CN103436511A - High temperature alkaline protease and preparation method thereof - Google Patents
High temperature alkaline protease and preparation method thereof Download PDFInfo
- Publication number
- CN103436511A CN103436511A CN2013102689334A CN201310268933A CN103436511A CN 103436511 A CN103436511 A CN 103436511A CN 2013102689334 A CN2013102689334 A CN 2013102689334A CN 201310268933 A CN201310268933 A CN 201310268933A CN 103436511 A CN103436511 A CN 103436511A
- Authority
- CN
- China
- Prior art keywords
- fppro1
- temperature alkaline
- proteinase gene
- preparation
- temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 108091005658 Basic proteases Proteins 0.000 title abstract description 13
- 108091005804 Peptidases Proteins 0.000 claims abstract description 22
- 239000013604 expression vector Substances 0.000 claims abstract description 13
- 241001200481 Flammeovirga pacifica Species 0.000 claims abstract description 8
- 239000012139 lysis buffer Substances 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 238000003259 recombinant expression Methods 0.000 claims abstract description 7
- 229920000936 Agarose Polymers 0.000 claims abstract description 5
- 238000000746 purification Methods 0.000 claims abstract description 5
- 239000000725 suspension Substances 0.000 claims abstract description 3
- 108010051873 alkaline protease Proteins 0.000 claims description 26
- 102000035195 Peptidases Human genes 0.000 claims description 21
- 241000588724 Escherichia coli Species 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 238000005336 cracking Methods 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 150000002460 imidazoles Chemical class 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 230000000968 intestinal effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000004365 Protease Substances 0.000 abstract description 2
- 238000005119 centrifugation Methods 0.000 abstract description 2
- 241000672609 Escherichia coli BL21 Species 0.000 abstract 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 34
- 108090000790 Enzymes Proteins 0.000 description 34
- 102000018120 Recombinases Human genes 0.000 description 19
- 108010091086 Recombinases Proteins 0.000 description 19
- 239000000243 solution Substances 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000009413 insulation Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000013016 damping Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 108010076119 Caseins Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
- 235000021240 caseins Nutrition 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 235000019833 protease Nutrition 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 3
- 241000193388 Bacillus thuringiensis Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229940097012 bacillus thuringiensis Drugs 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical class [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 108010046845 tryptones Proteins 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108010088842 Fibrinolysin Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 101000713102 Mus musculus C-C motif chemokine 1 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- 102000005158 Subtilisins Human genes 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000010200 folin Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 101150040063 orf gene Proteins 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000003870 refractory metal Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Images
Abstract
The invention provides a high temperature alkaline protease and a preparation method thereof, and relates to a protease, particularly to a high temperature alkaline protease Fppro1 and a preparation method thereof, and high temperature alkaline protease gene Fppro1. The production strain of the high temperature alkaline protease Fppro1 is Flammeovirga Pacifica H2. During preparation, the high temperature alkaline protease gene Fppro1 is cloned, and is inserted into an expression vector to construct a recombinant expression vector carrying the high temperature alkaline protease gene Fppro1; the recombinant expression vector carrying the high temperature alkaline protease gene Fppro1 is transformed into escherichia coli BL21, and positive clones are selected and cultured in a culture medium; and the fermented escherichia coli BL21 cells are collected through centrifugation, the escherichia coli BL21 cells are resuspended in a lysis buffer, the lysed suspension is centrifuged, the supernatant is collected and is mixed with Ni-NTA Agarose, and purification is performed.
Description
Technical field
The present invention relates to proteolytic enzyme, particularly a kind of high-temperature alkaline proteolytic enzyme and preparation method thereof.
Background technology
Sumizyme MP refers to the enzyme of protein hydrolysate peptide bond in pH value meta-alkalescence scope, many from the bacillus protein enzyme at present, major part belongs to serine protease, and it can generate polypeptide or amino acid by protein hydrolysate molecule peptide chain, has the ability of stronger decomposing protein.
Sumizyme MP is found in Pancreas Sus domestica the earliest, and 1913, at first Rohm was used for washing soaking agent by pancreas islet proteolytic enzyme.Jaag in 1945 etc. have found Sumizyme MP in microorganism, make the research of this enzyme and application be rapidly developed.1963, Novo Nordisk Co.,Ltd (existing Novozymes Company) found efficiently, has been applicable to the Sumizyme MP Alcalase of washing composition.Also more and more for the Sumizyme MP commodity of washing composition at present, such as Sabinase, the Kannase of Novozymes Company, Durazyme etc., the KAP of KAO. Corp. SA, the Properase CT of GENENCOR company, the Savinase4.OT high-temperature alkaline proteolytic enzyme of NOVO company etc.At present, proteolytic enzyme is the class of enzymes of applying at most in industrial enzymes, accounts for 60% of enzyme total amount, and wherein Sumizyme MP has just accounted for 25%.Except washing composition, the industry such as it also is widely used in food, medical treatment, brewages, silk, process hides.Its application prospect in business, and the vital role in scientific research makes it obtain researchist and the long-term concern of company both at home and abroad.
Optimum temperature is the most common in the patent of current research report and application/mandate lower than the low-temperature alkaline protease of 30 ℃ at middle temperature Sumizyme MP and the optimum temperature of 30～40 ℃.And optimum temperature is less in the patent of the research report of the high-temperature alkaline proteolytic enzyme more than 50 ℃ and apply for/mandate.Chinese patent CN103013960A provides a kind of high-temperature alkaline proteolytic enzyme and recombinant expressed engineering bacteria thereof, and its optimum temperature is 60 ℃, and optimum pH is 12.Enzyme, after having added various stablizers and making mixed preparation, is deposited the residual enzyme vigor that still has 95% in 30 days, but the thermostability feature of protoenzyme is not provided under 40 ℃.Chinese patent ZL200620111116.8 provides a kind of refractory metal prolease gene engineering bacterium and preparation method thereof, and its optimum temperature is 65 ℃, and optimum pH is 7.2, does not belong to Sumizyme MP, and the feature of thermostability is not provided.Chinese patent ZL200610069339.2 provides a kind of bacillus thuringiensis screening and cultural method of high-yield thermostable proteinase, wherein said proteolytic enzyme optimum temperature is 55 ℃, the basic non-inactivation of 1h under 55 ℃ of conditions, under 60 ℃ of conditions, 1h still has the vigor more than 60%, and the feature of action pH value is not provided.
High-temperature alkaline proteolytic enzyme at high temperature can be brought into play maximum catalytic efficiency, has thermostability preferably, than middle gentle low-temperature alkaline protease, has using value widely.In actual applications, except the vigor of enzyme under differing temps, condition of different pH, the stability of enzyme is one of key index of evaluate application potentiality, comprises thermostability, to tolerance of organic solvent etc.Therefore, alkalescence, thermostable high-temperature proteolytic enzyme have important development and application values.
Summary of the invention
The present invention aims to provide a kind of high-temperature alkaline proteinase gene Fppro1.
Second purpose of the present invention is to provide a kind of recombinant high temperature Sumizyme MP Fppro1 by described high-temperature alkaline proteinase gene Fppro1 coding.
The 3rd purpose of the present invention is to provide the preparation method of a kind of described recombinant high temperature Sumizyme MP Fppro1.
Described high-temperature alkaline proteolytic enzyme called after Fppro1, it produces bacterial strain is Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2, this bacterial strain has been preserved in Chinese Typical Representative culture collection center on June 13rd, 2012, address: China. Wuhan. Wuhan University, deposit number is: CCTCC NO:M2012229.
The sequence of described high-temperature alkaline proteinase gene Fppro1 is as follows:
1 ATGAAAAATA CAATTTCAAA AATCTTATTG GGTGCTGCTA TTTTTACCCA TATAGGTTTT
61 ACTGCAAACG CACAAGAAGA ACCTCGTAAA GAAGCTCCAA AGAATTGGTT TAACCTTAGC
121 TATGAACAAG ATGGTGTTTA TGGAGTTGGA ACCGAAAGAG CATACGACGA GATTTTAAAA
181 AATAAAAAAT CAAAAAAAGT TGTGGTTGCA GTGATTGATA GCGGAATCGA TATCGATCAT
241 GAAGATCTTA AGGACGTTAT CTGGAAAAAT AAAAAGGAAG TTGCAGGTAA CGGTAAAGAT
301 GATGATAACA ACGGTTATGT TGATGACGTA AACGGTTGGA ACTTTATTGG TGGTGCTGAT
361 GGATCAATGG TGAATGAAGA AAACCTTGAG GTAGCTCGTC TTTATGGAAA ACTTAGTAAG
421 AAGTTTGAAG GAGTAGCTGA AGGTGATGTA GCAAAAGCGG ATAAAAAAGA ATACACTTTG
481 TGGTTAGAAG TGAAAAAAGC TTTCGAAGAA GGCTACACTA AAGCAGAAGA AAACTATACA
541 CGTTACTCTA CATATTTACA CCAATTTCAA AGAGGAAAAG CATTATTTAT GGCTTATTTC
601 GATTTAGAAG ATGAAGGTGA AATTGTTGGT GCGTTAGAAT CATTTGATTC TAGTGATGAA
661 GTACTGATGG GAATGAAGGA GATGACATTA GGTCTTCTTT CACAAGGTGT TACAGATGAT
721 CAGCTTCAAG AAGGTGTTGA CTACTTTGAA GGTCAGGTGA AATCCAATTA TAATTATGAG
781 TTAAATACTC GTGAAATTGT TGGTGATGAT ATCGACAATA AGTCTCAAAG AGATTATGGT
841 AATAATAAAG TAACAGGTCC TGATGCTCTT CATGGTACTC ATGTTGCTGG AATTATTGCT
901 GCCTCAAGAG GAAATGAAAT TGGTATGGAT GGTGTTGCCG ACAATGTAGA AATTATGGTT
961 GTCAGAACTG TACCTAACGG TGATGAACGT GATAAAGATG TTGCAAATTC AATTATTTAT
1021 GCTGTTGATA ATGGTGCACA AATCATTAAT ATGAGCTTTG GTAAACCTTA TTCGCCATAC
1081 AAAGGAACAG TAGATAAAGC TGTTAAGTAT GCTGAATCAA AAGGAGTACT TCTAGTACAT
1141 GCCGCTGGTA ATGACCATAA GAATACAGAT AAAGGAAATA ATTTCCCAAG AGATAAATAT
1201 GATTCTGGCA AAACTGCAAA GAATTGGATA GAGGTTGGAG CATCAACTTG GATGACTGAT
1261 GAGAAACTCC CTGCAACGTT TTCTAACTAT GCTAAGAAAA GTGTAGACTT ATTTGCTCCA
1321 GGTTTTGATA TTTACTCAAC AATTCCAGGT TCAGAATATA CTAATTTAAA TGGTACAAGT
1381 ATGGCATGTC CTGTTGTTGC AGGTTGTGCA GCTGTATTAA TGTCATATTA TCCGAACTTG
1441 TCAGCAGTAC AAGTGAAGAA GATTTTAATG AAAACAGTAA CTCCATTGAA GAGTAAGGAA
1501 GTATTGTTAC CTGGTTATAA TAGTTTAGAA GAAGGTGAAG AGCCTCAAAA AGTTAAATTT
1561 GGATCATTAT CAGTAAGTGG TGGTGTAGTT AACTTATATG AAGCTGTAAA ATACGCTGAA
1621 AAGATCTCAA AATAG
The aminoacid sequence of the high-temperature alkaline proteinase gene Fppro1 of described high-temperature alkaline proteinase gene Fppro1 coding is as follows:
1 MKNTISKILL GAAIFTHIGF TANAQEEPRK EAPKNWFNLS YEQDGVYGVG
51 TERAYDEILK NKKSKKVVVA VIDSGIDIDH EDLKDVIWKN KKEVAGNGKD
101 DDNNGYVDDV NGWNFIGGAD GSMVNEENLE VARLYGKLSK KFEGVAEGDV
151 AKADKKEYTL WLEVKKAFEE GYTKAEENYT RYSTYLHQFQ RGKALFMAYF
201 DLEDEGEIVG ALESFDSSDE VLMGMKEMTL GLLSQGVTDD QLQEGVDYFE
251 GQVKSNYNYE LNTREIVGDD IDNKSQRDYG NNKVTGPDAL HGTHVAGIIA
301 ASRGNEIGMD GVADNVEIMV VRTVPNGDER DKDVANSIIY AVDNGAQIIN
351 MSFGKPYSPY KGTVDKAVKY AESKGVLLVH AAGNDHKNTD KGNNFPRDKY
401 DSGKTAKNWI EVGASTWMTD EKLPATFSNY AKKSVDLFAP GFDIYSTIPG
451 SEYTNLNGTS MACPVVAGCA AVLMSYYPNL SAVQVKKILM KTVTPLKSKE
501 VLLPGYNSLE EGEEPQKVKF GSLSVSGGVV NLYEAVKYAE KISK
The preparation method of described high-temperature alkaline proteinase gene Fppro1 comprises the following steps:
1) clone's high-temperature alkaline proteinase gene Fppro1;
2) high-temperature alkaline proteinase gene Fppro1 is inserted to expression vector, build the recombinant expression vector that carries described high-temperature alkaline proteinase gene Fppro1;
3) recombinant expression vector is transformed in intestinal bacteria (E.coli) BL21;
4) choose the positive colony transformed in rear E.coli BL21 and carry out fermentation culture in substratum;
5) the E.coli BL21 cell after centrifugal collection fermentation, resuspended described E.coli BL21 cell carries out cracking in lysis buffer;
6) suspension after cracking in step 5) is centrifugal, collect supernatant liquor, then mix with Ni-NTA Agarose, carry out purifying according to the purification kit specification sheets, obtain high-temperature alkaline proteinase gene Fppro1.
In step 2) in, described expression vector can be selected from pCold I carrier etc.
In step 4), the optional LB substratum from containing 100 μ g/mL penbritins of described substratum, the condition of described fermentation culture can be: temperature is 37 ° of C, and shaking table is cultured to A
600=0.6 o'clock, then to add isopropylthio-β-D-galactoside (IPTG) to final concentration be 50 μ mol/L, under 16 ° of C conditions, induces 12h.
In step 5), described lysis buffer formula can be: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/LNaH
2pO
4, pH8.0.
In step 6), described centrifugal condition can be 18000 * g, centrifugal 20min, 4 ℃.
The present invention has following outstanding advantages:
1. described high-temperature alkaline Protease F ppro1 has new gene order, there is no similar complete ORF gene order in ncbi database.
Described high-temperature alkaline Protease F ppro1 can be in E.coli stably express, induce the vigor of enzyme in rear supernatant liquor can reach 38600U/mL.The optimum temperature of recombinase Fppro1 is 60 ℃, in 40～70 ℃ of scopes, can keep the vigor more than 60 ℃, keeps the approximately vigor more than 90% in 50～65 ℃ of scopes, has wider operative temperature scope.
3. recombinase Fppro1 has good thermostability: under the condition that does not add any stablizer, at least keep the 24h non-inactivation under 30 ℃; After 40 ℃ of lower 12h, the enzyme activity residue is more than 95%; Under 50 ℃ after insulation 1h vigor rising is slightly arranged on the contrary, after 2h, vigor can keep after 85%, 4h keeping approximately 75%; 60 ℃ of insulation 1h still can keep approximately 65% vigor.
4. Optimun pH is 9, in pH8～10 scopes, can keep the vigor more than 80%; Can keep approximately 65% vigor when pH7, there is wider pH sphere of action.
Due to above-mentioned feature, the Protease F of high-temperature alkaline described in the present invention ppro1 can be widely used in the industry such as washing, food, process hides.
The accompanying drawing explanation
The SDS-PAGE collection of illustrative plates that Fig. 1 is the recombinant expressed purifying of high-temperature alkaline proteinase gene Fppro1.In Fig. 1, M is albumen Marker, swimming lane 1 is the expression (induced) of recombinant vectors pColdI-Fppro1 in E.coli BL21, swimming lane 2 is the target protein Fppro1(～60kDa through Ni-NTA purifying gained), swimming lane 3 is the abduction delivering of recombinant vectors pColdI-Fppro1 in E.coli BL21.
The optimum temperature that Fig. 2 is recombinase Fppro1.In Fig. 2, X-coordinate be temperature (℃), the relative reactivity that ordinate zou is enzyme (%).
The thermostability that Fig. 3 is recombinase Fppro1.In Fig. 3, X-coordinate is soaking time (h), the relative activity that ordinate zou is enzyme (%); Respectively be labeled as: 30 ℃ of ■; ● 40 ℃; ◆ 50 ℃;
The Optimun pH that Fig. 4 is recombinase Fppro1.In Fig. 4, X-coordinate is the pH value, the relative reactivity that ordinate zou is enzyme (%).
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition as molecular cloning laboratory manual (New York:Cold Spring Harbor Laboratory Press, 2001) experiment condition described in, or the condition of advising according to reagent or instrument production firm.
Production bacterial strain for the preparation of described high-temperature alkaline proteinase gene Fppro1 is Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2.This bacterial strain has been preserved in Chinese Typical Representative culture collection center, address: China on June 13rd, 2012. Wuhan. and Wuhan University, deposit number is: CCTCC NO:M2012229.
1. the preparation of high-temperature alkaline Protease F ppro1
1) extraction of Flammeovirga Pacifica H2 genomic dna
Flammeovirga Pacifica H2 bacterial classification with transfering loop picking-80 ℃ preservation, at the LB culture medium flat plate, (a kind of bacteria culture medium, contain 1% Tryptones, 1% sodium-chlor, 0.5% yeast extract, 1.5% agarose) the single bacterium colony of upper line separation.Flat board after line is put 37 ℃ of overnight incubation, and single colony inoculation of picking is (a kind of bacteria culture medium, contain 1% Tryptones, 1% sodium-chlor, 0.5% yeast extract) in the LB nutrient solution pipe of 5mL, and 37 ℃ of wave and culture spend the night.Get the centrifugal 2min of culture 2000r/min of 1.5mL; Add again 500 μ L6mol/L Guanidinium hydrochlorides, mix gently, until cracking limpid (can consider and increase the Guanidinium hydrochloride consumption); Add isopyknic phenol/chloroform/primary isoamyl alcohol, mix, the centrifugal 4～5min of 15000r/min, move into new pipe mutually by upper water; Slowly add the equal-volume Virahol, mix rear placement 5min; The centrifugal 15s of 15000r/min, remove supernatant, and precipitation washes twice with 70% ethanol, dries up; Add 200 μ L TE solution (10mmol/L Tris-HCl, 1mmol/LEDTA), 2 μ L RNase A, 37 ℃ of enzymolysis 1h; Add 120 μ L Virahols, 2 μ L1mol/L MgCl
2, mix gently standing 10min; The centrifugal 5min of 15000r/min, remove supernatant, and precipitation washes twice with 70% ethanol, dries up; Be precipitated and dissolved in 50 μ L TE solution.
2) pcr amplification of high-temperature alkaline proteinase gene Fppro1 and sequential analysis
According to the gene annotation result after the order-checking of Flammeovirga Pacifica H2 strain gene group, design primers F: 5 '-CG
gGATCCaTG AAA AAT ACA ATT TCA-3 ' (being scribed ss BamH I restriction enzyme site) (SEQIDNo.3) and R:5 '-AC
aAGCTTcTA TTT TGA GAT CTT TTC-3 ' (being scribed ss Hind III restriction enzyme site) (SEQIDNo.4).The Flammeovirga Pacifica H2 genomic dna extracted of take is template, with the Fppro1 gene of primers F and R amplification total length.The PCR reaction conditions is: contain 100ng template, 400nmo/L primers F, 400nmo/L primer R, 200 μ mo/L dNTP, 2.5mmo/L Mg in the reaction system of 50 μ L
2+, 5U Primer Star HSTaq enzyme (purchased from TaKaRa company), 5mL10 * PCR reaction buffer; Reaction conditions: 94 ℃ of 5min denaturations; 98 ℃ of 10s, 55 ℃ of 45s, 72 ℃ of 2min circulation 30 circles; 72 ℃ are extended 10min.Carry out enzyme with BamH I and Hind III after gained PCR product purification and cut, and be connected with the pColdI carrier after Hind III enzyme is cut with BamH I with same, obtain the pColdI-Fppro1 expression vector, adopt CaCl
2method is transformed into pColdI-Fppro1 in E.coli DH5 α, and the picking positive colony is checked order.
3) expression of recombinase Fppro1 and purifying
By step 2) the middle correct recombinant expression vector pColdI-Fppro1 Transformed E .coli BL21 of sequence obtained, choose positive colony and shaking and training to A containing 37 ° of C in the LB substratum of 100 μ g/mL penbritins
600=0.6 o'clock, add isopropylthio-β-D-galactoside (IPTG) to final concentration 100 μ mol/L, 16 ° of C are collected into bacterium liquid in the centrifuge tube of 200mL after inducing 12h, 5000 * g centrifugation bacterial cell.By the bacterial cell Eddy diffusion, at the lysis buffer of 20mL, (the lysis buffer formula is: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/L NaH
2pO
4, pH8.0) in, ultrasonication to bacterium liquid becomes translucent, the centrifugal 20min of 18000 * g, supernatant with the Ni-NTA Agarose of lysis buffer balance, mix in advance, 4 ° of C were in conjunction with 1 hour, purge process illustrates and carries out according to purification kit (purchased from Qiagen company).The albumen of purifying is through 12% SDS-PAGE electrophoretic analysis, and its molecular weight is about 60kDa, and purity reaches (result is referring to Fig. 1) more than 95%
2. the embodiment of the basic zymetology property analysis of recombinase Fppro1
1) measuring method of proteinase activity
Take casein as substrate, adopt Forint phenol method to measure.Solution used comprises: forint is used solution (a commercially available folin solution mixes with two parts of water, shakes up), sodium carbonate solution (42.4g/L), trichoroacetic acid(TCA) (65.4g/L), gradient pH value damping fluid, casein solution (10.0g/L).Reaction process is as follows: add 800 μ L casein solution in the little centrifuge tube of 1.5mL, put into the modulated preheating of the water-bath to temperature of reaction 5min, add 200 μ L enzyme liquid, slowly shake up, put back in water-bath and react 10min.Add 270 μ L trichoroacetic acid(TCA)s, mix with termination reaction.
One control tube is set in addition, adds 800 μ L casein solution in control tube, put into the modulated preheating of the water-bath to temperature of reaction 5min, add 200 μ L water, slowly shake up, put back in water-bath and react 10min.Add 270 μ L trichoroacetic acid(TCA)s and, with the enzyme liquid after 100 ℃ of processing 5min, mix.
Experiment tube and control tube, with the centrifugal 2min of 13,000r/min, are got respectively to the 1mL supernatant liquor in the empty test tube of corresponding numbering, add successively more afterwards 5mL sodium carbonate solution and 0.5mL forint to use solution, after mixing in 40 ° of C water-baths colour developing 20min.Measure the OD of each pipe
680value.And the amount of from ready-made Tyr typical curve, reading the Tyr produced in reaction, calculate enzyme and live.
Protease activity unit of force (U) is defined as the per minute caseinhydrolysate and produces the required enzyme amount of 1 μ g tyrosine.
2) the optimum temperature analysis of recombinase Fppro1
Adopt Tris-HCl damping fluid (pH8.0), the recombinase Fppro1 after purified is carried out to enzymatic reaction under differing temps and measure enzyme and live.The highest enzyme work of take is 100%, calculates relative enzyme and lives, and draws temperature-relative enzyme curve (see figure 2) alive.Result shows that the optimum temperature of the recombinase Fppro1 in the present invention is 60 ℃, and has wider operative temperature scope: in 40 ℃～70 ℃ scopes, can keep the vigor more than 60 ℃; Keep the approximately vigor more than 90% in 50 ℃～65 ℃ scopes; Can keep approximately 40% vigor in the time of 30 ℃.
3) thermal stability analysis of recombinase Fppro1
Adopt Tris-HCl damping fluid (pH8.0), the recombinase Fppro1 after purified is incubated to certain hour under differing temps, then carry out enzymatic reaction, measure the vigor of residual protein enzyme, detect and react under optimal reactive temperature.Draw soaking time-relative activity curve (see figure 3).Result shows that the recombinase Fppro1 in the present invention has extraordinary thermostability.Under 30 ℃, after insulation 24h, vigor is not lost; After 40 ℃ of lower 12h, the enzyme activity residue is more than 95%; Under 50 ℃ after insulation 1h vigor rising is slightly arranged on the contrary, after 2h, vigor can keep after 85%, 4h keeping approximately 75%, can keep approximately 50% vigor after 8h; 60 ℃ of insulation 1h enzymes residue approximately 65% of living, the enzyme residue 40% of living after 2h.
Add 0.1mmol/L Ca in Tris-HCl damping fluid (pH8.0)
2+, then detecting the stability (Fig. 3) of recombinase Fppro1 under 60 ℃ by same steps as, result shows Ca
2+significantly improve the thermostability of recombinase Fppro1, after 60 ℃ of insulation 1h, enzyme is lived and is remained 76%, and after insulation 2h, residual enzyme work is 65%, all than there is no Ca
2+the time 65% and 40% height.
In research report so far and the patent of application/mandate, if do not add stablizer, initial proteolytic enzyme is all poor in the thermostability more than 40 ℃, best a kind of be the thermostable proteinase produced by bacillus thuringiensis that Chinese patent ZL200610069339.2 provides, its optimum temperature is 55 ℃, the basic non-inactivation of 1h under 55 ℃ of conditions, under 60 ℃ of conditions, the 1h residual activity is 60%.Therefore the recombinase Fppro1 in the present invention has very outstanding thermostability.
4) the suitableeest action pH analysis of recombinase Fppro1
Purified recombinase Fppro1, in the damping fluid of different pH values, is reacted to measure its optimal pH under 60 ℃, and the highest enzyme work of take is 100%, calculates relative enzyme and lives, and draws the relative enzyme of pH-curve (see figure 4) alive.Result shows that the Optimun pH of the recombinase Fppro1 in the present invention is 9, can keep the vigor more than 80% in pH8～10 scopes; Can keep approximately 65% vigor when pH7, there is wider pH sphere of action.
Claims (9)
1. a high-temperature alkaline proteolytic enzyme, it is characterized in that described high-temperature alkaline proteolytic enzyme called after Fppro1, it produces bacterial strain is Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2, this bacterial strain has been preserved in Chinese Typical Representative culture collection center on June 13rd, 2012, address: China. Wuhan. Wuhan University, deposit number is: CCTCC NO:M2012229.
2. a kind of high-temperature alkaline proteolytic enzyme as claimed in claim 1 is characterized in that the sequence of high-temperature alkaline proteinase gene Fppro1 is as follows:
1 ATGAAAAATA CAATTTCAAA AATCTTATTG GGTGCTGCTA TTTTTACCCA TATAGGTTTT
61 ACTGCAAACG CACAAGAAGA ACCTCGTAAA GAAGCTCCAA AGAATTGGTT TAACCTTAGC
121 TATGAACAAG ATGGTGTTTA TGGAGTTGGA ACCGAAAGAG CATACGACGA GATTTTAAAA
181 AATAAAAAAT CAAAAAAAGT TGTGGTTGCA GTGATTGATA GCGGAATCGA TATCGATCAT
241 GAAGATCTTA AGGACGTTAT CTGGAAAAAT AAAAAGGAAG TTGCAGGTAA CGGTAAAGAT
301 GATGATAACA ACGGTTATGT TGATGACGTA AACGGTTGGA ACTTTATTGG TGGTGCTGAT
361 GGATCAATGG TGAATGAAGA AAACCTTGAG GTAGCTCGTC TTTATGGAAA ACTTAGTAAG
421 AAGTTTGAAG GAGTAGCTGA AGGTGATGTA GCAAAAGCGG ATAAAAAAGA ATACACTTTG
481 TGGTTAGAAG TGAAAAAAGC TTTCGAAGAA GGCTACACTA AAGCAGAAGA AAACTATACA
541 CGTTACTCTA CATATTTACA CCAATTTCAA AGAGGAAAAG CATTATTTAT GGCTTATTTC
601 GATTTAGAAG ATGAAGGTGA AATTGTTGGT GCGTTAGAAT CATTTGATTC TAGTGATGAA
661 GTACTGATGG GAATGAAGGA GATGACATTA GGTCTTCTTT CACAAGGTGT TACAGATGAT
721 CAGCTTCAAG AAGGTGTTGA CTACTTTGAA GGTCAGGTGA AATCCAATTA TAATTATGAG
781 TTAAATACTC GTGAAATTGT TGGTGATGAT ATCGACAATA AGTCTCAAAG AGATTATGGT
841 AATAATAAAG TAACAGGTCC TGATGCTCTT CATGGTACTC ATGTTGCTGG AATTATTGCT
901 GCCTCAAGAG GAAATGAAAT TGGTATGGAT GGTGTTGCCG ACAATGTAGA AATTATGGTT
961 GTCAGAACTG TACCTAACGG TGATGAACGT GATAAAGATG TTGCAAATTC AATTATTTAT
1021 GCTGTTGATA ATGGTGCACA AATCATTAAT ATGAGCTTTG GTAAACCTTA TTCGCCATAC
1081 AAAGGAACAG TAGATAAAGC TGTTAAGTAT GCTGAATCAA AAGGAGTACT TCTAGTACAT
1141 GCCGCTGGTA ATGACCATAA GAATACAGAT AAAGGAAATA ATTTCCCAAG AGATAAATAT
1201 GATTCTGGCA AAACTGCAAA GAATTGGATA GAGGTTGGAG CATCAACTTG GATGACTGAT
1261 GAGAAACTCC CTGCAACGTT TTCTAACTAT GCTAAGAAAA GTGTAGACTT ATTTGCTCCA
1321 GGTTTTGATA TTTACTCAAC AATTCCAGGT TCAGAATATA CTAATTTAAA TGGTACAAGT
1381 ATGGCATGTC CTGTTGTTGC AGGTTGTGCA GCTGTATTAA TGTCATATTA TCCGAACTTG
1441 TCAGCAGTAC AAGTGAAGAA GATTTTAATG AAAACAGTAA CTCCATTGAA GAGTAAGGAA
1501 GTATTGTTAC CTGGTTATAA TAGTTTAGAA GAAGGTGAAG AGCCTCAAAA AGTTAAATTT
1561 GGATCATTAT CAGTAAGTGG TGGTGTAGTT AACTTATATG AAGCTGTAAA ATACGCTGAA
1621 AAGATCTCAA AATAG。
3. a kind of high-temperature alkaline proteolytic enzyme as claimed in claim 1 is characterized in that the aminoacid sequence of high-temperature alkaline proteinase gene Fppro1 of high-temperature alkaline proteinase gene Fppro1 coding is as follows:
1 MKNTISKILL GAAIFTHIGF TANAQEEPRK EAPKNWFNLS YEQDGVYGVG
51 TERAYDEILK NKKSKKVVVA VIDSGIDIDH EDLKDVIWKN KKEVAGNGKD
101 DDNNGYVDDV NGWNFIGGAD GSMVNEENLE VARLYGKLSK KFEGVAEGDV
151 AKADKKEYTL WLEVKKAFEE GYTKAEENYT RYSTYLHQFQ RGKALFMAYF
201 DLEDEGEIVG ALESFDSSDE VLMGMKEMTL GLLSQGVTDD QLQEGVDYFE
251 GQVKSNYNYE LNTREIVGDD IDNKSQRDYG NNKVTGPDAL HGTHVAGIIA
301 ASRGNEIGMD GVADNVEIMV VRTVPNGDER DKDVANSIIY AVDNGAQIIN
351 MSFGKPYSPY KGTVDKAVKY AESKGVLLVH AAGNDHKNTD KGNNFPRDKY
401 DSGKTAKNWI EVGASTWMTD EKLPATFSNY AKKSVDLFAP GFDIYSTIPG
451 SEYTNLNGTS MACPVVAGCA AVLMSYYPNL SAVQVKKILM KTVTPLKSKE
501 VLLPGYNSLE EGEEPQKVKF GSLSVSGGVV NLYEAVKYAE KISK。
4. the preparation method of high-temperature alkaline proteinase gene Fppro1 is characterized in that comprising the following steps:
1) clone's high-temperature alkaline proteinase gene Fppro1;
2) high-temperature alkaline proteinase gene Fppro1 is inserted to expression vector, build the recombinant expression vector that carries described high-temperature alkaline proteinase gene Fppro1;
3) recombinant expression vector is transformed in intestinal bacteria (E.coli) BL21;
4) choose the positive colony transformed in rear E.coli BL21 and carry out fermentation culture in substratum;
5) the E.coli BL21 cell after centrifugal collection fermentation, resuspended described E.coli BL21 cell carries out cracking in lysis buffer;
6) by step 5) in suspension after cracking centrifugal, collect supernatant liquor, then mix with Ni-NTA Agarose, carry out purifying according to the purification kit specification sheets, obtain high-temperature alkaline proteinase gene Fppro1.
5. the preparation method of high-temperature alkaline proteinase gene Fppro1 as claimed in claim 4, is characterized in that in step 2) in, described expression vector is selected from pCold I carrier.
6. the preparation method of high-temperature alkaline proteinase gene Fppro1 as claimed in claim 4, is characterized in that in step 4) in, described substratum is selected from the LB substratum that contains 100 μ g/mL penbritins.
7. the preparation method of high-temperature alkaline proteinase gene Fppro1 as claimed in claim 4, is characterized in that in step 4) in, the condition of described fermentation culture is: temperature is 37 ℃, and shaking table is cultured to A
600=0.6 o'clock, then to add isopropylthio-β-D-galactoside to final concentration be 50 μ mol/L, under 16 ℃ of conditions, induces 12h.
8. the preparation method of high-temperature alkaline proteinase gene Fppro1 as claimed in claim 4, is characterized in that in step 5) in, described lysis buffer formula is: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/L NaH
2pO
4, pH8.0.
9. the preparation method of high-temperature alkaline proteinase gene Fppro1 as claimed in claim 4, is characterized in that in step 6) in, described centrifugal condition is 18000 * g, centrifugal 20min, 4 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310268933.4A CN103436511B (en) | 2013-06-28 | 2013-06-28 | High temperature alkaline protease and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310268933.4A CN103436511B (en) | 2013-06-28 | 2013-06-28 | High temperature alkaline protease and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103436511A true CN103436511A (en) | 2013-12-11 |
| CN103436511B CN103436511B (en) | 2015-03-04 |
Family
ID=49690227
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310268933.4A Active CN103436511B (en) | 2013-06-28 | 2013-06-28 | High temperature alkaline protease and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103436511B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560914A (en) * | 2014-12-29 | 2015-04-29 | 国家海洋局第三海洋研究所 | Alpha-amylase Amy16, as well as gene and application thereof |
| CN108004220A (en) * | 2017-12-25 | 2018-05-08 | 刘丹妮 | Improve alkali protease BmP mutant and its gene and the application of heat endurance |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220361A (en) * | 2006-07-21 | 2008-07-16 | 汕头大学医学院 | Representation method for recombinant american cockroaches allergen protein |
| CN101270347A (en) * | 2008-05-08 | 2008-09-24 | 新疆大学 | High-temperature and strong base resistant clausii bacillus cereus, excretive proteolytic enzyme and preparation |
| CN101466827A (en) * | 2006-06-09 | 2009-06-24 | 宝生物工程株式会社 | Method of producing lymphocytes |
| CN101487002A (en) * | 2009-03-02 | 2009-07-22 | 华南理工大学 | Alkaline protease |
| CN102925388A (en) * | 2012-10-26 | 2013-02-13 | 天津科技大学 | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same |
-
2013
- 2013-06-28 CN CN201310268933.4A patent/CN103436511B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101466827A (en) * | 2006-06-09 | 2009-06-24 | 宝生物工程株式会社 | Method of producing lymphocytes |
| CN101220361A (en) * | 2006-07-21 | 2008-07-16 | 汕头大学医学院 | Representation method for recombinant american cockroaches allergen protein |
| CN101270347A (en) * | 2008-05-08 | 2008-09-24 | 新疆大学 | High-temperature and strong base resistant clausii bacillus cereus, excretive proteolytic enzyme and preparation |
| CN101487002A (en) * | 2009-03-02 | 2009-07-22 | 华南理工大学 | Alkaline protease |
| CN102925388A (en) * | 2012-10-26 | 2013-02-13 | 天津科技大学 | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same |
Non-Patent Citations (2)
| Title |
|---|
| REFSEQ: "Protease precursor [Fulvivirga imtechensis]", 《GENBANK DATABASE》 * |
| 舒丹等: "高温芽孢杆菌碱性蛋白酶发酵条件及酶性质研究", 《四川大学学报(自然科学版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560914A (en) * | 2014-12-29 | 2015-04-29 | 国家海洋局第三海洋研究所 | Alpha-amylase Amy16, as well as gene and application thereof |
| CN108004220A (en) * | 2017-12-25 | 2018-05-08 | 刘丹妮 | Improve alkali protease BmP mutant and its gene and the application of heat endurance |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103436511B (en) | 2015-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102183558B1 (en) | Gene encoding L-alanyl-L-glutamine biosynthetic enzyme and use thereof | |
| CN103756979B (en) | Method for improving thermal stability of enzyme | |
| CN105018402A (en) | Bacillus licheniformis capable of producing alkaline protease in large quantity and application of Bacillus licheniformis | |
| CN112391365A (en) | Starch branching enzyme mutant with improved catalytic activity and application thereof | |
| CN101629169B (en) | Purine nucleoside phosphorylase (PNP) with modified molecules and preparation method thereof | |
| CN114107266B (en) | Protease mutant with improved heat resistance, encoding gene and application thereof | |
| CN103436511B (en) | High temperature alkaline protease and preparation method thereof | |
| CN107267545A (en) | Recombinate the preparation method of Bsa I restriction enzymes | |
| CN102653743B (en) | Thermal stability improved lipase mutant constructed through orthogenesis | |
| CN113444714A (en) | Beta subunit mutant nitrile hydratase mutant and application thereof | |
| CN103352045B (en) | Arylsulfatase and preparation method and applications thereof | |
| CN108330079A (en) | The structure of Pullulanase and recombinant vector and application that a kind of bacillus amyloliquefaciens of production Pullulanase, its fermentation obtain | |
| CN102653742B (en) | High-temperature resistant rhizopuschinensis lipase mutant | |
| CN111944790A (en) | Neutral protease gene, neutral protease, preparation method and application thereof | |
| CN101942427B (en) | Alkyl sulfatase and preparation method thereof | |
| CN113308453B (en) | Amidohydrolase SaAH, and coding gene and application thereof | |
| CN102653741B (en) | High-thermal stability rhizopuschinensis lipase | |
| CN107201354A (en) | A kind of neutral proteinase and its gene and application | |
| CN103740661B (en) | Enzymatic thermal stability improving method | |
| CN103740659B (en) | Enzymatic thermal stability improving method | |
| CN105950595A (en) | (-)-[gamma]-lactamase, gene, mutant, vector as well as preparation method and application of (-)-[gamma]-lactamase | |
| CN101892228B (en) | Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof | |
| CN101613681B (en) | Method for renaturing and purifying recombinant extremely heat-resistant alpha-amylase | |
| CN115896138B (en) | Anaerobic sulfatase mature enzyme gene, anaerobic sulfatase mature enzyme, and preparation methods and application thereof | |
| CN116731989B (en) | Laccase mutant, genetically engineered bacterium and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 361005 Fujian Province University of Siming District of Xiamen City Road No. 178 Patentee after: THIRD INSTITUTE OF OCEANOGRAPHY, MINISTRY OF NATURAL RESOURCES Address before: 361005 Fujian Province University of Siming District of Xiamen City Road No. 178 Patentee before: THIRD INSTITUTE OF OCEANOGRAPHY, STATE OCEANIC ADMINISTRATION |
|
| CP01 | Change in the name or title of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231011 Address after: 361000 room 201B, plant 8, No. 3701, Xiang'an North Road, Xiamen Torch High tech Zone (Xiang'an) Industrial Zone, Xiamen, Fujian Patentee after: LANNAO TECHNOLOGY (XIAMEN) Co.,Ltd. Address before: 361005 Fujian Province University of Siming District of Xiamen City Road No. 178 Patentee before: THIRD INSTITUTE OF OCEANOGRAPHY, MINISTRY OF NATURAL RESOURCES |
|
| TR01 | Transfer of patent right |