CN1834236A - Lipase A variant of wild Bacillus subtilis and application thereof - Google Patents

Lipase A variant of wild Bacillus subtilis and application thereof Download PDF

Info

Publication number
CN1834236A
CN1834236A CN 200610066857 CN200610066857A CN1834236A CN 1834236 A CN1834236 A CN 1834236A CN 200610066857 CN200610066857 CN 200610066857 CN 200610066857 A CN200610066857 A CN 200610066857A CN 1834236 A CN1834236 A CN 1834236A
Authority
CN
China
Prior art keywords
lipase
variant
bacillus subtilis
wild
gene
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
Application number
CN 200610066857
Other languages
Chinese (zh)
Other versions
CN100417723C (en
Inventor
林章凛
蔡真
陈博
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
Original Assignee
Tsinghua University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Tsinghua University filed Critical Tsinghua University
Priority to CNB2006100668579A priority Critical patent/CN100417723C/en
Publication of CN1834236A publication Critical patent/CN1834236A/en
Application granted granted Critical
Publication of CN100417723C publication Critical patent/CN100417723C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

This invention discloses the application of the mutant of wild Bacillus subtilis lipase A in abluents. The mutant is obtained by subjecting the amino acid residue swquence to one or more of the following five mutations: (1) mutation of serine 16 to proline, (2) mutation of lysine 112 to glutamic acid, (3) mutation of arginine 142 to serine, (4) mutation of histidine 152 to glutamine, and (5) mutation of glycine 153 to aspartic acid. The mutant of wild Bacillus subtilis lipase A can be applied in preparing abluents.

Description

Lipase A variant of wild Bacillus subtilis and application thereof
Technical field
The present invention relates to lipase Variant and application thereof, particularly relate to variant and screening method and its application in the preparing washing agent of wild Bacillus subtilis lipase A (BSLA).
Background technology
Lipase (Lipase) is widely regarded as a kind of very useful biological catalyst, not only can be used as detergent additives, food ingredient use, also can be used for numerous areas such as papermaking, production of biodiesel, organic synthesis.Wherein, the lipase A that comes from Bacillus subtillis (Bacillus subtilis 168) has multiple distinguished character (Dartois V., B., A., Schanck, K., Colson, C., Cloning, nucleotide sequenceand expression in Escherichia coli of a lipase gene from Bacillus subtilis168, BIOCHIMICA ET BIOPHYSICA ACTA, 1992,1131:253-260; Jaeger, K., S.Ransac, et al., Bacterial lipases, FEMS MICROBIOLOGY REVIEWS, 1994,15 (1): 29-63; Reetz, M., Lipases as practical biocatalysts, CURRENT OPINION IN CHEMICALBIOLOGY, 2002,6 (2): 145-150), comprising: 1) be one of lipase of minimum known today, its maturation protein (mature protein) only is made up of 181 amino-acid residues, and molecular weight is about 19kDa; 2) do not have lid (lid) structure, do not need the interface to activate (Interfacial activation); 3) fat with medium chain lipid acid is shown high reactivity; 4) optimum pH is 10.More than these characteristics, especially because the characteristics that do not need the interface to activate (interfacial activation) make the hydrolytic activity of this enzyme not rely on appropriate water-oil interface (or suitable water-content), and prevent and the combining of residual fat mols, the present inventor thinks that this lipase is expected to realize washing (first-wash) first in detergent industry very much, and can use it for foodstuffs industry or other industrial biotechnology field.
The orientation plan of enzyme is a kind of means that change the character of enzyme by the gene order that changes the enzyme molecule.Specifically, it is to evolve at the laboratory natural imitation.At first the gene of enzyme is carried out random mutation (technology commonly used has fallibility PCR (Error-prone PCR), DNA shuffle (DNA shuffling) etc.), make up the variant library; Secondly, select suitable carriers and host to express; At last the library is screened (screening) or selected (selection) to obtain the variant that character changes.And then be template with this variant, repeat said process, reach desired character until variant.At present, orthogenesis successfully has been used to change substrate activity, substrate specificity, thermostability, enantio-selectivity of enzyme etc., and obtained significant achievement (Kuchner, O., F.Arnold, Directed evolution of enzyme catalysts, TRENDS IN BIOTECHNOLOGY, 1997,15 (12): 523-530; Petrounia, I., F.Arnold, Designed evolution of enzymatic properties, CURRENT OPINION IN BIOTECHNOLOGY, 2000,11 (4): 325-33; Arnold, F., combinatorial and computational challenges for biocatalyst design, NATURE, 2001,409 (6817): 253-257).
Summary of the invention
The variant that the purpose of this invention is to provide wild Bacillus subtilis (Bacillus subtilis) lipase A with improvement enzymic activity.
Described variant is that the amino acid residue sequence with the wild Bacillus subtilis lipase A carries out the protein that obtains after one or more sudden change combinations in following five kinds of sudden changes: 1) Serine (S) from the 16th of aminoterminal (N end) sports proline(Pro) (P), is designated as S16P; 2) the 112nd Methionin (K) sports L-glutamic acid (E), is designated as K112E; 3) the 142nd arginine (R) sports Serine (S), is designated as R142S; 4) the 152nd Histidine (H) sports glutamine (Q), is designated as H152Q; 5) the 153rd glycine (G) sports aspartic acid (D), is designated as G153D; Described wild Bacillus subtilis lipase A has SEQ ID № in the sequence table: 1 amino acid residue sequence.
Wherein, preferred lipase A variant of wild Bacillus subtilis can have one of following amino acid residue sequences:
1) SEQ ID № in the sequence table: 2;
2) SEQ ID № in the sequence table: 3.
SEQ ID №: 2-3 forms by 181 amino-acid residues in the sequence table.To have SEQ ID № in the sequence table: the variant called after BSLA-1-4 of 2 amino acid residue sequence, this variant have above-mentioned S16P and two kinds of sudden changes of G153D simultaneously; To have SEQ ID № in the sequence table: the variant called after BSLA-2-6 of 3 amino acid residue sequence, this variant have above-mentioned S16P, K112E, R142S, H152Q and five kinds of sudden changes of G153D simultaneously.
The gene of above-mentioned lipase A variant of wild Bacillus subtilis of encoding also belongs to protection scope of the present invention, and wherein, preferred lipase A variant of wild Bacillus subtilis gene can be one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 5;
2) SEQ ID № in the sequence table: 6.
SEQ ID №: 5-6 is by 543 based compositions in the sequence table, and above-mentioned lipase A variant of wild Bacillus subtilis BSLA-1-4 and BSLA-2-6 encode respectively.
Contain above-mentioned lipase A variant of wild Bacillus subtilis expression carrier, transgenic cell and host bacterium and also belong to protection scope of the present invention.
Another object of the present invention provides the method that a kind of screening has the active lipase Variant of high enzyme.
Screening method provided by the present invention may further comprise the steps:
1) the variant library of structure lipase gene;
2) the variant library is connected with carrier, the recombinant vectors that will carry the variant library then imports the host bacterium;
3) with step 2) transformant that obtains is inoculated in and carries out qualitative primary dcreening operation on the primary dcreening operation flat board, filters out the variant with lipase activity; Described primary dcreening operation is dull and stereotyped to add the marker that can filter out foreign vector on cultivating with the basis of solid medium in host bacterium routine, and the substrate of lipase;
4) the picking step 3) has the variant of lipase activity behind primary dcreening operation, it is inoculated in growth cultivates on the flat board, preserves as motherboard after growing single bacterium colony; Described growth is dull and stereotyped adds the marker that can filter out foreign vector on cultivating with the basis of solid medium in host bacterium routine;
5) bacterium colony on the step 4) motherboard is transferred to induce on the flat board induces with changeing embrane method; The described flat board of inducing adds the marker that can filter out foreign vector on cultivating with the basis of solid medium in host bacterium routine, and inductor;
6) cleavage step 5) in through the inductive bacterium colony, make the lipase of its expression be adsorbed on the surface of transfer film, transfer film is placed carry out qualitative multiple sieve on the multiple sieve flat board again, obtain the variant that improves than the wild-type lipase activity; Described multiple sieve is dull and stereotyped for only containing the flat board of the substrate of agarose and lipase;
7) activity of quantitative assay lipase Variant.
In above-mentioned screening method, can select for use any method of can be on a given lipase gene introducing sudden change randomly to make up lipase gene variant library in the described step 1), as fallibility PCR (Error-prone PCR), DNA shuffle (DNA shuffling) etc.
Step 2) carrier and host bacterium used in can be adopted any carrier and host bacterium that is applicable to lipase gene clone and expression in the biology field; Described carrier can be pET30a (+), pBR332, YEplac112, pRB473 etc.; The host bacterium can be e. coli bl21 (DE3), intestinal bacteria 5K, yeast 13bxV4, Bacillus subtillis MB216 etc.
The host bacterium difference of using, being used to prepare the dull and stereotyped selected solid medium of primary dcreening operation in the step 3) also may be different, is applicable to that such as the LB solid medium intestinal bacteria, YPD substratum are applicable to yeast etc.In addition, the marker that must determine to filter out foreign vector according to concrete used carrier and host bacterium, such as for the host bacterium that contains the plasmid of antibiotics resistance on those carriers and do not have this resistance, the host bacterium that can use this antibiosis usually to filter out to contain this plasmid, microbiotic commonly used is kantlex, penbritin, tsiklomitsin etc.For some auxotrophic host bacterium, then can select for use this nutritive substance to screen, for example to the yeast INVSc I of uridylic (Uracil) defective and contain uracil base because of plasmid pYES2, then can filter out the yeast INVSc I that contains plasmid pYES2 with uridylic.The substrate of lipase can be anyly can and easily judge the material whether this reaction takes place by lipase institute's catalysis.For example: colourless palmitinic acid p-nitrophenyl fat (p-nitrophenyl palmitate) is generated the xanchromatic p-NP behind the lipase hydrolysis; The material that sweet oil (Olive oil) is generated behind the lipase hydrolysis can be created on the compound that has fluorescent orange under the ultra violet lamp with orchil rhodamine B (Rhodamine B) reaction; Butyrin in solid medium (Tributyrin) can be formed transparent hydrolysis circle etc. behind the lipase hydrolysis.
Used transfer film can be any one film that can shift bacterium colony and adsorbed proteins, for example nitrocellulose filter, nylon membrane etc. in the step 5); Select suitable inductor according to concrete used carrier and host bacterium, as for the plasmid pET30a (+) that contains the T7 promotor, after this plasmid imports e. coli bl21 (DE3), (Isopropyl-β-D-thigalactopyranoside is IPTG) as inductor can to adopt isopropyl ss-D thiogalactoside.
Can adopt the bacterium colony cleavage method of any routine in the step 6), as contacting bacterium colony with the filter paper that contains lysate (as N,O-Diacetylmuramidase).
Can be in the step 7) with after in the variant liquid medium within that improves than the wild-type lipase activity that qualitative multiple sieve obtains, inducing in the step 6), its activity of quantitative assay.
The method that above-mentioned screening has the active lipase Variant of high enzyme is applicable to any one lipase, is particularly useful for the wild Bacillus subtilis lipase A.
The invention provides lipase A variant of wild Bacillus subtilis.The lipase activity determination result shows that this enzyme of lipase A variant of wild Bacillus subtilis of the present invention and wild-type compares, and the ratio of pNPP, pNPC and sweet oil is lived the highlyest can improve 4.8,3.0 and 2.6 times respectively.In conjunction with the kind specific character of the lipase of mentioning in the background technology, lipase A variant of wild Bacillus subtilis of the present invention has bigger advantage aspect the industrial preparation of washing composition.In addition, the screening method of lipase Variant of the present invention has simple to operate, and the advantage that the screening cycle is short easily realizes high flux screening.Based on above-mentioned advantage, the present invention has higher actual application value and vast market prospect.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is the physical map of recombinant plasmid pET30a-BSLA
Fig. 2 is the crystalline structure of wild-type BSLA
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, Dayid W., Molecular Cloning:A Laboratory Manual, 3 RdEdition, 2001, NY, Cold SpringHarbor).The primer is synthetic by Dalian precious biological (TakaRa) or the living worker in Shanghai.
The screening of embodiment 1, lipase A variant of wild Bacillus subtilis
Have the active lipase A variant of wild Bacillus subtilis of high enzyme with following method screening, detailed process may further comprise the steps:
1, the clone of wild Bacillus subtilis lipase A (BSLA) gene
Use the Wizard  Genomic DNA Purification test kit and the operation of reference reagent box specification sheets of Promega company to extract the genomic dna of Bacillus subtillis 168 (Bacillus subtilis 168), be template with the genomic dna that is extracted then, at primer P1 (upstream primer): 5 '
AT GAATTCATGGCTGAACACAATCCAGTCGTTATGG-3 ' (band underscore base is a restriction enzyme EcoR I recognition site) and primer P2 (downstream primer): 5 '-CCAGGGCG AAGCTTUnder the guiding of TTCATTAATTCGTATTCTGG-3 ' (band underscore base is a restriction enzyme Hind III recognition site), method amplification Bacillus subtillis lipase A gene with Touch Down PCR, the Deep VentR  archaeal dna polymerase of NEB company is used in this PCR reaction, and the PCR reaction conditions is: 94 ℃ of 2min of elder generation; 94 ℃ of 1min then, 62 ℃ of 1min, each circulation is successively decreased 0.5 ℃, 72 ℃ of 80sec, totally 21 circulations; 94 ℃ of 1min again, 52 ℃ of 1min, 72 ℃ of 80sec, totally 16 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, pcr amplification goes out the band of 575bp size as a result, conforms to expected results.Reclaim, this purpose fragment of purifying, with its with restriction enzyme EcoR I with (Novagen) be connected with plasmid pET30a (+) after Hind III carries out double digestion through same enzyme double digestion, to connect product electric shock transformed into escherichia coli Escherichia coliBL21 (DE3) competent cell, transformant is coated screening positive clone on the LB flat board that is added with 50 μ g/mL kantlex, extract plasmid, it is checked order, sequencing result shows correct (the SEQ ID № in the sequence table: 4) of the BSLA gene order of being cloned, and correctly be connected among the pET30a (+), with this recombinant plasmid called after pET30a-BSLA, its physical map as shown in Figure 1.
2, make up Bacillus subtillis lipase A variant library and import the host
Bacillus subtillis lipase A (BSLA) gene to step 1 clone carries out the fallibility pcr amplification, concrete grammar is: the recombinant plasmid that makes up with step 1 is a template, carry out the fallibility pcr amplification under the guiding of same primers as P1 and P2, the Taq of TaKaRa company is used in this PCR reaction TMArchaeal dna polymerase, MnCl 2Concentration is 0.5mM, and the PCR reaction conditions is: 94 ℃ of 2min of elder generation; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 40sec, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, the BSLA gene with sudden change of recovery, purifying pcr amplification, with its with restriction enzyme EcoR I with is connected with plasmid pET30a (+) after Hind III carries out double digestion through same enzyme double digestion, will connect product and shock by electricity among the importing intestinal bacteria Escherichia coli BL21 (DE3).
3, the screening enzyme BSLA variant that improves alive
1) qualitative primary dcreening operation
The conversion of step 2 there is the transformant of BSLA variant gene directly to coat to contain on the LB resistant panel of 50 μ g/mL kantlex, 10mg/L rhodamine B (Rhodamine B) and 31.25ml/L sweet oil (Olive oil) that (dull and stereotyped compound method is referring to Kouker, G., K.-E.Jaeger, Specific and Sensitive PlateAssay for Bacterial Lipases, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1987,53 (1): 211-213), under 37 ℃, cultivate and carried out preliminary screening in 12-16 hour.
2) growth
The bacterium colony of the dots in red shape that the picking step 1) grows at flat board behind primary dcreening operation is inoculated in it on LB resistant panel that contains 50 μ g/mL kantlex, cultivates 3-4 hour down at 37 ℃, preserves as motherboard.
3) dull and stereotyped abduction delivering
Bacterium colony on the motherboard is transferred on the LB resistant panel that contains 50 μ g/mL kantlex and 0.5mM IPTG with nitrocellulose filter, under 30C, induced 6 hours.
4) qualitative multiple sieve
With conventional cleavage method cracking somatic cells, the lipase of cell inner expression discharges by nitrocellulose filter and adsorbs.Then film is covered on the agarose plate that contains 10mg/L rhodamine B (Rhodamine B) and 31.25ml/L sweet oil (Oliveoil),, make the reaction of BSLA variant and sweet oil and rhodamine B 37 ℃ of warm down baths 10 hours.Take film then off, flat board is placed under the ultraviolet lamp observe, the bacterium colony that fluorescent orange is strong more represents that the activity of the lipase of wherein expressing is high more.
5) enzymic activity of quantitative assay BSLA variant
The picking fluorescent orange is better than the variant of wild-type BSLA, be inoculated in the LB liquid nutrient medium that contains 50 μ g/mL kantlex, add 0.5mM IPTG, under 30 ℃, induced 6 hours, and gathered in the crops 5 OD600 cells, broken wall, extract intracellular enzyme, behind the purifying, lipase activity is carried out detection by quantitative (is contrast with wild-type BSLA), determine the highest active variant.So far, finished one and taken turns evolution.Then can be active the highest BSLA variant gene repeats above-mentioned steps 2 and step 3 as the template of fallibility PCR, carries out the evolution of a new round.
Wherein, the method for quantitatively determining of lipase activity is as follows:
Measure wild-type BSLA and variant thereof to palmitinic acid p-nitrophenyl fat (p-nitrophenyl palmitate, pNPP), sad p-nitrophenyl fat (p-nitrophenyl caprylate, pNPC), the activity of three kinds of substrates of sweet oil (olive oil).Activity measurement method to pNPP and pNPC sees document (Winkler for details, U.K., M.Stuckmann, Glycogen, Hyaluronate, and Some Other Polysaccharides GreatlyEnhance the Formation of Exolipase by Serratia marcescens, JOURNAL OFBACTERIOLOGY, 1979,138 (3): 663-670), active measuring method to sweet oil sees document (N.W.Tietz for details, E.A.Fiereck, A specific method for serum lipase determination, Clinica Chimica acta, 1966,13:352-359).Activity is defined as: under condition determination, the above-mentioned substrate of hydrolysis produces 1 μ mol in 1 minute p-NP (p-nitrophenol) or the needed enzyme amount of lipid acid (fatty acid) are defined as 1 activity unit.
After the result evolved in the first round, obtain an active the highest variant, called after BSLA-1-4, the amino acid residue sequence that changes body and be the wild Bacillus subtilis lipase A sports proline(Pro) (P) (being designated as S16P) from the Serine (S) of the 16th of aminoterminal (N end), and the 153rd glycine (G) is sported aspartic acid (D) (being designated as G153D), have SEQ ID № in the sequence table: 2 amino acid residue sequence, its nucleotides sequence of encoding is classified SEQ ID № in the sequence table as: 5.With this variant be template carry out second take turns evolution after, with the highest variant called after BSLA-2-6 of activity that obtains, this variant is that the amino acid residue sequence with the wild Bacillus subtilis lipase A sports proline(Pro) (P) (being designated as S16P) from the Serine (S) of the 16th of aminoterminal (N end), the 112nd Methionin (K) sports L-glutamic acid (E) (being designated as K112E), the 142nd arginine (R) sports Serine (S) (being designated as R142S), the 152nd Histidine (H) sports glutamine (Q) (being designated as H152Q), and the 153rd glycine (G) sported aspartic acid (D) (being designated as G153D), have SEQ ID № in the sequence table: 3 amino acid residue sequence, its nucleotides sequence of encoding is classified SEQ ID № in the sequence table as: 6.Wild-type BSLA and variant BSLA-2-6 live as shown in table 1 to the ratio of pNPP, pNPC and sweet oil, variant BSLA-2-6 has improved 4.8,3.0 and 2.3 times than wild-type BSLA respectively to the activity of pNPP, pNPC and sweet oil.
Table 1 wild-type BSLA and variant BSLA-2-6 live relatively for the ratio of pNPP, pNPC and sweet oil
Ratio (U/mg protein) alive to pNPP Multiple Ratio (U/mg protein) alive to pNPC Multiple Ratio (U/mg protein) alive to sweet oil Multiple
BSLA BSLA-2-6 4.65 22.42 1.0 4.8 29.42 88.99 1.0 3.0 21.53 53.06 1.0 2.6
The amino-acid residue of embodiment 2, lipase A variant of wild Bacillus subtilis is substituted in the position analysis in the three-dimensional structure
The amino-acid residue of the lipase A variant of wild Bacillus subtilis that embodiment 1 is obtained replaces and carries out the position analysis of three-dimensional structure.The crystalline structure of wild-type BSLA can obtain (ID:1ISP) from PDB, as shown in Figure 2, the amino acid of ball-and-stick model mark is the catalysis triprotic acid (Ser77, Asp133 and His156) of BSLA among the figure, and the position of 5 aminoacid replacement of lipase A variant of wild Bacillus subtilis of the present invention (S16, K112, R142, H152 and G153) uses club model mark respectively, and this several amino acid replacement all occurs in this enzyme surface.
Sequence table
<160>6
<210>1
<211>181
<212>PRT
＜213〉Bacillus subtillis (Bacillus subtilis)
<400>1
Ala Glu His Asn Pro Val Val Met Val His Gly Ile Gly Gly Ala Ser
1 5 10 15
Phe Asn Phe Ala Gly Ile Lys Ser Tyr Leu Val Ser Gln Gly Trp Ser
20 25 30
Arg Asp Lys Leu Tyr Ala Val Asp Phe Trp Asp Lys Thr Gly Thr Asn
35 40 45
Tyr Asn Asn Gly Pro Val Leu Ser Arg Phe Val Gln Lys Val Leu Asp
50 55 60
Glu Thr Gly Ala Lys Lys Val Asp Ile Val Ala His Ser Met Gly Gly
65 70 75 80
Ala Asn Thr Leu Tyr Tyr Ile Lys Asn Leu Asp Gly Gly Asn Lys Val
85 90 95
Ala Asn Val Val Thr Leu Gly Gly Ala Asn Arg Leu Thr Thr Gly Lys
100 105 110
Ala Leu Pro Gly Thr Asp Pro Asn Gln Lys Ile Leu Tyr Thr Ser Ile
115 120 125
Tyr Ser Ser Ala Asp Met Ile Val Met Asn Tyr Leu Ser Arg Leu Asp
130 135 140
Gly Ala Arg Asn Val Gln Ile His Gly Val Gly His Ile Gly Leu Leu
145 150 155 160
Tyr Ser Ser Gln Val Asn Ser Leu Ile Lys Glu Gly Leu Asn Gly Gly
165 170 175
Gly Gln Asn Thr Asn
180
<210>2
<211>181
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Ala Glu His Asn Pro Val Val Met Val His Gly Ile Gly Gly Ala Pro
1 5 10 15
Phe Asn Phe Ala Gly Ile Lys Ser Tyr Leu Val Ser Gln Gly Trp Ser
20 25 30
Arg Asp Lys Leu Tyr Ala Val Asp Phe Trp Asp Lys Thr Gly Thr Asn
35 40 45
Tyr Asn Asn Gly Pro Val Leu Ser Arg Phe Val Gln Lys Val Leu Asp
50 55 60
Glu Thr Gly Ala Lys Lys Val Asp Ile Val Ala His Ser Met Gly Gly
65 70 75 80
Ala Asn Thr Leu Tyr Tyr Ile Lys Asn Leu Asp Gly Gly Asn Lys Val
85 90 95
Ala Asn Val Val Thr Leu Gly Gly Ala Asn Arg Leu Thr Thr Gly Lys
100 105 110
Ala Leu Pro Gly Thr Asp Pro Asn Gln Lys Ile Leu Tyr Thr Ser Ile
115 120 125
Tyr Ser Ser Ala Asp Met Ile Val Met Asn Tyr Leu Ser Arg Leu Asp
130 135 140
Gly Ala Arg Asn Val Gln Ile His Asp Val Gly His Ile Gly Leu Leu
l45 150 155 160
Tyr Ser Ser Gln Val Asn Ser Leu Ile Lys Glu Gly Leu Asn Gly Gly
165 170 175
Gly Gln Asn Thr Asn
180
<210>3
<211>181
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>3
Ala Glu His Asn Pro Val Val Met Val His Gly Ile Gly Gly Ala Pro
1 5 10 15
Phe Asn Phe Ala Gly Ile Lys Ser Tyr Leu Val Ser Gln Gly Trp Ser
20 25 30
Arg Asp Lys Leu Tyr Ala Val Asp Phe Trp Asp Lys Thr Gly Thr Asn
35 40 45
Tyr Asn Asn Gly Pro Val Leu Ser Arg Phe Val Gln Lys Val Leu Asp
50 55 60
Glu Thr Gly Ala Lys Lys Val Asp Ile Val Ala His Ser Met Gly Gly
65 70 75 80
Ala Asn Thr Leu Tyr Tyr Ile Lys Asn Leu Asp Gly Gly Asn Lys Val
85 90 95
Ala Asn Val Val Thr Leu Gly Gly Ala Asn Arg Leu Thr Thr Gly Glu
100 105 110
Ala Leu Pro Gly Thr Asp Pro Asn Gln Lys Ile Leu Tyr Thr Ser Ile
115 120 125
Tyr Ser Ser Ala Asp Met Ile Val Met Asn Tyr Leu Ser Ser Leu Asp
130 135 140
Gly Ala Arg Asn Val Gln Ile Gln Asp Val Gly His Ile Gly Leu Leu
145 150 155 160
Tyr Ser Ser Gln Val Asn Ser Leu Ile Lys Glu Gly Leu Asn Gly Gly
165 170 175
Gly Gln Asn Thr Asn
180
<210>4
<211>543
<212>DNA
＜213〉Bacillus subtillis (Bacillus subtilis)
<400>4
gctgaacaca atccagtcgt tatggttcac ggtattggag gggcatcatt caattttgcg 60
ggaattaaga gctatctcgt atctcagggc tggtcgcggg acaagctgta tgcagttgat 120
ttttgggaca agacaggcac aaattataac aatggaccgg tattatcacg atttgtgcaa 180
aaggttttag atgaaacggg tgcgaaaaaa gtggatattg tcgctcacag catggggggc 240
gcgaacacac tttactacat aaaaaatctg gacggcggaa ataaagttgc aaacgtcgtg 300
acgcttggcg gcgcgaaccg tttgacgaca ggcaaggcgc ttccgggaac agatccaaat 360
caaaagattt tatacacatc catttacagc agtgccgata tgattgtcat gaattactta 420
tcaagattag atggtgctag aaacgttcaa atccatggcg ttggacacat cggccttctg 480
tacagcagcc aagtcaacag cctgattaaa gaagggctga acggcggggg ccagaatacg 540
aat 543
<210>5
<211>543
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
gctgaacaca atccagtcgt tatggtccac ggtattggag gggcaccatt caattttgcg 60
ggaattaaga gctatctcgt atctcagggc tggtcgcggg acaagctgta tgcagttgat 120
ttttgggaca agacaggcac aaattataac aatggaccgg tattatcacg atttgtgcaa 180
aaggttttag atgaaacggg tgcgaaaaaa gtggatattg tcgctcacag catggggggc 240
gcgaacacac tttactacat aaaaaatctg gacggcggaa ataaagttgc aaacgtcgtg 300
acgcttggcg gcgcgaaccg tttgacgaca ggcaaggcgc ttccgggaac agatccgaat 360
caaaagattt tatacacatc catttacagc agtgccgata tgattgtcat gaattactta 420
tcaagattag atggtgctag aaacgttcaa atccatgacg ttggacacat cggccttctg 480
tacagcagcc aagtcaacag cctgattaaa gaagggctga acggcggggg ccagaatacg 540
aat 543
<210>6
<211>543
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
gctgaacaca atccagtcgt tatggtccac ggtattggag gggcaccatt taattttgcg 60
ggaattaaga gctatctcgt atctcagggc tggtcgcggg acaagttgta tgcagttgat 120
ttttgggaca agacaggcac aaattataac aatggaccgg tattatcacg atttgtgcaa 180
aaggttttag atgaaacggg tgcgaaaaaa gtggatattg tcgctcacag catggggggc 240
gcgaacacac tttactacat aaaaaatctg gacggcggaa ataaagttgc aaacgtcgtg 300
acgcttggcg gcgcgaaccg tttgacgaca ggcgaggcgc ttccgggaac agatccgaat 360
caaaagattt tatacacatc catttacagc agtgccgata tgattgtcat gaattactta 420
tcaagtttag atggtgctag aaacgttcaa atccaggacg ttggacacat cggccttctg 480
tacagcagcc aagtcaacag cctgattaaa gaagggctga acggcggggg ccagaatacg 540
aat 543

Claims (8)

1, lipase A variant of wild Bacillus subtilis, be that amino acid residue sequence with the wild Bacillus subtilis lipase A carries out the protein that obtains after one or more sudden change combinations in following five kinds of sudden changes: 1) mutant serine from the 16th of aminoterminal is a proline(Pro); 2) the 112nd lysine mutation is a L-glutamic acid; 3) the 142nd arginine sports Serine; 4) the 152nd Histidine sports glutamine; 5) the 153rd glycine mutation is an aspartic acid; Described wild Bacillus subtilis lipase A has SEQ ID № in the sequence table: 1 amino acid residue sequence.
2, lipase A variant of wild Bacillus subtilis according to claim 1 is characterized in that: described variant has one of following amino acid residue sequences:
1) SEQ ID № in the sequence table: 2;
2) SEQ ID № in the sequence table: 3.
3, the gene of the described lipase A variant of wild Bacillus subtilis of coding claim 1.
4, gene according to claim 3 is characterized in that: described gene is one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 5;
2) SEQ ID № in the sequence table: 6.
5, contain the described expression carrier of claim 3, transgenic cell and host bacterium.
6, a kind of method of screening lipase Variant may further comprise the steps:
1) the variant library of structure lipase gene;
2) the variant library is connected with carrier, the recombinant vectors that will carry the variant library then imports the host bacterium;
3) with step 2) transformant that obtains is inoculated in and carries out qualitative primary dcreening operation on the primary dcreening operation flat board, filters out the variant with lipase activity; Described primary dcreening operation is dull and stereotyped to add the marker that can filter out foreign vector on cultivating with the basis of solid medium in host bacterium routine, and the substrate of lipase;
4) the picking step 3) has the variant of lipase activity behind primary dcreening operation, it is inoculated in growth cultivates on the flat board, preserves as motherboard after growing single bacterium colony; Described growth is dull and stereotyped adds the marker that can filter out foreign vector on cultivating with the basis of solid medium in host bacterium routine;
5) bacterium colony on the step 4) motherboard is transferred to induce on the flat board induces with changeing embrane method; The described flat board of inducing adds the marker that can filter out foreign vector on cultivating with the basis of solid medium in host bacterium routine, and inductor;
6) cleavage step 5) in through the inductive bacterium colony, make the lipase of its expression be adsorbed on the surface of transfer film, transfer film is placed carry out qualitative multiple sieve on the multiple sieve flat board again, obtain the variant that improves than the wild-type lipase activity; Described multiple sieve is dull and stereotyped for only containing the flat board of the substrate of agarose and lipase;
7) activity of quantitative assay lipase Variant.
7, method according to claim 6 is characterized in that: the lipase gene in the described step 1) is a wild Bacillus subtilis lipase A gene.
8, the application of the described lipase A variant of wild Bacillus subtilis of claim 1 in the preparing washing agent.
CNB2006100668579A 2006-03-31 2006-03-31 Lipase A variant of wild Bacillus subtilis and application thereof Expired - Fee Related CN100417723C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB2006100668579A CN100417723C (en) 2006-03-31 2006-03-31 Lipase A variant of wild Bacillus subtilis and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB2006100668579A CN100417723C (en) 2006-03-31 2006-03-31 Lipase A variant of wild Bacillus subtilis and application thereof

Publications (2)

Publication Number Publication Date
CN1834236A true CN1834236A (en) 2006-09-20
CN100417723C CN100417723C (en) 2008-09-10

Family

ID=37002133

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB2006100668579A Expired - Fee Related CN100417723C (en) 2006-03-31 2006-03-31 Lipase A variant of wild Bacillus subtilis and application thereof

Country Status (1)

Country Link
CN (1) CN100417723C (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838634A (en) * 2010-05-25 2010-09-22 中国科学院武汉病毒研究所 Bacillus subtilis lipase, preparation method and application
CN101857856A (en) * 2010-05-25 2010-10-13 中国科学院武汉病毒研究所 Bacillus subtilis lipase, and preparation method and application thereof
CN102337253A (en) * 2011-10-13 2012-02-01 浙江大学 Isolated polypeptide, polynucleotide, carrier and host cell
CN102851263A (en) * 2011-07-01 2013-01-02 丰益(上海)生物技术研发中心有限公司 High-throughout screening method of lipase gene mutation database and lipase mutation gene
CN104450866A (en) * 2014-10-24 2015-03-25 中国科学院广州能源研究所 Membrane blotting high-throughput screening method for secreted lipase gene engineering bacteria
CN109182299A (en) * 2018-10-30 2019-01-11 福建师范大学 It is resistant to the Bacillus subtillis lipase mutant and preparation method thereof of hydrogen peroxide

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658871A (en) * 1989-07-07 1997-08-19 Lever Brothers Company, Division Of Conopco, Inc. Microbial lipase muteins and detergent compositions comprising same
EP0755442B1 (en) * 1994-05-04 2002-10-09 Genencor International, Inc. Lipases with improved surfactant resistance
US7303904B2 (en) * 2003-01-30 2007-12-04 Council Of Scientific & Industrial Research Stable gene variants of lipases

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838634A (en) * 2010-05-25 2010-09-22 中国科学院武汉病毒研究所 Bacillus subtilis lipase, preparation method and application
CN101857856A (en) * 2010-05-25 2010-10-13 中国科学院武汉病毒研究所 Bacillus subtilis lipase, and preparation method and application thereof
CN101857856B (en) * 2010-05-25 2012-02-01 中国科学院武汉病毒研究所 Bacillus subtilis lipase, and preparation method and application thereof
CN102851263A (en) * 2011-07-01 2013-01-02 丰益(上海)生物技术研发中心有限公司 High-throughout screening method of lipase gene mutation database and lipase mutation gene
CN102851263B (en) * 2011-07-01 2015-04-01 丰益(上海)生物技术研发中心有限公司 High-throughout screening method of lipase gene mutation database and lipase mutation gene
CN102337253A (en) * 2011-10-13 2012-02-01 浙江大学 Isolated polypeptide, polynucleotide, carrier and host cell
CN102337253B (en) * 2011-10-13 2017-08-22 浙江大学 Polypeptide, polynucleotides, carrier and the host cell of separation
CN104450866A (en) * 2014-10-24 2015-03-25 中国科学院广州能源研究所 Membrane blotting high-throughput screening method for secreted lipase gene engineering bacteria
CN109182299A (en) * 2018-10-30 2019-01-11 福建师范大学 It is resistant to the Bacillus subtillis lipase mutant and preparation method thereof of hydrogen peroxide
CN109182299B (en) * 2018-10-30 2021-12-31 福建师范大学 Hydrogen peroxide-tolerant bacillus subtilis lipase mutant and preparation method thereof

Also Published As

Publication number Publication date
CN100417723C (en) 2008-09-10

Similar Documents

Publication Publication Date Title
CN1180082C (en) Production method of novel mutant N-acetylglutamate synthetase and L-arginine
CN1229500C (en) Alkaline cellulase and method for producing same
CN1249784A (en) In vitro method for construction of DNA library
CN1834236A (en) Lipase A variant of wild Bacillus subtilis and application thereof
CN101063104A (en) Engineering bacterium producing 5-glycyl ethylformic acid and construction method thereof
CN1250568C (en) Protein participating in activation of nitrile hydratase and gene encoding the same
CN1143678A (en) Gene encoding lacto-N-biosidase
CN1456672A (en) Glyphosate tolerance-5-enolpyruvoyl shikimic acid-3-phosphoric acid synthesizing enzyme and its coding genes
CN1699424A (en) Heparinase I fusion protein and genes encoding same and expression method thereof
CN1699573A (en) V. alginolyticus outer-membrane protein W gene, process for preparing the same and uses thereof
CN1082090C (en) Process for producing D -N -carbamoyl -alpha -amino acid
CN1732387A (en) Protein chip for analyzing interaction between protein and substrate peptide
CN100335638C (en) Thermophilic long chain paraffin hydrocarbon mono oxygenase, its coding gene and application
CN1052509C (en) Gene encoding polypeptide having nitrile hydratase activity, transformant containing gene and process for production of amides using transformant
CN1408885A (en) Mononucleotide polymorphism analyzing method for detecting chicken ventral fat character
CN101063113A (en) Clone and expression for novel phytase
CN1486367A (en) Novel fructosyl amino acid oxidase
CN1239142A (en) Cytochrome BD type quinol oxidase gene of brevibacterium lactofermentum
CN1153832C (en) A thermophilic alkaline phosphoesterase and its expression
CN1244700C (en) Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence
CN1571841A (en) Glucose dehydrogenase
CN1233832C (en) Microbiological method for producing L-carnitine
CN1101469C (en) Genes encoding enzymers of BalI restruction-modification system
CN1858211A (en) Serine proteinase gene coming from new nematophagous fungi and its use
CN1555416A (en) Nucleic acid sequences encoding enantioselective amidases

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
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20080910

Termination date: 20150331

EXPY Termination of patent right or utility model