CN108913675A - The lipase mutant and its application that a kind of thermal stability improves - Google Patents
The lipase mutant and its application that a kind of thermal stability improves Download PDFInfo
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- CN108913675A CN108913675A CN201810742230.3A CN201810742230A CN108913675A CN 108913675 A CN108913675 A CN 108913675A CN 201810742230 A CN201810742230 A CN 201810742230A CN 108913675 A CN108913675 A CN 108913675A
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- lipase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Abstract
The lipase mutant improved the invention discloses a kind of thermal stability and its application.The lipase mutant is the lipase mutant that lipase TTL increases that disulfide bond obtains；Wherein, the nucleotide sequence of lipase TTL is as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2.The present invention has obtained the lipase mutant TTL-108/162 of thermal stability raising by constructing disulfide bond, enzyme activity retention rate after mutant TTL-108/162 is heat-treated 30 minutes at 75 DEG C and 80 DEG C is 65% and 48%, the lipase mutant TTL-108/162 of acquisition i.e. of the invention has good thermal stability, can be widely applied in industrial circle.
Description
Technical field
The invention belongs to molecular biology field, in particular to lipase mutant and its answer that a kind of thermal stability improves
With.
Background technique
Lipase is the general name for the class of enzymes that catalyzing glycerol ester is decomposed or synthesized on oil-water interfaces, can be catalyzed natural
Substrate grease hydrolysis discharges the glyceride or glycerol and fatty acid of less ester bond.Acidolysis, transesterification, ester conjunction can be catalyzed again simultaneously
It is reacted at transesterification etc..Since the unique enzymatic property of lipase has a wide range of applications it in fields such as feed, papermaking
Potentiality.
Many industrial circles have certain requirement to the thermal stability of lipase, it is desirable to which lipase can be under the high temperature conditions
Good stability, such as feed and many process procedures of papermaking industrial circle is kept to be related to hot environment, thermal stability is poor
Lipase in above-mentioned technique easy in inactivation be denaturalized, limit lipase in the application of these industrial circles, therefore it is steady to develop heat
Qualitative good lipase is of great significance.In the experiment of early period, this seminar obtains lipase TTL, the enzyme pair
Middle long chain glyceride substrate has good hydrolysing activity, and substrate specificity is extensive, but TTL is unstable under high temperature environment
It is fixed, it is easy deactivation (the enzyme activity retention rate after TTL is heat-treated 30 minutes at 75 DEG C and 80 DEG C is only 25% and 16%), it is unfavorable
In its industrial application.Therefore, the lipase mutant of good thermal stability is constructed, can be answered for the industrialization of lipase TTL
With laying the foundation.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of rouge that thermal stability improves
Fat enzyme mutant.
Another object of the present invention is to provide the encoding genes for the lipase mutant that the thermal stability improves.
Another object of the present invention is to provide the application for the lipase mutant that the thermal stability improves.
The purpose of the invention is achieved by the following technical solution：A kind of lipase mutant that thermal stability improves, the rouge
Fat enzyme mutant is the lipase mutant that lipase TTL increases that disulfide bond obtains；Wherein, the nucleotide sequence of lipase TTL
As shown in SEQ ID NO.1, amino acid sequence is as shown in SEQ ID NO.2.
The lipase mutant is lipase mutant TTL-108/162, amino acid sequence such as SEQ ID NO.4
It is shown.
The encoding gene for the lipase mutant that the thermal stability improves, nucleotide sequence such as SEQ ID NO.3
It is shown.
The mutational site I108C and A162C that the lipase mutant TTL-108/162 includes, i.e. amino acid sequence
108 Ile (I) in the lipase TTL as shown in SEQ ID NO.4 sport Cys (C), 162 Ala (A) sport Cys
(C)。
Recombinant vector containing the encoding gene (mutant TTL-108/162 gene)
Recombinant bacterial strain containing the encoding gene (mutant TTL-108/162 gene).
Application of the lipase mutant that the thermal stability improves in industrial circle, including the works such as feed and papermaking
Industry field.
The present invention has the following advantages and effects with respect to the prior art：The present invention has obtained heat by constructing disulfide bond
Stability-enhanced lipase mutant TTL-108/162, compared with lipase TTL, mutant TTL-108/162 includes prominent
Displacement point is I108C and A162C.Mutant TTL-108/162 75 DEG C and 80 DEG C be heat-treated 30 minutes after enzyme activity retention rate
For 65% and 48%, and the enzyme activity retention rate after wild type TTL is heat-treated 30 minutes at 75 DEG C and 80 DEG C is only 25% and 16%.
Relative to lipase TTL, the thermal stability after mutant TTL-108/162 is heat-treated 30 minutes at 75 DEG C and 80 DEG C is improved
160% and 200%.Therefore, the lipase mutant TTL-108/162 of acquisition of the invention has good thermal stability, is
Its industrial application lays the foundation.
Detailed description of the invention
The optimal reactive temperature measurement result figure of Fig. 1 original lipase TTL and mutant TTL-108/162.
The optimal reaction pH measurement result figure of Fig. 2 original lipase TTL and mutant TTL-108/162.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiment《Molecular Cloning:A Laboratory guide》(third edition)
J. specific method listed in one book of Pehanorm Brooker carries out, or carries out according to kit and product description；The reagent
And biomaterial commercially obtains unless otherwise specified.The experimental material and reagent being related in the present invention：
1, bacterial strain and carrier
Purchase obtains from commercial channels by coli strain Top10, Pichia pastoris X33, expression vector pPICZ α A.
2, enzyme and kit
Q5 high-fidelity Taq enzyme MIX is purchased from NEB company；Plasmid extracts, and gel purification kit is purchased from Tiangeng biochemical technology (north
Capital) Co., Ltd；Restriction enzyme, which is purchased from, cures biotechnology (Beijing) Co., Ltd precious day；Zeocin is purchased from Invitrogen
Company.
3, culture medium
Escherichia coli culture medium be LB (1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl,
pH7.0).LBZ is that LB culture medium adds 25 μ g/mL Zeocin (bleomycin).
Yeast culture medium is YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose).Ferment
Female screening and culturing medium is YPDZ (YPD+100mg/L zeocin).
Yeast induced medium BMGY (1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB,
0.00004% (w/v) Biotin, 1% glycerol (V/V)) and BMMY culture medium (glycerol is replaced divided by 0.5% (v/v) methanol,
Remaining composition phase is identical as BMGY).
Note：YNB is that yeast nitrogen is basic (Yeast Nitrogen Base)；Biotin is biotin.
Embodiment 1, lipase TTL expression vector establishment
Analyze the lipase TTL gene (Genebank of the website NCBI report：JF414585.1), TTL lipase base is found
It include 4 exons and 3 intrones because of overall length 1083bp.It finds that its open reading frame overall length is 876bp by comparing, compiles
291 amino acid of code, wherein 17 amino acid are signal peptide before albumen n end.According to the TTL gene order found, by complete
Gene synthesis technology synthesizes TTL gene ttl.According to the sequence design pair of primers of synthesis gene ttl, (primer sequence is respectively
fw：5'-agtcgaattcTCTCC AGTCAGACGTGAGGTT-3' and rev：5'-
TtctctagaTTACAAACAAGTACCAATCAA-3' it) for expanding the mature peptide gene tll-1 without signal peptide, will expand
Obtained tll-1 is cloned on carrier pPICZ α A, obtains recombinant vector pPICZ α A-ttl-1.
Embodiment 2, building disulfide bond mutant
The protein three-dimensional structure of TTL is obtained by homology modeling software Modeller first, then soft by molecular dynamics
Part Gromacs carries out the molecular dynamics simulation of TTL, and the flex region of TTL albumen is found according to the result of molecular dynamics simulation
Domain.Increase disulfide bond in albumen flexible region.Website (http is designed by online disulfide bond://
Cptweb.cpt.wayne.edu/DbD2/index.php 5 pairs of disulfide bond) are devised, are E61C/N31C, E61C/ respectively
T69C, I108C/A162C, G166C/G195C and G230C/V233C.
The building of disulfide bond mutant is approximately as (by taking mutant E61C/N31C as an example, other and so on)：With building
Good pPICZ α A-ttl-1 is template, first carries out PCR amplification with upstream and downstream primer E61C-fw and E61C-rev, agarose electrophoresis
PCR amplification is detected as a result, purification and recovery PCR product, is decomposed original plasmid with restriction enzyme DpnI, the production that will have been decomposed
Object is transferred to Escherichia coli Top10 with heat shock method, verifies recombinant conversion by bacterium solution PCR, extracts and verify correct transformant
Plasmid is sequenced, so that it is determined that mutant E61C.In the same manner using E61C mutant plasmid as template, primer is used
N31C-fw and E31C-rev carries out PCR amplification, and purifying converts Escherichia coli Top10, extracts plasmid, and sequencing is finally dashed forward
Variant E61C/N31C.Correct mutant will be sequenced, linearized with SacI, be transferred to Pichia pastoris X33.
1 disulfide bond mutant primer of table
The screening of embodiment 3, disulfide bond mutant recombination yeast engineering strain
Yeast recombinant conversion in embodiment 2 is chosen to 24 holes for containing 2mL BMGY culture medium to every hole one by one with toothpick
In plate, 30 DEG C, 220rpm cultivates 36h or so, then is separately added into the methanol progress Fiber differentiation of 0.75% (v/v).30 DEG C,
After 220rpm is cultivated 24 hours, centrifuging and taking supernatant carries out enzyme activity determination.The measurement of lipase active uses acid-base titration, used
Instrument be pH-stat acid alkaline titrimeter (ten thousand Tong Zhong Co., Ltd of Switzerland).Specific step is as follows for enzyme activity determination：First will
100mL water is added in the olive oil of 5% (v/v), while the Arabic gum of 2% (w/v) of addition, as stabilizer, high speed homogenization 5 divides
Olive oil substrate is obtained after clock；Take 15mL olive oil substrate be added reaction cup in, to reaction temperature and pH reach setting value and
Stablize a period of time, the enzyme solution that 10 μ L have diluted is added；Reaction calculates enzyme activity after reaching balance.The definition of enzyme activity is certain
Under reaction condition, enzyme amount needed for generating 1 μm of ol fatty acid with substrate reactions per minute is defined as 1 enzyme activity unit, with U table
Show.Each disulfide bond mutant screens the highest recombined engineering strain of an enzyme activity and carries out shaking flask culture.
Shaking flask culture carries out in 500mL triangular flask, and the access of corresponding recombined engineering strain is contained 5mL BMGY first
In the 50mL centrifuge tube of culture medium, 30 DEG C, 220rpm is cultivated 24 hours or so, and cultured recombination yeast engineering strain is pressed
In 500mL triangular flask of the inoculum concentration access containing 100mL BMMY culture medium of 1% (v/v).Shake flask culture conditions are 30 DEG C,
220rpm is induced every the methanol that 24 hours are added 0.75% (v/v), while sampling the measurement for carrying out lipase active,
Original TTL and disulfide bond mutant recombinant bacterium shaking flask 72 hours enzyme activity of culture are as shown in table 2.
2 original lipase TTL of table and mutant recombinant bacterium shaking flask culture enzyme activity
| Strain/recombinant strain | Enzyme activity (U/ml) |
| TTL | 120 |
| E61C/N31C | 115 |
| E61C/T69C | 108 |
| I108C/A162C | 70 |
| G166C/G195C | 123 |
| G230C/V233C | 100 |
The thermal stability analysis of embodiment 4, original lipase TTL and disulfide bond mutant
The thermal stability determination of disulfide bond mutant is as follows：PCR reaction tube is added in the lipase enzyme solution diluted, then will
PCR reaction tube equipped with enzyme solution is put into PCR instrument, is heat-treated 30 minutes under the conditions of 75 DEG C and 80 DEG C respectively, referring to lipase activity
Property measuring method, measure the enzyme activity of remaining lipase, not do the lipase being heat-treated, as control, carry out remaining enzyme activity
It calculates.Residual enzyme motility rate, divided by control sample enzyme activity, is indicated with heat treated sample enzyme activity multiplied by 100%.
Original lipase TTL (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence such as SEQ ID NO.2 institute
Show) and thermal stability of disulfide bond mutant under the conditions of 75 DEG C and 80 DEG C it is as shown in table 3.This 5 pairs of disulfide bond are prominent as shown in Table 3
In variant, and only I108C/A162C (nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence such as SEQ ID NO.4 institute
Show) it can effectively promote the thermal stability of TTL, the mutant remaining enzyme activity point after being heat-treated 30 minutes under the conditions of 75 DEG C and 80 DEG C
Not Wei 65% and 48%, and original TTL is only 25% and 16% in the 75 DEG C and 80 DEG C enzyme activity retention rates after water-bath 30 minutes.Its
He 4 does not influence too much on disulfide bond on the thermal stability of TTL.Wherein：
SEQ ID NO.1 (original lipase TTL)：
AGTCCTGTCCGACGAGAGGTCTCGCAGGATCTGTTTGACCAGTTCAACCTCTTTGCGCAGTACTCGGCGGCCGCATA
CTGCGCGAAGAACAACGATGCCCCGGCAGGTGCGAACGTAACGTGCAGGGGAAGTATTTGCCCCGAGGTAGAGAAGG
CGGATGCAACGTTTCTCTACTCGTTTGAAGATTCTGGAGTTGGCGATGTCACCGGGTTCCTTGCTCTCGACAACACG
AACA GACTGATCGTCCTCTCTTTCCGCGGCTCTCGTTCCCTGGAAAACTGGATCGGGAATATCAACTTGGACTTGA
AAGGAATTGACGACATCTGCTCTGGCTGCAAGGGACATGACGGCTTCACTTCCTCCTGGAGGTCCGTTGCCAATACC
TTGACTCAGCAAGTGCAGAATGCTGTGAGGGAGCATCCCGACTACCGCGTCGTCTTCACTGGGCACAGCTTGGGTGG
GGCATTGGCAACTGTGGCCGGGGCATCTCTGCGTGGAAATGGGTACGATATAGATGTGTTCTCATATGGCGCTCCCC
GCGTCGGAAACAGGGCTTTTGCGGAATTCCTGACCGCACAGACCGGCGGCACCTTGTACCGCATCACCCACACCAAT
GATATTGTCCCCAGACTCCCGCCACGCGAATTGGGTTACAGCCATTCTAGCCCAGAGTATTGGATCACGTCTGGAAC
CCTCGTCCCAGTGACCAAGAACGATATCGTCAAGGTGGAGGGCATCGATTCCACCGATGGAAACAACCAGCCAAATA
CCCCGGACATTGCTGCGCACCTATGGTACTTCGGCCTCATCGGGACATGTCTTTAG。
SEQ ID NO.2 (original lipase TTL)：
SPVRREVSQDLFDQFNLFAQYSAAAYCAKNNDAPAGANVTCRGSICPEVEKADATFLYSFEDSGVGDVTGFLALDNT
NRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGG
ALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAVFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGT
LVPVTKNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL。
SEQ ID NO.3 (lipase mutant TTL-108/162)：
AGTCCTGTCCGACGAGAGGTCTCGCAGGATCTGTTTGACCAGTTCAACCTCTTTGCGCAGTACTCGGCGGCCGCATA
CTGCGCGAAGAACAACGATGCCCCGGCAGGTGCGAACGTAACGTGCAGGGGAAGTATTTGCCCCGAGGTAGAGAAGG
CGGATGCAACGTTTCTCTACTCGTTTGAAGATTCTGGAGTTGGCGATGTCACCGGGTTCCTTGCTCTCGACAACACG
AACAGACTGATCGTCCTCTCTTTCCGCGGCTCTCGTTCCCTGGAAAACTGGATCGGGAATATCAACTTGGACTTGAA
AGGAATTGACGACTGCTGCTCTGGCTGCAAGGGACATGACGGCTTCACTTCCTCCTGGAGGTCCGTTGCCAATACCT
TGACTCAGCAAGTGCAGAATGCTGTGAGGGAGCATCCCGACTACCGCGTCGTCTTCACTGGGCACAGCTTGGGTGGG
GCATTGGCAACTGTGGCCGGGTGCTCTCTGCGTGGAAATGGGTACGATATAGATGTGTTCTCATATGGCGCTCCCCG
CGTCGGAAACAGGGCTTTTGCGGAATTCCTGACCGCACAGACCGGCGGCACCTTGTACCGCATCACCCACACCAATG
ATATTGTCCCCAGACTCCCGCCACGCGAATTGGGTTACAGCCATTCTAGCCCAGAGTATTGGATCACGTCTGGAACC
CTCGTCCCAGTGACCAAGAACGATATCGTCAAGGTGGAGGGCATCGATTCCACCGATGGAAACAACCAGCCAAATAC
CCCGGACATTGCTGCGCACCTATGGTACTTCGGCCTCATCGGGACATGTCTTTAG。
SEQ ID NO.4 (lipase mutant TTL-108/162)：
SPVRREVSQDLFDQFNLFAQYSAAAYCAKNNDAPAGANVTCRGSICPEVEKADATFLYSFEDSGVGDVTGFLALDNT
NRLIVLSFRGSRSLENWIGNINLDLKGIDDCCSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVVFTGHSLGG
ALATVAGCSLRGNGYDIDVFSYGAPRVGNRAFAVFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSPEYWITSGT
LVPVTKNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL。
2 original lipase TDL of table and single-point mutants thermal stability analysis
| Number | 75 DEG C of remaining enzyme activity (%) | 80 DEG C of remaining enzyme activity (%) |
| Original lipase TTL | 25 | 16 |
| E61C/N31C | 23 | 14 |
| E61C/T69C | 26 | 16 |
| I108C/A162C | 65 | 48 |
| G166C/G195C | 27 | 15 |
| G230C/V233C | 23 | 15 |
The optimal reactive temperature of embodiment 5, lipase TTL and mutant TTL-108/162
Lipase TTL and mutant TTL-108/162 is measured under the conditions of 9.5 pH under 30~80 DEG C of different temperatures
Enzyme activity (embodiment of the method 3) calculates the opposite enzyme activity at a temperature of other to measure the enzyme activity under enzyme activity maximum temperature as 100%.
Experimental result：The optimal reactive temperature of lipase TTL and mutant TTL-108/162 are as shown in Figure 1, can by Fig. 1
Know, the optimal reactive temperature of original lipase TTL is 60 DEG C, and the optimal reactive temperature of mutant TTL-108/162 is 65 DEG C.
The optimal reaction pH of embodiment 6, lipase TTL and mutant TTL-108/162
The enzyme activity of lipase TTL and mutant TTL-108/162 under pH7~11 is measured under the conditions of 60 DEG C, and (method is real
Apply example 3), to measure the enzyme activity under enzyme activity highest pH as 100%, calculate the opposite enzyme activity under other pH.
Experimental result：The optimal reaction pH of lipase TTL and mutant TTL-108/162 as shown in Fig. 2, as shown in Figure 2,
The optimal reaction pH of TTL and mutant TTL-108/162 is 9.5, and opposite enzyme activity is all larger than 60% in the range of pH8~10.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>South China Science & Engineering University
<120>The lipase mutant and its application that a kind of thermal stability improves
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 825
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase TTL
<400> 1
agtcctgtcc gacgagaggt ctcgcaggat ctgtttgacc agttcaacct ctttgcgcag 60
tactcggcgg ccgcatactg cgcgaagaac aacgatgccc cggcaggtgc gaacgtaacg 120
tgcaggggaa gtatttgccc cgaggtagag aaggcggatg caacgtttct ctactcgttt 180
gaagattctg gagttggcga tgtcaccggg ttccttgctc tcgacaacac gaacagactg 240
atcgtcctct ctttccgcgg ctctcgttcc ctggaaaact ggatcgggaa tatcaacttg 300
gacttgaaag gaattgacga catctgctct ggctgcaagg gacatgacgg cttcacttcc 360
tcctggaggt ccgttgccaa taccttgact cagcaagtgc agaatgctgt gagggagcat 420
cccgactacc gcgtcgtctt cactgggcac agcttgggtg gggcattggc aactgtggcc 480
ggggcatctc tgcgtggaaa tgggtacgat atagatgtgt tctcatatgg cgctccccgc 540
gtcggaaaca gggcttttgc ggaattcctg accgcacaga ccggcggcac cttgtaccgc 600
atcacccaca ccaatgatat tgtccccaga ctcccgccac gcgaattggg ttacagccat 660
tctagcccag agtattggat cacgtctgga accctcgtcc cagtgaccaa gaacgatatc 720
gtcaaggtgg agggcatcga ttccaccgat ggaaacaacc agccaaatac cccggacatt 780
gctgcgcacc tatggtactt cggcctcatc gggacatgtc tttag 825
<210> 2
<211> 274
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase TTL
<400> 2
Ser Pro Val Arg Arg Glu Val Ser Gln Asp Leu Phe Asp Gln Phe Asn
1 5 10 15
Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Ala Lys Asn Asn Asp
20 25 30
Ala Pro Ala Gly Ala Asn Val Thr Cys Arg Gly Ser Ile Cys Pro Glu
35 40 45
Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly
50 55 60
Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Arg Leu
65 70 75 80
Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Leu Glu Asn Trp Ile Gly
85 90 95
Asn Ile Asn Leu Asp Leu Lys Gly Ile Asp Asp Ile Cys Ser Gly Cys
100 105 110
Lys Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asn Thr
115 120 125
Leu Thr Gln Gln Val Gln Asn Ala Val Arg Glu His Pro Asp Tyr Arg
130 135 140
Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala
145 150 155 160
Gly Ala Ser Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr
165 170 175
Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Val Phe Leu Thr Ala
180 185 190
Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val
195 200 205
Pro Arg Leu Pro Pro Arg Glu Leu Gly Tyr Ser His Ser Ser Pro Glu
210 215 220
Tyr Trp Ile Thr Ser Gly Thr Leu Val Pro Val Thr Lys Asn Asp Ile
225 230 235 240
Val Lys Val Glu Gly Ile Asp Ser Thr Asp Gly Asn Asn Gln Pro Asn
245 250 255
Thr Pro Asp Ile Ala Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr
260 265 270
Cys Leu
<210> 3
<211> 825
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase mutant TTL-108/162
<400> 3
agtcctgtcc gacgagaggt ctcgcaggat ctgtttgacc agttcaacct ctttgcgcag 60
tactcggcgg ccgcatactg cgcgaagaac aacgatgccc cggcaggtgc gaacgtaacg 120
tgcaggggaa gtatttgccc cgaggtagag aaggcggatg caacgtttct ctactcgttt 180
gaagattctg gagttggcga tgtcaccggg ttccttgctc tcgacaacac gaacagactg 240
atcgtcctct ctttccgcgg ctctcgttcc ctggaaaact ggatcgggaa tatcaacttg 300
gacttgaaag gaattgacga ctgctgctct ggctgcaagg gacatgacgg cttcacttcc 360
tcctggaggt ccgttgccaa taccttgact cagcaagtgc agaatgctgt gagggagcat 420
cccgactacc gcgtcgtctt cactgggcac agcttgggtg gggcattggc aactgtggcc 480
gggtgctctc tgcgtggaaa tgggtacgat atagatgtgt tctcatatgg cgctccccgc 540
gtcggaaaca gggcttttgc ggaattcctg accgcacaga ccggcggcac cttgtaccgc 600
atcacccaca ccaatgatat tgtccccaga ctcccgccac gcgaattggg ttacagccat 660
tctagcccag agtattggat cacgtctgga accctcgtcc cagtgaccaa gaacgatatc 720
gtcaaggtgg agggcatcga ttccaccgat ggaaacaacc agccaaatac cccggacatt 780
gctgcgcacc tatggtactt cggcctcatc gggacatgtc tttag 825
<210> 4
<211> 274
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase mutant TTL-108/162
<400> 4
Ser Pro Val Arg Arg Glu Val Ser Gln Asp Leu Phe Asp Gln Phe Asn
1 5 10 15
Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Ala Lys Asn Asn Asp
20 25 30
Ala Pro Ala Gly Ala Asn Val Thr Cys Arg Gly Ser Ile Cys Pro Glu
35 40 45
Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly
50 55 60
Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Arg Leu
65 70 75 80
Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Leu Glu Asn Trp Ile Gly
85 90 95
Asn Ile Asn Leu Asp Leu Lys Gly Ile Asp Asp Cys Cys Ser Gly Cys
100 105 110
Lys Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asn Thr
115 120 125
Leu Thr Gln Gln Val Gln Asn Ala Val Arg Glu His Pro Asp Tyr Arg
130 135 140
Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala
145 150 155 160
Gly Cys Ser Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr
165 170 175
Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Val Phe Leu Thr Ala
180 185 190
Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val
195 200 205
Pro Arg Leu Pro Pro Arg Glu Leu Gly Tyr Ser His Ser Ser Pro Glu
210 215 220
Tyr Trp Ile Thr Ser Gly Thr Leu Val Pro Val Thr Lys Asn Asp Ile
225 230 235 240
Val Lys Val Glu Gly Ile Asp Ser Thr Asp Gly Asn Asn Gln Pro Asn
245 250 255
Thr Pro Asp Ile Ala Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr
260 265 270
Cys Leu
<210> 5
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer fw
<400> 5
agtcgaattc tctccagtca gacgtgaggt t 31
<210> 6
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer rev
<400> 6
ttctctagat tacaaacaag taccaatcaa 30
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer E61C-fw
<400> 7
ctctactcgt tttgcgattc tggagtt 27
<210> 8
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer E61C-rev
<400> 8
aactccagaa tcgcaaaacg agtagag 27
<210> 9
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer N31C-fw
<400> 9
tgcgcgaaga actgcgatgc cccggca 27
<210> 10
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer E31C-rev
<400> 10
tgccggggca tcgcagttct tcgcgca 27
<210> 11
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer E61C-fw
<400> 11
ctctactcgt tttgcgattc tggagtt 27
<210> 12
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer E61C-rev
<400> 12
aactccagaa tcgcaaaacg agtagag 27
<210> 13
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer T69C-fw
<400> 13
gttggcgatg tctgcgggtt ccttgct 27
<210> 14
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer T69C-rev
<400> 14
agcaaggaac ccgcagacat cgccaac 27
<210> 15
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer I 108C-fw
<400> 15
ggaattgacg actgctgctc tggctgc 27
<210> 16
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer I 108C-rev
<400> 16
gcagccagag cagcagtcgt caattcc 27
<210> 17
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer A162C-fw
<400> 17
actgtggccg ggtgctctct gcgtgga 27
<210> 18
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer A162C-rev
<400> 18
tccacgcaga gagcacccgg ccacagt 27
<210> 19
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer G166C-fw
<400> 19
gcatctctgc gttgcaatgg gtacgat 27
<210> 20
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer G166C-rev
<400> 20
atcgtaccca ttgcaacgca gagatgc 27
<210> 21
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer G195C-fw
<400> 21
accgcacaga cctgcggcac cttgtac 27
<210> 22
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer G195C-rev
<400> 22
gtacaaggtg ccgcaggtct gtgcggt 27
<210> 23
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer G230C/V233C-fw
<400> 23
cacgtcttgc accctctgcc cagtg 25
<210> 24
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Primer G230C/V233C-rev
<400> 24
cactgggcag agggtgcaag acgtg 25
Claims (7)
1. the lipase mutant that a kind of thermal stability improves, it is characterised in that：The lipase mutant is lipase TTL increasing
Add the lipase mutant that disulfide bond obtains；Wherein, the nucleotide sequence of lipase TTL is as shown in SEQ ID NO.1, amino acid
Sequence is as shown in SEQ ID NO.2.
2. the lipase mutant that thermal stability according to claim 1 improves, it is characterised in that：The lipase is prominent
Variant is lipase mutant TTL-108/162, and amino acid sequence is as shown in SEQ ID NO.4.
3. the encoding gene for the lipase mutant that thermal stability described in claim 1 improves.
4. the encoding gene for the lipase mutant that thermal stability according to claim 3 improves, it is characterised in that：Its core
Nucleotide sequence is as shown in SEQ ID NO.3.
5. the recombinant vector containing the encoding gene of claim 3 or 4.
6. the recombinant bacterial strain containing the encoding gene of claim 3 or 4.
7. application of the lipase mutant that thermal stability of any of claims 1 or 2 improves in industrial circle.
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Cited By (3)
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|---|---|---|---|---|
| CN111909915A (en) * | 2020-07-28 | 2020-11-10 | 深圳润康生态环境股份有限公司 | Lipase gene, lipase and application thereof |
| WO2020239064A1 (en) * | 2019-05-31 | 2020-12-03 | 南京百斯杰生物工程有限公司 | Thermostable glucose oxidase |
| 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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| CN102839164A (en) * | 2012-09-06 | 2012-12-26 | 江南大学 | Disulfide bond reinforced folding based lipase mutant with high heat stability and construction method thereof |
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| CN107488647A (en) * | 2016-06-10 | 2017-12-19 | 广东溢多利生物科技股份有限公司 | The lipase TTL mutant TTL-Gly60Glu and its gene and application that a kind of heat endurance improves |
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| 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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| Publication number | Publication date |
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| CN108913675B (en) | 2020-05-22 |
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