CN108384771B - Alkaline protease mutant for improving specific activity and coding gene thereof - Google Patents
Alkaline protease mutant for improving specific activity and coding gene thereof Download PDFInfo
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- CN108384771B CN108384771B CN201810119791.8A CN201810119791A CN108384771B CN 108384771 B CN108384771 B CN 108384771B CN 201810119791 A CN201810119791 A CN 201810119791A CN 108384771 B CN108384771 B CN 108384771B
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- 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
Abstract
The invention relates to the field of genetic engineering, in particular to an alkaline protease BmP mutant for improving specific activity and a coding gene thereof. The mutation site of the alkaline protease BmP mutant is that the 178 th site of the alkaline protease BmP shown by the amino acid SEQ ID NO.7 is changed from S to D or M or G or Q or T. The mutant obtained by the invention has higher specific activity than the original enzyme.
Description
Technical Field
The invention relates to the field of protein molecule modification, in particular to an alkaline protease BmP mutant for improving specific activity and a coding gene and application thereof.
Background
Proteases are enzymes that catalyze the hydrolysis of proteins and can be classified into acid proteases, neutral proteases and alkaline proteases according to their optimal reaction pH environment. Alkaline protease is an enzyme for hydrolyzing protein under alkaline conditions, and the alkaline protease has strong hydrolysis capability and is widely applied to the industrial fields of food, washing, feed and the like. Currently, alkaline proteases are mainly derived from bacillus licheniformis, bacillus subtilis and bacillus circulans, and other bacillus alkaline proteases are relatively rarely reported.
In the previous experiments, alkaline protease BmP from Bacillus mojavensis is cloned, expressed and analyzed in the laboratory, and the BmP has strong capability of decomposing protein raw materials, and is suitable for the industrial fields of food, washing, feed and the like. However, the BmP has relatively low specific activity and high production cost, and the industrial application of the BmP is limited. Therefore, the specific activity of the BmP is improved, and the problem to be solved is urgently needed in the industrial application of the BmP.
Disclosure of Invention
The invention carries out protein molecule modification on the BmP which is derived from Bacillus mojavensis alkaline protease, thereby improving the specific activity of the BmP, reducing the production cost and laying a foundation for the industrial application of the BmP.
The nucleotide sequence and the amino acid sequence of the alkaline protease BmP of the bacillus mojavensis are respectively shown as SEQ ID No.1 and SEQ ID No. 7.
The invention aims to provide a mutant of alkaline protease BmP with improved specific activity. The mutant is a mutant of protease BmP with an amino acid sequence shown as SEQ ID NO.7, wherein the mutation site is 178 th site.
According to the invention, a three-dimensional structure diagram of the alkaline protease BmP is obtained through homologous modeling, and the applicant finds that the 178 th site has a large potential contribution to the hydrolytic activity of the alkaline protease BmP through experiments, so that the 178 th site is selected as a site of site-directed saturation mutation target. The 178 th site of the alkaline protease BmP shown in SEQ ID NO.7 is mutated by adopting a site-specific saturation mutagenesis method, and 5 mutants are obtained by screening, so that the specific activity of the alkaline protease BmP can be effectively improved. These 5 mutants were designated B1, B2, B3, B4 and B5, respectively. The specific activities of B1, B2, B3, B4 and B5 were increased by 35%, 21%, 28%, 19% and 25%, respectively, relative to the original alkaline protease BmP. The nucleotide sequences of mutants B1, B2, B3, B4 and B5 are shown as SEQ ID NO.2 to SEQ ID NO.6, and the amino acid sequences are shown as SEQ ID NO.8 to SEQ ID NO. 12. Wherein B1 contains a mutation site of S178D; b2 contains a mutation site of S178M; b3 contains a mutation site of S178G; b4 contains a mutation site of S178Q; b5 contains a mutation site of S178T.
According to the reported gene sequence of the alkaline protease of the bacillus mojavensis (Genebank: AY665611.1), a target gene is directly synthesized, and the alkaline protease BmP gene of the bacillus mojavensis is amplified. Purifying and recovering the amplified PCR product, and connecting the PCR product to an expression vector phyP43L, obtaining an expression vector phyP43L-BmP; obtaining the mutant of the alkaline protease BmP with improved specific activity through the 178 th position of the saturation mutation alkaline protease BmP.
The site-specific saturation mutagenesis specifically comprises the following steps: to construct a good phyP43Performing PCR amplification by using the corresponding mutation primer by using L-BmP as a template; and (4) carrying out agarose electrophoresis on the amplified PCR product, and purifying and recovering the PCR product. Decomposing original plasmid with restriction endonuclease DpnI, transferring the decomposed product into Escherichia coli Top10 by heat shock method, verifying recombinant transformant by PCR of bacterial liquid, extracting plasmid of verified transformant, and sequencing to obtain the final productThe corresponding mutants were identified. The correctly sequenced mutants were transformed into Bacillus subtilis WB600 by electrotransformation.
It is still another object of the present invention to provide a gene encoding the BmP mutant which has improved specific activity. The nucleotide sequence of the gene is shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.
The invention also claims a recombinant vector containing the gene coding the BmP mutant of the alkaline protease with improved specific activity.
The invention also claims a host cell containing the gene coding for the BmP mutant of alkaline protease with improved specific activity.
The invention also claims application of the alkaline protease BmP mutant for improving the specific activity.
The invention improves the specific activity of the Bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP through the protein rational design, reduces the production cost and lays a foundation for the industrial application of the Bacillus mojavensis alkaline protease BmP.
Drawings
FIG. 1 optimal reaction pH for original alkaline protease BmP and mutants B1 to B5.
FIG. 2 pH stability of original alkaline protease BmP and mutants B1 to B5.
FIG. 3 optimal reaction temperatures for original alkaline protease BmP and mutants B1 to B5.
FIG. 4 thermostability of original alkaline protease BmP and mutants B1 to B5.
Detailed Description
The molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available. Experimental materials and reagents:
1. bacterial strains and vectors
Escherichia coli strain Topl0, Bacillus subtilis WB600 and expression vector phyP43All of L were deposited in this laboratory。
2. Enzyme and kit
Q5 high fidelity Taq enzyme MIX was purchased from NEB company, plasmid extraction, gel purification, restriction enzyme, kit was purchased from Shanghai Biotech company.
3. Culture medium
The E.coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). The Bacillus subtilis culture medium is LBK LB culture medium plus kanamycin.
Example 1 cloning of Bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP Gene
The target gene was directly synthesized based on the reported alkaline protease gene sequence of Bacillus mojavensis (Genebank: AY 665611.1). Two primers (R: 5'-ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3' and F: 5'-TCTAGCGGCCGC TTATTGAGCGGCAGCTTCGAC-3') were designed based on the synthesized target gene for amplification of the BmP gene of Bacillus mojavensis alkaline protease. Purifying and recovering the amplified PCR product, and connecting the PCR product to an expression vector phyP43L, obtaining an expression vector phyP43L-BmP。
Example 2 rational design of site-directed saturation mutagenesis
Modeling is carried out on the alkaline protease BmP through homologous modeling software, and a three-dimensional spatial configuration diagram of the BmP is obtained. And carrying out butt joint analysis on the constructed BmP model through bioinformatics software to find the 178 th site of the key amino acid. The effect of position 178 on the specific activity of the alkaline protease BmP was investigated by saturation mutagenesis.
The process of site-directed saturation mutagenesis is as follows: to construct a good phyP43Performing PCR amplification by using the corresponding mutation primer by using L-BmP as a template; and (4) carrying out agarose electrophoresis on the amplified PCR product, and purifying and recovering the PCR product. Decomposing the original plasmid by using restriction endonuclease DpnI, transferring the decomposed product into escherichia coli Top10 by using a heat shock method, verifying a recombinant transformant by using a bacterial liquid PCR, extracting a plasmid of the transformant which is verified to be correct, and sequencing to determine a corresponding mutant. The correctly sequenced mutants were transformed into Bacillus subtilis WB600 by electrotransformation.
Recombinant transformants were screened as follows: firstly, inoculating recombinant bacteria growing on a kanamycin resistant plate into an LBK culture medium, and culturing for 24 hours at 37 ℃ and 200 rpm; centrifuging the cultured bacterial liquid, taking the supernatant, and performing enzyme activity determination according to GBT/28715-2012.
Example 3 analysis of the specific Activity of the original alkaline protease BmP and mutants
The alkaline protease BmP and the mutant were purified by nickel column purification. The specific activity of the purified alkaline protease BmP and the mutant is measured, the experimental result is shown in Table 1, the 178 th site is a key amino acid site influencing the hydrolysis activity of the alkaline protease BmP, and when the alkaline protease BmP is mutated into D, M, G, Q and T, the relative activity is respectively improved by 35%, 21%, 28%, 19% and 25%.
TABLE 1 analysis of the specific Activity of the original alkaline protease BmP and the mutants
| Numbering | Relative specific activity (%) |
| Original |
100 |
| S178D | 135 |
| S178M | 121 |
| S178G | 128 |
| S178Q | 119 |
| S178T | 125 |
Example 4 optimal reaction pH and pH stability of original alkaline protease BmP and mutant
The optimum reaction pH of the original alkaline protease BmP and the mutants B1 to B5 were determined by the national standard method, and the optimum reaction pH of the original alkaline protease BmP and the mutants B1 to B5 are shown in FIG. 1. As can be seen from FIG. 1, the optimum pH values of the mutants B1 to B5 were almost the same as that of the original alkaline protease BmP, and were all 10.0.
The original alkaline protease BmP and the mutants B1 to B5 are respectively treated for 2 hours at room temperature under the condition of pH6 to 11, and then the enzyme activity is determined by referring to the national standard method, and the result is shown in figure 2. From FIG. 2, it can be seen that the pH stability of mutants B1 to B5 is consistent with that of the original alkaline protease BmP.
Example 6 optimum reaction temperature and thermal stability of alkaline protease BmP and mutant
The optimum reaction temperatures of the original alkaline protease BmP and the mutants B1 to B5 were determined by the national standard method, and the results are shown in FIG. 3. As can be seen from FIG. 3, the optimum reaction temperature for the alkaline protease BmP was 65 ℃ and the optimum reaction temperature for the mutants B1 to B5 was 60 ℃.
The original alkaline protease BmP and the mutants B1 to B5 are respectively treated in water bath at 70 ℃ for 5 minutes, and then the residual enzyme activity is determined according to the national standard. As can be seen from FIG. 4, the thermostability of mutants B1 to B5 was reduced relative to that of alkaline protease BmP (retention rate of 40%), and the retention rates of the remaining enzyme activities were 31%, 32%, 36%, 33% and 39%, respectively.
Sequence listing
<110> Zhuhai City double ring investment Limited
<120> alkaline protease mutant for improving specific activity and coding gene thereof
<160>12
<170>SIPOSequenceListing 1.0
<210>1
<211>1053
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<213>Bacillus mojavensis
<400>1
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaacg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210>2
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accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg agattacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaacg gaacgtcaat ggcttctcct 900
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ggtctgatca atgtcgaagc tgccgctcaa taa 1053
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<213>Bacillus mojavensis
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gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
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cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaacg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210>4
<211>1053
<212>DNA
<213>Bacillus mojavensis
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gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg aggttacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
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<210>5
<211>1053
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<213>Bacillus mojavensis
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agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg acaatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaacg gaacgtcaat ggcttctcct 900
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<210>6
<211>1053
<212>DNA
<213>Bacillus mojavensis
<400>6
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg aacctacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaacg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210>7
<211>350
<212>PRT
<213>Bacillus mojavensis
<400>7
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210>8
<211>350
<212>PRT
<213>Bacillus mojavensis
<400>8
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Asp Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210>9
<211>350
<212>PRT
<213>Bacillus mojavensis
<400>9
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Met Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210>10
<211>350
<212>PRT
<213>Bacillus mojavensis
<400>10
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Gly Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210>11
<211>350
<212>PRT
<213>Bacillus mojavensis
<400>11
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Gln Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210>12
<211>350
<212>PRT
<213>Bacillus mojavensis
<400>12
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr GlyVal Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Thr Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu SerAla Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
Claims (4)
1. An alkaline protease BmP mutant for improving specific activity, which is characterized in that the mutant is a mutant of protease BmP with an amino acid sequence shown as SEQ ID NO.7, wherein the mutation site is that the 178 th site is changed from S to D or M or G or Q or T, and the amino acid sequence is shown as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO. 12.
2. A gene for coding the alkaline protease BmP mutant with the improved specific activity of claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.
3. A recombinant vector comprising the gene of claim 2.
4. A host cell comprising the gene of claim 2.
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| CN112725316B (en) * | 2021-03-04 | 2022-09-06 | 宁夏夏盛实业集团有限公司 | Alkallikrein 2018 mutant and preparation method thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| CN1539966A (en) * | 2003-04-10 | 2004-10-27 | 花王株式会社 | Alkali protease |
| CN101215534A (en) * | 2007-12-28 | 2008-07-09 | 南京工业大学 | Organic solvent resisting basified protease producing strain, gene and application thereof |
| EP2204450A1 (en) * | 2000-11-22 | 2010-07-07 | Kao Corporation | Alkaline proteases |
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| EP2204450A1 (en) * | 2000-11-22 | 2010-07-07 | Kao Corporation | Alkaline proteases |
| CN1539966A (en) * | 2003-04-10 | 2004-10-27 | 花王株式会社 | Alkali protease |
| CN101215534A (en) * | 2007-12-28 | 2008-07-09 | 南京工业大学 | Organic solvent resisting basified protease producing strain, gene and application thereof |
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| Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence;Junxia Li;《Biotechnol Lett》;20130515;第35卷(第5期);第743-749页 * |
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