CN113151330B - Acid protease mutant and preparation method and application thereof - Google Patents
Acid protease mutant and preparation method and application thereof Download PDFInfo
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Abstract
An acidic protease mutant, a preparation method and application thereof, wherein the acidic protease mutant is a double-site mutant PepA-N11R/N12S of acidic protease PepA, and the nucleotide sequence of the acidic protease mutant is shown as SEQ ID NO. 2. The acid protease mutant is a composite site mutant PepA-N11R/N12S-E110R of acid protease PepA, and the nucleotide sequence of the acid protease mutant is shown as SEQ ID NO. 3.
Description
Technical Field
The invention relates to the field of protein molecular modification of genetic engineering and enzyme engineering, in particular to an acidic protease mutant with improved thermal stability through site-directed mutagenesis modification.
Background
The acid proteinase is also called aspartic proteinase, mainly derived from aspergillus, trichoderma and other filamentous fungi, has good effect of hydrolyzing protein under acidic condition, and has wide application in leather processing, food, feed, medicine, aquatic product processing and other aspects.
At present, the research of acid protease is mainly focused on the expression and solid state fermentation of aspergillus niger, which causes the problems of long fermentation period, unstable quality, difficult purification, high labor intensity and the like in the production of acid protease, and the research of heterologous expression in other industrial fungus expression systems is relatively less. Pichia pastoris has the advantages of low fermentation cost, less production of mixed protein and the like, and is an excellent expression system for industrial production of enzyme preparations. The temperature plays a key role in the action efficiency of the enzymatic reaction, the good thermal stability determines the sustainability of the catalytic effect of the enzyme on the substrate, but the heat resistance of the PepA is poor, the half-life of the PepA is less than 30min at 45 ℃, and the enzyme activity is rapidly lost at 60 ℃, so that the industrialization application of the PepA is greatly limited, and the thermal stability of the PepA is improved through a certain means strategy, so that the problem to be solved in the current industrial application of the acid protease is urgent. Site-directed mutagenesis is an effective protein molecule modification and is widely used in the aspects of enzyme property improvement, enzyme activity improvement and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide an acid protease mutant and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
an acidic protease mutant with improved thermal stability is modified by site-directed mutagenesis, wherein the acidic protease mutant is a double-site mutant PepA-N11R/N12S of acidic protease PepA, and the nucleotide sequence of the acidic protease mutant is shown as SEQ ID NO. 2.
Furthermore, the acid protease mutant is a composite site mutant PepA-N11R/N12S-E110R of acid protease PepA, and the nucleotide sequence of the acid protease mutant is shown as SEQ ID NO. 3.
Further, the acid protease mutant is a double-site mutant PepA-N11R/N12S mutation primer of the acid protease PepA, and the mutation primer is as follows:
PepA-N11R/N12S-F
5'-GTTACTACTCCTCAAAGATCTGACGAAGAGTACC-3'
PepA-N11R/N12S-R
5'-AGATCTTTGAGGAGTAGTAACAGCAGAACCTTTAGAAGCTGCC-3'。
further, the mutant of the acid protease is a composite site mutant PepA-N11R/N12S-E110R of the acid protease, and the mutation primers of the composite site mutant PepA-N11R/N12S-E110R are as follows: pepA-N11R/N12S-E110R-F
5'-GTAAGATTTCAAGTAGATTTGTCCAAAACAC-3'
PepA-N11R/N12S-E110R-R
5'-TCTACTTGAAATCTTACTTGCTGCTTCAAC-3'。
Further, the acid protease mutant has an optimum pH of 2.5 and an optimum temperature of 55 ℃.
Furthermore, the construction method of the acidic protease mutant with improved thermal stability through site-directed mutagenesis comprises the following steps:
(1) Sequence optimization and cloning of acid protease gene PepA: the target sequence is directly synthesized after the sequence of Pichia pastoris expression is optimized by using DNAwork, PCR amplification is carried out by taking the sequence as a template, and the product is connected to a pPIC9K carrier to obtain a recombinant carrier pPIC9K-PepA;
(2) Site-directed mutagenesis: carrying out PCR amplification and digestion by using a vector pPIC9K-PepA as a template and a corresponding mutation primer; transferring the digested product into DMT competent cells by a heat shock method, carrying out bacterial liquid PCR verification, then carrying out sequencing, determining that the correct mutant extracts plasmids, and then transferring the plasmids into Pichia pastoris GS115 by an electrotransfer method for expression by SalI restriction endonuclease.
Further, the mutation primer of the acid protease mutant is as follows:
PepA-N11R/N12S-F
5'-GTTACTACTCCTCAAAGATCTGACGAAGAGTACC-3'
PepA-N11R/N12S-R
5'-AGATCTTTGAGGAGTAGTAACAGCAGAACCTTTAGAAGCTGCC-3'
PepA-N11R/N12S-E110R-F
5'-GTAAGATTTCAAGTAGATTTGTCCAAAACAC-3'
PepA-N11R/N12S-E110R-R
5'-TCTACTTGAAATCTTACTTGCTGCTTCAAC-3'。
the application of the mutant in the aspect of thermal stability detection of acid protease PepA.
Compared with the prior art, the invention has the beneficial effects that:
the experiment utilizes pichia pastoris to carry out heterologous expression on the acid protease PepA from Aspergillus niger, and provides a theoretical basis for industrial production of the acid protease by the pichia pastoris. The supernatant crude enzyme solution has good hydrolysis effect on casein, and has good application potential in leather, feed and food. This experiment found that the B-factor of 109-SE-110 was more than twice std by normalized B-factor calculation and structural biological analysis of the PepA gene sequence; the B-factor of 11-NN-12 exceeds three times std. The N11-E242 has the electric field force effect, and N11 can be replaced by R to strengthen the salt bridge effect; the backbond of N12 has hydrogen bond action with Y275, N12 is replaced by S, and a side chain is truncated to increase stability; after single mutation site E110R and N11R/N12S and compound mutation site N11R/N12S-E110R, single mutation site N11R/N12S is obtained, the single mutation site E is tolerant for 30min at 45 ℃ and the residual enzyme activity is improved by about 50% compared with that of wild acid protease PepA after being tolerant for 15min at 50 ℃; after the compound mutation N11R/N12S-E110R, the residual enzyme activity is improved by about 65% compared with the residual enzyme activity of the wild-type acid protease PepA under the condition of 45 ℃ and is improved by about 56% compared with the residual enzyme activity of the wild-type acid protease PepA under the condition of 50 ℃ for 15 min.
Drawings
FIG. 1 Multi-sequence alignment of acid protease PepA
FIG. 2 simulation of the crystal structure of acid protease PepA
FIG. 3 acid protease PepA and its mutant optimum pH
FIG. 4 pH stability of acid protease PepA and its mutants
FIG. 5 optimum temperature of acid protease PepA and its mutant
FIG. 6 thermal stability of acid protease PepA and its mutant
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed description:
as shown in figures 1-6 of the drawings,
the invention improves the temperature stability of the PepA by carrying out site-directed mutagenesis on the acid protease through the calculation of the normal-factor and the structural biological analysis, provides a new strategy for the transformation of the thermal stability of the acid protease PepA, and lays the foundation of the acid protease PepA in industrial application.
The invention provides a double-site mutant PepA-N11R/N12S of acid protease PepA with improved thermal stability;
the invention provides a composite site mutant PepA-N11R/N12S-E110R of acid protease PepA with improved thermal stability;
the invention provides a codon-optimized gene PepA-N11R/N12S for encoding a PepA double-site mutant with improved thermal stability, and the corresponding nucleotide sequence is shown as SEQ ID NO. 2.
The invention provides a codon optimized PenA composite site mutant coding gene PepA-N11R/N12S-E110R with improved thermal stability; the corresponding nucleotide sequence is shown in the figure SEQ ID NO. 3.
The optimal pH value of the acid protease PepA mutant with improved heat stability is 2.5, and the optimal temperature is 55 ℃.
The difference of the thermal stability of the wild type and the thermal stability of (4) 8 and the thermal stability of the mutant are compared and analyzed through the measurement of the enzymatic properties of the PepA and the mutant thereof, and the thermal stability of the mutated protease is obviously improved compared with that of the wild type;
the mutation method of the mutant of the acid protease PepA with improved thermostability comprises the following steps:
(1) Sequence optimization and cloning of acid protease gene PepA: and (3) performing sequence optimization on the Pichia pastoris expression of the PepA by using DNAwork, directly synthesizing a target sequence, performing PCR amplification by taking the sequence as a template, and connecting the product to a pPIC9K vector to obtain a recombinant vector pPIC9K-PepA.
(2) Site-directed mutagenesis: the vector pPIC9K-PepA is used as a template, and corresponding mutation primers are used for PCR amplification and digestion. Transferring the digested product into DMT competent cells by a heat shock method, carrying out bacterial liquid PCR verification, then carrying out sequencing, determining that the correct mutant extracts plasmids, and then transferring the plasmids into Pichia pastoris GS115 by an electrotransfer method for expression by SalI restriction endonuclease.
The mutation method for improving the site-directed mutation of the acid protease PepA comprises the following steps of:
PepA-N11R/N12S-F
5'-GTTACTACTCCTCAAAGATCTGACGAAGAGTACC-3'
PepA-N11R/N12S-R
5'-AGATCTTTGAGGAGTAGTAACAGCAGAACCTTTAGAAGCTGCC-3'
PepA-N11R/N12S-E110R-F
5'-GTAAGATTTCAAGTAGATTTGTCCAAAACAC-3'
PepA-N11R/N12S-E110R-R
5'-TCTACTTGAAATCTTACTTGCTGCTTCAAC-3'
test example:
1. strain and vector
Coli DH 5. Alpha. Competent cells (Whole gold biosystems), E.coli DMT competent cells (whole gold biosystems), pPIC9K expression plasmid (supplied in this laboratory)
2. Culture medium
Solid medium
YEPD solid medium: yeast powder (1%), peptone (2%), glucose (2%), agar (2.5%);
YEPD-Sorbitol solid medium: yeast powder (1%), peptone (2%), sorbitol (1 mol/L), agar (2.5%);
yepd+g418 solid medium: yeast powder (1%), peptone (2%), glucose (2%), agar (2.5%), G418 (0.8G/L);
a liquid medium;
FA medium: yeast powder (1%), peptone (2%), glycerin (2%), phosphoric acid stock solution, YNB solution were 10% and biotin (0.04 g/L)
FB medium: yeast powder (1%), peptone (2%), methanol (0.5%), phosphoric acid stock solution, YNB solution were 10% and biotin (0.04 g/L)
EXAMPLE 1 construction of acid protease mutant PepA-N11R/N12S, pepA-N11R/N12S-E110R
The sequence of the PepA gene sequence is directly synthesized after the sequence optimization of Pichia pastoris expression by using DNAwork on-line software, the sequence is shown as SEQ ID No.1, nested primers are designed by using restriction enzyme sites EcorI and NotI to amplify a target gene with a joint, and the target gene is connected with a pPIC9K vector after corresponding restriction enzyme digestion after gel recovery and purification to obtain a recombinant vector pPIC9K-PepA. The recombinant vector pPIC9K-PepA is transferred into DH5 alpha competent cells to extract plasmids. According to the Fast Mutagenesis System kit instruction, PCR was performed with the recombinant vector pPIC9K-PepA as a template and the mutation primer overlapped. 1ul DMT enzyme is added for digestion for 1h, and the product is transferred into DMT competent cells by a heat shock method and plated. The next day, single colonies were picked for positive verification, plasmids of the mutants were extracted, and transferred into GS115 pichia pastoris using the electrotransfer method.
Nested primers:
pPIC9K-PepA-F:5'-GAGGCTGAAGCTTACGTAGAATTCATGGCTCCAGCCCCAAC--3'
pPIC9K-PepA-R:5'-CTAAGGCGAATTAATTCGCGGCCGCAGCTTGAGCGGCGAATCC-3'
overlapping mutation primer:
PepA-N11R/N12S-F
5'-GTTACTACTCCTCAAAGATCTGACGAAGAGTACC-3'
PepA-N11R/N12S-R
5
'-AGATCTTTGAGGAGTAGTAACAGCAGAACCTTTAGAAGCTGCC-3'
PepA-N11R/N12S-E110R-F
5'-GTAAGATTTCAAGTAGATTTGTCCAAAACAC-3'
PepA-N11R/N12S-E110R-R
5'-TCTACTTGAAATCTTACTTGCTGCTTCAAC-3'
target gene amplification system: 38.75ul of double distilled water, 5ul of buffer, 2.5ul of dNTPMmix, 1.25ul of each of the upstream and downstream nested primers, 2.5ul of template and 0.625 of rTaq.
Target gene PCR amplification conditions:
94 ℃ 5min,94 ℃ 30s,62 ℃ 30s,72 ℃ 1min,72 ℃ 10min,4 ℃ 30min pPICC 9K carrier enzyme digestion system and conditions: ecori:2ul, notI:2ul, pPICC 9K plasmid: 20ul,10 XBufferH: 6ul, BSA:6ul, double distilled water: 24ul. And (3) enzyme cutting at 30 ℃ for 3.5 hours, and preserving at-20 ℃ for standby after gel recovery.
PepA and pPIC9K connection system and conditions: the target fragment pepA is 2ul, plasmid after pPICC 9K digestion: 2ul, recombinase 1ul,CE Buffer:2ul, double distilled water: 3ul. Incubation is carried out for 30min at 37 ℃, heat shock is transferred into DH5 alpha competent cells, and positive verification is carried out, and the plasmid is preserved at-20 ℃ for standby.
Mutant amplification system:
and (3) adding a system: 2X Transstart FastPfu Fly PCR SuperMix:25ul, double distilled water: 22ul, pPICC 9K-PepA plasmid: 1ul, and 1ul of each of the upstream and downstream primers of the overlap mutation.
Mutant amplification PCR procedure:
94℃:5min,94℃:30s,62℃:20s,72℃:6min,72℃:10min。
after amplification, 1ul DMT enzyme was added, mixed well, incubated at 37℃for 1h for digestion, heat shock transferred into DMT competent cells, plated, positive verified and sequenced. The bacterial sample extracting plasmid with correct mutation after sequencing is subjected to enzyme digestion by a salI restriction enzyme digestion system: plasmid: 100ul, salI endonuclease: 6ul, bufferH:10ul. Water bath at 37 deg.c for 3.5 hr, adding sodium acetate 40ul, and absolute alcohol 300ul, freezing overnight, and transferring into yeast.
Example 2 inducible expression of acid protease
Transferring mutant plasmid into GS115 yeast, coating on YEPD plate containing sorbitol, adding 2ml YEPD liquid culture medium after two days, mixing, scraping to remove thallus, gradient diluting, and collecting 10 -3 ,10 -4 The diluted bacterial solution is coated on a YEPD solid medium added with G418 antibiotics. After single colony grows out, the single colony is inoculated into a YEPD liquid culture medium overnight, the single colony is transferred into an FA liquid culture medium for enriching thalli on the next day, and then transferred into a 0.5% (v/v) FB culture medium for induction expression on the next day, methanol is supplemented once every 24 hours, and the medium is cultured at the constant temperature of 30 ℃ for 120 hours.
Example 3 pH and temperature-dependent Properties of acid protease PepA and its mutants the pH and temperature-dependent Properties of acid protease PepA and its mutants were determined by reference to the national standard method.
1. Acid protease PepA and mutant thereof with optimal pH and pH stability
As shown in FIG. 3 and FIG. 4, the optimum average pH of the acidic protease PEPA and its mutant is 2.5, and there is no significant change before and after the mutation of pH stability.
2 acid protease PepA and optimum temperature and thermal stability of mutant thereof
As can be seen from fig. 5 and 6: the optimal temperature of the wild type and the mutant is 55 ℃, after the wild type is mutated by double-site N11R/N12S, the thermal stability of the acid protease mutant N11R/N12R is improved, and after the acid protease mutant N11R/N12R is subjected to heat treatment under the same condition (45 ℃) for 30min, the activity of the acid protease mutant N11R/N12R is improved by about 50% compared with that of the wild type acid protease; the second composite mutation N11R/N12R-E110R is carried out on the basis of the N11R/N12S mutation, and the heat stability of the modified PEPA is improved by 11.01 percent compared with that of the acid protease mutant N11R/N12R after being treated for 30min under the same condition, and is improved by 65 percent compared with that of the wild PEPA. In conclusion, the mutation has obvious effect of modifying the thermal stability of PEPA.
Wherein, the nucleotide sequence of the sequence PepA after the optimization of the wild acid protease is shown as SEQ ID No. 1; the amino acid sequence of the acid protease PepA is shown as SEQ ID No. 4.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any changes or substitutions that do not undergo the inventive effort should be construed as falling within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.
Sequence listing
<110> university of Yunnan teachers and students
<120> an acid protease mutant, and preparation method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
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actgttccac agtctgttaa agaggcagct tctaaaggtt ctgctgttac tactcctcaa 180
aacaatgacg aagagtacct tactccagta actgttggaa agtctacatt gcatcttgat 240
ttcgacactg gctctgctga tttgtgggtt ttttcagacg aattgccaag tagtgaacaa 300
actggtcatg atctgtacac tccatcttca tctgctacta agttgtctgg ttacacttgg 360
gatatttctt acggagatgg atctagtgct tcaggtgatg tttacagaga tactgtaacc 420
gtcggcggtg ttacgactaa taaacaagct gttgaagcag caagtaagat ttcaagtgaa 480
tttgtccaaa acactgctaa cgatggtcta ctaggtttgg ctttttcctc tatcaacact 540
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ggattttcta ctgatggtta ctctattggt gatggaagtt cttccagttc aggtttctca 780
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tacgaacaag tttctggtgc tcaagaatct gaagaggctg gtggttacgt gttttcttgt 900
tctactaacc caccagactt cactgtggtt ataggtgatt acaaggcagt tgttccaggt 960
aagtacatta actacgctcc aatttcaact ggatcttcca catgtttcgg aggtattcaa 1020
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atggctccag ccccaactag aaagggtttc acgattaacc aaattgcaag accagctaac 60
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gatatttctt acggagatgg atctagtgct tcaggtgatg tttacagaga tactgtaacc 420
gtcggcggtg ttacgactaa taaacaagct gttgaagcag caagtaagat ttcaagtgaa 480
tttgtccaaa acactgctaa cgatggtcta ctaggtttgg ctttttcctc tatcaacact 540
gttcaaccta aggctcagac tactttcttt gacaccgtta agtctcaatt ggattctccc 600
ttgtttgctg tgcaattaaa gcatgatgct ccaggagttt acgatttcgg ctacatcgac 660
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tctactaacc caccagactt cactgtggtt ataggtgatt acaaggcagt tgttccaggt 960
aagtacatta actacgctcc aatttcaact ggatcttcca catgtttcgg aggtattcaa 1020
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gtttttaact ctgaaggacc aaagttggga ttcgccgctc aagct 1125
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actgttccac agtctgttaa agaggcagct tctaaaggtt ctgctgttac tactcctcaa 180
agatctgacg aagagtacct tactccagta actgttggaa agtctacatt gcatcttgat 240
ttcgacactg gctctgctga tttgtgggtt ttttcagacg aattgccaag tagtgaacaa 300
actggtcatg atctgtacac tccatcttca tctgctacta agttgtctgg ttacacttgg 360
gatatttctt acggagatgg atctagtgct tcaggtgatg tttacagaga tactgtaacc 420
gtcggcggtg ttacgactaa taaacaagct gttgaagcag caagtaagat ttcaagtaga 480
tttgtccaaa acactgctaa cgatggtcta ctaggtttgg ctttttcctc tatcaacact 540
gttcaaccta aggctcagac tactttcttt gacaccgtta agtctcaatt ggattctccc 600
ttgtttgctg tgcaattaaa gcatgatgct ccaggagttt acgatttcgg ctacatcgac 660
gattctaaat acactggttc tatcacatat acggatgcag attcatctca aggatattgg 720
ggattttcta ctgatggtta ctctattggt gatggaagtt cttccagttc aggtttctca 780
gccattgctg atactggtac gactttgatt ttgttggatg acgagatcgt atctgcttac 840
tacgaacaag tttctggtgc tcaagaatct gaagaggctg gtggttacgt gttttcttgt 900
tctactaacc caccagactt cactgtggtt ataggtgatt acaaggcagt tgttccaggt 960
aagtacatta actacgctcc aatttcaact ggatcttcca catgtttcgg aggtattcaa 1020
agtaactccg gattgggact ttctatcttg ggtgacgttt ttttgaagtc acagtacgtt 1080
gtttttaact ctgaaggacc aaagttggga ttcgccgctc aagct 1125
<210> 4
<211> 375
<212> PRT
<213> Aspergillus niger (Aspergillus niger)
<400> 4
Met Ala Pro Ala Pro Thr Arg Lys Gly Phe Thr Ile Asn Gln Ile Ala
1 5 10 15
Arg Pro Ala Asn Lys Thr Arg Thr Ile Asn Leu Pro Gly Met Tyr Ala
20 25 30
Arg Ser Leu Ala Lys Phe Gly Gly Thr Val Pro Gln Ser Val Lys Glu
35 40 45
Ala Ala Ser Lys Gly Ser Ala Val Thr Thr Pro Gln Asn Asn Asp Glu
50 55 60
Glu Tyr Leu Thr Pro Val Thr Val Gly Lys Ser Thr Leu His Leu Asp
65 70 75 80
Phe Asp Thr Gly Ser Ala Asp Leu Trp Val Phe Ser Asp Glu Leu Pro
85 90 95
Ser Ser Glu Gln Thr Gly His Asp Leu Tyr Thr Pro Ser Ser Ser Ala
100 105 110
Thr Lys Leu Ser Gly Tyr Thr Trp Asp Ile Ser Tyr Gly Asp Gly Ser
115 120 125
Ser Ala Ser Gly Asp Val Tyr Arg Asp Thr Val Thr Val Gly Gly Val
130 135 140
Thr Thr Asn Lys Gln Ala Val Glu Ala Ala Ser Lys Ile Ser Ser Glu
145 150 155 160
Phe Val Gln Asn Thr Ala Asn Asp Gly Leu Leu Gly Leu Ala Phe Ser
165 170 175
Ser Ile Asn Thr Val Gln Pro Lys Ala Gln Thr Thr Phe Phe Asp Thr
180 185 190
Val Lys Ser Gln Leu Asp Ser Pro Leu Phe Ala Val Gln Leu Lys His
195 200 205
Asp Ala Pro Gly Val Tyr Asp Phe Gly Tyr Ile Asp Asp Ser Lys Tyr
210 215 220
Thr Gly Ser Ile Thr Tyr Thr Asp Ala Asp Ser Ser Gln Gly Tyr Trp
225 230 235 240
Gly Phe Ser Thr Asp Gly Tyr Ser Ile Gly Asp Gly Ser Ser Ser Ser
245 250 255
Ser Gly Phe Ser Ala Ile Ala Asp Thr Gly Thr Thr Leu Ile Leu Leu
260 265 270
Asp Asp Glu Ile Val Ser Ala Tyr Tyr Glu Gln Val Ser Gly Ala Gln
275 280 285
Glu Ser Glu Glu Ala Gly Gly Tyr Val Phe Ser Cys Ser Thr Asn Pro
290 295 300
Pro Asp Phe Thr Val Val Ile Gly Asp Tyr Lys Ala Val Val Pro Gly
305 310 315 320
Lys Tyr Ile Asn Tyr Ala Pro Ile Ser Thr Gly Ser Ser Thr Cys Phe
325 330 335
Gly Gly Ile Gln Ser Asn Ser Gly Leu Gly Leu Ser Ile Leu Gly Asp
340 345 350
Val Phe Leu Lys Ser Gln Tyr Val Val Phe Asn Ser Glu Gly Pro Lys
355 360 365
Leu Gly Phe Ala Ala Gln Ala
370 375
Claims (3)
1. The acidic protease mutant with improved thermal stability is modified by site-directed mutagenesis, and is characterized by being a double-site mutant PepA-N11R/N12S of acidic protease PepA, and the nucleotide sequence of the acidic protease mutant is shown as SEQ ID NO. 2;
2. the acidic protease mutant with improved thermal stability is modified by site-directed mutagenesis, and is characterized by being a composite site mutant PepA-N11R/N12S-E110R of acidic protease PepA, and the nucleotide sequence of the acidic protease mutant is shown as SEQ ID NO. 3.
3. Use of the mutant according to claim 1 or 2 in the leather, feed or food sector.
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| CN114908109B (en) * | 2022-06-14 | 2023-11-10 | 中农华威生物制药(湖北)有限公司 | Construction of acid protease expression strain suitable for feed and batch fermentation process |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3132936A1 (en) * | 1981-08-20 | 1983-03-03 | Henkel KGaA, 4000 Düsseldorf | "METHOD FOR PRODUCING ACID PROTEASE AND THESE FORMING MUTANTS OF THE RHIZOPUS GENUS" |
| WO1996016177A1 (en) * | 1994-11-24 | 1996-05-30 | Novo Nordisk A/S | A process for producing polypeptides with reduced allergenicity |
| WO1998000528A1 (en) * | 1996-06-28 | 1998-01-08 | Novo Nordisk A/S | A recombinant enzyme with mutanase activity |
| WO2005070407A1 (en) * | 2004-01-21 | 2005-08-04 | Elan Pharmaceuticals, Inc. | Methods of treatment of amyloidosis using aspartyl-protease inihibitors |
| WO2018113429A1 (en) * | 2016-12-20 | 2018-06-28 | 广州格拉姆生物科技有限公司 | Probiotic recombinant saccharomyces cerevisiae for assisting protein degradation and antimicrobial peptide secretion |
| CN108893458A (en) * | 2018-07-19 | 2018-11-27 | 中国农业科学院饲料研究所 | Acid protease Bs2688 and its gene and application |
| CN109161489A (en) * | 2018-07-18 | 2019-01-08 | 青岛蔚蓝生物集团有限公司 | A kind of Aspergillus niger strain of high yield acid protease |
| CN109371004A (en) * | 2018-12-11 | 2019-02-22 | 中国农业科学院饲料研究所 | The acid protease Bs2688 mutant K203E and its gene and application that thermal stability improves |
| CN109810967A (en) * | 2018-11-08 | 2019-05-28 | 中国农业科学院饲料研究所 | The acid protease Bs2688 mutant Y282L and its gene and application that thermal stability improves |
| CN112680371A (en) * | 2019-10-18 | 2021-04-20 | 广东利世康低碳科技有限公司 | Candida utilis double-gene co-expression strain for hydrolyzing protein components in kitchen waste and construction method thereof |
| CN113151329A (en) * | 2021-03-30 | 2021-07-23 | 云南师范大学 | Neutral protease mutant and construction method and application thereof |
| CN113403209A (en) * | 2021-07-30 | 2021-09-17 | 西南大学 | Application of aspartic protease gene in improving beauveria bassiana variety |
| CN113632878A (en) * | 2021-08-09 | 2021-11-12 | 云南师范大学 | Enzyme-bacterium composite biological agent for degrading mycotoxin in fermented unconventional feed and application |
| CN113647510A (en) * | 2021-08-09 | 2021-11-16 | 云南师范大学 | Enzyme-bacterium composite additive capable of degrading mycotoxin in fermented feed and application of enzyme-bacterium composite additive |
-
2021
- 2021-03-30 CN CN202110342505.6A patent/CN113151330B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3132936A1 (en) * | 1981-08-20 | 1983-03-03 | Henkel KGaA, 4000 Düsseldorf | "METHOD FOR PRODUCING ACID PROTEASE AND THESE FORMING MUTANTS OF THE RHIZOPUS GENUS" |
| WO1996016177A1 (en) * | 1994-11-24 | 1996-05-30 | Novo Nordisk A/S | A process for producing polypeptides with reduced allergenicity |
| WO1998000528A1 (en) * | 1996-06-28 | 1998-01-08 | Novo Nordisk A/S | A recombinant enzyme with mutanase activity |
| WO2005070407A1 (en) * | 2004-01-21 | 2005-08-04 | Elan Pharmaceuticals, Inc. | Methods of treatment of amyloidosis using aspartyl-protease inihibitors |
| WO2018113429A1 (en) * | 2016-12-20 | 2018-06-28 | 广州格拉姆生物科技有限公司 | Probiotic recombinant saccharomyces cerevisiae for assisting protein degradation and antimicrobial peptide secretion |
| CN109161489A (en) * | 2018-07-18 | 2019-01-08 | 青岛蔚蓝生物集团有限公司 | A kind of Aspergillus niger strain of high yield acid protease |
| CN108893458A (en) * | 2018-07-19 | 2018-11-27 | 中国农业科学院饲料研究所 | Acid protease Bs2688 and its gene and application |
| CN109810967A (en) * | 2018-11-08 | 2019-05-28 | 中国农业科学院饲料研究所 | The acid protease Bs2688 mutant Y282L and its gene and application that thermal stability improves |
| CN109371004A (en) * | 2018-12-11 | 2019-02-22 | 中国农业科学院饲料研究所 | The acid protease Bs2688 mutant K203E and its gene and application that thermal stability improves |
| CN112680371A (en) * | 2019-10-18 | 2021-04-20 | 广东利世康低碳科技有限公司 | Candida utilis double-gene co-expression strain for hydrolyzing protein components in kitchen waste and construction method thereof |
| CN113151329A (en) * | 2021-03-30 | 2021-07-23 | 云南师范大学 | Neutral protease mutant and construction method and application thereof |
| CN113403209A (en) * | 2021-07-30 | 2021-09-17 | 西南大学 | Application of aspartic protease gene in improving beauveria bassiana variety |
| CN113632878A (en) * | 2021-08-09 | 2021-11-12 | 云南师范大学 | Enzyme-bacterium composite biological agent for degrading mycotoxin in fermented unconventional feed and application |
| CN113647510A (en) * | 2021-08-09 | 2021-11-16 | 云南师范大学 | Enzyme-bacterium composite additive capable of degrading mycotoxin in fermented feed and application of enzyme-bacterium composite additive |
Non-Patent Citations (1)
| Title |
|---|
| 司晓光等.米曲霉3.042突变株高产 酸性蛋白酶的发酵条件优化.食品科技.2014,第39卷(第9期),第12-17页. * |
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