CN108239648A - The method of high efficient expression rhizomucor miehei lipase - Google Patents
The method of high efficient expression rhizomucor miehei lipase Download PDFInfo
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- CN108239648A CN108239648A CN201611217262.9A CN201611217262A CN108239648A CN 108239648 A CN108239648 A CN 108239648A CN 201611217262 A CN201611217262 A CN 201611217262A CN 108239648 A CN108239648 A CN 108239648A
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- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
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Abstract
The present invention relates to the methods of high efficient expression rhizomucor miehei lipase.Specifically, the present invention provides a kind of polynucleotide sequence, the polynucleotide sequence is selected from：(1)SEQ ID NO:Sequence shown in 1 the 229th~1035 nucleotide sequence；(2) complementary series of (1) described sequence.The present invention also provides a kind of polynucleotide sequence, the polynucleotide sequence is selected from：(1) polynucleotide sequence of peptide sequence shown in following formula is encoded：A‑L1‑B‑L2‑RML；In formula, A may be present or be not present, and be rhizomucor miehei lipase leader peptide when it is present；L1 exists in the presence of A, is the restriction enzyme site of protease kex2 and ste13；B is rhizomucor miehei lipase leader peptide；L2 is the restriction enzyme site of protease kex2 and ste13；RML represents the ripe peptide sequence of rhizomucor miehei lipase；(2) complementary series of (1) described sequence.
Description
Technical field
The present invention relates to the methods of high efficient expression rhizomucor miehei lipase.
Background technology
Lipase (Lipase EC 3..1.1.3) i.e. Lipase, it is catalyzed natural substrate grease water
Solution generation aliphatic acid, glycerine and monoglyceride or diester, are widely used in the industry such as fats and oils processing, food, medicine, daily use chemicals, are
One of important industrial enzyme preparation.Its catalytic activity is dependent only on its protein structure, thus the lipase of separate sources
With different catalysis characteristics and catalysis activity.
At present it is known that about 2% microorganism yielding lipase, the microorganism of energy yielding lipase includes at least 65 categories,
Wherein 28, bacterium belongs to, 4, actinomyces belong to, 10, saccharomycete belongs to, other fungies 23 belong to (Hasan, 2006).Wherein rice black root
For miehei lipase because having stronger sn-1, the characteristic of 3 selective and high vigor has been widely used for structured lipid
The enzymatic clarification of (Structured lipids), such as the synthesis of SOS, OPO, DAG, the how unsaturateds fat such as processing fish oil enrichment DHA
Preparation (Fujikih, 2009) of fat acid (Pedersen, 1995) and chiral medicinal intermediate and certain biomaterials etc..
Natural rhizomucor miehei aliphatic acid yield is low, ingredient is unstable, extraction difficulty etc. there are it is larger the defects of so that
It can not industrialized production.Therefore, its zymologic property, application range are concentrated mainly on and in gene work for the research of RML
High efficient expression in journey bacterium.In report in relation to RML, the research of letter (Novo) company of Novi is the most full and accurate.1977
(Moskowitz, 1977), Novo companies researcher has made intensive studies natural RML, the results showed that：RML is a kind of sugared egg
In vain, can hydrolysis of animal fat and vegetable oil extensively, in the range of pH4-9, keep in certain time stablizing at room temperature, wherein pH
Stablize the most when 6.0.1987, the Huge-jensen of Novo report in natural rhizomucor miehei zymotic fluid there are RML-A and
Two kinds of enzymes for having high degree of immunogenicity consistent of RML-B, and illustrate the relationship of the two and primary structure.1988 (Boel, 1988),
Novo companies Boel and Huge-jensen has found the cDNA sequence of coding RML, and determines that RML precursor proteins are by RML, 70
The signal peptide of the precursor peptide chain of a amino acid residue and 24 amino acid residues is formed, and passes through the MET- of cleavage zymogen protein
Peptide bond between SER and obtain the RML of 269 amino acid residues.1989, Huge-jensen was by the precursor protein gene of RML
It is inserted into the carrier of aspergillus oryzae, is expressed to obtain using the promoter of alpha-amylase gene and the terminator of carbohydrase extracellular
The rRML of secretion.The n terminal amino acid sequence for having 70% in the rRML obtained using the expression vector is consistent with native enzyme, and another
A 30% outer recombinase silk threonine residues fewer than native enzyme.In addition, the isoelectric point of recombinase is consistent with RML for 4.3,
Sugar content is 1.2%, and immune property is also similar to native enzyme height.The RML sold on Novo houses markets at present also with
The gene-modified lipase that technique for gene engineering is expressed using aspergillus oryzae as carrier, liquid enzymes trade name20000L,
Immobilised enzymes trade name Lipozyme RM IM.A kind of excellent expression vector although fungi aspergillus oryzae be can yet be regarded as, from
Body can secrete various non-targeted albumen, such as：The content of non-destination protein amylase is far above in the Lipozyme RM enzyme solutions of Novo
Destination protein RML lipase, in addition, also a lot of other foreign proteins are such as：Protease etc. causes its application to be very limited.
At present, it is purified again although Novo passes through RML liquid, obtains trade name388 RML, but because price is held high
It is expensive, further limit its application in the industry.
Invention content
First aspect present invention provides a kind of coded sequence of rhizomucor miehei lipase, and the sequence is selected from：
(1)SEQ ID NO:Sequence shown in 1 the 229th~1035 nucleotide sequence；With
(2) complementary series of (1) described sequence.
Second aspect of the present invention provides a kind of polynucleotide sequence, and the polynucleotide sequence is selected from：
(1) polynucleotide sequence of the peptide sequence shown in following formula I is encoded：
A-L1-B-L2-RML (Formulas I)
In formula,
A may be present or be not present, and be rhizomucor miehei lipase leader peptide when it is present；
L1 exists in the presence of A, is the restriction enzyme site of protease kex2 and ste13；
B is rhizomucor miehei lipase leader peptide；
L2 is the restriction enzyme site of protease kex2 and ste13；With
RML represents the ripe peptide sequence of rhizomucor miehei lipase；With
(2) complementary series of (1) described sequence.
In one or more embodiments, there are A and L1 for the peptide sequence shown in the Formulas I.
In one or more embodiments, the leading peptide-coding sequence of the rhizomucor miehei lipase such as SEQ ID NO:
Shown in 1 the 1st~210.
In one or more embodiments, the rhizomucor miehei lipase mature polypeptide coding sequence such as SEQ ID NO:
Shown in 1 the 229th~1035.
In one or more embodiments, the nucleotide sequence of the L1 and L2 are respectively independent such as SEQ ID NO:1
Shown in 211~228 or such as SEQ ID NO:Shown in 3 the 211st~234.
In one or more embodiments, the rhizomucor miehei lipase leader peptide such as SEQ ID NO:2 the 1st~70
Shown in position.
In one or more embodiments, the rhizomucor miehei lipase mature peptide such as SEQ ID NO:2 the 77th~
Shown in 345.
In one or more embodiments, the amino acid sequence of the L1 and L2 are respectively independent such as SEQ ID NO:2
Shown in 71~76 or such as SEQ ID NO:Shown in 4 the 71st~78.
In one or more embodiments, the peptide sequence such as SEQ ID NO shown in Formulas I:Shown in 2 or 4.
In one or more embodiments, the polynucleotide sequence is selected from：
(i)SEQ ID NO:Polynucleotide sequence shown in 1 or 3；With
(ii) complementary series of (i) described polynucleotide sequence.
Third aspect present invention provides a kind of nucleic acid constructs, which contains polynucleotides of the present invention
Sequence.
In one or more embodiments, the nucleic acid constructs contains the polynucleotides described in first aspect present invention
Sequence.
In one or more embodiments, the nucleic acid constructs contains the polynucleotides described in second aspect of the present invention
Sequence.
In one or more embodiments, the nucleic acid constructs is cloning vector or expression vector.
In one or more embodiments, the expression vector is the carrier expressed suitable for Pichia pastoris, including
But it is not limited to pPIC, pPICZ, pAO, pGAP and pGAPZ.
In one or more embodiments, the expression vector is using pAO815 plasmids as skeleton.
Fourth aspect present invention provides a kind of genetically engineered host cell, contains polynucleotides of the present invention
Sequence or nucleic acid constructs.
In one or more embodiments, the cell is yeast.
In one or more embodiments, the yeast is Pichia pastoris.
Fifth aspect present invention provides a kind of method for preparing rhizomucor miehei lipase, and this method includes structure containing this hair
The expression vector of the bright polynucleotide sequence is transferred in host cell and is cultivated the host cell to express
The step of stating lipase.
Fifth aspect present invention provides the side that a kind of structure can improve the expression vector of rhizomucor miehei lipase expression quantity
Method, the method includes inserting egg between the leading peptide-coding sequence of rhizomucor miehei lipase and mature polypeptide coding sequence
The gene order of the restriction enzyme site of white enzyme kex2 and ste13 is inserted into the step in expression vector.
It is leading in the rhizomucor miehei lipase in the gene order being inserted into one or more embodiments
Further include before peptide-coding sequence copy the leading peptide-coding sequence of rhizomucor miehei lipase and protease kex2 and
The restriction enzyme site of ste13.
In one or more embodiments, the gene order has carried out excellent according to the preferences of Pichia pastoris codon
Change.
In one or more embodiments, the expression vector is suitable for expressing in yeast.
In one or more embodiments, the expression vector is suitable for expressing in Pichia pastoris.
In one or more embodiments, the expression vector is using pAO815 plasmids as skeleton.
In one or more embodiments, the leading peptide-coding sequence of the rhizomucor miehei lipase such as SEQ ID NO:
Shown in 1 the 1st~210.
In one or more embodiments, the rhizomucor miehei lipase mature polypeptide coding sequence such as SEQ ID NO:
Shown in 1 the 229th~1035.
In one or more embodiments, the nucleotide sequence of the restriction enzyme site of the protease kex2 and ste13 is such as
SEQ ID NO:Shown in 1 the 211st~228 or such as SEQ ID NO:Shown in 3 the 211st~234.
In one or more embodiments, the rhizomucor miehei lipase leader peptide such as SEQ ID NO:2 the 1st~70
Shown in position.
In one or more embodiments, the rhizomucor miehei lipase mature peptide such as SEQ ID NO:2 the 77th~
Shown in 345.
In one or more embodiments, the amino acid sequence of the restriction enzyme site of the protease kex2 and ste13 is such as
SEQ ID NO:Shown in 2 the 71st~76 or such as SEQ ID NO:Shown in 4 the 71st~78.
In one or more embodiments, the gene order coding such as SEQ ID NO:Amino acid sequence shown in 2 or 4
Row.
In one or more embodiments, the gene order is selected from：
(i)SEQ ID NO:Polynucleotide sequence shown in 1 or 3；With
(ii) complementary series of (i) described polynucleotide sequence.
Sixth aspect present invention provides a kind of rhizomucor miehei lipase preparation method, and the method includes the fermentation present invention
The step of host cell.
Description of the drawings
Fig. 1：The fermentation enzyme activity of pro/RML and 2pro/RML.
Fig. 2：The SDS-PAGE electrophoresis of pro/RML-1 and 2pro/RML-1, No. 1 swimming lane are 2pro/RML-1；No. 2 swimming lanes are
pro/RML-2。
Specific embodiment
The present invention optimizes the rhizomucor miehei gene with leader peptide according to the preferences of Pichia pastoris codon, and
And increase the restriction enzyme site of protease kex2 and ste13 between leader peptide and mature peptide, by the gene cloning to Pichia pastoris
In expression vector such as pAO815, corresponding expression vector is obtained, converts Pichi strain, obtained positive colony carries out shaking flask
Fermentation measures its enzyme activity as 311U/ml.The present invention is also by the leader peptide of the rhizomucor miehei lipase gene of 2 copies and maturation
Peptide is attached, and between two leader peptides and second between leader peptide and mature peptide increase protease kex2 and
The restriction enzyme site of ste13 in the gene cloning to yeast expression vector such as pAOm-PLC, will obtain expressing carrying accordingly
Body converts Pichi strain, and obtained positive colony carries out shake flask fermentation, measures its enzyme activity as 464U/ml, the yield of RML
Expression way compared to single leader peptide improves 50%.
Therefore, the present invention provides a kind of method and high efficient expression rice black root of high efficient expression rhizomucor miehei lipase
The Pichia pastoris of miehei lipase.
In the present invention, rhizomucor miehei lipase can be the various fat from rhizomucor miehei well known in the art
Enzyme, the various mutant including wild type rhizomucor miehei lipase as long as the lipase activity described in the mutant, have work
Industry application value.In general, rhizomucor miehei lipase of the present invention refers to mature peptide.In certain embodiments, originally
The method of invention is used to express SEQ ID NO:Rhizomucor miehei lipase shown in 2 the 77th~345 amino acids sequences.
The present invention by expression rhizomucor miehei lipase vector encoded rhizomucor miehei lipase leader peptide and into
The restriction enzyme site of protease kex2 and ste13 are inserted between the coded sequence of ripe peptide, so as to realize rhizomucor miehei lipase
Via high expression of the expression vector in host cell.It can be inserted into several (such as 1~3) kex2 protease cleavage sites
With several (such as 1~3) stel3 protease cleavage sites.In certain embodiments, the enzyme of protease kex2 and ste13
The amino acid sequence of enzyme site can be such as SEQ ID NO:Shown in 2 the 71st~76.In certain embodiments, it can be inserted into 1
Kex2 and 2 stel3 restriction enzyme site.In these embodiments, the amino acid sequence of illustrative restriction enzyme site such as SEQ ID
NO:Shown in 4 the 71st~78.
Each aspect of the present invention will be described in detail below.
Polynucleotide sequence
The polynucleotide sequence of the present invention is selected from：
(1) polynucleotide sequence of the peptide sequence shown in following formula I is encoded：
A-L1-B-L2-RML (Formulas I)
In formula,
A may be present or be not present, and be rhizomucor miehei lipase leader peptide when it is present；
L1 exists in the presence of A, is the restriction enzyme site of protease kex2 and ste13；
B is rhizomucor miehei lipase leader peptide；
L2 is the restriction enzyme site of protease kex2 and ste13；With
RML represents the ripe peptide sequence of rhizomucor miehei lipase；With
(2) complementary series of (1) described sequence.
In the present invention, rhizomucor miehei lipase leader peptide can be the various leading of the lipase obtained from rhizomucor miehei
Peptide.In certain embodiments, the sequence of the leader peptide such as SEQ ID NO:Shown in 2 the 1st~70.
In the present invention, the restriction enzyme site of protease kex2 and ste13 are often referred to such as SEQ ID NO:Shown in 2 the 71st~76
Amino acid sequence.In certain embodiments, it can be inserted into 1 kex2 and 2 stel3 restriction enzyme site.In these embodiments
In, the amino acid sequence such as SEQ ID NO of illustrative restriction enzyme site:Shown in 4 the 71st~78.
Therefore, L1 of the invention and L2 can respectively stand alone as SEQ ID NO:Amino acid sequence shown in 2 the 71st~76
Or SEQ ID NO:Amino acid sequence shown in 4 the 71st~78.
In certain embodiments of the invention, there are A and L1 for the peptide sequence shown in the nucleotide sequence coded Formulas I.
As specific embodiment, the peptide sequence shown in Formulas I can be such as SEQ ID NO:Shown in 2 or 4.As polynucleotides sequence
The specific embodiment of row, the present invention provide SEQ ID NO:Polynucleotide sequence shown in 1 or 3, also including SEQ ID NO:1 He
3 complementary series.
The application further includes the degeneracy variant for the nucleotide sequence for encoding Formulas I polypeptide of the present invention.As used herein, " letter
And variant " refer to encode identical amino acid sequence, but the differentiated nucleotide sequence of nucleotide sequence in the present invention.
In preferred embodiments, the maturation of polynucleotide sequence of the invention, especially rhizomucor miehei lipase
The coded sequence of peptide optimizes according to the preferences of Pichia pastoris codon.For example, in certain embodiments, it is described
The coded sequence of mature peptide such as SEQ ID NO:Shown in 1 the 77th~345 nucleotide.
Therefore, in certain embodiments, the present invention also provides a kind of coding of the mature peptide of rhizomucor miehei lipase
Sequence is selected from：
(1)SEQ ID NO:Sequence shown in 1 the 229th~1035 nucleotide sequence；With
(2) complementary series of (1) described sequence.
The polynucleotide sequence of the present invention can usually use PCR amplification method, recombination method or artificial synthesized method to obtain.It is right
In PCR amplification method, can be drawn according to related nucleotide sequence, especially open reading frame sequence disclosed in this invention to design
Object, and by the use of commercially available cDNA libraries or the cDNA libraries as prepared by conventional method well known by persons skilled in the art are as template, expand
Increase and obtain related sequence.When sequence is longer, it is often necessary to twice or multiple PCR amplification, then again amplify each time
Segment be stitched together by proper order.
Nucleic acid constructs
The present invention also relates to the one or more regulation and control including being connect with polynucleotide sequence operability of the present invention
The nucleic acid constructs of sequence.
Herein, " operability connection " or the arrangement of similar description finger element, wherein the ingredient be ordered in it is certain
Shape, to perform the function needed for them.Thus, operability is connected to the given promoter of coded sequence, correct
Transcription factor etc. in the presence of, the coded sequence effective expression can be made.The promoter does not need to abut with the coded sequence, as long as
It plays the function that the sequence is instructed to express.Thus, for example the sequence that is not involved in translating but transcribe may be present in promoter
Between sequence and coded sequence, as transcribed introne；And still it is believed that promoter sequence " operability company
Connect " in the coded sequence.
The polynucleotides of coding polypeptide of the present invention can be operable to ensure in many ways the expression of the polypeptide.In its insertion
Before carrier the operation of the polynucleotide sequence may according to the expression vector but cater to the need or required.Utilize recombination
DNA methods are come to change the technology of polynucleotide sequence be known in the art.
Regulating and controlling sequence can be suitable promoter sequence, be for expressing the multinuclear for encoding polypeptide shown in Formulas I of the present invention
The nucleotide sequence that the host cell of thuja acid is identified.Promoter sequence includes the transcription regulating nucleotide sequence for being connected to polypeptide expression.It opens
Mover can be any nucleotide sequence that transcriptional activity is shown in selected host cell, including mutation, truncated
And hybrid promoter, and can be obtained from the gene for encoding the extracellular or intracellular polypeptide homologous or heterologous with the host cell.
In yeast host, useful promoter is available from saccharomyces cerevisiae enolase (ENO-1), saccharomyces cerevisiae galactolipin
Kinases (GAL1), Ethanol in Saccharomyces cerevisiae dehydrogenase, glyceraldehyde 3-phosphate dehydro-genase, saccharomyces cerevisiae phosphotriose isomerase, wine brewing
Gene, the pichia pastoris alcohol oxidase gene of yeast glycerol 3-phosphate acid kinase.For the other of yeast host cell
Useful promoter is by Romanos et al., and 1992, Yeast 8:423-488 is described.
Regulating and controlling sequence can also be suitable transcription terminator sequences, and the sequence to terminate transcription is identified by host cell.
3 ' ends of nucleotide sequence of the terminator sequence with encoding the polypeptide are operatively connected.It is functional in the host cell of selection
Any terminator can be used in the present invention.
For yeast host cell preferred terminator obtained from saccharomyces cerevisiae enolase, S. cerevisiae cytochrome C, make
Brewer yeast glyceraldehyde-3-phosphate dehydrogenase, pichia pastoris alcohol oxidase gene etc..
Carrier
The present invention also relates to the carrier for including polynucleotide sequence of the present invention, including but not limited to expression vector and clone carries
Body.For example, in certain embodiments, nucleic acid constructs of the present invention is expression vector or cloning vector.
In expression vector, various nucleic acid and regulating and controlling sequence can be joined together may include one or more hold to generate
Perhaps in the insertion of this site or the recombinant expression carrier for facilitating restriction site for the nucleotide sequence for replacing the coding polypeptide.It is standby
Selection of land, nucleotide sequence of the invention can be by being inserted into nucleotide sequence or including the nucleic acid of the sequence into appropriate expression vector
Construction and be expressed.When manufacturing expression vector, coded sequence is located in carrier so that the coded sequence operationally connects
It connects to express appropriate regulation sequence.
Recombinant expression carrier can allow to easily be subjected to recombinant DNA method and can lead to interested nucleotides sequence
Any carrier (such as plasmid or virus) that list reaches.The selection of carrier is generally dependent on carrier and is wherein imported into the place of the carrier
The compatibility of chief cell.The carrier can be circular plasmids that are linear or being closed.
Carrier can be the carrier of autonomous replication, i.e., exist as extrachromosomal entity, replicate independent of chromosome
The carrier of duplication, such as plasmid, extra-chromosomal element, minichromosome or artificial chromosome.Carrier may include ensureing certainly
Any mode that I replicates.Alternatively, carrier can be when being imported into host cell, be integrated into genome and with it
The carrier that chromosome through being be integrated into replicates together.Host cell gene group will be imported into addition, can be used and include together
The single carrier of total DNA or plasmid or two or more carriers or plasmid or transposons.
The present invention carrier preferably comprise one or more allow easily select convert, transfect, transduce it is isocellular optional
Select label.Selectable label is gene, and product provides the resistance to antibiotic or virus, the resistance to heavy metal, original and supports
Type is to auxotroph etc..
The carrier of the present invention, which preferably comprises, allows the vector integration to enter host cell gene group or the carrier in cell
Independently of autonomous element replicated of genome.
The polynucleotides of the present invention of more than one copy can be inserted into host cell to increase the yield of the gene outcome.
Increasing for polynucleotide copies number can be by being integrated into host cell gene group by the sequence of at least one additional copies or leading to
It crosses and is obtained including amplifiable selectable marker gene and the polynucleotides, wherein the selectable marker gene comprising amplification copy is simultaneously
And the cell thus comprising additional copies polynucleotides can be by there are cultivate the cell during appropriate selective agent to screen.
The expression vector of the present invention more preferably selects the carrier that can be used for expressing in Pichia pastoris.The carrier of the present invention
It is preferred that a series of carrier such as the carrier used in the Pichia pastoris of commercialization such as pPIC, pPICZ, pAO, pGAP or pGAPZ.
Cloning vector containing polynucleotide sequence of the present invention can be used for replicating enough target plasmids.Therefore, this hair
Bright cloning vector carries stronger self-replacation element, such as replication origin.In general, the cloning vector of the present invention does not have
There is Expression element.
Host cell
The present invention also relates to the recombinant host cells of the polynucleotides of the present invention including being used for recombinant production polypeptide.Including
The carrier of polynucleotides of the present invention is imported into host cell so that the composition portion as chromosome of the carrier as explained earlier
Divide or maintained as extrachromosomal self-replacation carrier.The selection of host cell is heavily dependent on coding polypeptide
Gene and its source.
Preferably, the cell that host cell is Ascomycota, such as saccharomyces are suitable for the invention
(Saccharomyces), pichia (Pichia), Ye Shi saccharomyces (Yarrowia), candida (Candida) with
And Komagataella categories etc..
At most preferred aspect, host cell is pichia pastoris yeast (Pichia pastoris), saccharomyces cerevisiae
(Saccharomyces cerevisiae), Yarrowia lipolytica (Yarrowia lipolytica) etc..In addition most preferably square
Face, host cell are pichia pastoris yeast (Pichia pastoris) cells.
Production method
After the coded sequence for obtaining polypeptide, following method can be used and produce polypeptide of the present invention, this method includes：(a) exist
Contribute under conditions of production polypeptide to cultivate the host cell containing the expression vector for expressing the polypeptide；And (b) recycles the polypeptide.
In the production method of the present invention, cell can be used a method known in the art in the culture medium suitable for production polypeptide
Middle culture.For example, cell can be by the shaking flask culture that is carried out in laboratory or industrial fermentation tank and small-scale or large-scale
Fermentation (including it is continuous, in batches, batch feeding or solid state fermentation), suitable culture medium and allow the polypeptide expression and/
Or it is cultivated under conditions of separation.Culture, which is happened at, to be used a method known in the art including carbon source and nitrogen source and inorganic salts
In suitable culture medium.Suitable culture medium is available from commercial provider or can be prepared according to disclosed composition.It if should
Polypeptide secretion enters culture medium, which can directly recycle from culture medium.If the polypeptide is not secreted into culture medium, it can be from
Cell lysate recycles.
In certain embodiments, host cell of the invention is yeast, preferably Pichia pastoris.It therefore, can be routinely
Yeast fermentation process carry out polypeptide production.For example, a specific embodiment as fermentation, it can be in the ferment for obtaining the present invention
After mother cell, the yeast cells is first activated in liquid YPD, is then inoculated in BMGY, 30 DEG C, 220rpm is incubated overnight
Afterwards, it is forwarded in BMMY culture mediums, initial OD 600 is 6, is initially induced with 2% methanol, is respectively added with after 32h for 24 hours
1% is respectively added after 1%, 48h and 56h.
Polypeptide described in the invention can be recycled using methods known in the art.For example, polypeptide can pass through conventional side
Method, including but not limited to centrifugation, filtering, ultrafiltration, extraction, chromatography, spray drying, freeze-drying, evaporation or precipitation etc. are from culture
Base recycles.
The present invention polypeptide can be purified by a variety of methods known in the art, including but not limited to chromatography (such as from
Sub- exchange, compatibility, hydrophobicity, chromatofocusing, molecular exclusion), electrophoresis (such as isoelectric focusing), differential solubility (such as salt
Analysis precipitation), SDS-PAGE or extraction to be to obtain substantially pure polypeptide.
Hereafter the present invention will be illustrated in a manner of specific embodiment.The experiment side of actual conditions is not specified in the following example
Method, such as Sambrook usually according to normal condition,《Molecular cloning：Lab guide》(New York, United States：Cold spring harbor laboratory
Publishing house (Cold Spring Harbor Laboratory Press), 1989) condition described in or according to institute of manufacturer
It is recommended that condition carry out.For the usage and dosage of reagent, unless otherwise stated, making according to conventional usage and dosage
With.
Experiment material
1. experimental strain and plasmid
Bacterial strain：Pichia pastoris GS115 (Invitrogen, article No. C175-00), escherichia coli DH5a (TAKARA：
Catalog#.D9057A)。
Plasmid：PAO815 plasmids (Invitrogen, article No. V180-20).
2. culture medium and solution
Unless otherwise indicated, chemical reagent use herein is purchased from raw work (Shanghai) bioengineering Co., Ltd.
LB fluid nutrient mediums：0.5% yeast extract, 1% tryptone, 1%NaCl, pH7.0.
LB solid mediums：Agar concentration 1.5% is added in LB fluid nutrient mediums.
YPD fluid nutrient mediums：1% yeast extract, 2% peptone, 2% glucose.
YPD solid mediums：Agar concentration 2% is added in LB fluid nutrient mediums.
MGYS solid mediums：1.34% yeast nitrogen alkali (YNB) liquid containing ammonium sulfate is free of amino acid, 1% glycerine, 1M sorbs
Alcohol, 4 × 10-5%D- biotins, 2% agar.
BMMY- olive oil screening and culturing mediums：Component A：1% yeast extract, 2% peptone, 1.34% yeast nitrogen alkali
(YNB) liquid containing ammonium sulfate be free of amino acid, 4 × 10-5%D- biotins, 0.5% methanol (add in) after sterilizing, 0.1M citric acids-lemon
Lemon acid sodium pH of buffer 6.6,2% agar.B component：Component B olive oil substrate solutions：4%PVA solution 150ml are measured, add olive
Olive oil 50ml emulsifies 3min with high-speed homogenization machine 8000rpm, emulsifies 3min again after suspending 1min, prepare substrate solution.Sterilizing
100ml component As are mixed with 12ml B components, add in 0.1% rhodamine Bs of 1ml.
BMGY fluid nutrient mediums：1% yeast extract, 2% peptone, 1.34% yeast nitrogen alkali (YNB) liquid containing ammonium sulfate
Without amino acid, 1% glycerine, 4 × 10-5%D- biotins, 0.1M citric acid-sodium citrate buffer solutions pH6.6.
BMMY fluid nutrient mediums：1% yeast extract, 2% peptone, 1.34% yeast nitrogen alkali (YNB) liquid containing ammonium sulfate
Without amino acid, 0.5% methanol (adds in) after sterilizing, and 4 × 10-5%D- biotins (add in) after sterilizing, 0.1M citric acids-lemon
Sour sodium pH of buffer 6.6.
Modified form Bradford method determination of protein concentration kit (is purchased from Shanghai Sheng Gong bioengineering Co., Ltd)
Restriction enzyme HindIII, EcoRI, AvrII (are purchased from knob Great Britain biotechnology (Beijing) Co., Ltd)
PCR enzymes：TaKaRa Taq, PrimeHS DNA Polymerase (have purchased from precious bioengineering (Dalian)
Limit company)
T4DNA ligases (are purchased from Fu Meitaisi Co., Ltds)
Embodiment 1：Rhizomucor miehei lipase gene Pichiapastoris expression strain structure with leader peptide and protease point of contact
It builds
According to the amino acid sequence (GenBank of rhizomucor miehei lipase gene：A02536.1) and Pichia pastoris is close
Numeral preferences, design obtain the DNA sequence dna of pro/RML, and in leader peptide (its coded sequence such as SEQ ID NO:1 1-
Shown in 210 bit bases) and mature peptide (its coded sequence such as SEQ ID NO:Shown in 1 229-1035 bit bases) between increase
The restriction enzyme site of protease kex2 and ste13 (its coded sequence such as SEQ ID NO:Shown in 1 211-228 bit bases).It should
DNA sequence dna such as SQE ID NO:Shown in 1, the amino acid sequence such as SEQ ID NO of coding:Shown in 2.
By SQE ID NO:1 sequence is supplied to Shanghai life work biology Co., Ltd, carries out full genome synthesis and Direct Cloning
Into pAO815 expression vectors, the NO of ID containing SQE are obtained:The yeast expression vector pAO-pro/RML of 1 sequence.
PAO-pro/RML is linearized with SalI, the competence that Pichia pastoris GS115 bacterial strain is prepared using LiAC methods is thin
Born of the same parents, then the pAO-pro/RML segments of linearisation are converted by GS115 competent cells by electricity conversion, conversion product is coated on MGYS
On tablet, 30 DEG C are cultivated 3 days, the monoclonal on a large amount of tablets are chosen in BMMY- olive oil screening flat boards, therefrom picking vigor
The positive colony to behave oneself best, is named as pro/RML-1.
It simultaneously will be such as SQE ID NO:Rhizomucor miehei lipase gene original gene sequence shown in 5 is supplied to Shanghai to give birth to
Work biology Co., Ltd is carried out in full genome synthesis and Direct Cloning to pAO815 expression vectors.Obtain the NO of ID containing SQE:5 sequences
Yeast expression vector pAO-pro/NOP-RML (the amino acid sequence of the pro/NOP-RML of the coding such as SEQ ID NO of row:
Shown in 6).PAO-pro/NOP-RML with SalI is linearized, the competence of Pichia pastoris GS115 bacterial strain is prepared using LiAC methods
Cell, then the pAO-pro/NOP-RML segments of linearisation are converted by GS115 competent cells, conversion product coating by electricity conversion
In on MGYS tablets, 30 DEG C are cultivated 3 days, and the monoclonal on a large amount of tablets is chosen in BMMY- olive oil screening flat boards, is therefrom chosen
The positive colony that vigor behaves oneself best is taken, is named as pro/NOP-RML-1.
Embodiment 2：Rhizomucor miehei lipase gene Pichia anomala expression bacterium with 2 copy leader peptides and protease point of contact
Strain structure
Take the amino acid sequence (GenBank of rhizomucor miehei lipase gene：A02536.1 leader peptide sequences), according to
Pichia pastoris codon-bias, design obtain the DNA of the rhizomucor miehei lipase gene leader peptide sequence of two copies series connection
Sequence 2rmlPRO both increases the restriction enzyme site of protease kex2 and 2 ste13, such as SQE ID in the end of two leader peptides
NO:Shown in 3, the amino acid sequence such as SEQ ID NO of coding:Shown in 4.
By SQE ID NO:3 sequences provide Shanghai life work biology Co., Ltd and carry out full genome synthesis, obtain pUC57-
2PRO carriers.
PAO-proRML is cut the ripe peptide sequence of RML with HindIII and EcoRI, and with HindIII and EcoRI
(construction method refers to described in 201510946696.1 embodiments 1 of CN the pmAO-PLC carriers of digestion.) connection, it obtains
PAOmu-RML carriers.The two copies leader peptide 2PRO sequences on pUC57-2PRO carriers are cut with AvrII and HindIII, with
It is connected with the pmAO-RML carriers of AvrII with HindIII digestions, obtains pmAO-2pro/RML carriers.By pAOmu-2pro/RML
It is linearized with SalI, the competent cell of Pichia pastoris GS115 bacterial strain is prepared using LiAC methods, then will linearisation by electricity conversion
PmAO-2pro/RML segments conversion GS115 competent cells, conversion product is coated on MGYS tablets, and 30 DEG C are cultivated 3 days, will
Monoclonal on a large amount of tablets is chosen in the positive colony that in BMMY- olive oil screening flat boards, therefrom picking vigor behaves oneself best, life
Entitled 2pro/RML-1.
Embodiment 3：Rhizomucor miehei lipase gene Pichiapastoris expression strain shake flask fermentation
Pro/NOP-RML-1, pro/RML-1 and 2pro/RML-1 bacterial strain are taken, is first activated in liquid YPD, is connected to BMGY
In, 30 DEG C, 220rpm is incubated overnight, then is forwarded in BMMY culture mediums, and initial OD 600 is 6, is initially carried out with 2% methanol
Induction respectively adds 1%, 72h samplings with after respectively adding 1%, 48h and 56h after 32h for 24 hours, by the use of olive oil as substrate, carries out fat
Fat enzyme activity detects.
Enzyme activity of fermenting is as shown in Figure 1.In Fig. 1, the fermentation enzyme activity of pro/NOP-RML-1 is 229 ± 11U/ml, pro/RML-
1 fermentation enzyme activity is 310 ± 35U/ml, and the fermentation enzyme activity of 2pro/RML-1 is 464 ± 4U/ml.
The protein electrophoresis of pro/RML-1 and 2pro/RML-1 zymotic fluids is as shown in Fig. 2, arrow meaning is RML destination proteins.
Fig. 2 shows that the destination protein amount of 2pro/RML-1 is apparently higher than pro/RML-1, improves about 50%.
Sequence table
<110>Feng Yi（Shanghai）Co., Ltd of biotechnology research and development centre
<120>The method of high efficient expression rhizomucor miehei lipase
<130> 165681
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1038
<212> DNA
<213>Artificial sequence
<220>
<223>Pro/RML nucleotide sequences
<400> 1
gttccaatca agagacaatc taattccact gtcgattctt tgcctccatt gattccttct 60
agaactagtg caccttcatc ctctccatct acaactgacc ctgaggctcc agctatgtca 120
agaaatggtc cacttccttc tgatgttgag accaagtacg gaatggccct gaatgctact 180
tcttatccag attctgtcgt tcaagctatg aaaagagagg ctgaagcttc catcgacgga 240
ggtattagag ccgctacttc tcaggaaatc aacgaactta cttactatac aactttgtca 300
gctaattctt actgtagaac tgttattcct ggtgctactt gggattgcat acattgtgac 360
gccactgaag atttaaagat aattaaaacc tggtctactt tgatttacga cactaacgct 420
atggttgcta gaggagattc cgagaagact atttatatcg tgtttagagg ttcttcatct 480
attcgtaatt ggatcgctga tttgacattc gttccagtct cttaccctcc agtttctggt 540
actaaggttc acaaaggatt tcttgattct tatggtgaag ttcaaaacga gttggttgct 600
actgtcttgg atcagtttaa acaataccca tcttataagg ttgctgtcac tggtcactct 660
ttgggaggtg ctactgcctt gctgtgtgct ttaggtttat accagagaga ggaaggattg 720
tcttcaagta acctattctt gtacactcaa ggtcagccta gagttggaga tccagcattt 780
gctaattatg tggtttctac tggtattcca tatagacgta ctgttaacga aagagacata 840
gtaccacact tgcctccagc tgccttcgga tttctgcatg ccggtgaaga gtactggatc 900
acagataatt ctcctgaaac cgttcaagtg tgtacatctg atttagagac ttccgactgc 960
tctaacagta ttgttccatt tacttcagtt cttgatcatt tgtcttattt tggaattaac 1020
accggtttgt gtacttaa 1038
<210> 2
<211> 345
<212> PRT
<213>Artificial sequence
<220>
<223>Pro/RML amino acid sequences
<400> 2
Val Pro Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro
1 5 10 15
Leu Ile Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr
20 25 30
Asp Pro Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp
35 40 45
Val Glu Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp
50 55 60
Ser Val Val Gln Ala Met Lys Arg Glu Ala Glu Ala Ser Ile Asp Gly
65 70 75 80
Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr
85 90 95
Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala
100 105 110
Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile
115 120 125
Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala Met Val Ala Arg
130 135 140
Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser
145 150 155 160
Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro Val Ser Tyr Pro
165 170 175
Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser Tyr Gly
180 185 190
Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe Lys Gln
195 200 205
Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly Gly Ala
210 215 220
Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg Glu Glu Gly Leu
225 230 235 240
Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly
245 250 255
Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg
260 265 270
Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro Ala Ala
275 280 285
Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser
290 295 300
Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys
305 310 315 320
Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu Ser Tyr
325 330 335
Phe Gly Ile Asn Thr Gly Leu Cys Thr
340 345
<210> 3
<211> 1278
<212> DNA
<213>Artificial sequence
<220>
<223>2pro/RML nucleotide sequences
<400> 3
gtgcccataa agagacaatc caactccaca gtcgattccc ttccaccatt aattccttcc 60
aggacatcag caccttcttc ttctccttct accaccgacc ctgaagcacc tgctatgtca 120
agaaacggac ctttgccatc agatgttgaa acgaagtacg gtatggcttt aaacgctacc 180
tcttacccag acagtgtcgt tcaggctatg aaacgagagg ctgaggctga agctgttcca 240
atcaaacgtc aatctaattc tactgttgac tcactgccac ccctgattcc ctctcgtaca 300
agtgctccat ctagtagtcc ttctactact gatccagagg cccctgccat gtcaagaaat 360
gggccattgc caagtgatgt tgaaactaaa tatggcatgg ccttgaatgc cacttcatat 420
cccgattcag tagtacaggc catgaagagg gaggctgaag ccgaagcttc catcgacgga 480
ggtattagag ccgctacttc tcaggaaatc aacgaactta cttactatac aactttgtca 540
gctaattctt actgtagaac tgttattcct ggtgctactt gggattgcat acattgtgac 600
gccactgaag atttaaagat aattaaaacc tggtctactt tgatttacga cactaacgct 660
atggttgcta gaggagattc cgagaagact atttatatcg tgtttagagg ttcttcatct 720
attcgtaatt ggatcgctga tttgacattc gttccagtct cttaccctcc agtttctggt 780
actaaggttc acaaaggatt tcttgattct tatggtgaag ttcaaaacga gttggttgct 840
actgtcttgg atcagtttaa acaataccca tcttataagg ttgctgtcac tggtcactct 900
ttgggaggtg ctactgcctt gctgtgtgct ttaggtttat accagagaga ggaaggattg 960
tcttcaagta acctattctt gtacactcaa ggtcagccta gagttggaga tccagcattt 1020
gctaattatg tggtttctac tggtattcca tatagacgta ctgttaacga aagagacata 1080
gtaccacact tgcctccagc tgccttcgga tttctgcatg ccggtgaaga gtactggatc 1140
acagataatt ctcctgaaac cgttcaagtg tgtacatctg atttagagac ttccgactgc 1200
tctaacagta ttgttccatt tacttcagtt cttgatcatt tgtcttattt tggaattaac 1260
accggtttgt gtacttaa 1278
<210> 4
<211> 425
<212> PRT
<213>Artificial sequence
<220>
<223>2pro/RML amino acid sequences
<400> 4
Val Pro Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro
1 5 10 15
Leu Ile Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr
20 25 30
Asp Pro Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp
35 40 45
Val Glu Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp
50 55 60
Ser Val Val Gln Ala Met Lys Arg Glu Ala Glu Ala Glu Ala Val Pro
65 70 75 80
Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro Leu Ile
85 90 95
Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr Asp Pro
100 105 110
Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp Val Glu
115 120 125
Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp Ser Val
130 135 140
Val Gln Ala Met Lys Arg Glu Ala Glu Ala Glu Ala Ser Ile Asp Gly
145 150 155 160
Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr
165 170 175
Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala
180 185 190
Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile
195 200 205
Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala Met Val Ala Arg
210 215 220
Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser
225 230 235 240
Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro Val Ser Tyr Pro
245 250 255
Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser Tyr Gly
260 265 270
Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe Lys Gln
275 280 285
Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly Gly Ala
290 295 300
Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg Glu Glu Gly Leu
305 310 315 320
Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly
325 330 335
Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg
340 345 350
Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro Ala Ala
355 360 365
Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser
370 375 380
Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys
385 390 395 400
Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu Ser Tyr
405 410 415
Phe Gly Ile Asn Thr Gly Leu Cys Thr
420 425
<210> 5
<211> 1038
<212> DNA
<213>Artificial sequence
<220>
<223>Pro/NOP-RML nucleotide sequences
<400> 5
gtgccaatca agagacaatc aaacagcacg gtggatagtc tgccacccct catcccctct 60
cgaacctcgg caccttcatc atcaccaagc acaaccgacc ctgaagctcc agccatgagt 120
cgcaatggac cgctgccctc ggatgtagag actaaatatg gcatggcttt gaatgctact 180
tcctatccgg attctgtggt ccaagcaatg aaaagagagg ctgaagctag cattgatggt 240
ggtatccgcg ctgcgacctc gcaagaaatc aatgaattga cttattacac tacactatct 300
gccaactcgt actgccgcac tgtcattcct ggagctacct gggactgtat ccactgtgat 360
gcaacggagg atctcaagat tatcaagact tggagcacgc tcatctatga tacaaatgca 420
atggttgcac gtggtgacag cgaaaaaact atctatatcg ttttccgagg ttcgagctct 480
atccgcaact ggattgctga tctcaccttt gtgccagttt catatcctcc ggtcagtggt 540
acaaaagtac acaagggatt cctggacagt tacggggaag ttcaaaacga gcttgttgct 600
actgttcttg atcaattcaa gcaatatcca agctacaagg ttgctgttac aggtcactca 660
ctcggtggtg ctactgcgtt gctttgcgcc ctgggtctct atcaacgaga agaaggactc 720
tcatccagca acttgttcct ttacactcaa ggtcaaccac gggtaggcga ccctgccttt 780
gccaactacg ttgttagcac cggcattcct tacaggcgca cggtcaatga acgagatatc 840
gttcctcatc ttccacctgc tgcttttggt tttctccacg ctggcgagga gtattggatt 900
actgacaata gcccagagac tgttcaggtc tgcacaagcg atctggaaac ctctgattgc 960
tctaacagca ttgttccctt cacaagtgtt cttgaccatc tctcgtactt tggtatcaac 1020
acaggcctct gtacttaa 1038
<210> 6
<211> 345
<212> PRT
<213>Artificial sequence
<220>
<223>Pro/NOP-RML amino acid sequences
<400> 6
Val Pro Ile Lys Arg Gln Ser Asn Ser Thr Val Asp Ser Leu Pro Pro
1 5 10 15
Leu Ile Pro Ser Arg Thr Ser Ala Pro Ser Ser Ser Pro Ser Thr Thr
20 25 30
Asp Pro Glu Ala Pro Ala Met Ser Arg Asn Gly Pro Leu Pro Ser Asp
35 40 45
Val Glu Thr Lys Tyr Gly Met Ala Leu Asn Ala Thr Ser Tyr Pro Asp
50 55 60
Ser Val Val Gln Ala Met Lys Arg Glu Ala Glu Ala Ser Ile Asp Gly
65 70 75 80
Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu Leu Thr Tyr Tyr
85 90 95
Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val Ile Pro Gly Ala
100 105 110
Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp Leu Lys Ile Ile
115 120 125
Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala Met Val Ala Arg
130 135 140
Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg Gly Ser Ser Ser
145 150 155 160
Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro Val Ser Tyr Pro
165 170 175
Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu Asp Ser Tyr Gly
180 185 190
Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp Gln Phe Lys Gln
195 200 205
Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser Leu Gly Gly Ala
210 215 220
Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg Glu Glu Gly Leu
225 230 235 240
Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln Pro Arg Val Gly
245 250 255
Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly Ile Pro Tyr Arg
260 265 270
Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu Pro Pro Ala Ala
275 280 285
Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile Thr Asp Asn Ser
290 295 300
Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu Thr Ser Asp Cys
305 310 315 320
Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp His Leu Ser Tyr
325 330 335
Phe Gly Ile Asn Thr Gly Leu Cys Thr
340 345
Claims (10)
1. a kind of polynucleotide sequence, which is characterized in that the polynucleotide sequence is selected from：
(1)SEQ ID NO:Sequence shown in 1 the 229th~1035 nucleotide sequence；With
(2) complementary series of (1) described sequence.
2. a kind of polynucleotide sequence, which is characterized in that the polynucleotide sequence is selected from：
(1) polynucleotide sequence of the peptide sequence shown in following formula I is encoded：
A-L1-B-L2-RML (Formulas I)
In formula,
A may be present or be not present, and be rhizomucor miehei lipase leader peptide when it is present；
L1 exists in the presence of A, is the restriction enzyme site of protease kex2 and ste13；
B is rhizomucor miehei lipase leader peptide；
L2 is the restriction enzyme site of protease kex2 and ste13；With
RML represents the ripe peptide sequence of rhizomucor miehei lipase；With
(2) complementary series of (1) described sequence.
3. polynucleotide sequence as claimed in claim 2, which is characterized in that there are A and L1 for the peptide sequence shown in Formulas I；With/
Or
The amino acid sequence of the L1 and L2 is respectively independent such as SEQ ID NO:Shown in 2 the 71st~76 or such as SEQ ID NO:4
Shown in 71st~78；
Preferably, such as SEQ ID NO of the peptide sequence shown in Formulas I:Shown in 2 or 4.
4. polynucleotide sequence as claimed in claim 2 or claim 3, which is characterized in that the polynucleotide sequence is selected from：
(i)SEQ ID NO:Polynucleotide sequence shown in 1 or 3；With
(ii) complementary series of (i) described polynucleotide sequence.
5. a kind of nucleic acid constructs, which is characterized in that the nucleic acid constructs contains described in any one of claim 1-4
Polynucleotide sequence；
Preferably, the nucleic acid constructs is cloning vector or expression vector；
It is highly preferred that the expression vector is the carrier expressed suitable for Pichia pastoris, including but not limited to pPIC, pPICZ,
PAO, pGAP and pGAPZ；
It is highly preferred that the expression vector is using pAO815 plasmids as skeleton.
6. a kind of genetically engineered host cell, which is characterized in that the host cell contains any in claim 1-4
Polynucleotide sequence described in or the nucleic acid constructs described in claim 5；
Preferably, the cell is yeast cells；
It is highly preferred that the yeast is Pichia pastoris.
A kind of 7. method for preparing rhizomucor miehei lipase, which is characterized in that the method includes building 1- containing claim
The expression vector of any one of 4 polynucleotide sequences, be transferred in host cell and cultivated the host cell with
The step of expressing the lipase.
8. a kind of structure can improve the method for the expression vector of rhizomucor miehei lipase expression quantity, which is characterized in that the side
Method includes that protease kex2 will be inserted between the leading peptide-coding sequence of rhizomucor miehei lipase and mature polypeptide coding sequence
The step being inserted into the gene order of the restriction enzyme site of ste13 in expression vector；
Preferably, it in the gene order being inserted into, is further included before the leading peptide-coding sequence of the rhizomucor miehei lipase
The restriction enzyme site of one leading peptide-coding sequence of rhizomucor miehei lipase copied and protease kex2 and ste13.
9. method as claimed in claim 8, which is characterized in that the method has following one or more features：
The gene order optimizes according to the preferences of Pichia pastoris codon；
The expression vector is suitable for expressing in yeast, is preferably expressed in Pichia pastoris；
The expression vector suitable for being expressed Pichia pastoris, including but not limited to pPIC, pPICZ, pAO, pGAP and
pGAPZ；
The leading peptide-coding sequence of the rhizomucor miehei lipase such as SEQ ID NO:Shown in 1 the 1st~210；
The rhizomucor miehei lipase mature polypeptide coding sequence such as SEQ ID NO:Shown in 1 the 229th~1035；
The nucleotide sequence of the restriction enzyme site of the protease kex2 and ste13 such as SEQ ID NO:Shown in 1 the 211st~228,
Or such as SEQ ID NO:Shown in 3 the 211st~234；
The rhizomucor miehei lipase leader peptide such as SEQ ID NO:Shown in 2 the 1st~70；
The rhizomucor miehei lipase mature peptide such as SEQ ID NO:Shown in 2 the 77th~345；With
The amino acid sequence of the restriction enzyme site of the protease kex2 and ste13 such as SEQ ID NO:Shown in 2 the 71st~76 or
Such as SEQ ID NO:Shown in 4 the 71st~78.
10. a kind of rhizomucor miehei lipase preparation method, the method includes the steps of host cell described in fermentation claim 6
Suddenly.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923216A (en) * | 2018-09-19 | 2020-03-27 | 江苏师范大学 | Method for producing rhizomucor miehei lipase pRML enzyme powder |
| CN110923215A (en) * | 2018-09-19 | 2020-03-27 | 江苏师范大学 | Method for producing rhizomucor miehei lipase mRML enzyme powder |
| CN111363734A (en) * | 2018-12-25 | 2020-07-03 | 丰益(上海)生物技术研发中心有限公司 | Lipase mutant, composition and application thereof |
| CN113046338A (en) * | 2019-12-27 | 2021-06-29 | 宜昌东阳光生化制药有限公司 | High-selectivity lipase from trichoderma reesei and application thereof |
| JP2022515531A (en) * | 2018-12-28 | 2022-02-18 | 豊益（上海）生物技術研発中心有限公司 | Pichia pastoris mutant strain for expressing foreign genes |
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| CN110923215B (en) * | 2018-09-19 | 2021-08-10 | 江苏师范大学 | Method for producing rhizomucor miehei lipase mRML enzyme powder |
| CN111363734A (en) * | 2018-12-25 | 2020-07-03 | 丰益(上海)生物技术研发中心有限公司 | Lipase mutant, composition and application thereof |
| JP2022515531A (en) * | 2018-12-28 | 2022-02-18 | 豊益（上海）生物技術研発中心有限公司 | Pichia pastoris mutant strain for expressing foreign genes |
| JP7430189B2 (en) | 2018-12-28 | 2024-02-09 | 豊益（上海）生物技術研発中心有限公司 | Pichia pastoris mutants for expressing foreign genes |
| CN113046338A (en) * | 2019-12-27 | 2021-06-29 | 宜昌东阳光生化制药有限公司 | High-selectivity lipase from trichoderma reesei and application thereof |
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