CN108795959A - A kind of stearothermophilus soil gemma lipase gene secretory expression method - Google Patents
A kind of stearothermophilus soil gemma lipase gene secretory expression method Download PDFInfo
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Abstract
The invention discloses a kind of stearothermophilus soil gemma lipase gene secretory expression methods, search obtains stearothermophilus soil gemma lipase maturation peptide gene in Genbank, and codon optimization is carried out to stearothermophilus soil gemma lipase maturation peptide gene to the preference of genetic codon according to Pichia pastoris；According to the requirement of rite-directed mutagenesis PCR, three pairs of primers are separately designed；Stearothermophilus soil gemma lipase maturation peptide gene after three-wheel Kex2 rite-directed mutagenesis is sequenced；The wild type of synthesis or the stearothermophilus soil gemma lipase maturation peptide gene of Kex2 rite-directed mutagenesis are subjected to digestion respectively with Pichia anomala expression plasmid；Extract the plasmid through sequencing identification in above-mentioned bacterial strains, the present invention passes through rite-directed mutagenesis, change three potential serine stretch protein enzyme recognition site Kex2 in stearothermophilus soil gemma lipase maturation peptide sequence, stearothermophilus soil gemma lipase that overall length expressed intact is obtained in pichia yeast expression system, having more enzymatic activity high.
Description
Technical field
The present invention relates to Biochemistry and Molecular Biology technical field, more particularly to a kind of stearothermophilus soil gemma fat
Fat enzyme gene secretory expression method.
Background technology
Lipase (lipase, E.C.3.1.1.) is called glyceride hydrolase (triacylglycerol
Acylhydrolase), be it is a kind of can reduce fat, the protease with degradation of lipid ability.Lipase can be sweet by trigalloyl
Grease is hydrolyzed to aliphatic acid, diacylglycerol (DGDG), monoglyceride and glycerine.The natural substrate of lipase is usually not soluble in water
Long chain fatty acids acyl ester, reaction characteristics are to play catalytic action (1) in oil-water interfaces.According to the property of substrate, can be divided into
Lipase, esterase, phosphatidase or cutinase.Lipase is one of most important industrial enzymes, is widely used in food, raises
The fields such as material, washing, process hides, medicine, grease chemical industry.Due to biological enzyme biodiesel synthesis, that is, utilize lipase non-aqueous
Animal and plant fat and low-carbon alcohol catalysis are prepared into corresponding fatty acid methyl ester or fatty-acid ethyl ester by the transesterification in phase, tool
It has ready conditions mild and the advantages that without exhaust emission, lipase is also more and more applied in recent years in green energy resource field.
From Geobacillus stearothermophilus T1 bacterial strains (Geobacillus stearothermophilus strain
T1 lipase (GENBANK accession number JC8061)) is a kind of heat resistance alkaline lipase (E.C.3.1.1.3).It is to difference
The natural or synthetic substrate of carbochain all shows activity, especially has a preference for the substrate of C4-C16, most suitable substrate is the substrate of C8.It is thermophilic
The hot native gemma lipase of fat is in the presence of various detergent ingredients, such as surfactant or other detergent ingredients exist
Under, still there is carboxyester hydrolysis enzymatic activity, it can be with hydrolyze lipid.This characteristic makes the lipase be highly suitable to be applied for washing
Agent industry.In addition, the lipase also has transesterification ability, it can be by aliphatic acid and alcohol catalysis reaction at fatty-acid ethyl ester.
Pichia pastoris yeast (Pichia Pastoris) is an efficient eukaryon being increasingly taken seriously in recent years
Expression system.The expression system has the advantages that many uniquenesses, and oneself rapidly develops as extensive in molecular biology field
One of main means for recombinant protein production.Pichia pastoris has the advantage that as eukaryotic expression system:(1) finish red ferment
Mother's growth is rapid, and High Density Cultivation can be achieved in simple synthetic media；(2) Pichia pastoris has the startup of accuracy controlling
Son, including methanol dependent form promoter or non-methanol dependent form constitutive promoter, can high efficient expression purpose foreign protein；
(3) exogenous protein expression is efficient, and itself secretory protein is few, and the foreign protein of expression can account for total secreting, expressing albumen
90% or more, be conducive to isolating and purifying for external source destination protein；(4) after Pichia pastoris has the distinctive protein translation of eucaryote
Modification folds and efficient secretion, and the soluble protein expression quantity of prokaryotic expression system can be overcome low and unit mass albumen
The defects such as enzymatic activity is low.Therefore, Pichia pastoris becomes in recent years as the expression most promising expression system of external source destination protein.
But the report (2) of stearothermophilus soil gemma lipase successful expression only in bacterium.It was found that thermophilic
When fatty soil gemma lipase is expressed in pichia pastoris yeast expression system, the main problem encountered is exactly that expression product goes out
Different degrees of degradation is showed.The size of the complete stearothermophilus soil gemma lipase of secreting, expressing should be 46kDa, but we
It is found that the product of other small molecule quality.These small-molecular-weight products have N- identical with stearothermophilus soil gemma lipase
Terminal amino acid sequence, and the C-terminal of these segments contains pairs of alkaline amino acid residue.Further investigation revealed that should
The characteristic degradation of lipase is calcium ion dependence serine endoprotease (kexin, the EC present in yeast cells
3.4.21.61 caused by).Serine endoprotease is a kind of prerequisite knowledge of yeast cells itself coding, is located at yeast
On cell membrane, the action site of the enzyme is double alkaline amino acid residues, it be mainly responsible for the secretions of most of Yeast proteins at
It is ripe, such as signal peptide or leader peptide working process (3-5).We are in the ripe peptide sequence of stearothermophilus soil gemma lipase
It is found that the specific recognition site Kex2 (6) of 3 potential serine endoproteases.
Invention content
Invention is designed to provide a kind of stearothermophilus soil gemma lipase gene secretory expression method, prominent by pinpointing
The method of change changes 3 potential serine stretch protein enzyme recognition sites in stearothermophilus soil gemma lipase maturation peptide sequence
Kex2 obtains expressed intact, the higher stearothermophilus soil gemma lipase of enzymatic activity in Pichia pastoris, overcomes the fat
The defects of fat enzyme expression quantity in bacterial expression system is relatively low, unit mass lipase active is relatively low, to solve above-mentioned background
The problem of being proposed in technology.
To achieve the above object, the present invention provides the following technical solutions：
A kind of stearothermophilus soil gemma lipase gene secretory expression method, includes the following steps：
Step 1：Search obtains stearothermophilus soil gemma lipase maturation peptide gene in Genbank, according to Pichia pastoris pair
The preference of genetic codon carries out codon optimization to stearothermophilus soil gemma lipase maturation peptide gene；
Step 2：According to the requirement of rite-directed mutagenesis PCR, three pairs of primers are separately designed；Respectively:
A1:GTTGCCAGAATTGAAGCAAGGTGGTAGAATCCA；
A2:TGGATTCTACCACCTTGCTTCAATTCTGGCAAC；
B1:TCGAAAGATTGAAGCAATCCCCAGTTTGGAC；
B2:GTCCAAACTGGGGATTGCTTCAATCTTTCGA；
C1:TGAACGGTCCAAAGCAAGGTTCTTCTGATAG；
C2:CTATCAGAAGAACCTTGCTTTGGACCGTTCA；
IDT is transferred to carry out primer synthesis；
Step 3：It using A1 and A2 as primer, carries out PCR and reacts rite-directed mutagenesis R103Q, using B1 and B2 as primer, carry out PCR
Rite-directed mutagenesis R230Q is reacted, then using C1 and C2 as primer, carries out PCR reaction rite-directed mutagenesis R330Q；
Step 4：Stearothermophilus soil gemma lipase maturation peptide gene after three-wheel Kex2 rite-directed mutagenesis is sequenced,
The sequence one that sequencing result shows the stearothermophilus soil gemma lipase gene sequence after passing through rite-directed mutagenesis PCR and is pre-designed
It causes；
Step 5：By the wild type of synthesis or the stearothermophilus soil gemma lipase maturation peptide gene of Kex2 rite-directed mutagenesis with
Pichia anomala expression plasmid carries out digestion respectively, and glue recycles endonuclease bamhi, and T4 ligases convert Escherichia coli after connecting the two, obtain
The expression plasmid containing promoter-alpha factor signal peptide-stearothermophilus soil gemma lipase maturation peptide gene bacterial strain, choose gram
It is sequenced after grand extraction plasmid；
Step 6：Extract the plasmid through sequencing identification in above-mentioned bacterial strains, electrotransformation pep4/prb1 egg-pairs after linearization for enzyme restriction
White deficient pichia pastoris yeast competent cell, the cell after conversion are coated on the zeocin resistances with various concentration
YPD tablets, choose clone, a small amount of fermented and cultureds carried out to clone, detect enzyme activity, to obtain new and effective stearothermophilus soil
The Pichia yeast engineering of gemma lipase gene.
Preferably, 3 potential serine stretch protein enzyme recognition site Kex2 are changed to other amino in ripe peptide sequence
Sour residue and the stearothermophilus soil gemma lipase gene designed by Pichia pastoris preference codon.
Preferably, expression vector is that the composing type containing high efficiency methanol evoked promoter AOX1 or non-methanol dependences starts
The plasmid of sub- GAP.
Preferably, the gene optimized is synthesized by GenScript in step 1, the gene order of synthesis by
Macrogen is sequenced, and sequencing result is consistent with the gene order being pre-designed.
Preferably, amino acid number is with first amino acid of stearothermophilus soil gemma lipase mature peptide in step 3
1。
Compared with prior art, the beneficial effects of the invention are as follows：The present invention using pichia yeast expression system and according to
Pichia pastoris preference optimizes the stearothermophilus soil gemma lipase gene after codon, after being more advantageous to the translation of the lipase
Modification, folding and efficient secretion.Pass through three serines in rite-directed mutagenesis stearothermophilus soil gemma lipase maturation peptide sequence
Protease site Kex2, the lipase for avoiding secretion are dropped by yeast cells endogenous serine endoprotease kexin
Solution obtains expressed intact, high enzyme activity stearothermophilus soil gemma lipase in Pichia pastoris.This is in stearothermophilus soil bud
The research field of spore lipase still belongs to the first time, and the large-scale industrial production for stearothermophilus soil gemma lipase provides reliably
It ensures.
Description of the drawings
Fig. 1 be the present invention different P-NP substrates in the presence of lipase active block diagram；
Fig. 2 is influence line charts of the pH of the present invention to lipase active.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
A kind of stearothermophilus soil gemma lipase gene secretory expression method, includes the following steps：
Step 1：Search obtains stearothermophilus soil gemma lipase maturation peptide gene in Genbank, according to Pichia pastoris pair
The preference of genetic codon carries out codon optimization to stearothermophilus soil gemma lipase maturation peptide gene；
Step 2：According to the requirement of rite-directed mutagenesis PCR, three pairs of primers are separately designed；Respectively:
A1:GTTGCCAGAATTGAAGCAAGGTGGTAGAATCCA；
A2:TGGATTCTACCACCTTGCTTCAATTCTGGCAAC；
B1:TCGAAAGATTGAAGCAATCCCCAGTTTGGAC；
B2:GTCCAAACTGGGGATTGCTTCAATCTTTCGA；
C1:TGAACGGTCCAAAGCAAGGTTCTTCTGATAG；
C2:CTATCAGAAGAACCTTGCTTTGGACCGTTCA；
IDT is transferred to carry out primer synthesis；
Step 3：It using A1 and A2 as primer, carries out PCR and reacts rite-directed mutagenesis R103Q, using B1 and B2 as primer, carry out PCR
Rite-directed mutagenesis R230Q is reacted, then using C1 and C2 as primer, carries out PCR reaction rite-directed mutagenesis R330Q；
Step 4：Stearothermophilus soil gemma lipase maturation peptide gene after three-wheel Kex2 rite-directed mutagenesis is sequenced,
The sequence one that sequencing result shows the stearothermophilus soil gemma lipase gene sequence after passing through rite-directed mutagenesis PCR and is pre-designed
It causes；
Step 5：By the wild type of synthesis or the stearothermophilus soil gemma lipase maturation peptide gene of Kex2 rite-directed mutagenesis with
Pichia anomala expression plasmid carries out digestion respectively, and glue recycles endonuclease bamhi, and T4 ligases convert Escherichia coli after connecting the two, obtain
The expression plasmid containing promoter-alpha factor signal peptide-stearothermophilus soil gemma lipase maturation peptide gene bacterial strain, choose gram
It is sequenced after grand extraction plasmid；
Step 6：Extract the plasmid through sequencing identification in above-mentioned bacterial strains, electrotransformation pep4/prb1 egg-pairs after linearization for enzyme restriction
White deficient pichia pastoris yeast competent cell, the cell after conversion are coated on the zeocin resistances with various concentration
YPD tablets, choose clone, a small amount of fermented and cultureds carried out to clone, detect enzyme activity, to obtain new and effective stearothermophilus soil
The Pichia yeast engineering of gemma lipase gene.
Embodiment 1
The codon optimization and synthesis of stearothermophilus soil gemma lipase gene
Search obtains stearothermophilus soil gemma lipase maturation peptide gene (SEQ ID No.4) in Genbank, in thermophilic fat
It is close using the heredity of Pichia pastoris preference on the basis of fat soil gemma lipase maturation peptide amino acid sequence (SEQ ID No.4)
Numeral replaces the lower codon of stearothermophilus soil gemma lipase gene frequency of use in Pichia pastoris, designs complete red
The high gene order of frequency of use in yeast, specific core former times acid sequence is as shown in SEQID No.3, to improve stearothermophilus
Expression quantity of the native gemma lipase in yeast.The gene optimized is synthesized by GenScript.The gene order of synthesis
It delivers Macrogen to be sequenced, sequencing result and the gene order being pre-designed are completely the same；
Embodiment 2
The structure of Pichia anomala expression plasmid
Using the stearothermophilus soil gemma lipase mature peptide of the above-mentioned synthesis of restriction enzyme Xho1/EcoR5 double digestions
Gene, and the expression plasmid of yeast (pD912, DNA2.0) containing high efficiency methanol evoked promoter AOX1, or contain non-methanol
The expression plasmid of yeast (pD915, DNA2.0) of dependent form constitutive promoter GAP.Glue recycles product after digestion, and by the two
(the stearothermophilus soil gemma lipase after digestion and yeast expression plasmid vector) is ligated and transformed into Escherichia coli with T4 ligases
TOP10 competent cells.It screens and obtains the positive colony of recombinant plasmid (containing promoter-alpha factor signal peptide-stearothermophilus
Native gemma lipase mature peptide expression casette), it delivers Macrogen and is sequenced, positive colony gene order and target
Gene order is completely the same；
Embodiment 3
The structure of stearothermophilus soil gemma lipase Pichia yeast engineering
With the above-mentioned recombinant plasmid that electric shocking method linearizes BamH 1, conversion wild type Pichia pastoris host (DNA2.0,
) or pep4/prb1 double protein deficient pichia pastoris yeast (DNA2.0, PPS-9016) competent cell PPS-9010.
Cell after conversion is coated on the YPD tablets of the 0.2mg/ml -0.8mg/ml zeocin resistances with various concentration, cultivates 3-5
It, the transformant picking monoclonal that will occur on high concentration zeocin-YPD tablets carries out a small amount of fermented and cultured identifications；
Embodiment 4
Express culture and the enzyme activity determination of the Pichia yeast engineering of stearothermophilus soil gemma lipase
The sub- monoclonal of recombinant conversion that picking contains high efficiency methanol evoked promoter AOX1 is inoculated in 20ml BMGY culture mediums
In, 28-30 DEG C, 250rpm/min shaken cultivations 160h to OD600 to 3, thalline were collected by centrifugation, then is suspended in BMMY cultures
In base, it is 1 to be diluted to OD600, continues shaken cultivation, methanol is added into BMMY culture mediums to final concentration of 1.0% every for 24 hours
Induced expression is carried out, is fermented 6 days, is taken supernatant daily, do SDS-PAGE detections.And for containing constitutive promoter GAP's
Recombinant conversion, is directly inoculated in from picking monoclonal on high concentration zeocin-YPD tablets in 2ml YPD culture mediums, 28-30
DEG C, 250rpm/min shaken cultivation 48-72h, zymotic fluid centrifuges 15 minutes in 5000rpm, 4 DEG C, abandons precipitation, takes supernatant, does
SDS-PAGE is detected.The method of lipase activity power in each clonal supernatants of high throughput assay is to utilize the artificial of different carbon chain
The p-nitrophenyl phenolic ester of synthesis makees substrate (being prepared with DMSO), and p-nitrophenyl phenolic ester can be released yellow after lipase hydrolysis
P-nitrophenol calculates reaction using spectrophotometer according to p-nitrophenol in the absorption curves of A405nm normal concentrations
The p-nitrophenol generated in regular hour range Inner in system, then calculate each sample decomposition p-nitrophenyl phenolic ester per minute
Generate the μ g numbers of p-nitrophenol, i.e. enzyme activity unit (U).Do not have in the supernatant of wild type Pichia yeast strain clone not only
Apparent enzyme activity is detected, even if in the supernatant in pep4/prb1 double protein deficient Pichia yeast strain clones
Apparent enzyme activity is not detected.SDS-PAGE shows that difference occurs in the stearothermophilus soil gemma lipase that should be 46kDa
The degradation of degree.These small-molecular-weight products have n terminal amino acid sequence identical with stearothermophilus soil gemma lipase, and
And the C-terminal of these segments contains pairs of alkaline amino acid residue, implies that the characteristic degradation of the lipase may be by ferment
Caused by calcium ion dependence serine endoprotease kexin present in mother cell.By further sequence analysis,
Three possible serine endoprotease kexin are rarely found that in the ripe peptide sequence of stearothermophilus soil gemma lipase
Specific recognition site Kex2；
Embodiment 5
Rite-directed mutagenesis is carried out to potential Kex2 in stearothermophilus soil gemma lipase maturation peptide gene
According to the requirement of rite-directed mutagenesis PCR, three pairs of primers (SEQ ID No.5-10) are separately designed.
Respectively:A1:GTTGCCAGAATTGAAGCAAGGTGGTAGAATCCA,；
A2:TGGATTCTACCACCTTGCTTCAATTCTGGCAAC；
B1:TCGAAAGATTGAAGCAATCCCCAGTTTGGAC；
B2:GTCCAAACTGGGGATTGCTTCAATCTTTCGA；
C1:TGAACGGTCCAAAGCAAGGTTCTTCTGATAG；
C2:CTATCAGAAGAACCTTGCTTTGGACCGTTCA。
IDT is transferred to carry out primer synthesis.Wherein (amino acid number is with thermophilic for rite-directed mutagenesis R103Q for A1 and A2 primers
1) first amino acid of fatty soil gemma lipase mature peptide is that B1 and B2 primers draw for rite-directed mutagenesis R230Q, C1 and C2
Object is used for rite-directed mutagenesis R330Q.Rite-directed mutagenesis PCR is using the Pichia anomala expression plasmid built in embodiment 2 as template, specifically
PCR reaction systems are：1 μ l of template；5 μ l, 2.5mmol/L dNTP of 10X pfu polymerase buffers, 4 μ l；10 μM of upstream and downstream
Each 1 μ l, pf u archaeal dna polymerases, 1 μ l (10U) of primer, it is 50 μ l to add sterile water to total volume.PCR reaction conditions are:95 DEG C pre-
It is denaturalized 20s；98 DEG C of denaturation 10s, 50 DEG C are returned fiery 15s, 72 DEG C of extension 6min, totally 15 cycles；72 DEG C of extensions after 15th cycle
10min.PCR product handles 1h at 37 DEG C by Dpn1, then converts Escherichia coli TOP10 competent cells.It screens and obtains
The positive colony of mutant plasmid extracts the plasmid after rite-directed mutagenesis and Macrogen is transferred to be sequenced.By three-wheel rite-directed mutagenesis
Afterwards, the stearothermophilus soil gemma lipase gene sequence after sequencing result display Kex2 rite-directed mutagenesis and the sequence being pre-designed are complete
It is complete consistent, i.e., it is consistent with nucleotide sequence shown in SEQ ID NO.1；
Embodiment 6
The structure and enzyme activity of the Pichia yeast engineering of stearothermophilus soil gemma lipase gene containing Kex2 mutation are surveyed
It is fixed
By the Pichia pastoris of the stearothermophilus soil gemma lipase gene being mutated containing Kex2 built in above-described embodiment 5
Expression plasmid carries out linearization process with restriction endonuclease BamH 1, is then converted the expression plasmid of yeast of linearisation with electric shocking method
Pep4/prb1 double protein deficient Pichia pastoris competent cells.Yeast cells after conversion is applied to different dense
The YPD tablets of the 0.2mg/ml -0.8mg/ml zeocin resistances of degree are cultivated 3-5 days, will be on high concentration zeocin-YPD tablets
The transformant picking monoclonal of appearance carries out a small amount of fermented and cultureds (incubation step is with reference to embodiment 4).Fermentation culture is through centrifugation
After take supernatant, do SDS-PAGE detections and high-throughput enzyme activity determination.As desired by us, almost each contain
The Pichia yeast engineering clone of the stearothermophilus soil gemma lipase gene of Kex2 mutation shows higher than bacterial expression very
More lipase hydrolysis vigor.The secretory protein that SDS-PAGE is showed more than 70% is the stearothermophilus soil bud of the 46kDa of overall length
Spore lipase, and without apparent protein degradation phenomenon.And the yeast of the constitutive promoter GAP containing non-methanol dependence
Express engineering bacteria clone has higher lipase table than the Yeast expression engineering bacteria clone containing methanol induction promoter AOX1
Up to amount and enzyme activity；
Embodiment 7
The biochemical characteristic of the stearothermophilus soil gemma lipase containing Kex2 mutation of Pichia yeast engineering expression
Choose the highest stearothermophilus soil gemma lipase ferment containing constitutive promoter GAP of a lipase activity power
Matrix expression engineered bacteria is cloned, and is made substrate using the artificial synthesized p-nitrophenyl phenolic ester of C2-C18 different carbon chains and (is matched with DMSO
System), spectrophotometer method measures the stearothermophilus soil gemma lipase containing Kex2 mutation of Pichia anomala expression to different carbon
The activity of chain substrate.As shown in the figure 1, the stearothermophilus soil gemma lipase of Yeast expression all shows the substrate of different carbon chain
Activity especially has a preference for the substrate of C2-C16, and most suitable substrate is the p-nitrophenyl phenolic ester of C10.And with the p-nitrophenyl phenolic ester of C10
Make substrate, the range Inner of the stearothermophilus soil gemma lipase of Yeast expression in pH3.4-10.0 is active, and as schemed institute
Show 2, pH value is higher, and enzyme activity is higher, is typical alkaline lipase.
Sequence table
The information of SEQ ID NO.1
<>A kind of stearothermophilus soil gemma lipase gene being mutated containing novel kex2 and its in Pichia pastoris
Expression
<>1167
<>DNA
<>Artificial sequence
GCTTCACTTAGAGCAAATGACGCACCTATCGTTCTTCTTCACGGTTTCACAGGATGGGGT
AGAGAGGAGATGTTCGGTTTCAAATACTGGGGTGGTGTTAGAGGAGATATTGAACAATGG
TTGAACGATAACGGTTACAGAACTTACACTTTGGCTGTTGGTCCATTGTCTTCTAACTGG
GATAGAGCTTGTGAAGCTTACGCTCAATTGGTTGGTGGTACTGTTGATTACGGTGCTGCT
CATGCTGCTAAGCATGGTCATGCTAGATTTGGTAGAACTTACCCAGGTTTGTTGCCAGAA
TTGAAGCAAGGTGGTAGAATCCATATTATTGCTCATTCTCAAGGTGGTCAAACTGCTAGA
ATGTTGGTTTCTTTGTTGGAAAACGGTTCTCAAGAAGAAAGAGAATACGCTAAGGCTCAT
AACGTTTCTTTGTCTCCATTGTTTGAAGGTGGTCATCATTTCGTTTTGTCTGTTACTACT
ATCGCTACTCCACATGATGGTACTACTTTGGTTAACATGGTTGATTTCACTGATAGATTT
TTCGATTTGCAAAAGGCTGTTTTGGAGGCTGCTGCTGTTGCTTCTAACGTTCCATACACT
TCTCAAGTTTACGATTTCAAGTTGGATCAATGGGGTTTGAGAAGACAACCTGGTGAATCT
TTCGATCATTACTTCGAAAGATTGAAGCAATCCCCAGTTTGGACTTCTACTGATACTGCT
AGATACGATTTGTCTGTTTCTGGTGCTGAAAAGTTGAACCAATGGGTTCAAGCTTCTCCA
AACACTTACTACTTGTCTTTTTCTACTGAAAGAACTTACAGAGGTGCTTTGACTGGTAAC
CACTACCCAGAATTGGGTATGAACGCTTTTTCTGCTGTTGTTTGTGCTCCATTTTTGGGT
TCTTACAGAAACCCTACTTTGGGTATTGATGATAGATGGTTGGAAAACGATGGTATTGTT
AACACTGTTTCTATGAACGGTCCAAAGCAAGGTTCTTCTGATAGAATTGTTCCATACGAT
GGTACTTTGAAGAAGGGTGTTTGGAACGATATGGGTACTTATAACGTTGATCATTTGGAA
ATTATTGGTGTTGATCCAAACCCATCCTTTGACATCAGAGCCTTTTATTTGAGATTGGCA
GAACAACTTGCTTCATTGCAGCCATAA
The information of SEQ ID NO.2
<>A kind of stearothermophilus soil gemma lipase gene being mutated containing novel kex2 and its in Pichia pastoris
Expression
<>388
<>PRT
<>Artificial sequence
ASLRANDAPIVLLHGFTGWGREEMFGFKYWGGVRGDIEQWLNDNGYRTYTLAVGPLSSNWDRACEAYAQ
LVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKQGGRIHIIAHSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSP
LFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRFFDLQKAVLEAAAVASNVPYTSQVYDFKLDQWGLRRQPGESFDH
YFERLKQSPVWTSTDTARYDLSVSGAEKLNQWVQASPNTYYLSFSTERTYRGALTGNHYPELGMNAFSAVVCAPFLG
SYRNPTLGIDDRWLENDGIVNTVSMNGPKQGSSDRIVPYDGTLKKGVWNDMGTYNVDHLEIIGVDPNPSFDIRAFYL
RLAEQLASLQP
The information of SEQ ID NO.3
<>A kind of native gemma lipase gene of wild-type thermophilic fat containing codon optimization and its in Pichia pastoris
Expression
<>1167
<>DNA
<>Artificial sequence
GCTTCACTTAGAGCAAATGACGCACCTATCGTTCTTCTTCACGGTTTCACAGGATGGGGTAGAGAGGAG
ATGTTCGGTTTCAAATACTGGGGTGGTGTTAGAGGAGATATTGAACAATGGTTGAACGATAACGGTTACAGAACTTA
CACTTTGGCTGTTGGTCCATTGTCTTCTAACTGGGATAGAGCTTGTGAAGCTTACGCTCAATTGGTTGGTGGTACTG
TTGATTACGGTGCTGCTCATGCTGCTAAGCATGGTCATGCTAGATTTGGTAGAACTTACCCAGGTTTGTTGCCAGAA
TTGAAGAGAGGTGGTAGAATCCATATTATTGCTCATTCTCAAGGTGGTCAAACTGCTAGAATGTTGGTTTCTTTGTT
GGAAAACGGTTCTCAAGAAGAAAGAGAATACGCTAAGGCTCATAACGTTTCTTTGTCTCCATTGTTTGAAGGTGGTC
ATCATTTCGTTTTGTCTGTTACTACTATCGCTACTCCACATGATGGTACTACTTTGGTTAACATGGTTGATTTCACT
GATAGATTTTTCGATTTGCAAAAGGCTGTTTTGGAGGCTGCTGCTGTTGCTTCTAACGTTCCATACACTTCTCAAGT
TTACGATTTCAAGTTGGATCAATGGGGTTTGAGAAGACAACCTGGTGAATCTTTCGATCATTACTTCGAAAGATTGA
AGAGATCCCCAGTTTGGACTTCTACTGATACTGCTAGATACGATTTGTCTGTTTCTGGTGCTGAAAAGTTGAACCAA
TGGGTTCAAGCTTCTCCAAACACTTACTACTTGTCTTTTTCTACTGAAAGAACTTACAGAGGTGCTTTGACTGGTAA
CCACTACCCAGAATTGGGTATGAACGCTTTTTCTGCTGTTGTTTGTGCTCCATTTTTGGGTTCTTACAGAAACCCTA
CTTTGGGTATTGATGATAGATGGTTGGAAAACGATGGTATTGTTAACACTGTTTCTATGAACGGTCCAAAGAGAGGT
TCTTCTGATAGAATTGTTCCATACGATGGTACTTTGAAGAAGGGTGTTTGGAACGATATGGGTACTTATAACGTTGA
TCATTTGGAAATTATTGGTGTTGATCCAAACCCATCCTTTGACATCAGAGCCTTTTATTTGAGATTGGCAGAACAAC
TTGCTTCATTGCAGCCATAA
The information of SEQ ID NO.4
<>A kind of native gemma lipase gene of wild-type thermophilic fat containing codon optimization and its in Pichia pastoris
Expression
<>388
<>PRT
<>Artificial sequence
ASLRANDAPIVLLHGFTGWGREEMFGFKYWGGVRGDIEQWLNDNGYRTYTLAVGPLSSNW
DRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRGGRIHIIAHSQGGQTAR
MLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIATPHDGTTLVNMVDFTDRF
FDLQKAVLEAAAVASNVPYTSQVYDFKLDQWGLRRQPGESFDHYFERLKRSPVWTSTDTA
RYDLSVSGAEKLNQWVQASPNTYYLSFSTERTYRGALTGNHYPELGMNAFSAVVCAPFLG
SYRNPTLGIDDRWLENDGIVNTVSMNGPKRGSSDRIVPYDGTLKKGVWNDMGTYNVDHLE
IIGVDPNPSFDIRAFYLRLAEQLASLQP
The information of SEQ ID NO.5
<>Carry out the primer A1 of kex2 rite-directed mutagenesis
<>33
<>DNA
<>Artificial sequence
GTTGCCAGAATTGAAGCAAGGTGGTAGAATCCA
The information of SEQ ID NO.6
<>Carry out the primer A2 of kex2 rite-directed mutagenesis
<>33
<>DNA
<>Artificial sequence
TGGATTCTACCACCTTGCTTCAATTCTGGCAAC
The information of SEQ ID NO.7
<>Carry out the primer B1 of kex2 rite-directed mutagenesis
<>31
<>DNA
<>Artificial sequence
TCGAAAGATTGAAGCAATCCCCAGTTTGGAC
The information of SEQ ID NO.8
<>Carry out the primer B2 of kex2 rite-directed mutagenesis
<>31
<>DNA
<>Artificial sequence
GTCCAAACTGGGGATTGCTTCAATCTTTCGA
The information of SEQ ID NO.9
<>Carry out the primer C1 of kex2 rite-directed mutagenesis
<>31
<>DNA
<>Artificial sequence
TGAACGGTCCAAAGCAAGGTTCTTCTGATAG
The information of SEQ ID NO.10
<>Carry out the primer C2 of kex2 rite-directed mutagenesis
<>31
<>DNA
<>Artificial sequence
CTATCAGAAGAACCTTGCTTTGGACCGTTCA
In conclusion stearothermophilus soil gemma lipase gene secretory expression method proposed by the present invention, using complete red ferment
Female expression system and according to Pichia pastoris preference optimize codon after stearothermophilus soil gemma lipase gene, more favorably
In the posttranslational modification, folding and efficient secretion of the lipase.Pass through rite-directed mutagenesis stearothermophilus soil gemma lipase mature peptide
Three serine stretch protein enzyme recognition site Kex2 in sequence avoid the lipase of secretion by yeast cells endogenous serine
Endo protease kexin degradation obtains expressed intact, high enzyme activity stearothermophilus soil gemma fat in Pichia pastoris
Enzyme.This still belongs to the first time in the research field of stearothermophilus soil gemma lipase, is the extensive of stearothermophilus soil gemma lipase
Industrialized production provides reliable guarantee.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (5)
1. a kind of stearothermophilus soil gemma lipase gene secretory expression method, it is characterised in that：Include the following steps：
Step 1：Search obtains stearothermophilus soil gemma lipase maturation peptide gene in Genbank, according to Pichia pastoris to heredity
The preference of codon carries out codon optimization to stearothermophilus soil gemma lipase maturation peptide gene；
Step 2：According to the requirement of rite-directed mutagenesis PCR, three pairs of primers are separately designed；Respectively:
A1:GTTGCCAGAATTGAAGCAAGGTGGTAGAATCCA；
A2:TGGATTCTACCACCTTGCTTCAATTCTGGCAAC；
B1:TCGAAAGATTGAAGCAATCCCCAGTTTGGAC；
B2:GTCCAAACTGGGGATTGCTTCAATCTTTCGA；
C1:TGAACGGTCCAAAGCAAGGTTCTTCTGATAG；
C2:CTATCAGAAGAACCTTGCTTTGGACCGTTCA；
IDT is transferred to carry out primer synthesis；
Step 3：It using A1 and A2 as primer, carries out PCR and reacts rite-directed mutagenesis R103Q, using B1 and B2 as primer, carry out PCR reactions
Rite-directed mutagenesis R230Q, then using C1 and C2 as primer, carry out PCR reaction rite-directed mutagenesis R330Q；
Step 4：Stearothermophilus soil gemma lipase maturation peptide gene after three-wheel Kex2 rite-directed mutagenesis is sequenced, is sequenced
As a result display is consistent with the sequence being pre-designed by the stearothermophilus soil gemma lipase gene sequence after rite-directed mutagenesis PCR；
Step 5：By the wild type of synthesis or the stearothermophilus soil gemma lipase maturation peptide gene of Kex2 rite-directed mutagenesis with finish it is red
Expression plasmid of yeast carries out digestion respectively, and glue recycles endonuclease bamhi, and T4 ligases convert Escherichia coli after connecting the two, contained
The bacterial strain for having the expression plasmid of promoter-alpha factor signal peptide-stearothermophilus soil gemma lipase maturation peptide gene, chooses clone and carries
It is sequenced after taking plasmid；
Step 6：Extract the plasmid through sequencing identification in above-mentioned bacterial strains, electrotransformation pep4/prb1 double protein enzymes after linearization for enzyme restriction
Deficiency pichia pastoris yeast competent cell, the cell after conversion are coated on the zeocin resistances with various concentration
YPD tablets choose clone, carry out a small amount of fermented and cultureds to clone, enzyme activity are detected, to obtain new and effective stearothermophilus soil bud
The Pichia yeast engineering of spore lipase gene.
2. a kind of stearothermophilus soil gemma lipase gene secretory expression method according to claim 1, it is characterised in that：
3 potential serine stretch protein enzyme recognition site Kex2 are changed to other amino acid residues and by complete red ferment in ripe peptide sequence
The stearothermophilus soil gemma lipase gene of female preference codon design.
3. a kind of stearothermophilus soil gemma lipase gene secretory expression method according to claim 1, it is characterised in that：
Expression vector is the plasmid of the constitutive promoter GAP containing high efficiency methanol evoked promoter AOX1 or non-methanol dependences.
4. a kind of stearothermophilus soil gemma lipase gene secretory expression method according to claim 1, it is characterised in that：
The gene optimized is synthesized by GenScript in step 1, the gene order of synthesis is sequenced by Macrogen, is surveyed
Sequence result is consistent with the gene order being pre-designed.
5. a kind of stearothermophilus soil gemma lipase gene secretory expression method according to claim 1, it is characterised in that：
In step 3 amino acid number with first amino acid of stearothermophilus soil gemma lipase mature peptide be 1.
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| CN110452892A (en) * | 2019-09-24 | 2019-11-15 | 杭州师范大学 | A kind of lipase of macro gene source, encoding gene, carrier, engineering bacteria and the application in lutein preparation |
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