CN107151660A - lipase variant Ala36Ser/Asp49His/Ala52Val/Asn55Asp - Google Patents
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- CN107151660A CN107151660A CN201710019846.3A CN201710019846A CN107151660A CN 107151660 A CN107151660 A CN 107151660A CN 201710019846 A CN201710019846 A CN 201710019846A CN 107151660 A CN107151660 A CN 107151660A
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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
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- 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)
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Abstract
The present invention relates to genetic engineering field, and in particular to lipase TTL mutant TTL Ala36Ser/Asp49His/Ala52Val/Asn55Asp and its gene and application that a kind of heat endurance is improved.The amino acid sequence of the lipase TTL mutant TTL Ala36Ser/Asp49His/Ala52Val/Asn55Asp is as shown in SEQ ID NO.4.Mutant remaining enzyme activity after 80 DEG C of water-bath 5min is 31.2%, is improved to 1.70 times of parent lipase.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to lipase mutant Ala36Ser/Asp49His/
Ala52Val/Asn55Asp and its gene and application.
Background technology
Lipase (EC 3.1.1.3) full name triacylglycerol acyl hydrolase, belongs to α/β and folds enzyme family, be a kind of silk ammonia
Sour hydrolase.Lipase can not only be catalyzed natural substrate grease hydrolysis on oil-water interfaces, discharge the glyceride of less ester bond
Or glycerine and aliphatic acid, and lipase can also be catalyzed the reaction such as acidolysis, transesterification and Lipase absobed in non-aqueous system.Industry is raised
Material is mainly derived from microorganism with lipase, and the lipase of microorganism secretion has wider action pH value and operative temperature.With
The continuous popularization of enzyme technology, the application conditions of enzyme preparation are also increasingly stricter, such as special bar of high temperature, strong acid and strong base
Part.Lipase is widely applied to food processing, feed, washing, medicine and other fields as a kind of important enzyme.Grease adds
The technique such as work and feed granulating need to generally be carried out at relatively high temperatures, the poor lipase of heat endurance easy in inactivation in above-mentioned technique
Denaturation, therefore exploitation high thermal stability lipase is always the target that academic and industrial circle is made great efforts.
In order to obtain more excellent enzyme preparation, on the one hand suitable enzyme gene can be screened from nature, another is by way of just
It is to be transformed the zymoprotein of existing industrialization by Protocols in Molecular Biology, to adapt to the demand of Different Industries.Change now
Making the strategy of zymoprotein mainly has two, one is nonideal explosives, the gene of enzyme are transformed by random mutation, further according to specific
Transformation purpose, filter out the zymoprotein for more meeting its action condition.This tactful advantage be need not further investigation enzyme structure and
The mechanism of action, but need great workload to go to complete to screen, efficiency is relatively low.
There is document report scientist from the southern thermophilic naked sufficient bacterium in isolated one plant of Volcanic Region in Tunisia, it secretes fat
Enzyme has remaining 50% enzyme activity after preferable heat resistance, 70 DEG C of processing 1h.Pichia pastoris condition of culture is simple, be easy to industrial metaplasia
Produce and efficient secretory expression foreign protein, the multiple outer lipase in a steady stream of successful expression.The present invention is efficiently divided using Pichia pastoris
The lipase for expressing thermophilic naked sufficient bacterium source is secreted, and template is based upon with thermophilic naked sufficient bacterium lipase, passes through site-directed mutagenesis technique
Obtain the lipase mutant that heat endurance is significantly improved.
The content of the invention
It is an object of the invention to provide the lipase TTL of high thermal stability.The high thermal stability lipase TTL, be
Rite-directed mutagenesis, the lipase mutant of acquisition are carried out by parent lipase TTL.For the table for the mutant for expressing the lipase
It is pPICZ α A and pPIC9K up to carrier；The host cell converted for the expression vector is Pichia pastoris X33 and GS115.Institute
Stating lipase mutant is：TTL-Ala36Ser/Asp49His/Ala52Val/Asn55Asp.
825 bases of lipase gene total length (SEQ ID No.1) of wild type and its 274 amino acid (SEQ of coding
ID No.2)
SEQ ID NO.1
TCTCCAGTCAGACGTGAGGTTTCTCAGGACTTGTTCGACCAGTTTAATTTGTTCGCTCAGTACTCTGCT
GCTGCCTACTGTGCCAAGAATAACGATGCCCCAGCTGGTGCTAACGTTACCTGTAGAGGTTCCATCTGCCCAGAGGT
TGAGAAGGCTGACGCCACCTTCTTGTACTCTTTCGAGGACTCTGGAGTTGGTGACGTTACCGGTTTCTTGGCTTTGG
ACAACACCAACAGATTGATCGTCTTGTCTTTCCGTGGTTCCCGTTCCCTTGAGAATTGGATCGGTAACATTAATTTG
GATTTGAAGGGTATCGACGACATCTGCTCTGGTTGCAAGGGACACGATGGTTTCACCTCCTCTTGGAGATCCGTTGC
CAACACCTTGACCCAACAGGTTCAGAACGCCGTTAGAGAGCATCCAGACTACCGTGTCGTCTTCACTGGTCACTCTT
TGGGTGGTGCTTTGGCTACCGTTGCTGGTGCCTCTTTGAGAGGTAACGGTTACGACATCGACGTTTTCTCCTACGGT
GCTCCTCGTGTTGGTAACAGAGCCTTCGCTGTCTTCTTGACCGCTCAGACCGGTGGTACCTTGTACAGAATCACCCA
TACCAACGATATCGTCCCACGTTTGCCACCAAGAGAGCTTGGATACTCCCACTCTTCCCCAGAGTACTGGATCACCT
CCGGTACTTTGGTTCCAGTTACCAAGAACGATATTGTTAAGGTTGAGGGAATTGACTCCACCGACGGTAACAACCAG
CCAAATACCCCAGACATTGCTGCCCACTTGTGGTACTTCGGTTTGATTGGTACTTGTTTGTAA
SEQ ID NO.2
SPVRREVSQDLFDQFNLFAQYSAAAYCAKNNDAPAGANVTCRGSICPEVEKADATFLYSFEDSGVGDVT
GFLALDNTNRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVV
FTGHSLGGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSP
EYWITSGTLVPVTKNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL
825 bases of lipase gene total length (as shown in nucleotide sequence SEQ ID NO.3) after mutation, what it was encoded
274 amino acid (as shown in amino acid sequence SEQ ID NO.4).
SEQ ID NO.3
TCTCCAGTCAGACGTGAGGTTTCTCAGGACTTGTTCGACCAGTTTAATTTGTTCACCCAGTACTCTGCT
GCTGCCTACTGTGCCAAGAATAACCACGCCCCAGTCGGTGCTGACGTTACCTGTAGAGGTTCTATCTGCCCAGAGGT
TGAGAAGGCTGACGCCACCTTCTTGTACTCTTTCGAGGACTCTGGAGTTGGTGACGTTACCGGTTTCTTGGCTTTGG
ACAACACCAACAGATTGATCGTCTTGTCTTTCCGTGGTTCCCGTTCCCTTGAGAATTGGATCGGTAACATTAATTTG
GATTTGAAGGGTATCGACGACATCTGCTCTGGTTGCAAGGGACACGATGGTTTCACCTCCTCTTGGAGATCCGTTGC
CAACACCTTGACCCAACAGGTTCAGAACGCCGTTAGAGAGCATCCAGACTACCGTGTCGTCTTCACTGGTCACTCTT
TGGGTGGTGCTTTGGCTACCGTTGCTGGTGCCTCTTTGAGAGGTAACGGTTACGACATCGACGTTTTCTCCTACGGT
GCTCCTCGTGTTGGTAACAGAGCCTTCGCTGTCTTCTTGACCGCTCAGACCGGTGGTACCTTGTACAGAATCACCCA
TACCAACGATATCGTCCCACGTTTGCCACCAAGAGAGCTTGGATACTCCCACTCTTCCCCAGAGTACTGGATCACCT
CCGGTACTTTGGTTCCAGTTACCAAGAACGATATTGTTAAGGTTGAGGGAATTGACTCCACCGACGGTAACAACCAG
CCAAATACCCCAGACATTGCTGCCCACTTGTGGTACTTCGGTTTGATTGGTACTTGTTTGTAA
SEQ ID NO.4
SPVRREVSQDLFDQFNLFAQYSAAAYCAKNNHAPVGADVTCRGSICPEVEKADATFLYSFEDSGVGDVT
GFLALDNTNRLIVLSFRGSRSLENWIGNINLDLKGIDDICSGCKGHDGFTSSWRSVANTLTQQVQNAVREHPDYRVV
FTGHSLGGALATVAGASLRGNGYDIDVFSYGAPRVGNRAFAEFLTAQTGGTLYRITHTNDIVPRLPPRELGYSHSSP
EYWITSGTLVPVTKNDIVKVEGIDSTDGNNQPNTPDIAAHLWYFGLIGTCL
Parent lipase TTL remaining enzyme activity after 80 DEG C of water-bath 5min is 18.3%.The present invention utilizes molecular biology skill
Art rite-directed mutagenesis lipase TTL, obtains the lipase mutant TTL-Ala36Ser/Asp49His/ of heat endurance raising
Ala52Val/Asn55Asp.Mutant TTL-Ala36Ser/Asp49His/Ala52Val/Asn55Asp is in 80 DEG C of water-bath 5min
Remaining enzyme activity is 31.2% afterwards, is improved to 1.70 times of parent lipase.
Brief description of the drawings
Fig. 1 α containing pPICz A-TTL Pichia pastoris X33 recombinant bacterium 50L tank fermentographs.
The protein electrophoresis figure of each period enzyme liquid of Fig. 2 fermentations.
Fig. 3 lipase TTL zymologic property figure.
The zymologic property figure of Fig. 4 lipase mutants
Embodiment
In order to increase the industrial application value of lipase, lipase gene TTL is carried out table by the present invention in Pichia pastoris
Reach, further study after this lipase three-dimensional structure, for the amino acid mutation of its key characteristic to promote its heat endurance.With
Under will be described the method for present invention transformation lipase and its lipase of resulting improvement.
Do not make the experimental methods of molecular biology illustrated, equal reference in following examples《Molecular Cloning:A Laboratory guide》
Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or is carried out according to kit and product description；
The reagent and biomaterial, unless otherwise specified, are commercially obtained.
Experiment material and reagent：
1st, bacterial strain and carrier
Coli strain Topl0, Pichia pastoris X33, GS115, carrier pPICz α A, Ppic9K, Zeocin are purchased from
Invitrogen companies.
2nd, enzyme and kit
PCR enzymes, restriction enzyme, plasmid extraction and gel purification kit are purchased from Shanghai Sheng Gong companies.
The high efficient expression of embodiment 1, lipase TTL in Pichia pastoris X33
1. it regard lipase gene TTL as target gene, 825 base (SEQ ID of lipase gene total length of wild type
No.1) and its coding 274 amino acid (SEQ ID No.2), after restricted enzyme cutting EcoRI and XbaI enzyme cutting, build
Expression vector pPICz α A-TTL are obtained into pPICz α A carriers, then pPICz α A-TTL are converted into the competent cells of Top 10, warp
After antibiotic Zeocin screenings, positive colony is obtained.Above-mentioned recombinant expression carrier is linearized with SacI, after linearisation
The electroporated Pichia pastoris X33 of recombinant vector, obtains Pichia pastoris recombinant bacterial strain transformant.Above-mentioned recombinant bacterium single bacterium colony is carried out
High density fermentation culture.Timing sampling during the fermentation determines enzyme activity, and the expression of lipase is as shown in figure 1, fermentation
Lipase activity is 31500U/mL during 189h.By the lipase in above-mentioned zymotic fluid by ammonium sulfate precipitation, fully thoroughly
Analysis, purified again by ion-exchange chromatography, protein liquid after purification is subjected to 12%SDS-PAGE detections, as a result such as Fig. 2.
2. lipase activity, which is determined, uses olive oil emulsion hydrolytic titration method, the definition of enzyme activity is：Lipase 40 DEG C,
Hydrolysis of Olive Oil emulsion produces the enzyme amount that 1 μm of ol/min aliphatic acid is consumed, as 1 lipase activity under the conditions of pH 7.0
Unit.Lipase heat treatment uses immersion method, is placed in after enzyme liquid is suitably diluted in teat glass, in different temperatures water-bath
Insulation 5min in (70 DEG C~90 DEG C).
Embodiment 2, lipase TTL zymologic property are determined
1. lipase TTL optimal reactive temperature and thermal stability determination
At 30 DEG C~80 DEG C, with 10 DEG C for interval, respectively determine lipase vigor, using the enzyme activity at 50 DEG C as pair
According to the relative enzyme activity under measure different temperatures.As a result such as Fig. 3 (a), the most suitable temperature of fatty enzyme effect is 50 DEG C.By fat
Enzyme enzyme liquid is placed in teat glass, and (70 DEG C~90 DEG C) heat treatment 5min, determine residual fat enzyme activity at different temperatures, with
Untreated enzyme activity is control, and the enzyme activity after heat treatment obtains remaining relative enzyme activity at this temperature by comparison, that is,.As a result
Such as Fig. 3 (b), it is 18.3% that 80 DEG C, which are heat-treated the remaining enzyme activity of lipase after 5min,.
2. lipase TTL optimal reaction pH and pH Stability Determination
The enzyme activity of lipase is determined respectively in the buffer solution system of different pH (3.0~9.0), with the enzyme activity of pH value 7.0
For control, the relative enzyme activity under different pH value is calculated.As a result such as Fig. 3 (c), the optimum pH of fatty enzyme effect is 7.0.To above-mentioned
The enzyme liquid diluted is separately added into different pH buffer solution system, room temperature treatment 2h determines residual fat enzyme activity, not locate
The enzyme activity of reason is control, calculates remaining relative enzyme activity under the pH.The pH stability ranges of measurement result such as Fig. 3 (d) enzymes compared with
Extensively, enzyme activity is stable in the range of pH5.0~9.0.
The lipase mutant TTL-Ala36Ser/Asp49His/Ala52Val/ that embodiment 3, heat endurance are improved
Asn55Asp
1st, using plasmid pPICZ α A-TTL-A36SD49HA52V as template, respectively with primers F 1, PIC-R and primer R1,
PIC-F enters performing PCR amplification, then will obtain 2 DNA fragmentations progress fusion DNA vaccines, obtains recombinant vector pPICZ α A-TTL-
A36SD49HA52VN55D.Above-mentioned recombinant vector is converted into the competent cells of Top 10, after being screened through antibiotic Zeocin, obtained
Positive colony.Above-mentioned recombinant expression carrier is linearized with SacI, the recombinant vector after linearisation is electroporated to finish red ferment
Female X33, obtains Pichia pastoris recombinant bacterial strain transformant.825 bases of lipase gene total length, thus derive 274 after mutation
Individual amino acid.
The primer is as follows：
F1:5’-CCAGCTGGTGCTGATGTTACCTGTAGAGGT-3’
R1:5’-CAGGTAACATCAGCACCAGCTGGGGCATC-3’
PIC-F：5’-CTTGCTTGAGAAGGTTTTGGGACGC-3’
PIC-R：5’-CTTGGAGCGAACGACCTACACCGAA-3’
2nd, the optimal reactive temperature and thermal stability determination of recombinant lipase
At 30 DEG C~80 DEG C, with 10 DEG C for interval, respectively determine lipase vigor, using the enzyme activity at 50 DEG C as pair
According to the relative enzyme activity under measure different temperatures.As a result such as Fig. 4 (a), the most suitable temperature of fatty enzyme effect is 50 DEG C.By fat
Enzyme enzyme liquid determines residual fat enzyme activity in (70 DEG C~90 DEG C) of different temperatures heat treatment 5min, using untreated enzyme activity as
Control, calculating obtains remaining relative enzyme activity at this temperature.As a result such as Fig. 4 (b), lipase residual enzyme after 80 DEG C of heat treatment 5min
Living is 31.2%.
3rd, optimal reaction pH and the pH Stability Determination of recombinant lipase
The enzyme activity of lipase is determined respectively in the buffer solution system of different pH (3.0~9.0), with the enzyme activity of pH value 7.0
For control, the relative enzyme activity under different pH value is determined.As a result as shown in Fig. 4 (c)：The optimum pH of the fatty enzyme effect is 7.0.
The enzyme liquid diluted is separately added into above-mentioned different pH buffer solution system, room temperature treatment 2h determines residual fat enzyme activity,
Using untreated enzyme activity as control, the enzyme activity of enzyme liquid obtains remaining relative enzyme activity under the pH by comparison, that is, after processing.Survey
Determine result such as Fig. 4 (d), the pH stability ranges of the enzyme are wider, enzyme activity is stable in the range of pH 5.0~9.0.
<110>GuangDong YiDuoLi Biology Science Co., Ltd
<120>Lipase Variant Ala36Ser/Asp49His/Ala52Val/Asn55Asp
<160>4
<210> 1
<211> 825
<212> DNA
<213>Artificial sequence
<400> 1
tctccagtca gacgtgaggt ttctcaggac ttgttcgacc agtttaattt gttcgctcag 60
tactctgctg ctgcctactg tgccaagaat aacgatgccc cagctggtgc taacgttacc 120
tgtagaggtt ccatctgccc agaggttgag aaggctgacg ccaccttctt gtactctttc 180
gaggactctg gagttggtga cgttaccggt ttcttggctt tggacaacac caacagattg 240
atcgtcttgt ctttccgtgg ttcccgttcc cttgagaatt ggatcggtaa cattaatttg 300
gatttgaagg gtatcgacga catctgctct ggttgcaagg gacacgatgg tttcacctcc 360
tcttggagat ccgttgccaa caccttgacc caacaggttc agaacgccgt tagagagcat 420
ccagactacc gtgtcgtctt cactggtcac tctttgggtg gtgctttggc taccgttgct 480
ggtgcctctt tgagaggtaa cggttacgac atcgacgttt tctcctacgg tgctcctcgt 540
gttggtaaca gagccttcgc tgtcttcttg accgctcaga ccggtggtac cttgtacaga 600
atcacccata ccaacgatat cgtcccacgt ttgccaccaa gagagcttgg atactcccac 660
tcttccccag agtactggat cacctccggt actttggttc cagttaccaa gaacgatatt 720
gttaaggttg agggaattga ctccaccgac ggtaacaacc agccaaatac cccagacatt 780
gctgcccact tgtggtactt cggtttgatt ggtacttgtt tgtaa 825
<210> 2
<211> 274
<212> PRT
<213>Artificial sequence
<400> 2
SPVRREVSQD LFDQFNLFAQ YSAAAYCAKN NDAPAGANVT CRGSICPEVE KADATFLYSF 60
EDSGVGDVTG FLALDNTNRL IVLSFRGSRS LENWIGNINL DLKGIDDICS GCKGHDGFTS 120
SWRSVANTLT QQVQNAVREH PDYRVVFTGH SLGGALATVA GASLRGNGYD IDVFSYGAPR 180
VGNRAFAEFL TAQTGGTLYR ITHTNDIVPR LPPRELGYSH SSPEYWITSG TLVPVTKNDI 240
VKVEGIDSTD GNNQPNTPDI AAHLWYFGLI GTCL 274
<210> 3
<211> 825
<212> DNA
<213>Artificial sequence
<400> 3
tctccagtca gacgtgaggt ttctcaggac ttgttcgacc agtttaattt gttcacccag 60
tactctgctg ctgcctactg tgccaagaat aaccacgccc cagtcggtgc tgacgttacc 120
tgtagaggtt ctatctgccc agaggttgag aaggctgacg ccaccttctt gtactctttc 180
gaggactctg gagttggtga cgttaccggt ttcttggctt tggacaacac caacagattg 240
atcgtcttgt ctttccgtgg ttcccgttcc cttgagaatt ggatcggtaa cattaatttg 300
gatttgaagg gtatcgacga catctgctct ggttgcaagg gacacgatgg tttcacctcc 360
tcttggagat ccgttgccaa caccttgacc caacaggttc agaacgccgt tagagagcat 420
ccagactacc gtgtcgtctt cactggtcac tctttgggtg gtgctttggc taccgttgct 480
ggtgcctctt tgagaggtaa cggttacgac atcgacgttt tctcctacgg tgctcctcgt 540
gttggtaaca gagccttcgc tgtcttcttg accgctcaga ccggtggtac cttgtacaga 600
atcacccata ccaacgatat cgtcccacgt ttgccaccaa gagagcttgg atactcccac 660
tcttccccag agtactggat cacctccggt actttggttc cagttaccaa gaacgatatt 720
gttaaggttg agggaattga ctccaccgac ggtaacaacc agccaaatac cccagacatt 780
gctgcccact tgtggtactt cggtttgatt ggtacttgtt tgtaa 825
<210> 4
<211> 274
<212> PRT
<213>Artificial sequence
<400> 4
SPVRREVSQD LFDQFNLFAQ YSAAAYCAKN NHAPVGADVT CRGSICPEVE KADATFLYSF 60
EDSGVGDVTG FLALDNTNRL IVLSFRGSRS LENWIGNINL DLKGIDDICS GCKGHDGFTS 120
SWRSVANTLT QQVQNAVREH PDYRVVFTGH SLGGALATVA GASLRGNGYD IDVFSYGAPR 180
VGNRAFAEFL TAQTGGTLYR ITHTNDIVPR LPPRELGYSH SSPEYWITSG TLVPVTKNDI 240
VKVEGIDSTD GNNQPNTPDI AAHLWYFGLI GTCL 274
Claims (5)
1. the lipase TTL mutant TTL-Ala36Ser/Asp49His/Ala52Val/Asn55Asp that heat endurance is improved, its
It is characterised by, the amino acid sequence of the lipase TTL mutant TTL-Ala36Ser/Asp49His/Ala52Val/Asn55Asp
Row are as shown in SEQ ID NO.4.
2. the lipase TTL mutant genes that heat endurance is improved, it is characterised in that thermally-stabilised described in coding claim 1
Property improve lipase TTL mutant TTL-Ala36Ser/Asp49His/Ala52Val/Asn55Asp.
3. the lipase TTL mutant genes that heat endurance according to claim 2 is improved, it is characterised in that its nucleosides
Acid sequence is as shown in SEQ ID NO.3.
4. the lipase TTL mutant TTL-Ala36Ser/Asp49His/ that the heat endurance described in claim 1 is improved
Ala52Val/Asn55Asp application.
5. the application for the lipase TTL mutant genes that the heat endurance described in claim 2 is improved.
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|---|---|---|---|
| CN201710019846.3A CN107151660B (en) | 2017-01-11 | 2017-01-11 | lipase variant Ala36Ser/Asp49His/Ala52Val/Asn55Asp |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047838A (en) * | 2016-06-07 | 2016-10-26 | 华南农业大学 | Heatproof mutation lipase with high catalytic activity as well as preparation method and application of heatproof mutation lipase |
| CN106085975A (en) * | 2016-06-16 | 2016-11-09 | 上海交通大学 | A kind of general policies of efficient raising enzyme thermodynamic stability |
-
2017
- 2017-01-11 CN CN201710019846.3A patent/CN107151660B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047838A (en) * | 2016-06-07 | 2016-10-26 | 华南农业大学 | Heatproof mutation lipase with high catalytic activity as well as preparation method and application of heatproof mutation lipase |
| CN106085975A (en) * | 2016-06-16 | 2016-11-09 | 上海交通大学 | A kind of general policies of efficient raising enzyme thermodynamic stability |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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