CN103627685B - Higher-activity partial glyceride lipase mutant and application thereof - Google Patents

Higher-activity partial glyceride lipase mutant and application thereof Download PDF

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CN103627685B
CN103627685B CN201310586753.0A CN201310586753A CN103627685B CN 103627685 B CN103627685 B CN 103627685B CN 201310586753 A CN201310586753 A CN 201310586753A CN 103627685 B CN103627685 B CN 103627685B
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mutant
partial glyceride
lipase
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seq
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CN103627685A (en
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王永华
蓝东明
刘璐
王卫飞
杨博
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South China University of Technology SCUT
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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
    • 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 invention discloses a higher-activity partial glyceride lipase mutant and application thereof. The mutant is an enzyme mutant prepared by carrying out site-directed mutagenesis on Malassezia globosa partial glyceride lipase, wherein the mutant site is Phe at the 278th site, and the Phe at the 278th site is mutated into Ile or Gly. The obtained mutant Phe278Ile or Phe278Gly has higher partial glyceride hydrolysis activity, and is more suitable for application in biochemical engineering industry. The partial glyceride hydrolysis activities of the modified SMG1Phe278Ile and SMG1Phe278Gly mutants are enhanced to different degrees, which are respectively 1.84 times and 1.68 times of the wild type SMG1 lipase hydrolysis activity; and the modified SMG1Phe278Ile and SMG1Phe278Gly mutants have higher partial glyceride activity, and can be used for removing partial glyceride in grease.

Description

The partial glyceride lipase mutant that a kind of activity improves and application thereof
Technical field
The invention belongs to biological technical field, relate to a kind of partial glyceride lipase mutant and application thereof of activity raising.
Background technology
Lipase (EC3.1.1.3) i.e. Lipase, can generate lipid acid, glycerine and monoglyceride or diester by catalytic hydrolysis natural substrate grease.And partial glyceride lipase is a kind of only hydrolysis monoglyceride and triglyceride, and a kind of lipase of not hydrolyzing triglyceride.The catalyzed reaction type that lipase participates in is extensive, comprises the reactions such as catalysis solution fat, transesterify, Lipase absobed, is widely used in the fields such as fodder additives, fats and oils processing, food service industry, biological medicine, daily-use chemical industry.
The special substrate selectivity characteristic of partial glyceride lipase can be used in optionally to be removed the monoglyceride in grease and triglyceride.Patent JP62061590 discloses a kind of firm butter utilizing the catalyzer Lipase G of mixing and Rhizopus deremer lipase to prepare low diglyceride content.Chinese patent 201210549885.1 disclose a kind of utilize mixing inclined glycerin fatty enzyme and single glycerin fatty enzyme for removing the partial glyceride in grease.Although these methods can reduce the content of partial glyceride in grease effectively, need use two kinds of dissimilar lipase preparation, the cost added in production process drops into, thus causes the rising of final product price.Using the inclined glycerin fatty enzyme of the activity of high hydrolysis monoglyceride and triglyceride, be a kind of feasible method, but the lipase reagent of this type still lacks with removal partial glyceride.
Protein engineering is a kind of feasible method can improving zymoprotein characteristic, method by molecular biology and information biology carries out orthomutation and rationality transformation to albumen, and obtain performance raising albumen or enzyme mutant, because the method has, workload is little and to obtain the probability of Positive mutants body large, is therefore widely used in the Application Areas of enzyme molecular modification.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of SMG1 lipase mutant improved partial glyceride hydrolytic activity, can be applied to the partial glyceride in selective removal oil and fat product.By rational choice mutational site, by Marxist philosophy lesson (Malassezia globosa) SMG1 lipase gene (Genbank accession number XM_001732152.1), by the analysis to its protein structure (PDB:3UUE), use the method for rite-directed mutagenesis and the lipase mutant that obtains.Carry out rite-directed mutagenesis and obtain enzyme mutant, undergo mutation in aminoacid replacement position in parent SMG1 aminoacid sequence SEQ ID NO:1, adopt " amino acid of Original amino acid-position-replacement " to represent the amino acid suddenlyd change in lipase mutant, described partial glyceride lipase mutant is: Phe278Ile and Phe278Gly.
Technical scheme of the present invention is specific as follows:
The partial glyceride lipase mutant that a kind of activity improves, this mutant is enzyme mutant Malassezia (Malassezia globosa) partial glyceride lipase being carried out to rite-directed mutagenesis acquisition, wherein mutational site is the Phe of 278, and the Phe of described 278 sports Ile or Gly.
The DNA sequence dna of described mutant is SEQ NO.2 or SEQ NO.3.
The aminoacid sequence of described mutant is SEQ NO.4 or SEQ NO.5.
Said mutation body is removing the application of partial glyceride in oil and fat product.This application mainly comprises the steps:
(1) partial glyceride lipase Mutant Preparation mutant expression plasmid is utilized:
A, utilize Overlap extension PCR method to introduce object mutating acid site, the upstream and downstream gene fragment of first segmentation amplification containing mutating acid site, is then spliced into the gene fragment with mutational site containing mutational site full-length gene fragment;
B, by above-mentioned amplification gene PCR primer after DNA purifying, restriction enzyme Kpn I and SalI carries out double digested to the gene fragment of purifying and plasmid pGAPZ α A respectively, connect, be converted into E. coli DH5 α competent cell, obtain mutant expression plasmid;
(2) mutant expression plasmid is utilized to prepare recombinant bacterial strain:
A, by the mutant expression plasmid of step (1) after restriction enzyme Bln I linearizing, electricity goes to host cell;
B, coat conversion fluid containing 100mg/ml bleomycin YPD flat board, cultivate after 3 days for 30 DEG C, flat board growing yeast list bacterium colony is recombinant bacterial strain;
(3) partial glyceride lipase recombinant expressed for recombinant bacterial strain is used for the partial glyceride removed in oil and fat product.
The primer sequence of step (1) described PCR is as follows:
SMG For5’-GGGGTACCAGCAGTATTTACGCCCGTGGCCG-3’
3'AOX5’-GGCAAATGGCATTCTGACAT-3’
Phe278Ile For5'-GCTCGCGAGTTCAACATTGACGACCACCAAG-3'
Phe278Ile Rev5'-CTTGGTGGTCGTCAATGTTGAACTCGCGAGC-3'
Phe278Gly For5'-GTTGCTCGCGAGTTCAACGGTGACGACCACCA
AGGTAT-3'
Phe278Gly Rev5'-ATACCTTGGTGGTCGTCACCGTTGAACTCGCGA
GCAAC-3'
Step (2) described host cell is pichia pastoris phaff X-33(Pichia pastoris).
Compared with prior art, the present invention has following beneficial effect:
Gained mutant Phe278Ile and Phe278Gly of the present invention is the mutant that partial glyceride hydrolytic activity improves, and is more suitable for the application of biochemical lines.Activity through the hydrolyzes partial glycerides of improved SMG1Phe278Ile and SMG1Phe278Gly mutant has raising in various degree, 1.84 times and 1.68 times of the hydrolytic activity of the SMG1 lipase of wild-type respectively, there is the activity of better hydrolyzes partial glycerides, can be applied to and remove partial glyceride in grease.
Accompanying drawing explanation
Fig. 1 is lipase SMG1 tomograph, arrow points be the Phe site of 278 amino acids.
Fig. 2 is the protein purification figure of lipase SMG1 and mutant, and swimming lane one is molecular weight of albumen marker, and swimming lane two is the SMG1 after purifying, swimming lane three SMG1-Phe278Gly that to be the SMG1-Phe278Ile swimming lanes four after purifying be after purifying.
Fig. 3 is the relative content of lipase SMG1 and the acid of mutant hydrolyzes partial glycerides release fat.
Embodiment
Below in conjunction with specific embodiment, the present invention is more specifically described in detail, but embodiments of the present invention are not limited thereto, for the processing parameter do not indicated especially, can refer to routine techniques and carry out.
Embodiment 1
The structure of lipase SMG1 wild-type yeast expression vector
According to spherical horse traction colour cast glycerin fatty enzyme smg1 gene (accession number: XM_001732152) of Genbank, entrust Shanghai Sheng Gong bio-engineering corporation to utilize the method for synthetic, and according to pichia spp codon preference, sequence is optimized (sequence SEQ ID NO:1).After gene chemical synthesis, primer SMG For:5 '-GGGGTACCAGCAGTATTTACGCCCGTGGCCG-3 ' and SMG Rev:5 ' ACGCGTCGACTTAATGAGCACCAACCTGAGCT-3 ' is utilized to increase mature peptide sequence, pcr amplification condition: 94 DEG C of 5min; 94 DEG C of 20s, 53 DEG C of 30s, 72 DEG C of 80s, 25 circulations; 72 DEG C of 7min.Pcr amplification product is after DNA Purification Kit, and restriction enzyme Kpn I and Sal I carries out double digested to the gene fragment of purifying and plasmid pGAPZ α A respectively, connects, is converted into E. coli DH5 α competent cell.Coat LB(containing 25ug/ml zeocion) dull and stereotyped.Picking positive colony is identified and gene sequencing by Kpn I and SalI double digestion, and result shows to obtain wild-type pGAPZ α A-SMG1 expression plasmid.
Embodiment 2
Lipase SMG1278 amino acid sites mutation construction
Adopt site-directed mutagenesis technique to build mutant SMG1Phe278Gly and SMG1Phe278Ile, utilize Overlap extension PCR method to introduce object mutational site, with SMG1 wild type gene pGAPZ α A-SMG1 for template, rite-directed mutagenesis reaction conditions:
Amplification sudden change fragment upstream:
10X pfu buffer： 5
d NTP： 2.5
SMG for： 2.5
Phe278Ile rev： 2.5
Pfu archaeal dna polymerase: 1.5
Distilled water: 40
PGAPZαA-SMG1： 2
Amplification sudden change segments downstream:
10X pfu buffer 5
dNTP： 2.5
Phe278Ile for： 2.5
3’AOX： 2.5
Pfu archaeal dna polymerase: 1.5
Distilled water: 40
PGAPZαA-SMG1： 2
Increase with the full-length gene fragment in mutational site:
10Xpfu buffer： 10
dNTP： 5
SMG for： 5
3’AOX： 5
Pfu archaeal dna polymerase: 3
Distilled water: 8
Fragment upstream: 5
Segments downstream: 5
The primer used, sees the following form:
Pcr amplification condition: 94 DEG C of 5min; 94 DEG C of 20s, 53 DEG C of 30s, 72 DEG C of 80s, 25 circulations; 72 DEG C of 7min.Overlap extension PCR amplified production is after DNA Purification Kit, and restriction enzyme Kpn I and Sal I carries out double digested to the gene fragment of purifying and plasmid pGAPZ α A respectively, connects, is converted into E. coli DH5 α competent cell.Coat LB(containing 25ug/mlzeocion) dull and stereotyped.After growth 15h, dull and stereotyped upper 10 the single bacterium colonies of picking carry out bacterium colony PCR, picking positive colony is identified and gene sequencing by Kpn I and SalI double digestion, and result shows to obtain correct mutation expression plasmid pGAPZ α A-SMG1Phe278Gly and pGAPZ α A-SMG1Phe278Ile.
Embodiment 3
Recombinant expressed and the purifying of lipase SMG1 and mutant thereof
By mutant expression plasmid after restriction enzyme Bln I linearizing, electricity goes to pichia pastoris X-33 competence state cell.Conversion fluid is coated YPD(100ug/ml Zeocin) dull and stereotyped, cultivate after 3 days for 30 DEG C, on picking flat board, single bacterium colony ferments after 72 hours in 100ml YPD substratum, centrifugal and concentrated broth carries out SDS-PAGE detection, and acquisition can express the positive recombinant bacterial strain of lipase SMG1 mutant.
By wild type strain and each lipase mutant bacterial strain, be seeded in 100ml YPD substratum, 30 DEG C, after 200rpm shaking culture 48-72 hour, collect fermented supernatant fluid (10,000rpm, centrifugal 20min, 4 DEG C).
By the filter membrane suction filtration of the fermented supernatant fluid 0.22um of SMG1 wild-type and mutants which had thereof.After use 10KD film born of the same parents (Vivaflow200, Sartorius) fermented liquid to be replaced as 20mM pH8.0Tris-HCl damping fluid, and fermented liquid is concentrated to about 30ml.Concentrated fermentation supernatant utilizes Q Sepharose tMfastFlow anion-exchange chromatography post, flow velocity 5ml/min, finally by the Tris-HCl buffer solution for gradient elution of pH8.0 containing 1M NaCl, target protein is eluted at 80mM salt ionic concentration place.SDS-PAGE electrophoresis is utilized to carry out Purity (see figure 2) to the recombinant protein of purifying.
Embodiment 4
The hydrolysis of partial glyceride
The partial glyceride substrate of 3.5mL is added in 25mL tool plug triangular flask, add purified after SMG1-WT, SMG1-Phe278Gly or SMG1-Phe278Ile, enzyme concentration is 100U/mL (U/v, relative to grease volume) enzyme liquid, and enzyme liquid utilizes 50mM Na 2hPO 4-NaH 2pO 4buffered soln is diluted to 1.5mL, mixes rear preheating 20min.Temperature of reaction is 25 DEG C, and shaking speed is 200rpm, samples respectively in reaction process at 1h, 3h, 5h, 7h and 16h.By get 100 μ L samples and be dissolved in the moving phase of 1mL, the ratio of moving phase is: normal hexane/Virahol/formic acid=15:1:0.003(volume ratio), after concussion evenly, centrifugal 5min under 10000rpm, removing lower floor aqueous phase, by upper organic phase with after the organic phase membrane filtration of 0.45 μm, get 10 μ L and carry out HPLC-IR analysis.
The condition that HPLC detects is: chromatographic column is Luna5u Silica (2) 100A, 250 × 4.60mm, (phenomenex), and moving phase is normal hexane: Virahol: formic acid=15:1:0.003 (volume ratio); Flow rate of mobile phase is 1.0mL/min, and column temperature keeps 30 DEG C; Sample size is 10 μ L, and each sample detection time is about 30min, and data processing software is Breeze workstation.The burst size (see figure 3) of lipid acid after different ferment treatment, the lipid acid burst size through reaction SMG1-Phe278Ile, SMG1-Phe278Gly and SMG1-WT of 16 hours is respectively 92.54%, 84.16% and 50.06%.The efficiency of mutant SMG1-Phe278Ile and SMG1-Phe278Gly hydrolyzes partial glycerides is 1.84 times and 1.68 times of SMG1-WT.

Claims (6)

1. the active partial glyceride lipase mutant improved, it is characterized in that, this mutant is enzyme mutant Malassezia (Malassezia globosa) partial glyceride lipase being carried out to rite-directed mutagenesis acquisition, wherein mutational site is the Phe of 278, and the Phe of described 278 sports Ile or Gly; The DNA sequence dna of described mutant is SEQ NO.2 or SEQ NO.3.
2. mutant according to claim 1, is characterized in that, the aminoacid sequence of described mutant is SEQ NO.4 or SEQ NO.5.
3. claim 1 ~ 2 any one mutant is removing the application of partial glyceride in oil and fat product.
4. application according to claim 3, is characterized in that, comprises the steps:
(1) partial glyceride lipase Mutant Preparation mutant expression plasmid is utilized:
A, utilize Overlap extension PCR method to introduce object mutating acid site, the upstream and downstream gene fragment of first segmentation amplification containing mutating acid site, is then spliced into the gene fragment with mutational site containing mutational site full-length gene fragment;
B, by above-mentioned amplification gene PCR primer after DNA purifying, restriction enzyme Kpn I and SalI carries out double digested to the gene fragment of purifying and plasmid pGAPZ α A respectively, connect, be converted into E. coli DH5 α competent cell, obtain mutant expression plasmid;
(2) mutant expression plasmid is utilized to prepare recombinant bacterial strain:
A, by the mutant expression plasmid of step (1) after restriction enzyme Bln I linearizing, electricity goes to host cell;
B, coat conversion fluid containing 100mg/ml bleomycin YPD flat board, cultivate after 3 days for 30 DEG C, flat board growing yeast list bacterium colony is recombinant bacterial strain;
(3) partial glyceride lipase recombinant expressed for recombinant bacterial strain is used for the partial glyceride removed in oil and fat product.
5. application according to claim 4, is characterized in that, the primer sequence of step (1) described PCR is as follows:
SMG For 5’-GGGGTACCAGCAGTATTTACGCCCGTGGCCG-3’
3'AOX 5’-GGCAAATGGCATTCTGACAT-3’
Phe278Ile For 5'-GCTCGCGAGTTCAACATTGACGACCACCAAG-3'
Phe278Ile Rev 5'-CTTGGTGGTCGTCAATGTTGAACTCGCGAGC-3'
Phe278Gly For 5'-GTTGCTCGCGAGTTCAACGGTGACGACCACCA
AGGTAT-3'
Phe278Gly Rev 5'-ATACCTTGGTGGTCGTCACCGTTGAACTCGCGA
GCAAC-3'。
6. application according to claim 4, is characterized in that, step (2) described host cell is pichia pastoris phaff X-33 (Pichia pastoris).
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