CN102102093B - High temperature lipase, preparation for mutants thereof and application thereof - Google Patents

High temperature lipase, preparation for mutants thereof and application thereof Download PDF

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CN102102093B
CN102102093B CN2009102012093A CN200910201209A CN102102093B CN 102102093 B CN102102093 B CN 102102093B CN 2009102012093 A CN2009102012093 A CN 2009102012093A CN 200910201209 A CN200910201209 A CN 200910201209A CN 102102093 B CN102102093 B CN 102102093B
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lipase
sequence
phe
leu
glu
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CN102102093A (en
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叶秀云
靳伟刚
张洋
罗鋆琳
陈萍
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Fujian Fuda Biotech Co Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

The invention discloses high temperature lipase, preparation for mutants thereof, and application thereof, and in particular discloses a separated lipase with an amino acid sequence as SEQ ID NO:4, a coding sequence thereof, a carrier, a cell and a composition containing the separated lipase, a preparation method for the separated lipase, and application of the separated lipase in catalyzing ester synthesis, performing chiral separation or transforming plant oil into biodiesel.

Description

Preparation of a kind of high temperature lipase mutant and uses thereof
Technical field
The present invention relates to genetically engineered and enzyme engineering field, specifically, the separating out fat enzyme that relates to the bacterium Thermotoga petrophila source of the hot born of the same parents Pseudomonas of dwelling of a kind of sudden change, its encoding gene, and contain the recombinant plasmid of this encoding gene and be used for to express the recombinant bacterial strain of target lipase albumen; The invention still further relates to the purposes of expressed thermal stable lipase.
Background technology
Lipase (lipase, EC3.1.1.3, GEH) is lipolytic enzyme, ubiquity in animal vegetable tissue and multiple-microorganism, and its natural substrate is biogenic natural fats and oils, is one of studied the earliest enzyme.Activity from rabbit steapsase in 1834 is reported so far, the existing century-old history of the research of relevant lipase.20 beginnings of the century, external scientific research personnel found microbial lipase first.The microbial lipase kind is many, action pH, operative temperature and wider to the specificity type of substrate than animals and plants enzyme, be convenient to carry out suitability for industrialized production and obtain the high purity zymin, these factors have promoted the research of microbial lipase, have also greatly driven the basis of lipase in each field and the research of the aspect such as practical application.Lipase has become one of important industrial enzymes at present.
Current, industrial fatty enzyme is mainly obtained by microorganisms producing.Occurring in nature has many microorganisms can the synthetic fat enzyme.The mushroom of yielding lipase mainly contains black-koji mould, Pseudomonas fluorescens, geotrichum candidum rhizopus arrhizus, Mucor cylinder candidiasis, the mould De Shi rhizopus of nest palpus, many coccuses wool shape detritus mould, penicillium cyclopium clayey look bacillus.According to statistics, the microorganism of yielding lipase has 65 genus, 28 genus of bacterium wherein, 4 genus of actinomycetes, 10 genus of yeast, 4 genus of other actinomycetes, 10 genus of yeast, 23 genus of other fungi.
Lipase reacts at normal temperatures and pressures, and reaction conditions is gentle, transformation efficiency is high and high specificity, is difficult for producing by product, avoid the objectionable impurities brought because of chemical catalysis, thereby the lipase Application Areas is extensive, and it can be used as digestive ferment and clinical diagnosis zymin, is applied in pharmaceutical industries; Also can be used as the flavour improvement that foodstuff additive are used for food; In the washing industry, alkaline lipase is widely used to improve the clean ability of washing composition; In paper industry, can be used to remove " be in harmony fat " in the paper pulp; In leather processing procedure, can be used to remove residual fat and the protein that closely links to each other with leather or fur; In addition, along with deepening continuously of lipase research, lipase has been used in the biological degradation research of plastics, and this research has presented wide application prospect.What is more important lipase also has the functions such as esterification, transesterification and isomers fractionation, also has been subject to growing interest.
Lipase optimum temperature, optimal pH etc. are all different and different with substrate with the source of enzyme, so optimum temperuture or pH only have just meaningful under certain condition.Lipase inhibitor can be divided into reversible and irreversible inhibitor because of mechanism of action difference, and metal ion can be used as different lipase activator or inhibitor.Lipase alpha-helix lid structure plays a protective role to the triplet active centre, and the water-oil interface activation phenomenon makes alpha-helix directed again, and lipase-catalyzed being easy to carried out.In addition, lipase activity division center difference makes them also different to different substrate specificities.Lipase source is different, causes the problems such as diversity, unstable, substrate water-insoluble of structure and character, make the lipase progress compare with proteolytic enzyme, amylase will be slowly many.Lipase structures research, dynamics research, molecular biology research are to answer from now on emphasis to strengthen fundamental research, also will strengthen applied research simultaneously.
At present, industrial production is mainly passed through mutagenic obtained with the lipase superior strain.Except known microorganisms is carried out the high yielding lipase mutagenesis screening, extreme microorganism has also caused investigator's attention.Extreme environment microbial lipase energy anti-adverse environment, as low temperature resistant, acid and alkali-resistance, high pressure resistant etc., important using value is arranged in industrial production.Polar Research Institute of China is separated to low temperature yielding lipase bacterial strain from South Pole soil, Alaska frozen soil and deep phreatic water etc., and has studied its gene and enzymatic property.Begun screening yielding lipase bacterial strain from marine microorganism abroad.
In recent years, gene engineering has been simplified Downstream processing, makes the output of microbial lipase increase manyfold.And introduce denaturing agent and inductor makes us might obtain the lipase with advantage three-dimensional structure, because the product that uses inductor and lipase to produce has specificity directional property and special application.Along with going deep into of research, the industrial application of lipase will constantly increase.Therefore, the novel method acquisition is novel to have the lipase of industrial value that potential needs are arranged for adopting at present.
Summary of the invention
Therefore, the purpose of this invention is to provide a kind of new lipase, so that it satisfies industrial needs.
In one aspect of the invention, provide a kind of lipase of separation, it contains just like the aminoacid sequence shown in the SEQ ID NO:4.In a preference aspect this, the aminoacid sequence of described lipase and SEQ IDNO:4 have and have at least 50% at least, and preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, at least 95% homogeny most preferably.In another preference aspect this, this lipase also comprises its functional fragment.At one aspect this most preferably in the example, the aminoacid sequence of this lipase is SEQ ID NO:4.
In another aspect of the present invention, provide a kind of nucleic acid molecule of the above-mentioned lipase of encoding.In a preference, it has the nucleotide sequence shown in SEQ ID NO:2.In a preference aspect this, described nucleic acid molecule and SEQ ID NO:2 have and have at least 50% at least, and preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, at least 95% homogeny most preferably.At one aspect this most preferably in the example, the nucleotide sequence of this nucleic acid molecule is SEQ ID NO:2.
Aspect the 3rd of the present invention, a kind of carrier is provided, it comprises above-mentioned polynucleotide.
Aspect the 4th of the present invention, a kind of cell is provided, it comprises above-mentioned carrier, or is integrated with above-mentioned polynucleotide in its genome.
Aspect the 5th of the present invention, the purposes of above-mentioned lipase is provided, be used for that the catalysis ester is synthetic, chiral separation or vegetables oil is converted into biofuel.
Aspect the 6th of the present invention, a kind of composition is provided, contain above-mentioned lipase and carrier in the said composition.In a preference aspect this, carrier is selected from the food, the carrier on industrial, medicinal.
Aspect the 7th of the present invention, a kind of method of producing above-mentioned lipase is provided, comprising: cultivate above-mentioned host cell, from culture, isolate expression product.
Aspect the 8th of the present invention, provide a kind of vegetables oil has been converted into the method for biofuel, comprise with above-mentioned lipase treatment vegetables oil.
Aspect the 9th of the present invention, provide a kind of catalysis ester synthetic method, be included in anhydrous or a small amount of aqueous systems with above-mentioned lipase-catalyzed esterification.
Aspect the of the present invention ten, a kind of method of chiral separation is provided, comprise with above-mentioned lipase-catalyzed chiral separation.
Description of drawings
Fig. 1 a: from the nucleotide sequence (SEQ ID NO:1) of the coding lipase of Thermotoga petrophila and the aminoacid sequence (SEQ ID NO:3) of coding thereof.
Fig. 1 b: the aminoacid sequence of the lipase TP-Esterase of sudden change of the present invention (SEQ ID NO:4) and encoding gene (SEQ ID NO:2) thereof.
Fig. 2: mutant lipase (SEQ ID NO:4), from the lipase (SEQID NO:3) of Thermotoga petrophila and from the comparison of the lipase aminoacid sequence (SEQ ID NO:5) of Marinitoga piezophila KA-3.
Fig. 3: lipase and the recon structure iron of mutant lipase on plasmid pET28a (+) in Thermotoga petrophila source.
Fig. 4: the lipase in escherichia coli expression Thermotoga petrophila source and the optimal reaction pH of mutant lipase, map to enzyme activity (%) with pH.
Fig. 5: the lipase in escherichia coli expression Thermotoga petrophila source and the optimal reactive temperature of mutant lipase, with temperature enzyme activity (%) is mapped.
Fig. 6: the pH stability of the lipase in escherichia coli expression Thermotoga petrophila source and mutant lipase, with pH residual enzyme activity (%) is mapped.
Fig. 7 a: the escherichia coli expression Thermotoga petrophila fatty Thermostability of originating, with temperature residual enzyme activity (%) is mapped.
Fig. 7 b: escherichia coli expression mutant fat Thermostability, with temperature residual enzyme activity (%) is mapped.
Fig. 8 a: the lipase in escherichia coli expression Thermotoga petrophila source is to stomach en-and the time dependent figure of tryptic resistance.
Fig. 8 b: escherichia coli expression mutant lipase is to stomach en-and the time dependent figure of tryptic resistance.
Fig. 9: the SDS-PAGE of sample in the lipase in escherichia coli expression Thermotoga petrophila source and the mutant lipase production by fermentation.M:SDS-PAGE protein standard specimen wherein; 1:Thermotoga petrophila lipase is induced 2hr; 2:Thermotoga petrophila lipase is induced 4hr; 3: mutant lipase is induced 2hr; 4: mutant lipase is induced 4hr.
Figure 10: the lipase in escherichia coli expression Thermotoga petrophila source and the PAGE of mutant lipase.Wherein: M:PAGE protein standard specimen; 1:Thermotoga petrophila lipase (coomassie brilliant blue staining); 2: mutant lipase (coomassie brilliant blue staining); 3:Thermotoga petrophila lipase (active coloring); 4: mutant lipase (active coloring).
Figure 11: the lipase in escherichia coli expression Thermotoga petrophila source and the SDS-PAGE of mutant lipase behind the purifying.M:SDS-PAGE protein standard specimen; 1:Thermotoga petrophila lipase; 2: mutant lipase.
Figure 12: the reference colour spectrogram that lipase-catalyzed ester is synthetic.
Figure 13: the reference colour spectrogram of lipase-catalyzed chiral separation.
Figure 14: the reference colour spectrogram of lipase-catalyzed generation biofuel.
Embodiment
By the screening to Thermotoga petrophila, the contriver has found a kind of novel lipase, and its Heat stability is good has application performance highly.Finish on this basis the present invention.
As used herein, " separation " refers to that material separates (if natural substance, primal environment namely is natural surroundings) from its primal environment.Do not have separation and purification such as the polynucleotide under the native state in the active somatic cell and albumen, but same polynucleotide or albumen as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " lipase of separation " refers to that this lipase is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein Purification of Lipase of standard.Basically pure albumen can produce single master tape on non-reduced polyacrylamide gel.The purity of lipase can be used amino acid sequence analysis.
Lipase of the present invention can be recombinant protein (polypeptide), native protein, synthetic proteins, preferred recombinant protein.Lipase of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, lipase of the present invention can be glycosylated, maybe can be nonglycosylated.Lipase of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analogue of lipase.As used herein, term " fragment ", " derivative " refer to basically keep the identical biological function of native lipase of the present invention or active albumen with " analogue ".Lipase fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted albumen of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has the albumen of substituted radical, or (iii) maturation protein and another compound (such as the compound that prolongs the albumen transformation period, polyoxyethylene glycol for example) merges formed albumen, or (iv) additional aminoacid sequence is fused to this protein sequence and the albumen that forms (such as leader sequence or secretion sequence or be used for sequence or the proteinogen sequence of this albumen of purifying, or with the fusion rotein of the formation of antigen I gG fragment).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " lipase " refers to wild-type Thermotoga petrophila lipase, or has the albumen of aminoacid sequence of the SEQ ID NO:4 of lipase activity.This term also comprises having and the variant form lipase identical function, SEQ ID NO:4 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-30, preferably 1-20, more preferably 1-10,1-5 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing with the close or similar amino acid of performance, usually can not change the function of protein.Again such as, add the function that or several amino acid also can not change protein usually at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of lipase.
The variant form of this lipase comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringency condition can with the coded albumen of the DNA of the DNA hybridization of lipase and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-lipase to obtain.The present invention also provides other albumen, as comprises the fusion rotein of lipase or its fragment.Except the albumen of total length almost, the present invention has also comprised the soluble fragments of lipase.Usually, this fragment have the lipase sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of lipase.The difference of these analogues and native lipase can be the difference on the aminoacid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps haves both at the same time.These albumen comprise genetic variant natural or that induce.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(such as D-amino acid), and the analogue with that non-natural exists or synthetic amino acid (such as β, gamma-amino acid).Should be understood that lipase of the present invention is not limited to the above-mentioned representational albumen that exemplifies.
(usually the not changing primary structure) form of modification comprises: chemically derived form such as the acetylize or carboxylated of the albumen that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and albumen that produce in the procedure of processing such as those in the synthetic and processing of albumen or further.This modification can be carried out glycosylated enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishes by albumen is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (such as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the albumen that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " lipase conservative property variant protein (polypeptide) " refers to compare with the aminoacid sequence of SEQ ID NO:4, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed albumen at the most best.These conservative property variant protein are preferably carried out amino acid substitution according to table 1 and are produced.
Table 1
Initial residue Representational replacement The preferred replacement
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
As optimal way of the present invention, described lipase be from the lipase of Thermotoga petrophilia by the mutant that point mutation produces, be called as TPEsterase (TP lipase), have the aminoacid sequence shown in the SEQ ID NO:4.Simultaneously, lipase of the present invention comprises that also those have functionally active, and the aminoacid sequence shown in the SEQ IDNO:4 has at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, the variant of at least 95% aminoacid sequence most preferably.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature albumen can be identical with the coding region sequence shown in SEQ ID NO:1 is arbitrary or the varient of degeneracy.As used herein, " varient of degeneracy " refers in the present invention encode and has the protein of SEQ ID NO:4, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:2.
The polynucleotide of the maturation protein of coding SEQ ID NO:4 comprise: the encoding sequence of an encoding mature albumen; The encoding sequence of maturation protein and various additional code sequence; The encoding sequence of maturation protein (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of proteins encoded " can be the polynucleotide that comprise this albumen of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the albumen of identical aminoacid sequence or fragment, analogue and the derivative of albumen with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from the function of the albumen that changes in fact its coding.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " refers to: (1) than the hybridization under low ionic strength and the comparatively high temps and wash-out, such as 0.2 * SSC, and .1%SDS, 0 ℃; Or in (2) when hybridization, be added with denaturing agent, 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the albumen of interfertile polynucleotide encoding has identical biological function and activity with the maturation protein shown in the SEQ ID NO:2.
The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding lipase.
As optimal way of the present invention, the polynucleotide of the described lipase of encoding separate from Thermotogapetrophila, have the nucleotide sequence shown in the SEQ ID NO:2.
Albumen among the present invention preferably provides with the form of separating with polynucleotide, more preferably is purified to homogeneous.
Lipase Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell over to again, then separates obtaining relevant sequence from the host cell after the propagation by ordinary method.
In addition, also can synthesize relevant sequence, especially fragment length more in short-term with the method for synthetic.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.Then this dna sequence dna can be introduced in various existing dna moleculars as known in the art (or such as carrier) and the cell.In addition, also can will suddenly change by chemosynthesis and introduce in the protein sequence of the present invention.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is such as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
As one of embodiment, the inventor extracts the genomic dna of Thermotoga petrophila, is cloned into the full length sequence of coding lipase by the method for the good screening active ingredients of genomic library construction.The result obtains the nucleotide sequence shown in SEQ ID NO:1.Then, by point mutation, the amino acid mutation that makes 1 place of aminoacid sequence is that phenylalanine, 6 amino acid mutation are methionine(Met) (the Thermotoga petrophila lipase sequence among Fig. 2 after the first behavior sudden change, separating from Thermotoga petrophila before the second behavior sudden change obtains the lipase sequence, the third line is the sequence from the lipase of Marinitoga piezophila KA-3), the amino acid mutation at the 277th place is Serine, the 324th amino acid mutation is L-glutamic acid, shown in the sequence alignment result of Fig. 2.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or lipase encoding sequence, and the method that produces albumen of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), polymerized nucleoside acid sequence of the present invention can be used to express or the lipase of Restruction.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding lipase of the present invention, or transform or transduction appropriate host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the lipase polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral such as adenovirus, retrovirus or other carriers.Applicable carrier includes but not limited in the present invention: escherichia coli plasmid.As long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is usually to contain replication orgin, promotor, marker gene and translation controlling elements.
Method well-known to those having ordinary skill in the art can be used for making up fatty enzyme DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can be effectively connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P LPromotor; But eukaryotic promoter comprises CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, the LTRs of retrovirus and the promotor that some other known controlling gene is expressed in protokaryon or eukaryotic cell or its virus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, with the phenotypic character of the host cell that is provided for selecting transforming, cultivate Tetrahydrofolate dehydrogenase, the neomycin resistance of usefulness such as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
In one embodiment, with not with the lipase encoding sequence of the present invention of endogenous signal coding sequence through NcoI be connected pET28a (+) carrier with the NotI double digestion with NcoI after the NotI enzyme is connected and be connected, obtain large intestine recombinant expression vector pET-28a (+)-TPEsterase.
Comprise above-mentioned suitable dna sequence dna and the suitable carrier of promotor or control sequence, can be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, such as bacterial cell; Or the eukaryotic cell such as low, such as yeast cell; Or higher eucaryotic cells, such as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.The preferred various cells that are beneficial to gene product expression or fermentative production of described host cell, this type of cell has been well known and commonly used, for example various Bacillus coli cells and yeast cell.In one of embodiments of the present invention, select the reconstitution cell of e. coli bl21 and JM109 construction expression lipase.
Persons skilled in the art are all known the suitable carrier of How to choose, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can in exponential growth after date results, be used CaCl 2Method is processed, and used step is well-known in this area.Another kind method is to use MgCl 2If necessary, transforming also the method for available electroporation carries out.When the host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses lipase of the present invention.According to used host cell, substratum used in the cultivation can be selected from various conventional mediums.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (such as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular be expressed or be secreted into to recombinant protein in the above methods can in cell or at cytolemma.If necessary, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.
As one of embodiment, by intestinal bacteria (for example e. coli bl21) the fermentation yielding lipase in next life that comprises lipase encoding sequence of the present invention, and by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography have obtained the target protein of pure enzyme form.
The purposes of lipase of the present invention includes, but is not limited to: be used for that the catalysis ester is synthetic, chiral separation or vegetables oil is converted into the purposes of biofuel.Lipase of the present invention has that excellent catalysis ester is synthetic, chiral separation or vegetables oil is converted into the effect of biofuel, and has good thermostability and pH stability, and application prospect is good.
The present invention also provides a kind of composition, and it contains on the lipase of significant quantity and the bromatology or industrial acceptable carrier or vehicle.This class carrier comprises (but being not limited to): damping fluid, water etc.It can be made into solution or pulvis etc.Described " significant quantity " refers to bring into play the function of lipase or active and can received amount.In use, described significant quantity can be determined easily according to the enzymic activity of described lipase in this area.
The present invention utilizes genetic engineering means to prepare can express the recombinant bacterial strain of lipase, and has obtained the lipase of high-quality.
In one of embodiments of the present invention, obtain a kind of lipase, its optimum pH is 9.0, optimum temperuture is 60 ℃, its specific activity, pH stability, thermostability and be applied to that the catalysis ester is synthetic, efficient that chiral separation and biofuel transform all increases than the wild-type lipase in Thermotoga petrophila source, these character make it have widely application and higher using value in the industries such as medicine, chemical industry, papermaking, weaving.
The genetic recombination of using learns a skill and is routine techniques in this area among the present invention.In following examples, unless stated otherwise, all experimental implementation are all carried out according to the related Sections in following laboratory manual or the document or part, comprising: the people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocols in Molecular Biology (people such as Ausubel compiles, 1994), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.
Embodiment
I. experiment material and reagent:
1. bacterial strain and carrier:
Intestinal bacteria Escherichia coli BL21 (DE3), DH5 α and JM109, expression vector pET-28a (+) (available from Novagen company), pMD 18-T is available from TakaRa company.Primer is synthetic to be finished by JaRa company (China, Shanghai).
2. enzyme and other biochemical reagents: restriction endonuclease is available from Fermentas company, and ligase enzyme is available from Invitrogen company.The substrates such as vanay, tributyrin, triolein, AOT (two-ethylhexyl succinate sodium sulfonate) is available from Sigma company.Other all is commercially available.
3. the substratum that uses: LB, YPD, YPAD, BMDY, BNNY, MM, MD substratum are all with reference to Invitrogen intestinal bacteria operational manual.
4. method:
4.1 lipase activity is measured
All involved enzyme work, enzyme activity, enzymic activity all refer to lipase activity in the various embodiments of the present invention, and available triglyceride level such as vanay, tributyrin, triolein etc. check as substrate and measure.Wherein, acid base titration is adopted in the enzyme activity quantitative assay.
Specifically, carry out according to following step:
1) principle: the lipase in the sample is hydrolyzed to the substrate triglyceride level, and the lipid acid that hydrolysis produces reduces the pH of reaction system, with the lipid acid of standard caustic soda solution drop reaction generation.
2) reagent preparation: agents useful for same in present method when dated other do not require, all refers to analytical reagent; Solvent for use and water all refer to distilled water when dated.
1. damping fluid: 50mM Tris-HCl pH9.0
2. 50mM NaOH standardized solution: demarcate with Potassium Hydrogen Phthalate, redilution is 1 times during use.
3. substrate emulsion: take by weighing 15.0g polyvinyl alcohol (PVA), add 450ml distilled water, heat while stirring, until fully dissolving, stopped heating continues to stir 30min, regulate pH to 9.0 with concentrated hydrochloric acid or sodium hydroxide after, be settled to 500mL with distilled water.4 ℃ of preservations, a week effectively.
Measure above-mentioned PVA solution 150ml, add triglyceride level substrate 50ml, stir 6min with high-speed tissue mashing machine, 4000rpm/min, each 3min of front and back, midfeather 5min is the substrate emulsion, places 4 ℃ of preservations, and is effective in 3 days.
3) sample determination:
(50mM Tris-HCl pH9.0) does 10 times of gradient dilutions with sample with phosphate buffered saline buffer, adjust enzyme activity between 20-50U/ml, then operate by following reaction sequence, in reaction process, from adding damping fluid (50mM Tris-HCl pH9.0), the timed interval to the middle adding of every triangular flask (250ml) reagent wants unanimously absolute, and 60 ℃ are incubated respectively 10min.Reactions steps is as shown in table 1.
Table 1
Reaction sequence Sample, reference liquid Sample blank
Tris-HCl damping fluid (ml) 4 4
Substrate emulsion (ml) 5 5
60 ℃ of preheating 5min √ √
Add successively sample (ml) 1 1
Dehydrated alcohol (ml) -- 15
Mix √ √
60 ℃ of insulation reaction 10min √ √
Dehydrated alcohol (ml) 15 --
Mix √ √
Cumulative volume (ml) 25 25
Annotate: " √ " expression must be carried out this operation
After reacted sample added 15ml distilled water and dilutes, the free fatty acids that produces with the sodium hydroxide solution titration hydrolysis of 0.05mol/L was with pH electrode indication titration end point.Then the Δ V between standard and the blank calculates the lipase activity of sample per sample.
4) enzyme is lived and defined: under pH9.0, temperature 60 C condition, the lipase per minute hydrolyzing triglyceride substrate of unit vol discharges the needed enzyme amount of 1 μ mol lipid acid, is defined as enzyme unit alive, represents with U.
5) enzyme work is calculated
Fat enzyme activity U calculates by following formula:
U = V - V 0 t × 1 × 50 × n
In the formula: U---sample lipase activity, U/ml;
V---sample standard consumption alkali milliliter number, ml;
V 0---sample blank consumption alkali milliliter number, ml;
T---the reaction times, min;
1---sample dosage, 1ml;
50---the micromolar number of 1ml 0.05N sodium hydroxide;
N---extension rate.
The measurement result of two parallel sample arithmetical mean value representation keeps integer, and the relative deviation of two replicate(determination) values of same sample is not more than 10%
6) calculate than vigor:
The formula that the ratio vigor of lipase is pressed face calculates:
U c＝U/c
Wherein:
U c---sample lipase specific activity, U/mg;
U---sample lipase activity, U/ml;
C---the protein content in the sample solution, mg/ml.
Purification X calculates according to following formula:
X＝U C/U C0
Wherein:
X---purification;
U C---the ratio vigor of lipase sample behind the purifying, U/mg;
U C0---the ratio vigor of lipase sample before the purifying, U/mg.
The cultivation of embodiment 1Thermotoga petrophila and the extraction of genomic dna
With anaerobism, heterotrophism, thermophilic, have a liking for oily bacterium Thermotoga petrophila (ATCC BAA-488) on the YM substratum, 85 ℃ cultivate 4-7d after, it is stand-by to collect bacterium liquid.Concrete operations and substratum are formulated as follows:
Consisting of of YM substratum: the ASW (artificial seawater) that contains 0.2% (w/v) yeast extract, with 6N salt acid for adjusting pH to 7.0, be distributed in the Heng Gaite pipe (Hungate tubes) of 30ml every pipe 9ml, then 121 ℃ of sterilization 20min in Autoclave.In the Heng Gaite pipe, pass into oxygenless nitrogen and hydrogen (nitrogen and hydrogen need be used the aseptic membrane filtration of 0.2um), and heating, make the copper wire reduction in the pipe.Then use aseptic butyl rubber bung jam-pack pipe, pipe is sealed fully.The Na of preparation proper concn 2S is with the aseptic membrane filtration degerming of 0.2um, passing into nitrogen in pipe when, with Na 2S solution adds, and makes its final concentration be about 400uM, and the final pH of YM substratum should be in (under the room temperature) between the 6.9-7.1.The bacterium liquid of access 1ml is cultivated in the most backward pipe.
ASW consists of: 20g/L NaCl, 3g/L MgCl 26H 2O, 6g/L MgSO 47H 2O, 1g/L (NH 4) 2SO 4, 0.3g/L CaCl 22H 2O, 0.2g/L KH 2PO 4, 0.5g/L KCl, 0.05g/L NaBr, 0.025g/LH 3BO 3, 0.02g/L SrCl 26H 2O, the 0.01g/L ferric citrate amine, 2.25g/L 1, two ((trishydroxymethyl) methylamino) propane of 3-, 10ml/L trace element solution, 10ml/L vitamin solution, 0.6mg/L resazurin.
Consisting of of trace element solution: 1.5g/L nitrilotriacetic acid, 3g/L MgSO 47H 2O, 500mg/LMnSO 4H 2O, 1g/L NaCl, 100mg/L FeSO 47H 2O, 100mg/L Co (NO 3) 26H 2O, the anhydrous CaCl of 100mg/L 2, 100mg/L ZnSO 47H 2O, 10mg/L CuSO 45H 2O, the anhydrous AlK (SO of 10mg/L 4) 2, 10mg/L H 3BO 3, 10mg/L Na 2MoO 42H 2O, the 1mg/L anhydrous Na 2SeO 3
Consisting of of vitamin solution: 40mg/L vitamin H, 100mg/L para-amino benzoic acid, 40mg/L folic acid, 100mg/L pantothenic acid calcium salt, 100mg/L nicotinic acid, 2mg/K vitamin H B12,10mg/L thiamine hydrochloride, the 200mg/L pyridoxine hydrochloride, 100mg/L Thioctic Acid, 10mg/L riboflavin.
The extraction of genomic dna is carried out according to following step:
1) in super clean bench, 10ml bacterium liquid is transferred in the 50ml round bottom centrifuge tube 4 ℃ of centrifugal 20min of 5000rpm;
2) abandon supernatant, add 1.2ml 10mM Tris-HCl (pH8.0)/25% sucrose (m/v), vortex oscillation, make the thalline Eddy diffusion after, minute install to (0.6ml/EP pipe) in 2 1.5ml EP pipes;
3) add the 10mg/ml N,O-Diacetylmuramidase, making its final concentration is 1mg/ml, mixing, 37 ℃ of water-bath 10min;
4) add 20%SDS (m/v), making its final concentration is 0.5% (m/v), adds Proteinase K, and making its final concentration is 100ug/ml, soft mixing, 50 ℃ of water-bath 3hr, during frequently put upside down mixing;
5) be chilled to room temperature, and adding equal-volume phenol/chloroform/primary isoamyl alcohol (25: 4: 1, v/v/v), fully put upside down mixing 10min;
6) the centrifugal 15min of room temperature 5000rpm gets supernatant;
7) repeating step 5), 6);
8) add the equal-volume chloroform, fully put upside down mixing 10min, the centrifugal 15min of room temperature 5000rpm gets supernatant;
9) 2 EP pipes are merged, divide to 3 EP pipes (about 0.4ml/EP pipe);
10) every EP pipe adds the dehydrated alcohol of 2 times of volume-20 ℃ precoolings and the 3M sodium acetate (making its final concentration is 0.3M) of 1/10 volume ,-20 ℃ of sedimentation 2hr or spend the night;
11) 0 ℃ of centrifugal 10min of 12000rpm abandons supernatant;
12) add semicanal (about 700ul) 70% ethanol (v/v) washing, 4 ℃ of centrifugal 10min of 12000rpm abandon supernatant;
13) drying at room temperature;
14) be dissolved in suitable TE (pH 7.410mM Tris-HCl+1mM EDTA) (20-50ul), add 10mg/ml RNase, making its final concentration is 20ug/ml, 37 ℃ of water-bath 2hr;
15) repeat phenol/chloroform/primary isoamyl alcohol extracting, dehydrated alcohol/3M sodium acetate precipitation, 70% washing with alcohol (step and method are the same);
16) with an amount of ultrapure water dissolving ,-20 ℃ of preservations are stand-by.
Clone and the acquisition of embodiment 2Thermotoga petrophila lipase encoding gene
Synthesize degenerated primer according to anaerobism, conserved sequence design thermophilic, that have a liking for oily bacterium lipase:
Forward primer: 5 '-RTGGCCTTYTTCGATWTRCCMCTYGARGAACTGA-3 ' (SEQ ID NO:6)
Reverse primer: 5 '-CTAGCCTTYCTCAAATAGTCTCTTCA-3 ' (SEQ ID NO:7).
Wherein, W is A or T, and Y is C or T or U, and M is A or C, and R is A or G.
Carry out the degenerate pcr amplification take Thermotoga petrophila bacterium genomic dna (prepared among the embodiment 1) as template.The PCR reaction parameter is: be cooled to 4 ℃ behind 94 ℃ of sex change 3min; Then 94 ℃ of sex change 30sec, 50 ℃ of annealing 30sec, 72 ℃ are extended 1min, 32 rear 72 ℃ of insulation 10min of circulation.Obtain the full gene fragment of lipase, carry out agarose electrophoresis and reclaim test kit with this fragment recovery (the E.Z.N.A. gel extraction agent box of OMEGA company) with glue.
Getting 10ul reclaims the dna fragmentation obtain and is connected with plasmid pMD 18-T, change among the DH5 α, (LB-agar is dull and stereotyped to be coated with flat board, contain 100ug/ml Amp), 37 ℃ just putting 1h after, be inverted overnight incubation, picking (contains 100ug/ml Amp in the positive colony access 2ml LB substratum that resistant panel is grown, the 10ml test tube), 37 ℃, 200rpm are cultivated 4-6h, collect nutrient solution, the centrifugal 10min of 10000rpm room temperature collects thalline, extracts plasmid (the E.Z.N.A.Plasmid Mini Kit I of OMEGA company) with plasmid extraction kit.
Design primer 5 '-CCATGGATGGCCTTTTTCGATTTACCACT-3 ' (SEQ ID NO:8) and 5 '-GCGGCCGCCTAGCCTTTCTCAAATAGTCT-3 ' (SEQ ID NO:9), introduce restriction enzyme site NcoI and NotI, take the top plasmid that is extracted as template, carry out PCR, products therefrom is carried out agarose electrophoresis, and reclaim dna fragmentation (the E.Z.N.A. gel extraction agent box of OMEGA company).
Fetch and receive the dna fragmentation 10ul that obtains, carrying out enzyme with restriction enzyme NcoI and NotI cuts, then be connected with pET28a (+) plasmid after the NotI enzyme is connected with NcoI with same, obtain the recon (Fig. 3) of Thermotoga petrophila lipase on pET28a (+), this recon is joined in the competent cell (e. coli bl21 and JM109) that 100ul prepares, shake up and place on ice ice bath 30min; Place 42 ℃ of water-bath thermal shock 90s; With centrifuge tube rapid moving ice bath 2min to the mixture of ice and water; Every pipe adds 400ul SOC substratum (2% peptone m/v, 0.5% yeast powder m/v, 10mM NaCl, 2.5mM KCl, 10mM MgCl2,10mM MgSO4,20mM glucose, pH7.0～7.2), with pipettor light inhale break up after on 37 ℃ of shaking tables recovery 1h (80rpm～200rpm); Centrifugal, 4000rpm * 5min removes the 400ul supernatant, the remainder mixing; Be coated with flat board (LB-agar dull and stereotyped, contain 80ug/ml Kan), 37 ℃ just putting 1h after, be inverted overnight incubation, resistant panel grow for containing positive colony of recombinant plasmid.
Getting recombinant escherichia coli strain JM109 is inoculated in the 2ml LB substratum and (contains 80ug/ml Kan, the 10ml test tube), 37 ℃, 200rpm are cultivated 4-6h, collect nutrient solution, the centrifugal 10min of 10000rpm room temperature collects thalline, extracts plasmid (the little preparation test kit of the E.Z.N.A. plasmid I of OMEGA company) with plasmid extraction kit.Enzyme is cut the coding DNA fragment of rear acquisition Thermotoga petrophila lipase, it is carried out determined dna sequence (Invitrogen company), the encoding sequence of resulting lipase has 978bp (Fig. 1 a thus, SEQ ID NO:1), wherein the 976-978 position is terminator codon TAA, 1-975 position coding does not contain the mature protein (Fig. 2) of signal peptide, and this mature protein contains 325 amino acid (SEQ ID NO:3).
The acquisition of embodiment 3 mutant lipase encoding genes
Design primer 5 '-CCATGGTTCGCTTTCTTTGACTTACCGCT-3 ' (SEQ ID NO:10), 5 '-GCGGCCGCTTCCTGAAACGCCTGTT-3 ' (SEQ ID NO:11) and 5 '-CCATGGTTCGCTTTCTTTGACATGCCGCT-3 ' (SEQ ID NO:12), introduce mutational site M1F, L6M and K344E (restriction enzyme site that contains NcoI and NotI in the primer).The restructuring pET28a (+) that is obtained in the embodiment 2 is as template, carry out PCR with primer sequence SEQ ID NO:10 and SEQ ID NO:11, products therefrom is carried out agarose electrophoresis, and reclaim dna fragmentation (the E.Z.N.A. gel extraction agent box of OMEGA company).Take the dna fragmentation that reclaims as template, carry out PCR with primer sequence SEQ ID NO:12 and SEQ ID NO:11 again, carry out agarose electrophoresis and reclaim dna fragmentation (the E.Z.N.A. gel extraction agent box of OMEGA company).Fetch and receive the dna fragmentation 10ul that obtains, carrying out enzyme with restriction enzyme NcoI and NotI cuts, then be connected with pET28a (+) plasmid after the NotI enzyme is connected with NcoI with same, change over to increase among the DH5 α after, extract recombinant plasmid dna (the little preparation test kit of the E.Z.N.A. plasmid I of OMEGA company).
Use Quick-Change transgenation test kit (Stratagene company), the design primer:
C277S (+) 5 '-TGTGGGTCTCATGGACAACATTAGTCCTCCTTCAA-3 ' (SEQ IDNO:13) and C277S (-) 5 '-TTGAAGGAGGACTAATGTTGTCCATGAGACCCACA-3 ' (SEQ ID NO:14), take the top recombinant plasmid dna that is extracted as template, carry out PCR (actual conditions is with reference to described the carrying out of specification sheets in the Quick-Change transgenation test kit of Stratagene company).After purifying reclaims, obtain the recombinant plasmid (Fig. 3) of mutant lipase on pET28a (+), this recombinant plasmid is changed respectively among e. coli bl21 and the JM109 over to (that narrates among method for transformation and the embodiment 2 is identical), obtain the recombinant escherichia coli strain of mutant lipase.
Getting mutant lipase recombinant escherichia coli strain JM109 is inoculated in the 2ml LB substratum and (contains 80ug/ml Kan, the 10ml test tube), 37 ℃, 200rpm are cultivated 4-6h, collect nutrient solution, the centrifugal 10min of 10000rpm room temperature collects thalline, extracts plasmid (the little preparation test kit of the E.Z.N.A. plasmid I of OMEGA company) with plasmid extraction kit.Enzyme is cut the coding DNA fragment of rear acquisition mutant lipase, it is carried out determined dna sequence (Invitrogen company), the encoding sequence of resulting mutant lipase has 978bp (Fig. 1 b thus, SEQ ID NO:2), wherein the 976-978 position is terminator codon TAA, 1-975 position coding does not contain the mature protein (Fig. 2) of signal peptide, this mature protein contains 325 amino acid (SEQ IDNO:4), wherein the 1st is F, the 6th is M, the 277th is S, and the 344th is E.
The preparation of embodiment 4 recombinant lipases
Get respectively the recombinant escherichia coli strain BL21 of Thermotoga petrophila lipase prepared among embodiment 2 and the embodiment 3 and mutant lipase recombinant plasmid positive colony, respectively be inoculated in (250ml triangular flask in 2 bottles of 50ml LB nutrient solutions, contain 50ug/ml Kan), 37 ℃ of 250rpm shaking culture are to OD600nm=0.3～0.5 (about 2-3hr), then respectively 2 bottles of seeds are inoculated in separately 3L fermentation minimum medium (10g/L peptone, the 5g/L yeast powder, 1g/L NaCl, 6g/L Na 2HPO 412H 2O, 3g/L KH 2PO 4, 6g/L (NH 4) 2SO 4, 1g/L MgSO 47H 2O, 0.01g/L CaCl 2, 15g/L Glucose, 0.05g/L Kan, 0.1g/L FeSO 4) in, in the 5L fermentor tank, ferment.
At initial period---in the thalli growth stage, the ammoniacal liquor (v/v) with 25% in the fermenting process is regulated pH, makes it maintain 7.0-7.2, and the speed stream with 2.8ml/hr adds trace element solution (3.5mM copper sulfate, 0.06mM sodium iodide, 1.8mM manganous sulfate, 0.08mM Sodium orthomolybdate, 0.04mM boric acid, 0.5mM cobalt chloride, 0.02mM zinc chloride, 0.03mM ferrous sulfate, 0.17mM vitamin H) carry out continuous flow feeding, until OD600=15 (3-4hr).
Enter induction period, add the IPTG that a certain amount of concentration is 1mol/L, making its final concentration is 1mmol/L, begins to induce, and adds TY (500g/L glucose, 12.5g/L MgSO with the speed stream of 15-20ml/hr 47H 2O), the speed stream with 10-15ml/hr adds DY (100g/L yeast powder, 10g/L (NH 4) 2SO 4), make the final concentration of reducing sugar and amino nitrogen in the substratum between 0.4-%0.6% and 0.05%-0.15%.
In the fermenting process, from inducing, every the 1hr sampling once, with the centrifugal 10min of nutrient solution sample 10000rpm that gets, collect thalline, add again isopyknic sterilized water Eddy diffusion thalline, the centrifugal 10min of 12000rpm, get the pH9.0 that precipitates with 10 times of volumes, the Tris-HCl damping fluid suspension thalline of 50mM carries out ultrasonic disruption, broken condition is: 60% power, 5s broken 10min in interval stops 10min, more broken 10min.12000rpm is centrifugal, collects supernatant liquor, and measures lipase activity and carry out SDS-PAGE (Fig. 9), to enzyme activity when obviously increasing, stop fermentation (7-8hr).After the fermentation ends (8hr), record restructuring Thermotoga petrophila lipase and mutant fat enzyme activity and can reach respectively 928U/ml and 1260U/ml (take vanay as substrate).
The purifying of embodiment 5 recombinant lipases
Respectively embodiment 42 kinds of prepared fermentation cultures (are reclaimed gained behind the 8hr, Thermotogapetrophila lipase and mutant lipase) the centrifugal 10min of 10000rpm, collect separately thalline, with 4 times of pH9.0 to the thalline volume, the Tris-HCl damping fluid Eddy diffusion thalline of 50mM, after fully mixing, the centrifugal 10min of 10000rpm, the collecting precipitation thing, again use the pH9.0 of 4 times of volumes, the Tris-HCl damping fluid of 50mM fully suspends it, the centrifugal 10min of 10000rpm, the collecting precipitation thing, continue to use the pH9.0 of 10 times of volumes, the Tris-HCl damping fluid of 50mM fully suspends it, then carries out cell wall breaking with high pressure homogenizer again, the broken wall condition is: pressure 800-1000bar, cool off with 0-4 ℃ of frozen water in the process.With the centrifugal 10min of suspension 10000rpm after the cytoclasis, 4 ℃ of preservations of collection supernatant liquor are stand-by.
Get supernatant liquor as crude enzyme liquid, crude enzyme liquid placed 80 ℃ of water-baths, insulation 30min, be cooled to room temperature after, the centrifugal 10min of 10000rpm collects supernatant liquor.Supernatant liquor is placed ice bath, slowly add while stirring ammonium sulfate to 75% (w/v), the centrifugal 15min of 13000rpm, get precipitation, pH9.0, the Tris-HCl damping fluid of 50mM dissolves again, and placing molecular weight cut-off is the dialysis tubing of 8000Da, with pH9.0, the Tris-HCl damping fluid of 20mM is extracellular fluid dialysis, the volume ratio of extracellular fluid dialysis and interior liquid is greater than 50,4 ℃ of dialysis 12-16h, and extracellular fluid dialysis is changed once every 4h in the centre, after having dialysed, get dialyzed solution and concentrate with vacuum rotary evaporator, carry out lyophilize again after, place-20 ℃ cryogenic refrigerator to preserve stand-by.
Get above the 50mg resulting lyophilized powder in centrifuge tube, add 2ml pH7.0, the PBS damping fluid of 50mM (containing 150mM NaCl), it is fully dissolved after, (Φ 1.6 * 100cm) for upper Sephacryl S-300 gel column.Use first pH7.0, behind the abundant balance pillar of the PBS damping fluid of 50mM (containing 150mM NaCl), then loading is used 2 column volumes of identical buffer solution elution again, and flow velocity is 0.5ml/min, collects every pipe 3ml with Fraction Collector.Then to the measured in solution lipase activity in the collection tube and protein electrophoresis analysis.
Collect the Peak Activity after the normal pressure gel-filtration separates, after concentrated, desalination, the freeze-drying, use again pH9.0, the Tris-HCl damping fluid dissolving of 50mM, upper Mono Q TMThe 5/50GL anion-exchange column, use first pH9.0, the Tris-HCl damping fluid balance pillar of 50mM, then loading, NaCl with the same buffer preparation carries out wash-out according to following gradient again: 0M NaCl 2CV (column volume), 0-0.6M NaCl 4CV, 0.6-1.0M NaCl2CV, 1.0M NaCl 1CV.Flow velocity is 1ml/min, presses the peak with full-automatic Fraction Collector and collects.Then to the measured in solution lipase activity in the collection tube and protein electrophoresis analysis.
Collect the Peak Activity after the high-efficiency anion exchange separates, after concentrated, desalination, the freeze-drying, use again pH7.0, the dissolving of 50mM PBS damping fluid, upper Superdex TM200 preparation scale gel columns (column volume 25ml).Use first pH7.0,50mM PBS damping fluid balance pillar, then loading is used pH7.0,1.5 column volumes of 50mM PBS buffer solution elution, flow velocity is 0.5ml/min, presses the peak with full-automatic Fraction Collector and collects.Then to the measured in solution lipase activity in the collection tube and protein electrophoresis analysis.
Resulting cytoclasis liquid supernatant (Thermotogapetrophila lipase and mutant lipase) in the present embodiment is carried out PAGE, and carry out respectively coomassie brilliant blue staining and active coloring, coomassie brilliant blue staining is identical with ordinary method, active coloring carries out according to the methods below, be specially: take the glycerine triethyl as substrate, bromothymol blue is that staining agent detects the enzymic activity (principle: under the lipase effect that gel electrophoresis separates rear lipase district band, the ethyl hydrolysis of glycerine triethyl forms acetic acid, make the pH of latex gel decline of lipase location band and make bromothymol blue change yellow into by blue-greenish colour, thereby the substrate hydrolysis that shows lipase is active.)
Step:
1) non-sex change PAGE electrophoresis (usually using the 4-12% gradient gel).
2) gel 10 minutes behind the 50ml water rinse electrophoresis places on the shaking table and shakes gently.
3) repeat water rinse once.
4) with 30ml 10mM KH 2PO 4PH7.0 solution rinsing one time 10 minutes.
5) add 30ml 10mM KH 2PO 4PH 7.0 solution and 0.4ml is saturated and the bromothymol blue solution that filters through 0.22um, room temperature insulation 10 minutes.(because solution is neutral pH, staining fluid is blue-greenish colour).
6) vanay of adding 1ml 99.5% purity.
7) the yellow region band presents, and shows the activity gels district band of lipase.
SDS-PAGE result (Figure 11) shows that the lipase albumen behind the lipase purifying in 2 kinds of sources only has single band, and molecular weight all is about 35kDa.PAGE result shows (Figure 10), and 2 kinds of recombinant lipases all have the active zone band, is 280KDa corresponding to the molecular weight of the protein band of this active zone band.Hence one can see that, and the bioactive molecule of 2 kinds of lipase exists with the form of 8 aggressiveness.
After purifying was finished, the specific activity of Thermotoga petrophila lipase had been brought up to the 104U/mg of pure enzyme from the 14U/mg of crude enzyme liquid, and purification is 7.4; The specific activity of mutant lipase has been brought up to the 265U/mg of pure enzyme from the 17U/mg of crude enzyme liquid, and purification is 15.6.And the specific activity of mutant lipase has improved 155% than the specific activity of Thermotoga petrophila lipase.
The zymologic property analysis of embodiment 6 recombinant lipases
Embodiment 4 prepared lipase (Thermotoga petrophila lipase and mutant lipase cytoclasis liquid supernatant) are carried out enzymatic reaction to measure its optimal pH under different pH.Used damping fluid is the Britton-Robinson damping fluid (citric acid, potassium primary phosphate, boric acid, sodium hydroxide, veronal) of pH4.0-11.0.Respectively 2 kinds of lipase are placed the damping fluid of different pH, measure down different pH to the impact of enzymatic reaction for 60 ℃, the result shows that the optimal pH of Thermotoga petrophila lipase and mutant lipase is respectively 10.0 and 9.0 (Fig. 4).
Lipase liquid is processed 60min in the Britton-Robinson of different pH values damping fluid, measure again the residual enzyme activity with the pH stability of research lipase under room temperature.The result shows (Fig. 6), between pH3.0-11.0, the residual activity of mutant lipase is all more than 80%, in the scope of pH6.0-11.0, the residual activity of Thermotogapetrophila lipase is all more than 80%, 2 kinds of lipase of this explanation have good pH stability, and mutant lipase is more acidproof than Thermotoga petrophila lipase.
(mutant lipase is at pH9.0, in the 50mMTris-HCl buffer system for the specific buffer system that is determined at of optimal reactive temperature; Thermotoga petrophila lipase is at pH10.0, in the 50mM Gly-NaOH buffer system) and differing temps under (30 ℃-80 ℃) carry out, carry out enzymatic reaction and vitality test according to aforementioned acid base titration.Result (Fig. 5) shows that the optimal reactive temperature of mutant lipase and Thermotoga petrophila lipase is 60 ℃.
THERMAL STABILITY is at the lower 10-60min of processing of differing temps (60-85 ℃), carries out enzyme assay again.The results are shown in Figure 7a and Fig. 7 b, show in 60 ℃-70 ℃ scope to be incubated 60min, the residual enzyme activity of mutant lipase and Thermotoga petrophila lipase all can maintain more than 95%; At 80 ℃ of lower insulation 30min, the residual enzyme activity of mutant lipase and Thermotoga petrophila lipase is all more than 80%; At 85 ℃ of lower insulation 30min, the residual enzyme activity of mutant lipase can maintain more than 60%, the residual enzyme activity of Thermotogapetrophila lipase can maintain more than 40%, this explanation mutant lipase and Thermotoga petrophila lipase have good thermostability, and mutant lipase is more heat-resisting.
In mutant lipase and Thermotoga petrophila lipase solution, add respectively separately 0.05ml trypsin 0.1mg/ml, with the configuration of pH7.0PBS damping fluid) and stomach en-(0.1mg/ml, with pH2.0 glycine-HCL damping fluid configuration) in 37 ℃ of processing 30-240min, measure again lipase activity after the dilution.Behind trypsin treatment 240min, the residual enzyme activity of mutant lipase and Thermotoga petrophila lipase all still maintains more than 95%, without significantly loss; Behind pepsin 240min, the enzyme residual enzyme activity of mutant lipase still maintains about 80% (Fig. 8 b), the residual enzyme activity of Thermotoga petrophila lipase is that about 50% (Fig. 8 a), this explanation mutant lipase and Thermotoga petrophila lipase all have good protease inhibitor hydrolysis ability, and mutant lipase is stronger to pepsic resistance.
The preparation of embodiment 7 immobilization recombinant lipases
Adopt direct entrapping method, specifically: with massfraction 3.5% sodium alginate (or massfraction 3.5% sodium alginate and massfraction 3.0% gelatin mixture) and the enzyme liquid (lipase that embodiment 4 is prepared, mutant lipase and Thermotoga petrophila lipase cytoclasis liquid supernatant, enzyme dosage is 3000U/g over dry sodium alginate) mix by a certain percentage, in 37 ℃ of water-baths, sodium alginate is dissolved, and make sodium alginate soln and the abundant mixing of enzyme solution.Draw the mixed solution of carrier and enzyme with 5mL syringe (No. 5 syringe needles), dropwise in implantation quality mark 2% calcium chloride (m/v) solution, form immediately smooth gelled pill with the height about 10cm.Leach gelled pill, change calcium chloride solution, in 4 ℃ of refrigerators, leave standstill sclerosis 2h.Again leach gelled pill, after massfraction 0.9%NaCl (m/v physiological saline) washing, blot surface-moisture with thieving paper.Again this Immobilization in Sodium Alginate enzyme is placed 4 ℃ refrigerator ageing 24h, then be stored in 4 ℃ of refrigerators stand-by.The enzyme of immobilized enzyme yield alive is 45-50%, and the enzyme activity of immobilized enzyme is 45-50U/g.Can calculate according to following formula the enzyme yield alive of immobilized enzyme.
Figure G2009102012093D00211
In the formula: U 0---enzyme liquid vigor, U/ml;
V 0---enzyme liquid is long-pending, ml;
U 1---immobilized enzyme, U/g;
m 1---immobilized enzyme quality, g.
Embodiment 8 recombinant lipases are used for the catalysis ester synthesis reaction
In the allusion quotation measuring bottle of 100ml, acetic acid with 0.10mol/L, 0.04mol/L glycerine and 7.5ml normal heptane form nonaqueous phase esterification system, container is divided into two groups, then (mutant lipase is at pH9.0, in the 50mM Tris-HCl buffer system to be added in accordingly respectively different buffer systems; Thermotoga petrophila lipase is at pH10.0, in the 50mM Gly-NaOH buffer system) in the immobilized lipase (embodiment 7 is prepared) of pre-freeze-drying, every part of 1g, then this two group reactions system is placed under 50 ℃ of conditions, with the speed rotational oscillation 48h of 150rpm, then the GC analysis is carried out in sampling in the water bath with thermostatic control shaking table.
The Japan Shimadzu GC-2010 of company gas chromatograph, capillary column are (Φ 0.50mm * 25m), N 2Be carrier gas, injection port and detector temperature are respectively 270 ℃ and 300 ℃, temperature programming: 80 ℃ keep 2min, then are raised to 200 ℃ with the speed of 20 ℃/min and keep 1min, rise to 290 ℃ with the speed of 45 ℃/min at last and keep 2min.Take analytically pure vanay as standard substance, do reference colour spectrogram (Figure 12), and determine its retention time with marker method, dispose again the vanay standard substance difference loading of different concns, take peak area as X-coordinate, vanay concentration is ordinate zou, drawing standard curve, the then growing amount of vanay in the establishing criteria curve calculation sample.Experimental result is: the ester synthesis reaction of the lipase-catalyzed acetic acid of Thermotoga petrophila lipase and mutant and glycerine, the production rate of final product vanay is respectively 16mmol/L and 27mmol/L.The resulting mutant lipase of this explanation the present invention is the building-up reactions of catalysis short-chain ester class to a certain extent.
Embodiment 9 recombinant lipases are used for the reaction of catalysis chiral separation
The water activity of reaction system: just contain acry radical donor and (R; S)-organic solvent system of sec-n-octyl alcohol (boiling point of R-2-octanol and S-2-octanol all is 175 ℃) or ion liquid system and lipase places encloses container with the saturated salt solution that contains known water activity alpha w respectively, in 25 ℃ of lower 72h that place.Used saturated salt solution is respectively: LiCl (α w=0.11), MgCl 26H 2O (α w=0.33), Mg (NO 3) 26H 2O (α w=0.53), NaCl (α w=0.75), K 2SO 4(α w=0.97).
A. the catalyzed reaction in the organic solvent: the toluene (organic solvent all dewaters with the 4A molecular sieve after activating before use) that in the allusion quotation measuring bottle of 25ml, adds 5ml known water activity; add respectively again 50mmol/L acry radical donor ethyl chloroacetate and 25mmol/L (R, S)-sec-n-octyl alcohol.Container is divided into 2 groups, and (mutant lipase is at pH9.0, in the 50mM Tris-HCl buffer system to be added in accordingly respectively different buffer systems; Thermotogapetrophila lipase is at pH10.0, in the 50mM Gly-NaOH buffer system) in the immobilized lipase (embodiment 7 is prepared) of pre-freeze-drying, every part of 1g.Then this 2 group reaction system is placed under 50 ℃, with sampling and measuring transformation efficiency c and enantiomeric excess rate behind the 200r/min reaction 72-96h.
B. the enzymic catalytic reaction in the reverse micelle system: prepare reverse micelle system with injection.In the allusion quotation measuring bottle of 25ml, add respectively 5mL and contain the octane-iso (A liquid) of 380mmol/L AOT, above-mentioned reaction vessel is divided into 2 groups, (B liquid, mutant lipase are the pH9.0Tris-HCl damping fluid correspondingly to add respectively the enzyme liquid (embodiment 4 is prepared) of 580 μ L Thermotoga petrophila lipase and mutant lipase and 50mM damping fluid; Thermotoga petrophila lipase is the pH10.0Gly-NaOH damping fluid), make Wo (mol ratio of water and tensio-active agent) and be 17 AOT reverse micelle system.In each container, all add 25mmol/L (R, S)-sec-n-octyl alcohol and 50mmol/L acry radical donor ethyl chloroacetate, and this 2 group reaction system is placed under 50 ℃, with sampling and measuring transformation efficiency and enantiomeric excess rate behind the 200r/min reaction 120h.
Measure the amount of residual substrate sec-n-octyl alcohol with gas chromatograph (GC-2010, Japanese Shimadzu company).Detector is hydrogen flame (FID), adopts the derivative gas chromatography method to measure the enantiomeric excess rate of sec-n-octyl alcohol.Get the 1mL sample, add 100 μ l toluene and extract, get extraction liquid 50 μ, l adds 2 μ L (S)-(+)-α-styroyl isocyanic ester and 50 μ L dry toluene, and reactant is put 45 ℃ of lower derivatize 2h.Capillary column is SE30 (Φ 0.25mm * 30m, Australia SGE), N2 is carrier gas, injection port and detector temperature are respectively 250 ℃ and 270 ℃, Temperature Programmed Processes is: 110 ℃ keep 1min, then be raised to 210 ℃ and keep 2min with the speed of 10 ℃/min, continue to be raised to 222 ℃ with the speed of 3 ℃/min.Take analytically pure (R, S)-sec-n-octyl alcohol, R-2-octanol and S-2-octanol as standard substance, do the reference colour spectrogram, determine the retention time (Figure 13) of each component.
1. (S)-sec-n-octyl alcohol enantiomeric excess rate e.e.S or e.e.R calculate according to following formula:
E.e.S=(A S-A R)/(A S+ A R) * 100% or
e.e.R＝(A R-A S)/(A S+A R)×100％
A wherein SBe the peak area of (S)-sec-n-octyl alcohol derivative, A RPeak area for (R)-sec-n-octyl alcohol derivative.
2. the calculating of transformation efficiency c:
c＝(A S+A R)/A×100％
Wherein A is the total peak area of pure and mild ester.
3. the pass of mapping selection rate E and transformation efficiency c and e.e.S or e.e.R is:
E=ln[(1-c) (1-e.e.S)]/ln[(1-c) (1+e.e.S)] or
E＝ln[(1-c)(1-e.e.R)]/ln[(1-c)(1+e.e.R)]
Experimental result is: immobilization Thermotoga petrophila lipase and mutant lipase catalysis (R in organic phase, S)-and the enantiomeric excess rate of the chiral separation of sec-n-octyl alcohol reaction is respectively e.e.R 75.4% and e.e.R 83.5%, and transformation efficiency c is respectively 31% and 43%; The enantiomeric excess rate of the chiral separation reaction of Thermotoga petrophila lipase and mutant lipase catalysis (R, S)-sec-n-octyl alcohol in reverse micelle is respectively e.e.R12.8% and e.e.R 25.3%, and transformation efficiency is respectively 8.9% and 23.2%.The resulting mutant lipase of this explanation the present invention can the catalysis chiral separation react, and higher efficient is arranged, particularly in the organic reaction system.
Embodiment 10 recombinant lipases are used for catalysis and produce biofuel
With 3.00g Oleum Gossypii semen and 0.3g methyl alcohol (oleyl alcohol mol ratio 1: 3), 4.50g normal hexane, 1.0g (embodiment 7 is prepared, respectively accordingly pre-freeze-drying in different buffer systems: mutant lipase is at pH9.0, in the 50mM Tris-HCl buffer system for immobilized lipase; Thermotoga petrophila lipase is at pH10.0, in the 50mMGly-NaOH buffer system), mix 50 ℃ of airtight vibrations (200rpm) reaction 4h in the 50ml allusion quotation of packing into the measuring bottle, add again 0.3g methyl alcohol and continue reaction 4h, add for the third time at last methyl alcohol 0.3g and continue reaction 4h.Reaction is separated enzyme after finishing from product, again with the centrifugal 10min of reaction product 12000rpm, behind the standing demix, isolate the raw glycerine of lower floor and the distillate on upper strata, and organic solvent is reclaimed in final evaporation, obtains simultaneously refined biometric diesel oil.It is as follows that lipase-catalyzed vegetables oil is converted into the reaction equation of biofuel:
Figure G2009102012093D00241
R wherein 1, R 2, R 3Be C 7-17Alkyl or alkylene.
1. the transformation efficiency of biofuel calculates according to following formula:
Figure G2009102012093D00242
2. oil fatty acid content analysis: with reference to GB/T 17376-1998eqv ISO 5509.
3. the mensuration of biofuel:
Lipase-catalyzed reacted product biofuel is passed through gas chromatograph for determination.System is Shimadzu GC-2010 gas chromatograph, PEG-20M capillary column (long 30m, internal diameter 0.25mm), and fid detector, split stream sampling, temperature programming: 120 ℃ of column temperatures rise to 200 ℃ with 8 ℃/min, insulation 3min; Rise to 220 ℃ with 4 ℃/min again, insulation 3min; Rise to 240 ℃ with 5 ℃/min again, insulation 1min.Injector temperature is 240 ℃, and detector temperature is 270 ℃.Sample thief 0.1mL, concentration is per sample suitably diluted with normal hexane, gets 1 μ L sample feeding.Under above testing conditions, each component in the product all can access preferably separates.Analytically pure Uniphat A60, methyl stearate, Witconol 2301 and methyl linoleate are made into the mixed solution of concentration known with normal hexane, analyze according to above condition loading, do reference colour spectrogram (shown in Figure 14), and adopt marker method to determine the retention time of each component in this mixture, the then content of these 4 kinds of fatty acid methyl esters in conjunction with the external standard method sample.Acquired results is: the ester conversion rate that the lipase-catalyzed Oleum Gossypii semen of Thermotoga petrophila lipase and mutant is converted into biofuel is respectively 86% and 93%, this explanation the present invention in resulting mutant lipase effectively the catalysis vegetables oil be converted into biofuel.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Sequence table
＜110〉Fujian Fudabaite Sci-Tech Devpt Co., Ltd.
＜120〉high temperature lipase, preparation of its mutant and uses thereof
<130>096154 1CNCN
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<170>PatentIn version 3.3
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<222>(1)..(1)
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<222>(9)..(9)
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<210>14
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Claims (10)

1. the lipase of a separation is characterized in that, its aminoacid sequence is shown in SEQ ID NO:4.
2. a nucleic acid molecule is characterized in that, the lipase claimed in claim 1 of encoding.
3. nucleic acid molecule as claimed in claim 2 is characterized in that, its nucleotide sequence is shown in SEQ ID NO:2.
4. a carrier is characterized in that, described carrier comprises polynucleotide claimed in claim 2.
5. a cell is characterized in that, it comprises carrier claimed in claim 4, or is integrated with polynucleotide claimed in claim 2 in its genome.
6. the purposes of lipase as claimed in claim 1 is characterized in that, is used for that the catalysis ester is synthetic, chiral separation or vegetables oil is converted into biofuel.
7. a method of producing lipase claimed in claim 1 is characterized in that, comprising: cultivate cell claimed in claim 5, isolate expression product from culture.
8. one kind is converted into the method for biofuel with vegetables oil, it is characterized in that, comprises with lipase treatment vegetables oil claimed in claim 1.
9. the synthetic method of a catalysis ester is characterized in that, in anhydrous system with lipase-catalyzed esterification claimed in claim 1.
10. the method for a chiral separation is characterized in that, with lipase-catalyzed chiral separation claimed in claim 1.
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