
According to MAGDA method, by skimmed milk powder 0.01mol/L CaCl2It prepares 10% and restores 35 DEG C of items of degreasing milk solution
It takes enzyme solution 0.2mL to be measured that 2mL is added under part to restore in skimmed milk and mix, starts timing, until cotton-shaped agglutinating particle occurs
(i.e. enzyme amount needed for the skimmed milk of 40min solidification 1mL 100g/L is defined as an enzyme activity unit).
Enzyme activity (soxhelt unit, SU)=(degreasing breast amount mL/ curdled milk enzyme amount mL) × D × 2400/T
Note: D is enzyme solution extension rate；T is the curdled milk time.
2) proteolysis vitality test
2min is preheated in the 40 DEG C of water-baths of first enzyme solution and casein solution, 1mL casein solution and 1mL enzyme solution is taken to mix, 40
DEG C accurate heat preservation 10min, the time terminates is added 0.4mol/L trichloroacetic acid 2mL immediately, terminates reaction, continues to make it in a water bath
20min is kept the temperature, centrifugation removal precipitating takes supernatant 1min, the Folin reagent of 0.4mol/L sodium carbonate 5mL 0.67moL/L is added
1mL shakes up, and reacts optical density (OD after 20min660) measurement.0.4mol/L trichloroacetic acid is added in blank before adding casein
2mL inactivates enzyme, adds casein.
3) thermal stability determination
Sample to be tested is individually placed to keep the temperature 120min at a temperature of 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, every
10min sampling is primary, measures the remaining curd vigor of renin after ice bath is cooling at 35 DEG C, living with not thermally treated curdled milk
Power is 100%.
4) culture medium
BMGY culture medium: 10g/L yeast powder, 20g/L peptone, 3g/L dipotassium hydrogen phosphate, 11.8g/L potassium dihydrogen phosphate,
13.4g/L YNB, 4 × 10-4G/L biotin, 10g/L glycerol.
BMMY culture medium: 10g/L yeast powder, 20g/L peptone, 3g/L dipotassium hydrogen phosphate, 11.8g/L potassium dihydrogen phosphate,
13.4g/L YNB, 4 × 10-4G/L biotin, 5ml/L anhydrous methanol.
MM culture medium: 13.4g/L YNB, 4 × 10-4G/L biotin, 5ml/L methanol, 15g/L agar.
MD culture medium: 13.4g/L YNB, 4 × 10-4G/L biotin, 20g/L glucose, 0.4g/L histidine, 15g/L
Agar.
Embodiment 1
(1) bacillus amyloliquefaciens source renin site-directed point mutation site is determined
By computer to bacillus amyloliquefaciens source rennet-based because carry out tertiary structure simulation, while with ox renin
Tertiary structure and nucleic acid sequence comparison, select 3 critical sites and carry out rite-directed mutagenesis, and selecting respectively may be with hydrolysis or thermostabilization
The relevant site 75 of property, 81 sites and 222 sites.
75 sites are leucine originally, and 81 sites are valine originally, and 222 sites are methionine originally, and the present invention is in advance by 75 sites
Sport glutamine, 81 site mutations are aspartic acid, and 222 site mutations are threonine, the one-step method mediated by PCR into
Row point mutation.
By DNAMAN to selected mutational site design primer, as shown in table 1.
Primer sequence used in 1 rite-directed mutagenesis of table
(2) the one-step method rite-directed mutagenesis that PCR is mediated
The recombination of e. coli jm109-cMCE is extracted with the kit (PE01) of Hangzhou Bao Sai Biotechnology Co., Ltd
Plasmid pPIC9K-cMCE (is disclosed in the application for a patent for invention application No. is 201611157248.4).With Leu75Gln-F and
Leu75Gln-R is that the 75th leucine of primer pair renin is mutated, construction recombination plasmid pPIC9K-cMCE-
Leu75Gln；Using Val81Asp-F and Val81Asp-R as primer, the 81st valine of renin is mutated, building recombination
Plasmid pPIC9K-cMCE-Val81Asp；Using Met222Thr-F and Met222Thr-R as primer, to the 222nd egg ammonia of renin
Acid is mutated, construction recombination plasmid pPIC9K-cMCE-Met222Thr.
PCR reaction system is as follows: 50 50 μ L, 20 μM/L of μ L, ddH2O of super pfu PCR Master Mix are upper and lower
Swim each 2 μ L of primer, 2 μ L of template DNA, 100 μ L of total volume, 10 pipe of packing, every 10 μ L of pipe.Reaction condition: 94 DEG C of initial denaturations
3min；98 DEG C of denaturation 40s, 60 DEG C of annealing 1min, 68 DEG C of extension 11min；18 circle；72 DEG C of extension 10min.PCR is produced
Object carries out agarose gel electrophoresis, as a result as shown in Figure 1.
After single-site mutant success, using single-site mutant plasmid as template, with another pair mutant primer, according to same
PCR system introduces second mutational site, i.e. building double mutations site Leu75Gln-Val81Asp, Leu75Gln-
Met222Thr, Val81Asp-Met222Thr.
After reaction, 1 μ L Dpn I is directly added in PCR in PCR reaction system, and 37 DEG C of metal baths are incubated for after mixing
1h。
With kit (PE03) purified pcr product of Hangzhou Bao Sai Biotechnology Co., Ltd, recombinant vector is transformed into greatly
Enterobacteria JM109 competent cell, obtains recombinant bacterium, and picking recombinant bacterium monoclonal extracts plasmid pPIC9K-cMCE-
Leu75Gln, pPIC9K-cMCE-Val81Asp, pPIC9K-cMCE-Met222Thr, pPIC9K-cMCE-Leu75Gln-
Val81Asp, pPIC9K-cMCE-Leu75Gln-Met222Thrp and PIC9K-cMCEVal81Asp-Met222Thr.
With EcoR I and Not I double digestion recombinant vector pPIC9K-cMCE-Leu75Gln, pPIC9K-cMCE-
Val81Asp, pPIC9K-cMCE-Met222Thr, pPIC9K-cMCE-Leu75Gln-Val81Asp, pPIC9K-cMCE-
After Leu75Gln-Met222Thrp and PIC9K-cMCEVal81Asp-Met222Thr, agarose gel electrophoresis is carried out, is such as schemed
1, the length of carrier and target fragment is all equal respectively.
It selects digestion and identifies that correct recombinant vector send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced, amplification
Rennet-based because nucleotide sequence and with the consistency of former sequence alignment result reach 99.82%, in addition to mutational site
Nucleotide sequence it is completely the same.
Embodiment 2
(1) expression of the curdled milk enzyme mutant in Pichia pastoris
Expression step of the curdled milk enzyme mutant in Pichia pastoris is referring to application No. is 201611157248.4 patent Shens
Please embodiment one associated operating steps.
Recombinant vector pPIC9K-cMCE-Leu75Gln, pPIC9K-cMCE-Val81Asp, pPIC9K-cMCE-
Met222Thr, pPIC9K-cMCE-Leu75Gln-Val81Asp, pPIC9K-cMCE-Leu75Gln-Met222Thrp, and
After PIC9K-cMCEVal81Asp-Met222Thr Sac I linearization for enzyme restriction, 2500V, 5ms electrotransformation Pichia pastoris GSll5
Competent cell is successively applied on MM and MD plate, filters out His+And Mut+Type transformant P-cMCE.
Select transformant carry out bacterium colony PCR verifying, PCR product is subjected to agarose gel electrophoresis, select transformant in
In BMGY culture medium, 30 DEG C/300rpm is cultivated to 0D600=2~6；Thallus is collected, thallus is resuspended with BMMY, makes OD600=
5.0 or so, anhydrous methanol is added into culture medium to final concentration of 0.5% (v/v, 5mL methanol/L culture medium)；Every 12h addition
The methanol of final concentration 0.5% (v/v) terminates induction, concentration induction 96h post-fermentation liquid supernatant after 96h.
(2) purifying of recombinant chymosin
It takes Pichia pastoris P-cMCE to induce 96 hours fermentation liquid 100mL, saturation ammonium persulfate solution is then added to mistake
Ammonium sulfate final concentration of 60%, 4 DEG C stand overnight.10000rpm is centrifuged 10min, collects albumen precipitation, and vacuum freeze drying obtains
To P-cMCE enzyme preparation 0.566g.10mL phosphate buffered saline solution is redissolved, and 20ul sample is taken to carry out SDS-PAGE electrophoresis detection.
It is solidifying to substantially conform to opportunistic pathogen as shown in Fig. 2, there is apparent band at molecular weight 27kDa for SDS-PAGE electrophoresis result
Galactenzyme albumen size.
Embodiment 3
(1) curdled milk enzyme activity, the comparison of protease activity and C/P value property
C/P value refers to the ratio of pure enzyme powder its milk-curdling activity and proteinase activity of unit mass.It can conscientiously accurately
Characterize the curdled milk ability of renin.
Mutant plasmid Leu75Gln, Val81Asp, Met222Thr, Leu75Gln-Val81Asp, Leu75Gln-
Met222Thr, Val81Asp-Met222Thr conversion Pichia pastoris GS115,30 transformants of random picking, inoculum concentration 0.5%,
Starting strain P-cMCE not to be mutated is cultivated through BMGY as control and methanol induction, pH is in 6.5~6.7, fermentation
30 DEG C of the temperature 96h that ferment in BMMY culture medium, centrifuging and taking supernatant measure crude enzyme liquid curdled milk enzyme activity, proteolysis vigor, meter
C/P ratio is calculated, the results are shown in Table 2.
As can be seen from Table 2, in single-site mutant, Leu75Gln mutation rennet curdling activity increases at most, reaches
25.0SU/mL improves 138.9%, Val81Asp mutation renin proteolytic activity compared to starting strain and is substantially reduced most
To be significant, but milk-clotting activity is extremely low, is unfavorable for curdled milk effect；In double mutations site, Leu75Gln-Met222Thr is dual
Mutation rennet curdling activity increases at most, reaches 28.0SU/mL, and proteolytic activity reduces, meanwhile, C/P ratio substantially has
It improves, improves 188.2% compared to starting strain.
Table 2 is three kinds of rite-directed mutagenesis transformants compared with the enzyme activity of starting strain
(2) comparison of thermal stability
The mutant that curdled milk enzyme mutant Leu75Gln and Met222Thr and the two mutational sites are combined
Leu75Gln-Met222Thr carries out the secondary screening of thermal stability, as a result as shown in Figure 3.
As seen from Figure 3, the mutation in two sites Leu75Gln and Met222Thr makes to be mutated renin thermostabilization
Property dies down, and is weaker than starting strain.Characteristic by renin in application aspect is influenced, when renin thermal stability be deteriorated,
Its at a lower temperature there is a phenomenon where inactivating, thus be not hydrolyzed into dairy products (such as cheese etc.) during storage for
Whey.Relative to Leu75Gln mutation renins, Met222Thr mutation renins have weaker thermal stability.
After 45 DEG C of processing 60min of Met222Thr mutation renins, opposite condenser water level is 62.0%.Leu75Gln and Met222Thr
Though double mutations curdled milk enzyme heat stability Weakening degree is not so good as Met222Thr single-site mutant renin, starting strain is compared
P-cMCE weakens 16%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>the curdled milk enzyme mutant that a kind of enzyme activity and thermal stability improve
<160> 10
<170> PatentIn version 3.3
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195 200 205
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Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala
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165 170 175
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210 215 220
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Claims (10)
1. a kind of curdled milk enzyme mutant, which is characterized in that shown in amino acid sequence such as NO.1~3 SEQ ID are any.
2. encoding the gene of curdled milk enzyme mutant described in claim 1.
3. expressing the carrier or cell line of curdled milk enzyme mutant described in claim 1.
4. a kind of genetic engineering bacterium of producing lab ferment, which is characterized in that with Pichia pastoris be host, express NO.1~3 SEQ ID
Curdled milk enzyme mutant shown in any.
5. genetic engineering bacterium according to claim 4, which is characterized in that with Pichia pastoris GS115 be host.
6. the method for constructing genetic engineering bacterium described in claim 4, which is characterized in that the method is by carrier and coding right
It is required that the gene of the 1 curdled milk enzyme mutant connects, convert into Pichia pastoris.
7. a kind of method for improving renin enzyme activity, which is characterized in that be bright by renin the 75th shown in SEQ ID NO.4
Histidine mutations are glutamine, or the 222nd methionine is replaced with threonine.
8. the method according to the description of claim 7 is characterized in that the method is by renin shown in SEQ ID NO.4
75th leucine sports glutamine, and the 222nd methionine is replaced with threonine.
9. a kind of method of producing lab ferment, which is characterized in that genetic engineering bacterium as claimed in claim 4 is seeded to fermented and cultured
In base, 25~30 DEG C of 24~96h of culture.
10. application of the curdled milk enzyme mutant in food, biology, field of medicaments described in claim 1.
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| CN109182311B (en) * | 2018-10-11 | 2021-07-27 | 山东隆科特酶制剂有限公司 | Chymosin for Yimeng black goat milk cheese |
| CN110862933A (en) * | 2019-12-31 | 2020-03-06 | 扬中酵诚生物技术研究有限公司 | Pichia pastoris organic medium and preparation process thereof |
| CN111172139B (en) * | 2020-02-27 | 2021-11-30 | 武汉轻工大学 | Calf abomasum rennin, gene, strain and application |
| CN114921513B (en) * | 2022-02-14 | 2024-05-31 | 上海理工大学 | Method for preparing cannabis protein peptide by combined solid-state fermentation of lactic acid bacteria and psychrophilic protease |
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