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CN107858337A - A kind of heat-resisting mutant lipase and preparation method and application - Google Patents

A kind of heat-resisting mutant lipase and preparation method and application Download PDF

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CN107858337A
CN107858337A CN201711220144.8A CN201711220144A CN107858337A CN 107858337 A CN107858337 A CN 107858337A CN 201711220144 A CN201711220144 A CN 201711220144A CN 107858337 A CN107858337 A CN 107858337A
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lipase
heat
disulfide bond
gly
val
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CN107858337B (en
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管武太
邓子潇
李力浪
吴炜坤
张世海
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South China Agricultural University
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    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

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Abstract

The invention discloses a kind of heat-resisting mutant lipase and preparation method and application.The heat-resisting mutant lipase is that the method combined in Yarrowia lipolytica lipase 2 by iteration introduces multipair disulfide bond mutation, the lipase 4s rich in the mutation of multipair disulfide bond obtained, lipase 5s and lipase 6s, wherein, lipase 4s amino acid sequence is as shown in SEQ ID NO.1；Lipase 5s amino acid sequence is as shown in SEQ ID NO.2；Lipase 6s amino acid sequence is as shown in SEQ ID NO.3.The present invention combines the strategy of multipair disulfide bond mutation using iteration, improve the heat endurance of lipase, compared with the strategy that tradition introduces disulfide bond, the heat endurance of medium temperature lipase is greatly improved in the number that disulfide bond is introduced by further increasing, and is particularly suitable for industrially applying.

Description

A kind of heat-resisting mutant lipase and preparation method and application
Technical field
The invention belongs to enzyme engineering field, more particularly to a kind of heat-resisting mutant lipase and preparation method and application.
Background technology
Traditional chemical reaction needs to carry out under conditions of HTHP, but catalyst is expensive, is not easily recycled, and instead Process is answered with a large amount of side reactions；Compared with chemical method, biological enzyme formulation catalytic reaction condition is gentle, species is various, price is low It is honest and clean.And biological enzyme formulation has the characteristics that Substratspezifitaet, the accessory substance of reaction generation is greatly reduced.Therefore, biology enzyme Preparation is widely used in industrial circle.As a kind of important biological enzyme formulation, Yarrowia lipolytica lipase 2 (Lip2) is A kind of lipase of function admirable, possess very high esterification, hydrolysis and transesterification activity, be widely applied to ester hydrolysis at present And the field such as synthesis, biodiesel preparation.But the hot heat resistance of the lipase is poor, it is unfavorable for producing and processing, store and transporting, It is the important bottleneck of production application, transforms the significant of its heat resistance.
Disulfide bond is that 2 sulfydryls are oxidized and the covalent bond between the sulphur atom of formation-S-S- forms.2 on peptide chain The mercapto groups of cysteine residues, oxidation reaction can occur and form disulfide bond；With the formation of disulfide bond, cysteine residues It is changed into cystine residue.For keeping protein active function to have extremely important influence, it matches somebody with somebody for the formation of disulfide bond It is the major reason of influence protein and peptide chain folding on correctness.Meanwhile many protein need disulfide bond to maintain it special Fixed higher structure.When disulfide bond, which is interrupted, is reduced to mercapto groups, protein structure will be likely occurred change, part or Original biological function is lost completely.Existing numerous studies obtain heat-resisting protein mutation by introducing the strategy of disulfide bond Body, such as lipase, amylase and mannase etc..But the disulfide bond number being generally artificially introduced is less, does not probe into multipair Influence of the disulfide bond to protein thermostability energy.
At present, to improve the research of its heat endurance to disulfide bond iterative evolution in document relatively fewer, therefore have very much must The iterative evolution rich in disulfide bond is carried out to it, inquires into its influence to heat endurance.
The content of the invention
The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, in Yarrowia lipolytica lipase 2 Multipair disulfide bond is introduced, to improve its heat resistance, and provides 3 kinds of thermostable lipases.
Another object of the present invention is to provide the preparation method of the heat-resisting mutant lipase.
It is still another object of the present invention to provide the application of the heat-resisting mutant lipase.
The purpose of the present invention is achieved through the following technical solutions：A kind of heat-resisting mutant lipase, it is to solve fat yeast in Ye Shi The method combined in lipase 2 by iteration introduces multipair disulfide bond and is mutated, and obtains the heat resistant grease rich in the mutation of multipair disulfide bond Fat enzyme.
Described disulfide bond includes disulfide bond S2-210, and (after mutation the 2nd and the 210th amino acids, two amino acid are formed Disulfide bond), S8-214 (mutation the 8th and the 214th amino acids after, two amino acids formed disulfide bond), S14-216 (dash forward After becoming the 14th and the 216th amino acids, two amino acids formed disulfide bond), S60-69 (mutation the 60th and the 69th bit amino After acid, two amino acids formed disulfide bond), (after mutation the 122nd and the 196th amino acids, two amino acid are formed S122-196 Disulfide bond) and S118-177 (mutation the 118th and the 177th amino acids after, two amino acids formed disulfide bond).
The multipair disulfide bond of described introducing is preferably to introduce 4~6 pairs of disulfide bond.
Described heat-resisting mutant lipase is preferably lipase 4s, lipase 5s or lipase 6s；
The method of described iteration combination is to combine disulfide bond S8-214, S60-69, S122-196 and S118-177 (4s), Combine disulfide bond S2-210, S8-214, S60-69, S122-196 and S118-177 (5s), or combination disulfide bond S2-210, S8- 214th, S14-216, S60-69, S122-196 and S118-177 (6s).
The amino acid sequence of described heat-resisting mutant lipase, it is as follows：
Lipase 4s amino acid sequence is as follows：
VYTSTETCHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFCDPRLIFDVCGYLAVDHA SKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTCTCDCCLVHNGFIQSYNNTYNQIGPKLDSVIEQYPDY QIAVTGHSLGGAAALLFGINLKCNGHDPLVVTLGQPIVGNACFANWVDKLFFGQENPDVCKVSKDRKLYRITHRGDI VPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI；
Lipase 5s amino acid sequence is as follows：
VCTSTETCHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFCDPRLIFDVCGYLAVDHA SKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTCTCDCCLVHNGFIQSYNNTYNQIGPKLDSVIEQYPDY QIAVTGHSLGGAAALLFGINLKCNGHDPLVVTLGQPIVGNACFANWVDKLFFGQECPDVCKVSKDRKLYRITHRGDI VPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI；
Lipase 6s amino acid sequence is as follows：
VCTSTETCHIDQECYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFCDPRLIFDVCGYLAVDHA SKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTCTCDCCLVHNGFIQSYNNTYNQIGPKLDSVIEQYPDY QIAVTGHSLGGAAALLFGINLKCNGHDPLVVTLGQPIVGNACFANWVDKLFFGQECPDVCKCSKDRKLYRITHRGDI VPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI。
The nucleotide sequence of the heat-resisting mutant lipase is encoded, it is as follows：
Lipase 4s nucleotide sequence is as follows：
gtgtacacctctaccgagacctgtcacattgaccaggagtcctacaacttctttgagaagtacgcccgactcgcaaa cattggatattgtgttggtcccggcactaagatcttcaagcccttcaactgtggcctgcaatgtgcccacttcccca acgttgagctcatcgaggagttctgtgacccccgtctcatctttgatgtttgtggttacctcgctgttgatcatgcc tccaagcagatctaccttgttattcgaggaacccactctctggaggacgtcataaccgacatccgaatcatgcaggc tcctctgacgaactttgatcttgctgctaacatctcttctacttgtacttgtgattgttgtcttgtccacaatggct tcatccagtcctacaacaacacctacaatcagatcggccccaagctcgactctgtgattgagcagtatcccgactac cagattgctgtcaccggtcactctctcggaggagctgcagcccttctgttcggaatcaacctcaagtgtaacggcca cgatcccctcgttgttactcttggtcagcccattgtcggtaacgcttgttttgctaactgggtcgataaactcttct ttggccaggagaaccccgatgtctgtaaggtgtccaaagaccgaaagctctaccgaatcacccaccgaggagatatc gtccctcaagtgcccttctgggacggttaccagcactgctctggtgaggtctttattgactggcccctgatccaccc tcctctctccaacgttgtcatgtgccagggccagagcaataaacagtgctctgccggtaacactctgctccagcagg tcaatgtgattggaaaccatctgcagtacttcgtcaccgagggtgtctgtggtatctaataa；
Lipase 5s nucleotide sequence is as follows：
gtgtgtacctctaccgagacctgtcacattgaccaggagtcctacaacttctttgagaagtacgcccgactcgcaaa cattggatattgtgttggtcccggcactaagatcttcaagcccttcaactgtggcctgcaatgtgcccacttcccca acgttgagctcatcgaggagttctgtgacccccgtctcatctttgatgtttgtggttacctcgctgttgatcatgcc tccaagcagatctaccttgttattcgaggaacccactctctggaggacgtcataaccgacatccgaatcatgcaggc tcctctgacgaactttgatcttgctgctaacatctcttctacttgtacttgtgattgttgtcttgtccacaatggct tcatccagtcctacaacaacacctacaatcagatcggccccaagctcgactctgtgattgagcagtatcccgactac cagattgctgtcaccggtcactctctcggaggagctgcagcccttctgttcggaatcaacctcaagtgtaacggcca cgatcccctcgttgttactcttggtcagcccattgtcggtaacgcttgttttgctaactgggtcgataaactcttct ttggccaggagtgtcccgatgtctgtaaggtgtccaaagaccgaaagctctaccgaatcacccaccgaggagatatc gtccctcaagtgcccttctgggacggttaccagcactgctctggtgaggtctttattgactggcccctgatccaccc tcctctctccaacgttgtcatgtgccagggccagagcaataaacagtgctctgccggtaacactctgctccagcagg tcaatgtgattggaaaccatctgcagtacttcgtcaccgagggtgtctgtggtatctaataa；
Lipase 6s nucleotide sequence is as follows：
gtgtgtacctctaccgagacctgtcacattgaccaggagtgttacaacttctttgagaagtacgcccgactcgcaaa cattggatattgtgttggtcccggcactaagatcttcaagcccttcaactgtggcctgcaatgtgcccacttcccca acgttgagctcatcgaggagttctgtgacccccgtctcatctttgatgtttgtggttacctcgctgttgatcatgcc tccaagcagatctaccttgttattcgaggaacccactctctggaggacgtcataaccgacatccgaatcatgcaggc tcctctgacgaactttgatcttgctgctaacatctcttctacttgtacttgtgattgttgtcttgtccacaatggct tcatccagtcctacaacaacacctacaatcagatcggccccaagctcgactctgtgattgagcagtatcccgactac cagattgctgtcaccggtcactctctcggaggagctgcagcccttctgttcggaatcaacctcaagtgtaacggcca cgatcccctcgttgttactcttggtcagcccattgtcggtaacgcttgttttgctaactgggtcgataaactcttct ttggccaggagtgtcccgatgtctgtaagtgttccaaagaccgaaagctctaccgaatcacccaccgaggagatatc gtccctcaagtgcccttctgggacggttaccagcactgctctggtgaggtctttattgactggcccctgatccaccc tcctctctccaacgttgtcatgtgccagggccagagcaataaacagtgctctgccggtaacactctgctccagcagg tcaatgtgattggaaaccatctgcagtacttcgtcaccgagggtgtctgtggtatctaataa。
The preparation method of described heat-resisting mutant lipase, comprises the following steps：
(1) using pPICZ α A-Lip2 as template, it is mutated by inverse PCR, selected amino acid sites is sported into half Guang Propylhomoserin, and be transferred in Escherichia coli, amplification cultivation, plasmid extraction and sequencing are carried out, obtains and introduces four pairs, five pairs or six pair two sulphur The mutant lipase recombinant plasmid of key；
(2) correct recombinant plasmid will be sequenced with the restriction enzyme linearization process of Pme I, and it is electroporated to experiencing In state Pichia pastoris X33, further by YPDS-Zeocin plate screenings, corresponding mutation engineering bacteria is obtained；
(3) mutation engineering bacteria is carried out in YPD fluid nutrient mediums after expanding numerous culture, is forwarded to BMGY fluid nutrient mediums and enters Capable culture of disinthibiting, finally it is inoculated with BMMY fluid nutrient mediums and is fermented, bacterium solution is centrifuged and obtains supernatant crude enzyme liquid；
(4) after crude enzyme liquid is concentrated by ultrafiltration using super filter tube, purified using nickel post one-step method, isolate band histidine The Lipase protein of label, and purity of protein is detected with reproducibility SDS-PAGE, lipase after purification is obtained, i.e., heat-resisting mutation Lipase.
Four couple of introducing described in step (1), five pairs and six pairs of disulfide bond be respectively disulfide bond S8-214, S60-69, S122-196 and S118-177, disulfide bond S2-210, S8-214, S60-69, S122-196 and S118-177, disulfide bond S2- 210th, S8-214, S14-216, S60-69, S122-196 and S118-177.
Colibacillus engineering described in step (1) is preferably Escherichia coli TOP10.
Zeocin concentration is preferably 100 μ g/mL in YPDS-Zeocin flat boards described in step (2).
The time of fermentation described in step (3) is preferably 96h.
Super filter tube described in step (4) is preferably 10kDa super filter tubes.
Mutant lipase described in step (4) is lipase 4s, lipase 5s or lipase 6s.
The preparation method of described heat-resisting mutant lipase, also comprises the following steps：
(5) melting temperature (T of lipase is determined by DSF fluoroscopic examinationsm), p-NPP colorimetric method for determining 15min half is inactivated Temperature (T50), the half-life period (t at 55 or 70 DEG C1/2), optimal reactive temperature (Topt), optimal reaction pH (pHopt) and/or pH it is steady It is qualitative.
Described heat resistant grease mutation fat enzyme heat stability is strong, acid and alkali-resistance, is particularly suitable for industrially being applied.
The present invention is had the following advantages relative to prior art and effect：
1st, the present invention is filtering out the single pair disulfide bond that can improve Lip2 heat resistances, disulfide bond S2-210, S8-214, S14-216, S60-69, S122-196 and S118-177 (make the T of lipasemValue is respectively increased 10.48,7.50,5.70,5.97, 4.55 and 3.33 DEG C) on the basis of, the strategy of multipair disulfide bond mutation is combined using iteration, improves the heat endurance of lipase. Compared with the strategy that tradition introduces disulfide bond, medium temperature fat is greatly improved in the number that disulfide bond is introduced by further increasing The heat endurance of fat enzyme.
2nd, the present invention obtains thermostable lipase 4s, fat by four pairs, the five pairs and six pairs disulfide bond of iteration combination in Lip2 Fat enzyme 5s and lipase 6s.Relative to parent lipase (Lip2), lipase 4s, lipase 5s and lipase 6s TmValue difference 16.45,20.23 and 22.53 DEG C are improved, its T50Value lifts 27.49,29.83 and 31.23 DEG C respectively, and it partly declines at 70 DEG C Phase is up to 11.95,24.76 and 101.93min respectively, greatly improves the heat-resisting of lipase 4s, lipase 5s and lipase 6s Performance.
Brief description of the drawings
Fig. 1 is Lip2, lipase 4s, lipase 5s and lipase 6s optimal reaction pH curve maps.
Fig. 2 is Lip2, lipase 4s, lipase 5s and lipase 6s pH stability curve figures.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.
Material and reagent：PPICZ α A-Lip2 yeast expression vectors are synthesized by Shanghai Jin Sirui biotech firms full genome With structure；Plasmid extraction kit is purchased from Omega trade Co., Ltds；KOD-PLUS mutagenesis kits spin company purchased from Japan； Protein Thermal Shift screening reagents box is purchased from Thermo companies；TOP10 competent escherichia coli cells are purchased from Tiangeng Biotech firm；Mutant primer is synthesized by Shanghai Sheng Gong bio-engineering corporations；The restriction enzymes of Pme I are purchased from New England Biolabs companies；PCR primer purifying QIAquick Gel Extraction Kit is purchased from Dalian treasured biotech firm；Electroporation is purchased from Bio-Rad companies； LLB, LLB+Zeocin, YPD, YPD+Zeocin, BMGY, BMMY culture medium are according to Invitrogen Pichia anomala expression reagents Box operation manual is prepared, and ni-sepharose purification kit is purchased from Shanghai Sheng Gong bio-engineering corporations, and remaining reagent is domestic and international purchase The pure rank of analysis.
Embodiment 1：The structure of mutant lipase expression plasmid
Using pPICZ α A-Lip2 as template, (" a kind of heat is steady with reference to Chinese patent application 201610279266.3 for construction method Fixed lipase and preparation method and application ") and table 1 in primer be mutated, obtain 3 sections of mutating acids.Wherein, 3 sections of ammonia Base acid sequence is as follows：
Lipase 4s amino acid sequence is as follows：
VYTSTETCHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFCDPRLIFDVCGYLAVDHA SKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTCTCDCCLVHNGFIQSYNNTYNQIGPKLDSVIEQYPDY QIAVTGHSLGGAAALLFGINLKCNGHDPLVVTLGQPIVGNACFANWVDKLFFGQENPDVCKVSKDRKLYRITHRGDI VPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI；
Lipase 5s amino acid sequence is as follows：
VCTSTETCHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFCDPRLIFDVCGYLAVDHA SKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTCTCDCCLVHNGFIQSYNNTYNQIGPKLDSVIEQYPDY QIAVTGHSLGGAAALLFGINLKCNGHDPLVVTLGQPIVGNACFANWVDKLFFGQECPDVCKVSKDRKLYRITHRGDI VPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI；
Lipase 6s amino acid sequence is as follows：
VCTSTETCHIDQECYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFCDPRLIFDVCGYLAVDHA SKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTCTCDCCLVHNGFIQSYNNTYNQIGPKLDSVIEQYPDY QIAVTGHSLGGAAALLFGINLKCNGHDPLVVTLGQPIVGNACFANWVDKLFFGQECPDVCKCSKDRKLYRITHRGDI VPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI。
The nucleotide sequence of the thermostable lipase is encoded, it is as follows：
Lipase 4s nucleotide sequence is as follows：
gtgtacacctctaccgagacctgtcacattgaccaggagtcctacaacttctttgagaagtacgcccgactcgcaaa cattggatattgtgttggtcccggcactaagatcttcaagcccttcaactgtggcctgcaatgtgcccacttcccca acgttgagctcatcgaggagttctgtgacccccgtctcatctttgatgtttgtggttacctcgctgttgatcatgcc tccaagcagatctaccttgttattcgaggaacccactctctggaggacgtcataaccgacatccgaatcatgcaggc tcctctgacgaactttgatcttgctgctaacatctcttctacttgtacttgtgattgttgtcttgtccacaatggct tcatccagtcctacaacaacacctacaatcagatcggccccaagctcgactctgtgattgagcagtatcccgactac cagattgctgtcaccggtcactctctcggaggagctgcagcccttctgttcggaatcaacctcaagtgtaacggcca cgatcccctcgttgttactcttggtcagcccattgtcggtaacgcttgttttgctaactgggtcgataaactcttct ttggccaggagaaccccgatgtctgtaaggtgtccaaagaccgaaagctctaccgaatcacccaccgaggagatatc gtccctcaagtgcccttctgggacggttaccagcactgctctggtgaggtctttattgactggcccctgatccaccc tcctctctccaacgttgtcatgtgccagggccagagcaataaacagtgctctgccggtaacactctgctccagcagg tcaatgtgattggaaaccatctgcagtacttcgtcaccgagggtgtctgtggtatctaataa；
Lipase 5s nucleotide sequence is as follows：
gtgtgtacctctaccgagacctgtcacattgaccaggagtcctacaacttctttgagaagtacgcccgactcgcaaa cattggatattgtgttggtcccggcactaagatcttcaagcccttcaactgtggcctgcaatgtgcccacttcccca acgttgagctcatcgaggagttctgtgacccccgtctcatctttgatgtttgtggttacctcgctgttgatcatgcc tccaagcagatctaccttgttattcgaggaacccactctctggaggacgtcataaccgacatccgaatcatgcaggc tcctctgacgaactttgatcttgctgctaacatctcttctacttgtacttgtgattgttgtcttgtccacaatggct tcatccagtcctacaacaacacctacaatcagatcggccccaagctcgactctgtgattgagcagtatcccgactac cagattgctgtcaccggtcactctctcggaggagctgcagcccttctgttcggaatcaacctcaagtgtaacggcca cgatcccctcgttgttactcttggtcagcccattgtcggtaacgcttgttttgctaactgggtcgataaactcttct ttggccaggagtgtcccgatgtctgtaaggtgtccaaagaccgaaagctctaccgaatcacccaccgaggagatatc gtccctcaagtgcccttctgggacggttaccagcactgctctggtgaggtctttattgactggcccctgatccaccc tcctctctccaacgttgtcatgtgccagggccagagcaataaacagtgctctgccggtaacactctgctccagcagg tcaatgtgattggaaaccatctgcagtacttcgtcaccgagggtgtctgtggtatctaataa；
Lipase 6s nucleotide sequence is as follows：
gtgtgtacctctaccgagacctgtcacattgaccaggagtgttacaacttctttgagaagtacgcccgactcgcaaa cattggatattgtgttggtcccggcactaagatcttcaagcccttcaactgtggcctgcaatgtgcccacttcccca acgttgagctcatcgaggagttctgtgacccccgtctcatctttgatgtttgtggttacctcgctgttgatcatgcc tccaagcagatctaccttgttattcgaggaacccactctctggaggacgtcataaccgacatccgaatcatgcaggc tcctctgacgaactttgatcttgctgctaacatctcttctacttgtacttgtgattgttgtcttgtccacaatggct tcatccagtcctacaacaacacctacaatcagatcggccccaagctcgactctgtgattgagcagtatcccgactac cagattgctgtcaccggtcactctctcggaggagctgcagcccttctgttcggaatcaacctcaagtgtaacggcca cgatcccctcgttgttactcttggtcagcccattgtcggtaacgcttgttttgctaactgggtcgataaactcttct ttggccaggagtgtcccgatgtctgtaagtgttccaaagaccgaaagctctaccgaatcacccaccgaggagatatc gtccctcaagtgcccttctgggacggttaccagcactgctctggtgaggtctttattgactggcccctgatccaccc tcctctctccaacgttgtcatgtgccagggccagagcaataaacagtgctctgccggtaacactctgctccagcagg tcaatgtgattggaaaccatctgcagtacttcgtcaccgagggtgtctgtggtatctaataa。
Inverse PCR is carried out by template of pPICZ α A-Lip2, often introducing a pair of disulfide bond needs to carry out inverse PCR twice. Due to disulfide bond S14-216 and S2-210, S8-214 be between two sections of loop (the 1st amino acids to the 14th amino acids and 207th amino acids to 221 amino acids), and disulfide bond S14-216 and S2-210 be from activity center's relative close, so choosing Select four disulfide bond of S8-214, S60-69, S122-196 and S118-177 as 4s, S2-210, S8-214, S60-69, Five disulfide bond of the S122-196 and S118-177 as 5s, S2-210, S8-214, S14-216, S60-69, S122-196 and Six disulfide bond of the S118-177 as 6s.Using the 122C-F of table 1 and 122-R as primer, obtain lipase 4s and expand for the first time Increase primer.
The mutant primer of table 1 collects
Note：It is mutational site at oblique line overstriking
PCR amplification conditions are：94℃2min；94 DEG C of 10s, 66 DEG C of 30s, 68 DEG C of 5min, 10 circulations.Reaction system is as follows Shown in table 2.
Table 2PCR reaction systems
Sense primer (10 μM) 1.5μL
Anti-sense primer (10 μM) 1.5μL
KOD-Plus high-fidelity enzymes 1μL
Template (50ng/ μ L) 1μL
Distilled water 35μL
5×SmartPCR buffer 5μL
5×dNTP(2M) 5μL
Total system 50μL
Amplified production after agarose gel electrophoresis detects mutation stripe size, is connected with the enzymic digestion templates of Dnp I with T4 Mutant plasmid overnight, is then transferred to TOP10 competent escherichia coli cells, and be coated on by enzyme connection cyclisation using heat shock method LLB+Zeocin (Zeocin concentration is 25 μ g/ml) 37 DEG C of flat board is incubated overnight, and selects the sequencing that positive transformant carries out plasmid.
It is overnight in LLB+Zeocin (Zeocin concentration is 25 μ g/ml) fluid nutrient medium that correct positive transformant will be sequenced After spreading cultivation, plasmid is extracted.Using the plasmid as template, after the same method, successively with the primer 196C-F and 196C-R of table 1, 196+118C-F and 196+118C-R, 177C-F and 177C-R, 8C-F and 8C-R, 214C-F and 214C-R, 60C-F and 60C-R, 69C-F and 69C-R carries out 7 mutation, obtains lipase 4s recombinant plasmid.
Using lipase 4s plasmid as template, successively with the primer 2 10+8C-F and 210+8C-R, 210+214C-F of table 1 and 210+214C-R carries out 2 mutation, obtains lipase 5s recombinant plasmid.
Using lipase 5s plasmid as template, successively with the primer 2 10+214+14C-F and 210+214+14C-R of table 1, 210+214+216C-F and 210+214+216C-R carries out 2 mutation, obtains lipase 6s recombinant plasmid.
Embodiment 2：Linearization plasmid electricity conversion Pichia pastoris, transformant screening and producing enzyme are screened
It is overnight in LLB+Zeocin (Zeocin concentration is 25 μ g/ml) fluid nutrient medium that correct positive transformant will be sequenced After amplification cultivation, extract plasmid, with the linearization process of Pme I and purify recovery, using total amount as 5 μ g plasmid linearization product and The mixing of X33 Pichia pastoris competence is electroporated.Pichia pastoris competence is prepared with reference to Invitrogen companies operation manual.Electricity Carryover sequence is set according to Bio-Rad companies recommended parameter.
Electricity turns to finish to be added immediately 1mL 1mol/L sorbitol solutions, by bacterium solution after 30 DEG C are incubated recovery 1 hour, uniformly It is coated in YPDS+Zeocin (Zeocin concentration is 100 μ g/ml) resistant panel and screens.
Embodiment 3：The fermentation of recombinant strain
With reference to Invitrogen companies Pichia anomala expression kit operation manual and it is modified slightly, modification content is as follows： Engineered strain single bacterium colony is inoculated into 2mL YPDS-Zeocin (Zeocin concentration is 100 μ g/mL) fluid nutrient medium carry out it is pure Change overnight incubation, centrifuge and cell is resuspended and is incubated overnight with BMGY fluid nutrient mediums, inoculate to 50mL BMMY Liquid Cultures Base, with 25 DEG C, 280r/min culture 96h, methanol is supplemented daily to final concentration of 1% (v/v).
Embodiment 4：The separation and purifying of lipase
1) it is concentrated by ultrafiltration
By 100mL zymotic fluids after 4 DEG C of 5000r/min are centrifuged 5 minutes Aspirate supernatant and with 10kDa super filter tubes in 4 DEG C of centrifugation 50min of 5000r/min, collect concentration enzyme liquid.
2) one-step method ni-sepharose purification
1. the Binding Buffer balance nickel posts of the 10mM containing imidazoles with 5mL, fully remove the ethanol of residual；
2. enzyme liquid is concentrated with the Binding Buffer of the 120mM containing imidazoles according to 1:It is added to after the mixing of 1 ratio in nickel post With reference to；
3. fully elute foreign protein with 15mL 60mM containing imidazoles Washing buffer；
4. elute target protein with 15mL 300mM containing imidazoles Elution Buffer；
5. purifying enzyme liquid is concentrated by ultrafiltration according to above-mentioned condition；
The mutant lipase purified, finally detects enzyme purity with reproducibility SDS-PAGE vertical electrophoresis, and purity exists More than 90%.
Embodiment 5：The zymologic property measure of lipase.
Using quantitative real time PCR Instrument, it is mutated according to Protein Thermal Shift kit recommendation responses program determination The T of lipasemValue, as a result as shown in table 3, the very big amplitude of multipair disulfide bond of introducing improves the T of lipasem, 6s TmValue compared with 4s and 5s are further improved.
Table 3DSF measurement results
Lipase Tm ΔTm
Lip2 48.32±0.25 -
4s 64.77±0.10 16.45
5s 68.55±0.15 20.23
6s 70.85±0.01 22.53
T50Assay method：With 0.1mg/mL purifying protein solution in PCR instrument respectively with 30,35,40,45,50,55, 60th, 65,70,75,80 DEG C of accurate insulation 15min, using p-NPPs of the 50mM containing 40mM Tris-Hcl buffer solutions as reaction system (pH=7.50), accurately after reaction 10min, 20% (w/v) trichloroacetic acid terminating reaction 5min is added, 20% (w/v's) Sodium carbonate liquor develops the color, and determines light absorption value at 410nm, after calculating different temperatures insulation, the remaining relative enzyme activity of lipase.
t1/2Assay method：With 0.1mg/mL purifying protein solution in PCR instrument 55 or 70 DEG C of accurate insulations 0,5,10, 15th, 30,45,60min is accurate anti-using p-NPP of the 50mM containing 40mM Tris-Hcl buffer solutions as reaction system (pH=7.50) After answering 10min, 20% (w/v) trichloroacetic acid terminating reaction is added, the colour developing of 20% (w/v) sodium carbonate liquor, is determined at 410nm Light absorption value, after calculating different temperatures insulation, lipase residual activity.
ToptAssay method：Added into 0.15mg/mL purifying protein solution 30,35,40,45,50,55,60,65 With 70 DEG C under the conditions of preheat after p-NPPs of the 50mM containing 40mM Tris-HCl buffer solutions (pH=7.50), accurately react 10min Afterwards, 20% (w/v) trichloroacetic acid terminating reaction is added, the colour developing of 20% (w/v) sodium carbonate liquor, determines light absorption value at 410nm, Calculate the relative enzyme activity of lipase under different temperatures.
pHoptThe measure of value：P-NPP standard mother liquors be added to pH be respectively 2.0,3.0,4.0,5.0,6.0,6.5, 7.0th, 7.5 and 8.0 citrate-phosphate sodium dihydrogen buffer solution (table 4), the 5 μ L 0.1mg/mL enzyme liquids purified are added thereto, with Colorimetric method determines residual activities in 30 DEG C of accurate reaction 10min, the enzyme activity under optimal reaction pH is defined as into 100%, with pH pairs Relative activity is mapped, and is obtained it and is determined curve.
The measure of pH stability：With distilled water the concentration dilution of enzyme liquid to 1mg/mL, then with pH be 2.0,3.0,4.0, 5.0th, 6.0,7.0,8.0,9.0,10.0 and 11.0 buffer solution dilutes enzyme liquid to final concentration of 0.1mg/ml；Wherein pH 2.0~ 8.0 scopes use citrate-phosphate sodium dihydrogen buffer solution (table 4), and the scope of pH9.0~11.0 is delayed using glycine-sodium hydroxide Fliud flushing (formula refers to table 5).24h is incubated at 25 DEG C, accurately reaction 10min measure lipase is remaining at 30 DEG C with colorimetric method Activity.
4 disodium hydrogen phosphates of table-citrate buffer solution (25 DEG C)
Note：Na2HPO4Molecular weight=141.98,0.2mol/L solution is 28.40g/L；With Na2HPO4·2H2O is prepared Na2HPO4Solution, Na2HPO4·2H2O molecular weight=178.05,0.2mol/L solution is 35.61g/L；Citric acid molecule amount= 210.14,0.1mol/L solution are 21.01g/L.
5 glycine of table-sodium hydrate buffer solution (0.05mol/L)
pH x/ml y/ml pH x/ml y/ml
8.6 50 4.0 9.6 50 22.4
8.8 50 6.0 9.8 50 27.2
9.0 50 8.8 10.0 50 32.0
9.2 50 12.0 10.4 50 38.6
9.4 50 16.8 10.6 50 45.5
Note：X ml 0.2mol/L glycine+y ml 0.2mol/L NaOH, are diluted with water to 200mL；Glycine molecule It is 15.01g/L to measure=75.07,0.2mol/L glycine solutions.
T50、t1/2And ToptAs shown in table 6, pHopT and pH stability difference is as depicted in figs. 1 and 2:
The thermal stability determination result of table 6
T50(℃) t1/2(min) Topt(℃)
Lip2 40.86 0.34 35
4s 68.35 11.95 40
5s 70.69 24.76 55
6s 72.06 101.93 55
Note：Lip2 measurement temperatures are 55 DEG C, and remaining is 70 DEG C.
It can be seen that lipase 4s, lipase 5s and lipase 6s heat endurance significantly increase, T50Significantly carry It is high.Lipase 4s optimal reactive temperature is promoted to 40 DEG C, and lipase 5s and lipase 6s is promoted to 55 DEG C.And fat Enzyme 5s and lipase 6s has longer half-life period at 70 DEG C, and its pH reaction interval is able to widen and acidproof alkali ability is increased By force.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>Agricultural University Of South China
<120>A kind of heat-resisting mutant lipase and preparation method and application
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 301
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase 4s amino acid sequence
<400> 1
Val Tyr Thr Ser Thr Glu Thr Cys His Ile Asp Gln Glu Ser Tyr Asn
1 5 10 15
Phe Phe Glu Lys Tyr Ala Arg Leu Ala Asn Ile Gly Tyr Cys Val Gly
20 25 30
Pro Gly Thr Lys Ile Phe Lys Pro Phe Asn Cys Gly Leu Gln Cys Ala
35 40 45
His Phe Pro Asn Val Glu Leu Ile Glu Glu Phe Cys Asp Pro Arg Leu
50 55 60
Ile Phe Asp Val Cys Gly Tyr Leu Ala Val Asp His Ala Ser Lys Gln
65 70 75 80
Ile Tyr Leu Val Ile Arg Gly Thr His Ser Leu Glu Asp Val Ile Thr
85 90 95
Asp Ile Arg Ile Met Gln Ala Pro Leu Thr Asn Phe Asp Leu Ala Ala
100 105 110
Asn Ile Ser Ser Thr Cys Thr Cys Asp Cys Cys Leu Val His Asn Gly
115 120 125
Phe Ile Gln Ser Tyr Asn Asn Thr Tyr Asn Gln Ile Gly Pro Lys Leu
130 135 140
Asp Ser Val Ile Glu Gln Tyr Pro Asp Tyr Gln Ile Ala Val Thr Gly
145 150 155 160
His Ser Leu Gly Gly Ala Ala Ala Leu Leu Phe Gly Ile Asn Leu Lys
165 170 175
Cys Asn Gly His Asp Pro Leu Val Val Thr Leu Gly Gln Pro Ile Val
180 185 190
Gly Asn Ala Cys Phe Ala Asn Trp Val Asp Lys Leu Phe Phe Gly Gln
195 200 205
Glu Asn Pro Asp Val Cys Lys Val Ser Lys Asp Arg Lys Leu Tyr Arg
210 215 220
Ile Thr His Arg Gly Asp Ile Val Pro Gln Val Pro Phe Trp Asp Gly
225 230 235 240
Tyr Gln His Cys Ser Gly Glu Val Phe Ile Asp Trp Pro Leu Ile His
245 250 255
Pro Pro Leu Ser Asn Val Val Met Cys Gln Gly Gln Ser Asn Lys Gln
260 265 270
Cys Ser Ala Gly Asn Thr Leu Leu Gln Gln Val Asn Val Ile Gly Asn
275 280 285
His Leu Gln Tyr Phe Val Thr Glu Gly Val Cys Gly Ile
290 295 300
<210> 2
<211> 301
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase 5s amino acid sequence
<400> 2
Val Cys Thr Ser Thr Glu Thr Cys His Ile Asp Gln Glu Ser Tyr Asn
1 5 10 15
Phe Phe Glu Lys Tyr Ala Arg Leu Ala Asn Ile Gly Tyr Cys Val Gly
20 25 30
Pro Gly Thr Lys Ile Phe Lys Pro Phe Asn Cys Gly Leu Gln Cys Ala
35 40 45
His Phe Pro Asn Val Glu Leu Ile Glu Glu Phe Cys Asp Pro Arg Leu
50 55 60
Ile Phe Asp Val Cys Gly Tyr Leu Ala Val Asp His Ala Ser Lys Gln
65 70 75 80
Ile Tyr Leu Val Ile Arg Gly Thr His Ser Leu Glu Asp Val Ile Thr
85 90 95
Asp Ile Arg Ile Met Gln Ala Pro Leu Thr Asn Phe Asp Leu Ala Ala
100 105 110
Asn Ile Ser Ser Thr Cys Thr Cys Asp Cys Cys Leu Val His Asn Gly
115 120 125
Phe Ile Gln Ser Tyr Asn Asn Thr Tyr Asn Gln Ile Gly Pro Lys Leu
130 135 140
Asp Ser Val Ile Glu Gln Tyr Pro Asp Tyr Gln Ile Ala Val Thr Gly
145 150 155 160
His Ser Leu Gly Gly Ala Ala Ala Leu Leu Phe Gly Ile Asn Leu Lys
165 170 175
Cys Asn Gly His Asp Pro Leu Val Val Thr Leu Gly Gln Pro Ile Val
180 185 190
Gly Asn Ala Cys Phe Ala Asn Trp Val Asp Lys Leu Phe Phe Gly Gln
195 200 205
Glu Cys Pro Asp Val Cys Lys Val Ser Lys Asp Arg Lys Leu Tyr Arg
210 215 220
Ile Thr His Arg Gly Asp Ile Val Pro Gln Val Pro Phe Trp Asp Gly
225 230 235 240
Tyr Gln His Cys Ser Gly Glu Val Phe Ile Asp Trp Pro Leu Ile His
245 250 255
Pro Pro Leu Ser Asn Val Val Met Cys Gln Gly Gln Ser Asn Lys Gln
260 265 270
Cys Ser Ala Gly Asn Thr Leu Leu Gln Gln Val Asn Val Ile Gly Asn
275 280 285
His Leu Gln Tyr Phe Val Thr Glu Gly Val Cys Gly Ile
290 295 300
<210> 3
<211> 301
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase 6s amino acid sequence
<400> 3
Val Cys Thr Ser Thr Glu Thr Cys His Ile Asp Gln Glu Cys Tyr Asn
1 5 10 15
Phe Phe Glu Lys Tyr Ala Arg Leu Ala Asn Ile Gly Tyr Cys Val Gly
20 25 30
Pro Gly Thr Lys Ile Phe Lys Pro Phe Asn Cys Gly Leu Gln Cys Ala
35 40 45
His Phe Pro Asn Val Glu Leu Ile Glu Glu Phe Cys Asp Pro Arg Leu
50 55 60
Ile Phe Asp Val Cys Gly Tyr Leu Ala Val Asp His Ala Ser Lys Gln
65 70 75 80
Ile Tyr Leu Val Ile Arg Gly Thr His Ser Leu Glu Asp Val Ile Thr
85 90 95
Asp Ile Arg Ile Met Gln Ala Pro Leu Thr Asn Phe Asp Leu Ala Ala
100 105 110
Asn Ile Ser Ser Thr Cys Thr Cys Asp Cys Cys Leu Val His Asn Gly
115 120 125
Phe Ile Gln Ser Tyr Asn Asn Thr Tyr Asn Gln Ile Gly Pro Lys Leu
130 135 140
Asp Ser Val Ile Glu Gln Tyr Pro Asp Tyr Gln Ile Ala Val Thr Gly
145 150 155 160
His Ser Leu Gly Gly Ala Ala Ala Leu Leu Phe Gly Ile Asn Leu Lys
165 170 175
Cys Asn Gly His Asp Pro Leu Val Val Thr Leu Gly Gln Pro Ile Val
180 185 190
Gly Asn Ala Cys Phe Ala Asn Trp Val Asp Lys Leu Phe Phe Gly Gln
195 200 205
Glu Cys Pro Asp Val Cys Lys Cys Ser Lys Asp Arg Lys Leu Tyr Arg
210 215 220
Ile Thr His Arg Gly Asp Ile Val Pro Gln Val Pro Phe Trp Asp Gly
225 230 235 240
Tyr Gln His Cys Ser Gly Glu Val Phe Ile Asp Trp Pro Leu Ile His
245 250 255
Pro Pro Leu Ser Asn Val Val Met Cys Gln Gly Gln Ser Asn Lys Gln
260 265 270
Cys Ser Ala Gly Asn Thr Leu Leu Gln Gln Val Asn Val Ile Gly Asn
275 280 285
His Leu Gln Tyr Phe Val Thr Glu Gly Val Cys Gly Ile
290 295 300
<210> 4
<211> 909
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase 4s nucleotide sequence
<400> 4
gtgtacacct ctaccgagac ctgtcacatt gaccaggagt cctacaactt ctttgagaag 60
tacgcccgac tcgcaaacat tggatattgt gttggtcccg gcactaagat cttcaagccc 120
ttcaactgtg gcctgcaatg tgcccacttc cccaacgttg agctcatcga ggagttctgt 180
gacccccgtc tcatctttga tgtttgtggt tacctcgctg ttgatcatgc ctccaagcag 240
atctaccttg ttattcgagg aacccactct ctggaggacg tcataaccga catccgaatc 300
atgcaggctc ctctgacgaa ctttgatctt gctgctaaca tctcttctac ttgtacttgt 360
gattgttgtc ttgtccacaa tggcttcatc cagtcctaca acaacaccta caatcagatc 420
ggccccaagc tcgactctgt gattgagcag tatcccgact accagattgc tgtcaccggt 480
cactctctcg gaggagctgc agcccttctg ttcggaatca acctcaagtg taacggccac 540
gatcccctcg ttgttactct tggtcagccc attgtcggta acgcttgttt tgctaactgg 600
gtcgataaac tcttctttgg ccaggagaac cccgatgtct gtaaggtgtc caaagaccga 660
aagctctacc gaatcaccca ccgaggagat atcgtccctc aagtgccctt ctgggacggt 720
taccagcact gctctggtga ggtctttatt gactggcccc tgatccaccc tcctctctcc 780
aacgttgtca tgtgccaggg ccagagcaat aaacagtgct ctgccggtaa cactctgctc 840
cagcaggtca atgtgattgg aaaccatctg cagtacttcg tcaccgaggg tgtctgtggt 900
atctaataa 909
<210> 5
<211> 909
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase 5s nucleotide sequence
<400> 5
gtgtgtacct ctaccgagac ctgtcacatt gaccaggagt cctacaactt ctttgagaag 60
tacgcccgac tcgcaaacat tggatattgt gttggtcccg gcactaagat cttcaagccc 120
ttcaactgtg gcctgcaatg tgcccacttc cccaacgttg agctcatcga ggagttctgt 180
gacccccgtc tcatctttga tgtttgtggt tacctcgctg ttgatcatgc ctccaagcag 240
atctaccttg ttattcgagg aacccactct ctggaggacg tcataaccga catccgaatc 300
atgcaggctc ctctgacgaa ctttgatctt gctgctaaca tctcttctac ttgtacttgt 360
gattgttgtc ttgtccacaa tggcttcatc cagtcctaca acaacaccta caatcagatc 420
ggccccaagc tcgactctgt gattgagcag tatcccgact accagattgc tgtcaccggt 480
cactctctcg gaggagctgc agcccttctg ttcggaatca acctcaagtg taacggccac 540
gatcccctcg ttgttactct tggtcagccc attgtcggta acgcttgttt tgctaactgg 600
gtcgataaac tcttctttgg ccaggagtgt cccgatgtct gtaaggtgtc caaagaccga 660
aagctctacc gaatcaccca ccgaggagat atcgtccctc aagtgccctt ctgggacggt 720
taccagcact gctctggtga ggtctttatt gactggcccc tgatccaccc tcctctctcc 780
aacgttgtca tgtgccaggg ccagagcaat aaacagtgct ctgccggtaa cactctgctc 840
cagcaggtca atgtgattgg aaaccatctg cagtacttcg tcaccgaggg tgtctgtggt 900
atctaataa 909
<210> 6
<211> 909
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Lipase 6s nucleotide sequence
<400> 6
gtgtgtacct ctaccgagac ctgtcacatt gaccaggagt gttacaactt ctttgagaag 60
tacgcccgac tcgcaaacat tggatattgt gttggtcccg gcactaagat cttcaagccc 120
ttcaactgtg gcctgcaatg tgcccacttc cccaacgttg agctcatcga ggagttctgt 180
gacccccgtc tcatctttga tgtttgtggt tacctcgctg ttgatcatgc ctccaagcag 240
atctaccttg ttattcgagg aacccactct ctggaggacg tcataaccga catccgaatc 300
atgcaggctc ctctgacgaa ctttgatctt gctgctaaca tctcttctac ttgtacttgt 360
gattgttgtc ttgtccacaa tggcttcatc cagtcctaca acaacaccta caatcagatc 420
ggccccaagc tcgactctgt gattgagcag tatcccgact accagattgc tgtcaccggt 480
cactctctcg gaggagctgc agcccttctg ttcggaatca acctcaagtg taacggccac 540
gatcccctcg ttgttactct tggtcagccc attgtcggta acgcttgttt tgctaactgg 600
gtcgataaac tcttctttgg ccaggagtgt cccgatgtct gtaagtgttc caaagaccga 660
aagctctacc gaatcaccca ccgaggagat atcgtccctc aagtgccctt ctgggacggt 720
taccagcact gctctggtga ggtctttatt gactggcccc tgatccaccc tcctctctcc 780
aacgttgtca tgtgccaggg ccagagcaat aaacagtgct ctgccggtaa cactctgctc 840
cagcaggtca atgtgattgg aaaccatctg cagtacttcg tcaccgaggg tgtctgtggt 900
atctaataa 909
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 122C-F
<400> 7
tgttgtcttg tccacaatgg cttcatc 27
<210> 8
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 122C-R
<400> 8
atcacaagta gcagtagaag agatgttagc agc 33
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 196C-F
<400> 9
acaagcgtta ccgacaatgg gctg 24
<210> 10
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 196C-R
<400> 10
tttgctaact gggtcgataa actcttcttt g 31
<210> 11
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 196+118C-F
<400> 11
acttgtgatt gttgtcttgt ccacaatggc t 31
<210> 12
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 196+118C-R
<400> 12
acaagtagaa gagatgttag cagcaagatc aaagttc 37
<210> 13
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 177C-F
<400> 13
tgtaacggcc acgatcccct cgt 23
<210> 14
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 177C-R
<400> 14
cttgaggttg attccgaaca gaagggc 27
<210> 15
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 8C-F
<400> 15
tgtcacattg accaggagtc ctacaacttc 30
<210> 16
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 8C-R
<400> 16
ggtctcggta gaggtgtaca catggtgat 29
<210> 17
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 214C-F
<400> 17
tgtaaggtgt ccaaagaccg aaagctcta 29
<210> 18
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 214C-R
<400> 18
gacatcgggg ttctcctggc ca 22
<210> 19
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 60C-F
<400> 19
tgtgaccccc gtctcatctt tgatgt 26
<210> 20
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 60C-R
<400> 20
gaactcctcg atgagctcaa cgttgg 26
<210> 21
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 69C-F
<400> 21
tgtggttacc tcgctgttga tcatgc 26
<210> 22
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 69C-R
<400> 22
aacatcaaag atgagacggg ggtcac 26
<210> 23
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+8C-F
<400> 23
tgtcacattg accaggagtc ctacaacttc 30
<210> 24
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+8C-R
<400> 24
ggtctcggta gaggtacaca catggtgatg 30
<210> 25
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+214C-F
<400> 25
tgtaaggtgt ccaaagaccg aaagctcta 29
<210> 26
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+214C-R
<400> 26
gacatcggga cactcctggc ca 22
<210> 27
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+214+14C-F
<400> 27
tacaacttct ttgagaagta cgcccgactc 30
<210> 28
<211> 28
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+214+14C-R
<400> 28
acactcctgg tcaatgtgac aggtctcg 28
<210> 29
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+214+216C-F
<400> 29
ccgaatcacc caccgaggag atatc 25
<210> 30
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> 210+214+216C-R
<400> 30
tagagctttc ggtctttgga acacttacag 30

Claims (8)

  1. A kind of 1. heat-resisting mutant lipase, it is characterised in that：It is the side combined in Yarrowia lipolytica lipase 2 by iteration Method introduces multipair disulfide bond mutation and obtained；The multipair disulfide bond of described introducing is 4~6 pairs of disulfide bond of introducing.
  2. 2. heat-resisting mutant lipase according to claim 1, it is characterised in that：Described heat-resisting mutant lipase is fat Enzyme 4s, lipase 5s or lipase 6s；
    Lipase 4s amino acid sequence is as shown in SEQ ID NO.1；
    Lipase 5s amino acid sequence is as shown in SEQ ID NO.2；
    Lipase 6s amino acid sequence is as shown in SEQ ID NO.3.
  3. 3. encode the nucleotide sequence of the heat-resisting mutant lipase described in claim 2, it is characterised in that：
    Described lipase 4s coding nucleotide sequence is as shown in SEQ ID NO.4；
    Described lipase 5s coding nucleotide sequence is as shown in SEQ ID NO.5；
    Described lipase 6s coding nucleotide sequence is as shown in SEQ ID NO.6.
  4. 4. the preparation method of the heat-resisting mutant lipase described in claim 1, it is characterised in that comprise the following steps：
    (1) using pPICZ α A-Lip2 as template, it is mutated by inverse PCR, selected amino acid sites is sported into cysteine, And be transferred in Escherichia coli, amplification cultivation, plasmid extraction and sequencing are carried out, obtains four pairs, five pairs or six pairs disulfide bond of introducing Mutant lipase recombinant plasmid；
    (2) correct recombinant plasmid will be sequenced with the restriction enzyme linearization process of Pme I, and electroporated finish to competence In red yeast X33, further by YPDS-Zeocin plate screenings, corresponding mutation engineering bacteria is obtained；
    (3) mutation engineering bacteria is carried out in YPD fluid nutrient mediums after expanding numerous culture, is forwarded to BMGY fluid nutrient mediums and is gone Suppress culture, be finally inoculated with BMMY fluid nutrient mediums and fermented, bacterium solution is centrifuged and obtains supernatant crude enzyme liquid；
    (4) after crude enzyme liquid is concentrated by ultrafiltration using super filter tube, purified, isolated with histidine-tagged using nickel post one-step method Lipase protein, and with reproducibility SDS-PAGE detect purity of protein, obtain heat-resisting mutant lipase after purification.
  5. 5. the preparation method of heat-resisting mutant lipase according to claim 4, it is characterised in that：Described in step (1) Escherichia coli are Escherichia coli TOP10.
  6. 6. the preparation method of heat-resisting mutant lipase according to claim 4, it is characterised in that：Described in step (2) Zeocin concentration is 100 μ g/mL in YPDS-Zeocin flat boards.
  7. 7. the preparation method of heat-resisting mutant lipase according to claim 4, it is characterised in that also comprise the following steps：
    (5) by DSF fluoroscopic examinations determine lipase melting temperature, the deactivation temperatures of p-NPP colorimetric method for determining 15min half, 55 Or the half-life period at 70 DEG C, optimal reactive temperature, optimal reaction pH, and pH stability.
  8. 8. the application of heat-resisting mutant lipase in the industry described in claim 1 or 2.
CN201711220144.8A 2017-11-29 2017-11-29 Heat-resistant mutant lipase, preparation method and application Active CN107858337B (en)

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CN110819609A (en) * 2019-12-13 2020-02-21 华南农业大学 Mutant lipase with improved thermal stability as well as preparation method and application thereof
CN111073876A (en) * 2020-01-18 2020-04-28 江南大学 Bacillus subtilis lipase A with improved heat stability
CN111117982A (en) * 2019-12-25 2020-05-08 武汉新华扬生物股份有限公司 Pyrethroid degrading enzyme, encoding gene thereof, recombinant strain and application
CN111117981A (en) * 2019-05-31 2020-05-08 江南大学 Lipase mutant and application thereof in decontamination

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Publication number Priority date Publication date Assignee Title
CN111117981A (en) * 2019-05-31 2020-05-08 江南大学 Lipase mutant and application thereof in decontamination
CN110819609A (en) * 2019-12-13 2020-02-21 华南农业大学 Mutant lipase with improved thermal stability as well as preparation method and application thereof
CN110819609B (en) * 2019-12-13 2022-05-10 华南农业大学 Mutant lipase with improved thermal stability as well as preparation method and application thereof
CN111117982A (en) * 2019-12-25 2020-05-08 武汉新华扬生物股份有限公司 Pyrethroid degrading enzyme, encoding gene thereof, recombinant strain and application
CN111117982B (en) * 2019-12-25 2023-05-12 武汉新华扬生物股份有限公司 Pyrethroid degrading enzyme, encoding gene, recombinant strain and application thereof
CN111073876A (en) * 2020-01-18 2020-04-28 江南大学 Bacillus subtilis lipase A with improved heat stability

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