CN101942422A - Genes of D-lactic dehydrogenase from serratia marcescens and research of cloning and expressing recombinant strains and recombinant enzymes - Google Patents
Genes of D-lactic dehydrogenase from serratia marcescens and research of cloning and expressing recombinant strains and recombinant enzymes Download PDFInfo
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Abstract
The invention relates to genes of D-lactic dehydrogenase from serratia marcescens and the research of cloning and expressing recombinant strains and recombinant enzymes, in particular provides new D-lactic dehydrogenase ('D-LDH protein' for short), polynucleotide for encoding the D-LDH protein and a method for generating the D-LDH protein through recombination technology. The invention also discloses application of the polynucleotide for encoding the D-LDH protein. The D-LDH protein has the function of catalyzing pyruvic acid to form D-lactic acid, and can be used for producing the D-lactic acid of which the price is 8 to 10 times higher than that of L-lactic acid efficiently, so the D-LDH protein has huge application prospect.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to new D-serum lactic dehydrogenase (D-LDH) polypeptide and the encoding gene thereof that come from serratia marcescens (Serratia marcescens).The invention still further relates to the recombinant vectors that contains this gene, with this carrier transformed host cells (recombinant bacterial strain), and generate purposes in the D-lactic acid at the catalysis pyruvic acid.
Background technology
Lactic acid extensively is present in human body, animal, plant and the microorganism.Lactic acid can be divided into D-lactic acid, L-lactic acid, D, three kinds of L-lactic acid according to its opticity difference.
D-lactic acid and derivative thereof are widely used in fields such as food, medicine, feed and chemical industry, and particularly at field of medicaments, the polymkeric substance of D-lactic acid can be used as the material of medicament slow release formulation.This is because the polymkeric substance of D-lactic acid can not be absorbed by human consumption, but can be parasitized the microbiological degradation of human body, thereby plays the effect of slow releasing pharmaceutical.In addition, D-lactic acid can utilize the chirality feature of optically pure D-lactic acid as the precursor of chiral drug, is used to develop chiral drug, improves the result of treatment of medicine and the security of medication greatly.In addition, D-lactic acid can be used as the precursor of agricultural chemicals, therefrom the synthetic agricultural chemicals easily decompose, noresidue, be a kind of environmental protection type agricultural chemical.Therefore, the demand to D-lactic acid increases day by day at present, and its price is 8-10 a times of L-lactic acid, has very big market potential.
In animal or microbe, serum lactic dehydrogenase (being called for short " LDH ") is to be coenzyme with NADH, pyruvic acid is generated lactic acid through biochemical reaction, so serum lactic dehydrogenase is the key enzyme of lactobacillus-fermented.Occurring in nature exists L and two kinds of serum lactic dehydrogenases that rely on NADH of D, and the catalysis pyruvic acid generates L-lactic acid and D-lactic acid respectively.
In recent years, chiral material is subjected to increasing attention with its special optical texture and application widely, slowly develops into a frontier of scientific research.Lactic acid is the chiral molecules of nature minimum, and the producer in the whole world nearly 80% adopts direct fermentation production optical purity lactic acid, mainly is L-lactic acid.D-lactic acid is the precursor of synthetic multiple chiral material as a chiral centre, and is very extensive in the application of aspects such as medicine, agricultural chemicals, chemical industry.Compare with the research scene of L-lactic acid prosperity, the exploitation of D-lactic acid shows slightly thin.The overall throughput of world's lactic acid is 250,000 tons/year at present, and ultimate production is 130,000 tons/year, and increases progressively with annual 6%～8%.At present D-lactic acid-producing ability is about 1.6 ten thousand tons, and about 2.6 ten thousand tons of the demand of D-lactic acid in the world, therefore, the demand to D-lactic acid increases day by day at present.
Different chiral material its preparation methods is roughly the same, mainly is divided into chemical resolution method and microbe fermentation method.The fossil resource that chemical resolution method need consume petroleum, coal etc. is deficient, expensive day by day gradually, and to the destruction of ecotope and serious; By contrast, microbe fermentation method with the renewable resources is raw material, and is with low cost, and environmental protection is a kind of pattern of biological processing efficiently.
Yet at present people understand very fewly for the D-lactic acid-producing, lack various and D-serum lactic dehydrogenase efficiently, and therefore, this area presses for develops new D-serum lactic dehydrogenase, for use in the fermentative production that is used for D-lactic acid.
Summary of the invention
The purpose of this invention is to provide a kind of new serratia marcescens D-serum lactic dehydrogenase (being D-LDH albumen) with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolating D-serum lactic dehydrogenase (being the D-LDH polypeptide) is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
Preferably, this D-serum lactic dehydrogenase is selected from down group:
(a) polypeptide of aminoacid sequence shown in SEQ ID NO:2;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have function that the catalysis pyruvic acid forms D-lactic acid by (a) polypeptides derived.
In another kind of preference, the aminoacid sequence of this D-serum lactic dehydrogenase is shown in SEQ ID NO:2, and perhaps this enzyme is the aminoacid sequence that has the SEQ ID NO:2 of 6His label.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of D-serum lactic dehydrogenase described in the coding first aspect present invention;
(b) with polynucleotide (a) complementary polynucleotide.
In another kind of preference, this polynucleotide encoding has the D-serum lactic dehydrogenase of aminoacid sequence shown in the SEQ ID NO:2.
In another kind of preference, the sequence of these polynucleotide is be selected from down group a kind of:
(a) sequence of 1-990 position among the SEQ ID NO:1;
(b) sequence of 1-993 position among the SEQ ID NO:1.
In a third aspect of the present invention, provide the carrier that contains above-mentioned polynucleotide.The host cell that transformed by this carrier or transduce also is provided or has directly been transformed by above-mentioned polynucleotide or the host cell of transduction, promptly contained above-mentioned carrier or in genome, be integrated with the host cell of the sequence of the polynucleotide described in the second aspect present invention.In another kind of preference, above-mentioned host cell is a serratia marcescens.
In a fourth aspect of the present invention, a kind of preparation method of D-serum lactic dehydrogenase of the present invention is provided, it is characterized in that this method comprises:
(a) under conditions suitable for the expression, cultivate the above-mentioned described host cell of the present invention;
(b) from culture, isolate serratia marcescens D-LDH polypeptide.
In a fifth aspect of the present invention, a kind of method of the D-of preparation lactic acid is provided, the method comprising the steps of:
In the presence of the host cell described in described D-serum lactic dehydrogenase of first aspect present invention or the third aspect present invention, catalysis pyruvic acid and coenzyme NAD H reaction, thus form D-lactic acid.
In another kind of preference, in described reaction, the pH in the reaction system is 7.0-8.0, and pyruvic acid concentration is 5-40mM, and/or coenzyme NAD H concentration is 0.3-0.7mM.
In another kind of preference, in the described reaction system, there be not the cupric ion or the ferrous ion of divalence basically.
In another kind of preference, temperature of reaction is 20-65 ℃, more preferably is 20-40 ℃.
In a sixth aspect of the present invention, provide and above-mentioned D-LDH polypeptid specificity bonded antibody.
In a seventh aspect of the present invention, the compound of simulation, promotion, antagonism D-LDH polypeptide active is provided, and the compound that suppresses the D-LDH polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of D-LDH polypeptide.
In a eighth aspect of the present invention, provide and whether had the proteic method of D-LDH in the test sample, it comprises: sample is contacted with the proteic specific antibody of D-LDH, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample D-LDH albumen.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.The proteic encoding sequence of serratia marcescens D-LDH for example of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described in below (eg embodiment) each technical characterictic can make up mutually, thereby constitute new or optimized technical scheme.For example, with regard to the upper limit of a certain (as 10-100) on a large scale can with another preferably more among a small circle the lower limit 20 of (as 20-80) make up, thereby constitute another scope (for example 20-100), vice versa.As space is limited, this tired no longer one by one stating.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is the proteic cDNA sequence of D-LDH of the present invention (SEQ ID NO:1).
Fig. 2 is the proteic full length amino acid sequence of D-LDH of the present invention (SEQ ID NO:2).
Fig. 3 has shown the ORF fragment (swimming lane 1) of the D-lactate dehydrogenase gene that utilizes degenerated primer LDHf (SEQ ID NO:3) and LDHr (SEQ ID NO:4) amplification, swimming lane M is the standard DNA molecular weight, the top-down band is respectively (kb): 17,9.3,5.4,4.1,3.1,2.7,1.6,1.1,0.5.
Fig. 4 is the building process of D-lactate dehydrogenase gene recombinant expression plasmid pET-ldh.
Fig. 5 has shown the agarose gel electrophoresis that recombinant plasmid pET-l dh double digestion is identified.Swimming lane 1 is cut back (positive findings) for the pET-ldh enzyme; Swimming lane 2 is cut back (negative findings) for pET-28a (+) enzyme; Swimming lane M is the standard DNA molecular weight, and the top-down band is respectively (kb): 17,9.3,5.4,4.1,3.1,2.7,1.6,1.1,0.5.
Fig. 6 has shown the whole cell SDS-PAGE electrophorogram behind original bacterium BL21 (DE3) (swimming lane 1) and E.coli BL21 (DE3)/pET-ldh (swimming lane 2) abduction delivering.Swimming lane M is that (top-down band is respectively 97.2,66.4,44.3,29.0,20.1 to the standard protein molecular weight, 14.3kDa) among the figure.
Fig. 7 has shown at the D-serum lactic dehydrogenase and has participated in the reaction that the catalysis pyruvic acid generates D-lactic acid in 1h, 3h, 17h sampling that the HPLC that utilizes the reverse post of SB-Aq to carry out detects.
Fig. 8 has shown at the D-serum lactic dehydrogenase and has participated in the reaction that the catalysis pyruvic acid generates D-lactic acid in 0h, 25h sampling that the HPLC that utilizes Chirobiotic R chiral column to carry out detects.
Fig. 9 is a D-serum lactic dehydrogenase Research on Thermal Stability.
Figure 10 is the research of D-serum lactic dehydrogenase enzyme reaction temperature.
Figure 11 is a D-serum lactic dehydrogenase pH stability study.
Figure 12 is the research of D-serum lactic dehydrogenase enzyme reaction pH.
Figure 13 is the influence of divalent metal to D-lactic dehydrogenase enzyme stability.
Figure 14 is the influence of pyruvic acid concentration to D-lactic dehydrogenase enzyme activity.
Figure 15 measures Km and the Vmax of D-serum lactic dehydrogenase to pyruvic acid for the double-reciprocal plot method.
Figure 16 is the influence of NADH concentration to D-lactic dehydrogenase enzyme activity.
Figure 17 measures Km and the Vmax of D-serum lactic dehydrogenase to coenzyme NAD H for the double-reciprocal plot method.
Embodiment
The inventor has screened a large amount of microorganism kinds through extensive and deep research, and unexpectedly serratia marcescens screens a kind of new D-serum lactic dehydrogenase.This D-serum lactic dehydrogenase catalysis pyruvic acid effectively forms D-lactic acid.Yet different with existing D-serum lactic dehydrogenase, D-serum lactic dehydrogenase of the present invention is insensitive (but for Cu for most of metallic cations
2+And Fe
2+And extremely responsive).Finished the present invention on the basis once more.
The main zymologic property and the enzymatic reaction kinetics of D-serum lactic dehydrogenase of the present invention are as follows:
(1) temperature stability of D-serum lactic dehydrogenase is being higher than in 50 ℃ the environment very easily inactivation.
(2) D-serum lactic dehydrogenase enzyme reaction optimum temperuture is 60 ℃.
(3) the pH stability of D-serum lactic dehydrogenase is the most stable about pH7.0.
(4) D-serum lactic dehydrogenase enzyme reaction optimal pH is 7.5
(5) divalent metal is to the stability influence of D-serum lactic dehydrogenase, Zn
2+, Mg
2+, Ca
2+, Mn
2+, Ba
2+, Ni
2+Enzyme only there are very weak restraining effect, Zn
2+And Ba
2+Restraining effect is the most weak, Cu
2+And Fe
2+Restraining effect stronger.
(6) the D-serum lactic dehydrogenase is to the kinetic parameter Km=3.39mmol/L of substrate pyruvic acid, Vmax=6.87mmol/ (mgmin).
(7) the D-serum lactic dehydrogenase is to the kinetic parameter Km=1.43mmol/L of coenzyme NAD H, Vmax=1.61mmol (mgmin).
In the present invention, term " D-LDH albumen ", " D-LDH polypeptide " or " D-serum lactic dehydrogenase D-LDH " are used interchangeably, and all refer to have the albumen or the polypeptide of serratia marcescens D-serum lactic dehydrogenase D-LDH aminoacid sequence (SEQ IDNO:2).They comprise the D-serum lactic dehydrogenase D-LDH that contains or do not contain initial methionine.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating D-LDH albumen or polypeptide " is meant that the D-LDH polypeptide 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 purifying D-LDH albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of D-LDH polypeptide can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide 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, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of serratia marcescens D-LDH, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function or the active polypeptide of natural serratia marcescens D-LDH albumen of the present invention basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide 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 a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence (as 6His) or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).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 " D-LDH polypeptide " refers to have the SEQID NO:2 polypeptide of sequence of serratia marcescens D-LDH protein-active.This term also comprises having and variant form serratia marcescens D-LDH albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 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, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of serratia marcescens D-LDH and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of D-LDH protein D NA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-D-LDH polypeptide to obtain.The present invention also provides other polypeptide, as comprises D-LDH polypeptide or its segmental fusion rotein (fusion rotein shown in SEQ ID NO:3).Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of D-LDH polypeptide.Usually, this fragment have the D-LDH peptide 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 serratia marcescens D-LDH albumen or polypeptide.The difference of these analogues and natural D-LDH polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.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-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " serratia marcescens D-LDH albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, there are 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 polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
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 |
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 polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide 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 polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.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 not changing the function of its encoded polypeptides in fact.
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 " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 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 polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to 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 (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation encoding D-LDH.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
D-LDH pyrenoids thuja acid 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 relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.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.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
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.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 as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
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 D-LDH albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the D-LDH polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of encoding D of the present invention-LDH polypeptide, or transform or the transduction proper 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, D-LDH albumen polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains D-LDH encoding histone dna sequence dna and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be 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; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.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, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, 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.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, 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 be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.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 (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, 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 handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The serratia marcescens D-LDH albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: be directly used in catalytic production D-lactic acid.
On the other hand, the present invention also comprises D-LDH protein D NA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into D-LDH protein gene product or fragment.Preferably, refer to that those can combine with D-LDH protein gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of serratia marcescens D-LDH, comprise that also those do not influence the antibody of serratia marcescens D-LDH protein function.The present invention also comprise those can with modify or without the D-LDH protein gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the D-LDH protein gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing serratia marcescens D-LDH albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerl ing, In Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block serratia marcescens D-LDH protein function and the antibody that does not influence serratia marcescens D-LDH protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of D-LDH protein gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of D-LDH protein gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-stick matter Serratia D-LDH can be used for detecting, and detects the serratia marcescens D-LDH albumen in the sample.
Whether having the proteic method of D-LDH in a kind of detection test sample is to utilize the proteic specific antibody of D-LDH to detect, and it comprises: sample is contacted with the D-LDH protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample D-LDH albumen.
The production of polyclonal antibody available serratia marcescens D-LDH albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with D-LDH albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.This polypeptide is the D-serum lactic dehydrogenase that the inventor is cloned into from the genomic dna of serratia marcescens (S.marcescens) H3010, its open reading frame (ORF) 993bp (SEQ IDNO:1 and Fig. 1), 330 amino acid (SEQ ID NO:2 and Fig. 2) of encoding.Show through bioinformatic analysis, contain 18 S atoms in this molecule, be made up of 12 beta sheets and 13 alpha-helixs, this enzyme molecular weight is about 36kDa (theoretical value 36466.8Da), and theoretical iso-electric point pI is 5.55.
The inventor also utilizes existing pET28a (+) expression system, success in E.coli BL21DE3, realized efficiently expressing to the D-serum lactic dehydrogenase, obtained most of D-serum lactic dehydrogenase that exists with soluble form, the N-terminal of the expression plasmid pET-ldh that makes up has 6 Histidines (His6) to merge mark, therefore select the method for Ni post affinity chromatography to carry out separation and purification, obtain highly purified D-serum lactic dehydrogenase (D-LDH) at last, enzyme is lived and is 322.9U/mg (condition determination is 37 ℃, and pH 7.0).
Measure the thermostability of enzyme and find, the D-serum lactic dehydrogenase is being higher than in 50 ℃ the environment very easily inactivation, and the measured reaction optimum temperuture is 60 ℃ (in reaction 30s).This enzyme is the most stable about pH7.0, and the reaction optimal pH is 7.5.Divalent metal Zn
2+, Mg
2+, Ca
2+, Mn
2+, Cu
2+, Fe
2+, Ba
2+, Ni
2+Enzyme only there are very weak restraining effect, Zn
2+And Ba
2+Restraining effect is the most weak, however Cu
2+And Fe
2+Restraining effect stronger.Dynamics research shows, restraining effect to enzyme when substrate pyruvic acid concentration surpasses 30mmol/L strengthens gradually, and coenzyme NAD H concentration has very strong restraining effect to enzyme when being higher than 0.5mmol/L, and the apparent vigor of enzyme becomes 0 when its concentration reaches 1.0mmol/L.The D-serum lactic dehydrogenase is to the Km=3.39mmol L of substrate pyruvic acid
-1, to the Km=1.43mmol L of coenzyme NAD H
-1, the D serum lactic dehydrogenase to the avidity of coenzyme NAD H greater than avidity to the substrate pyruvic acid.The D-LDH catalytic substrate pyruvic acid (pyruvate) that successfully detects heterogenous expression by HPLC generates chiral product D-lactic acid.
Major advantage of the present invention comprises:
(a) pyruvic acid that D-serum lactic dehydrogenase of the present invention can be cheap is as substrate, and production prices are than the expensive 8-10 of L-lactic acid D-lactic acid doubly efficiently, thereby have great application prospect.
(b) activity of D-serum lactic dehydrogenase of the present invention can be regulated and control easily with the cupric ion or the iron ion of divalence.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The extraction of embodiment 1 serratia marcescens genomic dna
The working instructions that provide according to the manufacturer, with Genomic DNA Purification Kit (Biodev, Beijing) extracting is in the genomic dna of serratia marcescens (Serratia marcescens) H3010 (available from ATCC) of logarithmic phase, and with 0.8% agarose gel electrophoresis the bacterial genomes that obtains is detected.
The clone of embodiment 2D-lactate dehydrogenase gene, the structure of expression plasmid and abduction delivering
1.D-the clone of lactate dehydrogenase gene
Synthetic following primer LDHf and LDHr, primer sequence is:
LDHf：5’-CCCATATGAAATTGGCGATATC-3’(SEQ?ID?NO：3)；
LDHr：5’-TCAGGCGTTCAGCTGGTTC-3’(SEQ?ID?NO：4)
The genomic dna of the serratia marcescens that obtains with embodiment 1 is a template, amplification Serratia gene.
The pcr amplification system is: genomic dna 2 μ l, each 2 μ l of primer LDHf and LDHr, dNTP1.6 μ l, 10 * Tag damping fluid, 2 μ l, TAKARA Tag polysaccharase 0.4 μ l, ddH
2O 10 μ l.
The PCR response procedures is: 97 ℃ of pre-sex change 5min, and 95 ℃ of sex change 30s, 56.5 ℃ of annealing 1.5min circulate 30 times, and 72 ℃ are extended 10min.
Pcr amplification product is carried out agarose gel electrophoresis, and partial results as shown in Figure 3.
Amplified band cut reclaim test kit with the pillar rubber tapping of vast Imtech behind the glue and reclaim, be connected in pMD18-T carrier, form carrier pMD-ldh, and transform conventional intestinal bacteria JM83 available from TaKaRa company.By cutting checking, identify positive colony, and carry out sequencing in Shanghai Ying Jun biotech company at dull and stereotyped enterprising row filter of penbritin LB and desmoenzyme.
Computer Analysis shows, the D-lactate dehydrogenase gene ORF that the clone obtains shown in SEQ ID NO:1, the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as D-LDH albumen, its encoding gene called after D-LDH protein gene.
D-LDH cDNA is 1757bp (Fig. 1 and SEQ ID NO:1), contains complete open frame 990bp, and coding contains the polypeptide (Fig. 2 and SEQ ID NO:2) of 330 amino-acid residues.This albumen contains 18 S atoms, is made up of 12 beta sheets and 13 alpha-helixs, and this enzyme molecular weight is about 36kDa (theoretical value 36466.8Da), and theoretical iso-electric point pI is 5.55.Lower with most of known D-serum lactic dehydrogenase homologys.
In sum, D-LDH albumen is a novel D-serum lactic dehydrogenase that does not appear in the newspapers.
The structure of embodiment 3D-serum lactic dehydrogenase expression system
Cultivate reorganization bacterium JM83/pMD-ldh, alkaline process extracting plasmid, with restriction enzyme BamHI, NdeI double digestion, the external source fragment that reclaims the 1.0kb size of test kit recovery through glue is connected under the effect of T4DNA Ligase with pET-28a (+) carrier that reclaims through restriction enzyme BamHI, NdeI double digestion glue again, can obtain plasmid pET-ldh, construction procedures as shown in Figure 4.
The pET-ldh that builds is carried out XbaI enzyme cutting (two XbaI enzyme cutting sites are arranged on the recombinant plasmid) checking, should obtain two fragments of 1.1kb and 5.2kb size, and serve as the linearizing fragment that 5.4kb the time should only appear in contrast with carrier pET-28a (+), as shown in Figure 5.For correctly having made up the pET-ldh expression plasmid.
Embodiment 4D-LDH is proteic recombinant expressed
1. the competent preparation of host bacterium e. coli bl21 (DE3)
Line on the LB solid medium with a little host bacterium BL21 (DE3) the preservative fluid dilution of toothpick picking, 37 ℃ of overnight incubation, picking list colony inoculation is in 30ml LB liquid nutrient medium, 37 ℃ of overnight incubation, by 1% inoculum size BL21 (DE3) nutrient solution is inoculated in the 30ml substratum, 37 ℃ of shaking tables are cultivated 1～2h to OD
600≈ 0.4, receives bacterium, 4 ℃ of constant temperature, and the centrifugal 10min of 6000rpm places ice-water bath with bacterial sediment, with the 10mmol/L CaCl of 5ml
2(freezing) mixing washs once, 4 ℃ of constant temperature, the centrifugal 10min of 6000rpm, the precipitation 75mmol/L CaCl of 1ml
2(freezing) mixing suspends, and places 8h for 4 ℃ and transforms.
2, pET-ldh transforms host's bacterium e. coli bl21 (DE3)
Take out e. coli bl21 (DE3) competent cell of placing more than the 8h from 4 ℃ of refrigerators, the palm of the hand melts, take out 100 μ L to new eppendorf pipe, place ice-water bath rapidly, add the plasmid pET-ldh that 10 μ L build, mixing gently, place 30min in the ice-water bath, 42 ℃ of water-bath pulse 2min are transferred to fast and place 1～2min in the ice-water bath, add 900 μ L LB substratum, 1～1.5h is cultivated in 37 ℃ of shaking table recoveries, draw 100 μ L conversion fluids and coat on the LB solid medium that penicillin final concentration of card is 50 μ g/ μ L, be inverted for 37 ℃ and cultivate 12～16h, pick out single bacterium colony line amplification.Further take out plasmid enzyme restriction and identify, will contain E.coli BL21 (DE3) order-checking of plasmid pET-ldh, sequencing result shows that open reading frame (ORF) sequence is correct.This shows, has obtained containing the expression strain of expression plasmid pET-ldh.
3, the abduction delivering of D-serum lactic dehydrogenase
Recombination bacillus coli BL21/pET-ldh is inoculated in adds in the suitable antibiotic LB liquid nutrient medium (the LB substratum consists of: yeast powder 0.5%, Tryptones 1%, NaCl 1%, pH7.0), 37 ℃ of shaking table overnight incubation; Be transferred to the 250mL that 30mL LB substratum is housed with 1% inoculum size again and shake in the bottle,, be cultured to OD respectively with the negative contrast of wild bacterium BL21 (DE3)
600During ≈ 0.6, add 0.8mmol/L inductor IPTG and cultivate in 37 ℃ of shaking tables, take a sample when inducing 4.5h, centrifugal collection thalline carries out full bacterium SDS-PAGE electrophoresis.
The result shows that genetic engineering bacterium has given expression to the D-serum lactic dehydrogenase as shown in Figure 6, and swimming lane M is that (top-down band is respectively 97.2,66.4,44.3,29.0,20.1 to the standard protein molecular weight, 14.3kDa) among the figure.Given expression to a tangible protein band between 29.0kDa and 44.3kDa, the molecular weight size conforms to predictor.This shows that enzyme is successful expression.
The separation and purification of embodiment 5D-serum lactic dehydrogenase
1. the acquisition of solubility D-serum lactic dehydrogenase crude enzyme liquid
The e. coli bl21 (DE3) that will contain recombinant expression plasmid pET-ldh is in the middle of the 30mL LB substratum of 50 μ g/mL with that penicillin of inoculum size access card (Kan) final concentration of 1%, 37 ℃ of shaking table overnight incubation (about 12～16h), be in the middle of the 150mL LB substratum of 50 μ g/mL with that penicillin of inoculum size access card (Kan) final concentration of 1% again, 37 ℃ of shaking tables are cultured to OD
600Add 0.8mmol/L inductor IPTG during ≈ 0.6, cultivate 4h (inductive condition after this optimization can guarantee that the recombinant protein major part of expressing exists with soluble form) in 20 ℃ of shaking tables again, collect thalline,, remove supernatant with the centrifugal 5min of 8000rpm.Use twice of phosphoric acid buffer (pH 7.0) washing thalline again, be 30mL phosphoric acid buffer (pH 7.0) the suspension thalline (albumen that reduces non-specific binding when containing certain density imidazoles in the crude enzyme liquid and helping affinity chromatography combines with post) of 20mmol/L at last with the imidazoles final concentration, with power 200w, ultrasonic 3s, interval 3s, the broken thalline of the condition that surpasses 100 times is penetrating to bacteria suspension, this moment again under 4 ℃ with 15000rpm high speed centrifugation 15min, keep supernatant and be target protein D-serum lactic dehydrogenase crude enzyme liquid.
2. the affinity chromatography of solubility D-serum lactic dehydrogenase crude enzyme liquid
According to constructed recombinant expression plasmid pET-ldh (6 * His) merge the characteristic of marks, select the Ni post as chromatography column, and post and Chromatography Data Acpuisition Unit CT-22 detector join with 6 Histidines.For the separation and purification effect that obtains imitating, the Ni post affinity chromatography of this target protein has been done the optimization of certain condition, finally select the Banding damping fluid to contain the 50mmol/L imidazoles, the Elution damping fluid contains the 300mmol/L imidazoles.The purer reorganization D-serum lactic dehydrogenase enzyme liquid that affinity chromatography conditional capture after optimizing with this elutes.
3. the pure enzyme liquid of ultrafiltration and concentration D-serum lactic dehydrogenase
Contain a certain amount of imidazoles in the pure enzyme liquid behind affinitive layer purification, NaCl salt, so need utilize the method for ultrafiltration removes these small molecule salts, in order to avoid the stability of enzyme is impacted, some small molecules foreign proteins less than the filter membrane aperture also are filtered off simultaneously, enzyme is further purified, the D-serum lactic dehydrogenase after can concentrating at last.
The functional response of embodiment 6D-serum lactic dehydrogenase is identified
1, reorganization D-serum lactic dehydrogenase is to the catalyzed reaction checking of substrate
Respectively go on foot the D-serum lactic dehydrogenase that purifying obtains higher degree through above-mentioned, adding final concentration in phosphoric acid buffer (pH 7.0) is the substrate pyruvic acid (Pyruvate) of 2.75mg/mL and the coenzyme NAD H that final concentration is 17.74mg/mL, the pure enzyme liquid (about 0.17mg) that adds 200 μ L again is to total reaction system 3.1mL, carry out the substrate catalyzed reaction in 37 ℃, when reaction 1h, 3h, 17h, take a sample respectively, utilize the reverse post of SB-Aq to carry out HPLC and detect.
The result be 659.4,1211.5,19160 progressively to increase successively at the peak area of 1h, 3h, 17h product lactic acid, and the peak area of reactant pyruvic acid obviously reduces as shown in Figure 7.Hence one can see that, and along with the carrying out of reaction, reaction product lactic acid constantly generates, and it progressively becomes greatly with the ratio of reaction substrate pyruvic acid, and especially behind reaction 17h, obviously increase is much for lactic acid production.Prove absolutely that thus this expressed D-serum lactic dehydrogenase of D-lactate dehydrogenase gene of finding has the function that pyruvic acid catalysis is generated lactic acid really from S.marcescens H3010.
2, the check of chiral product D-lactic acid generation
After having verified that this D-lactic dehydrogenase endonuclease capable transform to generate lactic acid with pyruvic acid, what need further to confirm is that institute's lactic acid producing is D type lactic acid but not L type lactic acid.0h (not enzyme-added) and 25h (adding enzyme reaction) sampling utilize Chirobiotic R chiral column to detect in the Yu Shangyi reaction.
The result as shown in Figure 8.By I width of cloth figure among the figure as can be known D-lactic acid appearance time be 3.7min, be 3765.2,21977.3 obviously to increase successively at the peak area of 0h and 25h product D-lactic acid.Because when using this post to detect, pyruvic acid all has interference at the place, peak that goes out of D-lactic acid, change moving phase or conversion dual-flow phase proportioning, and change flow velocity all can't be well on this post with pyruvic acid, the absorption peak of three kinds of materials of D-lactic acid separately, so detected peaks is just arranged when the 0h unreacted herein, and the detected result by reaction back 25h as can be seen, going out place, peak amount of substance at D-lactic acid increases greatly, and what detect that the conclusion draw as can be known should place's growth through the SB-Aq post thus is product lactic acid.With the comparison of racemic lactic acid mark product, can determine to react the lactic acid that is generated and be the D type but not the L type, because of it does not detect the existence of any material at L-lactic acid appearance time.
The research of embodiment 7D-serum lactic dehydrogenase zymologic property
1, the thermostability of enzyme
To be divided into several pipes of equivalent with the pure enzyme liquid of phosphoric acid buffer (pH 7.0) dilution, be positioned over respectively in 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and the 70 ℃ of water-baths, the enzyme liquid of getting equal quantities every 10min minute the water-bath under each temperature comes out, in 37 ℃, measure enzyme activity in pH 7.0 phosphoric acid buffers, last sample is got 40min, and the enzyme activity of being surveyed is 100% during with 0min.
The result as shown in Figure 9.D-LDH in 〉=50 ℃ temperature, during 10min just almost completely inactivation (because of the result of 60 ℃ and 70 ℃ and 50 ℃ with, so unlisted), in 40 ℃, preserve 10min, it is only surplus 50% that enzyme is lived, the enzyme last less than 10% of living during 40min.Preserve 40min down at 30 ℃, enzyme is lived and is lost half approximately.Preserve the basic not loss alive of 40min enzyme down at 20 ℃, remnant enzyme activity is more than 90%.Thereby the thermostability of this D-serum lactic dehydrogenase is not fine, need preserve below 20 ℃, otherwise inactivation easily.
2, temperature is to the influence of D-lactic dehydrogenase enzyme reaction
A certain amount of D-serum lactic dehydrogenase liquid is added preheating in the phosphoric acid buffer (pH7.0) that is mixed with substrate pyruvic acid and coenzyme NAD H of differing temps, be mixed, measure the enzyme activity in the 30s, the pairing enzyme activity of temperature is 100% when the highest with surveyed enzyme activity.
The result as shown in figure 10.In 20 ℃ of-60 ℃ of scopes, lactate dehydrogenase activity improves with the rising of temperature, its optimal reactive temperature is 60 ℃, when temperature of reaction surpassed 60 ℃, speed of response had reduction during than 60 ℃ slightly, but by before D-lactic dehydrogenase enzyme heat stability experimental result as can be known, raising along with temperature, thermoinducible enzyme deactivation speed is also improving, so react serum lactic dehydrogenase inactivation rapidly after the 30s under 〉=60 ℃ of high temperature.So temperature can change the speed of enzyme reaction itself, also can cause the zymoprotein sex change to be lost efficacy, therefore this " optimum temperuture " is closely related with the reaction times, is within the 30s in the time of this setting.This temperature effect characteristic of comprehensive serratia marcescens (S.marcescens H3010) D-serum lactic dehydrogenase, be not suitable under the condition of optimal reactive temperature, carrying out the catalyzed reaction production D-lactic acid of substrate pyruvic acid as can be known, be preferably in 30-40 ℃ and carry out, can guarantee that like this stability of enzyme makes enzyme reaction continue to carry out.
3, pH is to the influence of enzyme stability
The D-serum lactic dehydrogenase in the damping fluid of different pH, is preserved 24h for 4 ℃, at 37 ℃, measure enzyme activity under pH 7.0 conditions, living with enzyme, the pairing enzyme activity of pH is 100% when the highest.
The results are shown in shown in Figure 11.As seen this enzyme is the most stable at pH 6.5-8.0, and enzyme is lived loss all less than 5%, and wherein pH about 7.0 is the most stable.
4, pH is to the influence of D-lactic dehydrogenase enzyme reaction
The substrate pyruvic acid is mixed in the phosphoric acid buffer of different pH with coenzyme NAD H, in 37 ℃ of preheating 2min, again will be a certain amount of D-serum lactic dehydrogenase liquid add in the reaction system and be mixed, measure enzyme and live, the pairing enzyme activity of pH is 100% when the highest with enzyme activity.
The result as shown in figure 12.This D-serum lactic dehydrogenase is lived higher at pH 7.0-8.5 scope endoenzyme, all more than 70%, its optimal pH is 7.5, and when pH＜6 or pH＞8.5, enzymic activity sharply descends.
5, metal ion is to the influence of enzyme stability
Respectively at adding final concentration in the enzyme liquid is the divalent-metal ion Zn of 2mmol/L
2+, Mg
2+, Ca
2+, Mn
2+, Cu
2+, Fe
2+, Ba
2+, Ni
2+, room temperature places 15min, make it fully in conjunction with after, then respectively at 37 ℃, measure enzyme in the phosphoric acid buffer of pH7.0 and live, be contrast with the enzyme reaction that does not add metal ion, establishing its enzyme activity is 100%.
The result as shown in figure 13.Zn
2+, Mg
2+, Ca
2+, Mn
2+, Ba
2+, Ni
2+Enzyme only there is very weak restraining effect; Zn
2+And Ba
2+Restraining effect is the most weak, and enzyme is lived loss less than 5%, and Cu
2+And Fe
2+Stronger restraining effect is all arranged, wherein with Fe
2+Enzyme activity after the interaction has only 1.53% of contrast.
The influence of embodiment 8D-serum lactic dehydrogenase enzymatic reaction kinetics
1, substrate pyruvic acid concentration is to the influence of enzyme activity
Set different substrate pyruvic acid final concentration in the reaction system, a certain amount of enzyme is added reaction system,, measure enzyme in the phosphoric acid buffer of pH7.0 and live in 37 ℃.
The result as shown in figure 14.Enzyme activity can embody speed of reaction, and as seen, when substrate pyruvic acid concentration was lower than 5mmol/L, its concentration was the limiting factor of enzyme reaction, and the result is shown as first order reaction kinetics, and enzyme activity increases with the proportional example of concentration of substrate.When substrate pyruvic acid concentration surpasses 5mmol/L,, no longer be the limiting factor of enzyme reaction because that substrate begins is excessive gradually, this moment, enzyme reached capacity, and enzyme reaction rate is gradually slow, along with concentration of substrate further improves, enzyme reaction rate no longer increases, and presents zeroth order reaction kinetics.When substrate pyruvic acid concentration surpasses 30mmol/L,, discharge substrate thereby suppressed compound intermediate, and make enzyme present state of saturation, so enzyme reaction speed descends on the contrary because too much substrate combines with the compound intermediate of enzyme, substrate, coenzyme.
2, D-serum lactic dehydrogenase D-LDH is to the kinetic parameter of substrate pyruvic acid
Get some corresponding substrate pyruvic acid concentration speed of response corresponding in the linear extent in Figure 12 with it, by the Lineweaver-Burk method with 1/V to 1/[S] map Figure 15.Can try to achieve D-LDH at 37 ℃, pH=7.0, the Michaelis-Menton constant Km to pyruvic acid in the 0.2mol/L phosphoric acid buffer system is 3.39mmol/L, Vmax=0.94mmol/ (Lmin).The pure enzyme amount used with catalysis is expressed as Vmax=6.87mmol/ (mgmin)
3, coenzyme NAD H concentration is to the influence of enzyme activity
Set different coenzyme NAD H final concentration in the reaction system, a certain amount of enzyme is added reaction system,, measure enzyme in the phosphoric acid buffer of pH7.0 and live in 37 ℃.
The result as shown in figure 16.Enzyme activity can embody speed of reaction, and as seen, when coenzyme NAD H concentration was lower than 0.4mmol/L, its concentration was the limiting factor of enzyme reaction, and the result is shown as first order reaction kinetics, and enzyme activity increases with the proportional example of coenzyme NAD H concentration.When coenzyme NAD H concentration was between 0.4～0.5mmol/L, enzyme reaction rate was gradually slow.When coenzyme NAD H concentration surpasses 0.5mmol/L and further raising, enzyme activity descends rapidly, when coenzyme NAD H concentration surpasses 1.0mmol/L, enzyme activity is almost 0, be that enzyme no longer shows speed of response, this shows that the coenzyme NAD H that is higher than finite concentration (being 0.5mmol/L at this) has very strong restraining effect to enzyme, this is because too much coenzyme NAD H combines with the compound intermediate of enzyme, substrate, coenzyme, stop it to discharge substrate, and make enzyme present state of saturation, thereby the catalytic capability of enzyme is by strongly inhibited.
4, D-serum lactic dehydrogenase D-LDH is to the kinetic parameter of coenzyme NAD H
Get some corresponding coenzyme NAD H concentration speed of response corresponding in the linear extent in Figure 16 with it, by the Lineweaver-Burk method with 1/V to 1/[S] map Figure 17.Can try to achieve D-LDH at 37 ℃, pH=7.0, the Michaelis-Menton constant Km to coenzyme NAD H in the 0.2mol/L phosphoric acid buffer system is 1.43mmol/L, Vmax=0.22mmol/ (Lmin).The pure enzyme amount used with catalysis is expressed as Vmax=1.61mmol/ (mgmin).
The generation of embodiment 9 anti-D-LDH protein antibodies
The reorganization serratia marcescens D-LDH albumen that obtains among the embodiment 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation serratia marcescens D-LDH protein gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.
The preparation of D-serum lactic dehydrogenase varient
Repeat embodiment 3 and 4, difference is, changes the nucleotide sequence of SEQ ID NO:1 with the method for rite-directed mutagenesis, thereby makes following D-serum lactic dehydrogenase varient.
The varient that the 3rd Leu is replaced by Ile among the varient a:SEQ ID NO:2;
The varient that the 4th Ala is lacked among the varient b:SEQ ID NO:2;
Add the varient of Leu among the varient c:SEQ ID NO:2 between the 4th and the 5th.
The determination of activity of the above-mentioned varient a-c that obtains for recombinating shows that these varients can both form D-lactic acid by the catalysis pyruvic acid, and are active close with wild-type (SEQ ID NO:2), and it is 300-350U/mg (condition determination is 37 ℃, and pH 7.0) that enzyme is lived.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.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 institute restricted portion equally.
Sequence table
＜110〉East China University of Science
＜120〉serratia marcescens D-lactate dehydrogenase gene, the research of clone, express recombinant bacterial strain and recombinase
<130>092019
<160>4
<170>PatentIn?version?3.4
<210>1
<211>993
<212>DNA
＜213〉serratia marcescens (Serratia marcescens)
<220>
<221>CDS
<222>(1)..(990)
<400>1
atg?aaa?ttg?gcg?ata?tac?agt?acc?aaa?cag?tat?gac?cgt?aaa?tac?ctc 48
Met?Lys?Leu?Ala?Ile?Tyr?Ser?Thr?Lys?Gln?Tyr?Asp?Arg?Lys?Tyr?Leu
1 5 10 15
gaa?ctg?gtg?aac?cag?cag?ttt?ggc?tat?gag?ctg?gaa?ttc?ctc?gac?ttt 96
Glu?Leu?Val?Asn?Gln?Gln?Phe?Gly?Tyr?Glu?Leu?Glu?Phe?Leu?Asp?Phe
20 25 30
tta?ctc?agt?aaa?aaa?acc?gcc?aaa?acg?gcc?gcc?ggt?tgc?aag?gcg?gtg 144
Leu?Leu?Ser?Lys?Lys?Thr?Ala?Lys?Thr?Ala?Ala?Gly?Cys?Lys?Ala?Val
35 40 45
tgt?atc?ttc?gtc?gac?gac?gac?ggc?agc?cgt?gaa?gtg?ctc?gaa?gaa?ctg 192
Cys?Ile?Phe?Val?Asp?Asp?Asp?Gly?Ser?Arg?Glu?Val?Leu?Glu?Glu?Leu
50 55 60
gcg?gcg?ctg?ggc?gtc?gaa?att?ctc?gcc?ctg?cgc?tgc?gcc?ggt?ttt?aat 240
Ala?Ala?Leu?Gly?Val?Glu?Ile?Leu?Ala?Leu?Arg?Cys?Ala?Gly?Phe?Asn
65 70 75 80
aac?gtc?gat?ctg?gac?gcc?gcc?aaa?gaa?ctg?ggc?atc?aag?gtg?gtg?cgc 288
Asn?Val?Asp?Leu?Asp?Ala?Ala?Lys?Glu?Leu?Gly?Ile?Lys?Val?Val?Arg
85 90 95
gtg?ccg?gcc?tac?tcg?ccg?gag?gcc?gtg?gcg?gaa?cac?gcg?gta?ggc?atg 336
Val?Pro?Ala?Tyr?Ser?Pro?Glu?Ala?Val?Ala?Glu?His?Ala?Val?Gly?Met
100 105 110
atg?atg?tgc?ctg?aac?cgc?cgt?att?cac?cgc?gct?tat?cag?cgt?acc?cgt 384
Met?Met?Cys?Leu?Asn?Arg?Arg?Ile?His?Arg?Ala?Tyr?Gln?Arg?Thr?Arg
115 120 125
gac?gcc?aac?ttc?tcg?ctg?gaa?ggg?ctg?atc?ggt?ttt?aac?atg?cac?aat 432
Asp?Ala?Asn?Phe?Ser?Leu?Glu?Gly?Leu?Ile?Gly?Phe?Asn?Met?His?Asn
130 135 140
cgc?acc?gcc?ggc?gtg?atc?ggc?acc?ggt?aaa?atc?ggc?gtg?gcg?aca?atg 480
Arg?Thr?Ala?Gly?Val?Ile?Gly?Thr?Gly?Lys?Ile?Gly?Val?Ala?Thr?Met
145 150 155 160
cgc?att?ctg?aaa?ggc?ttc?ggc?atg?aag?ctg?ctg?gcc?tac?gat?ccg?ttc 528
Arg?Ile?Leu?Lys?Gly?Phe?Gly?Met?Lys?Leu?Leu?Ala?Tyr?Asp?Pro?Phe
165 170 175
ccg?agc?gaa?cag?gcg?ctg?gaa?ctg?ggc?gcg?gag?tat?gtc?gat?ctg?aag 576
Pro?Ser?Glu?Gln?Ala?Leu?Glu?Leu?Gly?Ala?Glu?Tyr?Val?Asp?Leu?Lys
180 185 190
acg?ctg?tac?gcg?caa?tcc?gac?gtg?atc?acc?ctg?cac?tgc?ccg?ctg?acg 624
Thr?Leu?Tyr?Ala?Gln?Ser?Asp?Val?Ile?Thr?Leu?His?Cys?Pro?Leu?Thr
195 200 205
ccg?gag?aac?cac?cat?ttg?ctg?aat?gcc?gac?gcc?ttc?gcg?atg?atg?aaa 672
Pro?Glu?Asn?His?His?Leu?Leu?Asn?Ala?Asp?Ala?Phe?Ala?Met?Met?Lys
210 215 220
aac?ggc?gta?atg?gtg?atc?aac?acc?agc?cgc?ggc?gcg?ctc?atc?gac?tcc 720
Asn?Gly?Val?Met?Val?Ile?Asn?Thr?Ser?Arg?Gly?Ala?Leu?Ile?Asp?Ser
225 230 235 240
acc?gcc?gcc?atc?gat?gcg?ctg?aag?cag?caa?aaa?atc?ggc?gcc?ctc?ggg 768
Thr?Ala?Ala?Ile?Asp?Ala?Leu?Lys?Gln?Gln?Lys?Ile?Gly?Ala?Leu?Gly
245 250 255
atg?gac?gtc?tac?gag?aac?gaa?cgc?gac?ctg?ttc?ttt?gag?gac?aag?tct 816
Met?Asp?Val?Tyr?Glu?Asn?Glu?Arg?Asp?Leu?Phe?Phe?Glu?Asp?Lys?Ser
260 265 270
aac?gac?gtg?att?cag?gat?gac?gtg?ttc?cgc?cgc?ctg?tcg?gcc?tgc?cac 864
Asn?Asp?Val?Ile?Gln?Asp?Asp?Val?Phe?Arg?Arg?Leu?Ser?Ala?Cys?His
275 280 285
aac?gtg?ctg?ttc?acc?ggc?cat?cgg?gct?ttc?ctg?acc?gaa?gag?gcg?ttg 912
Asn?Val?Leu?Phe?Thr?Gly?His?Arg?Ala?Phe?Leu?Thr?Glu?Glu?Ala?Leu
290 295 300
acc?agc?att?tcc?caa?acc?acg?ctg?cag?aac?atc?agc?cag?ttg?gat?cgc 960
Thr?Ser?Ile?Ser?Gln?Thr?Thr?Leu?Gln?Asn?Ile?Ser?Gln?Leu?Asp?Arg
305 310 315 320
ggc?gaa?gcc?tgc?ccg?aac?cag?ctg?aac?gcc?tga 993
Gly?Glu?Ala?Cys?Pro?Asn?Gln?Leu?Asn?Ala
325 330
<210>2
<211>330
<212>PRT
＜213〉serratia marcescens (Serratia marcescens)
<400>2
Met?Lys?Leu?Ala?Ile?Tyr?Ser?Thr?Lys?Gln?Tyr?Asp?Arg?Lys?Tyr?Leu
1 5 10 15
Glu?Leu?Val?Asn?Gln?Gln?Phe?Gly?Tyr?Glu?Leu?Glu?Phe?Leu?Asp?Phe
20 25 30
Leu?Leu?Ser?Lys?Lys?Thr?Ala?Lys?Thr?Ala?Ala?Gly?Cys?Lys?Ala?Val
35 40 45
Cys?Ile?Phe?Val?Asp?Asp?Asp?Gly?Ser?Arg?Glu?Val?Leu?Glu?Glu?Leu
50 55 60
Ala?Ala?Leu?Gly?Val?Glu?Ile?Leu?Ala?Leu?Arg?Cys?Ala?Gly?Phe?Asn
65 70 75 80
Asn?Val?Asp?Leu?Asp?Ala?Ala?Lys?Glu?Leu?Gly?Ile?Lys?Val?Val?Arg
85 90 95
Val?Pro?Ala?Tyr?Ser?Pro?Glu?Ala?Val?Ala?Glu?His?Ala?Val?Gly?Met
100 105 110
Met?Met?Cys?Leu?Asn?Arg?Arg?Ile?His?Arg?Ala?Tyr?Gln?Arg?Thr?Arg
115 120 125
Asp?Ala?Asn?Phe?Ser?Leu?Glu?Gly?Leu?Ile?Gly?Phe?Asn?Met?His?Asn
130 135 140
Arg?Thr?Ala?Gly?Val?Ile?Gly?Thr?Gly?Lys?Ile?Gly?Val?Ala?Thr?Met
145 150 155 160
Arg?Ile?Leu?Lys?Gly?Phe?Gly?Met?Lys?Leu?Leu?Ala?Tyr?Asp?Pro?Phe
165 170 175
Pro?Ser?Glu?Gln?Ala?Leu?Glu?Leu?Gly?Ala?Glu?Tyr?Val?Asp?Leu?Lys
180 185 190
Thr?Leu?Tyr?Ala?Gln?Ser?Asp?Val?Ile?Thr?Leu?His?Cys?Pro?Leu?Thr
195 200 205
Pro?Glu?Asn?His?His?Leu?Leu?Asn?Ala?Asp?Ala?Phe?Ala?Met?Met?Lys
210 215 220
Asn?Gly?Val?Met?Val?Ile?Asn?Thr?Ser?Arg?Gly?Ala?Leu?Ile?Asp?Ser
225 230 235 240
Thr?Ala?Ala?Ile?Asp?Ala?Leu?Lys?Gln?Gln?Lys?Ile?Gly?Ala?Leu?Gly
245 250 255
Met?Asp?Val?Tyr?Glu?Asn?Glu?Arg?Asp?Leu?Phe?Phe?Glu?Asp?Lys?Ser
260 265 270
Asn?Asp?Val?Ile?Gln?Asp?Asp?Val?Phe?Arg?Arg?Leu?Ser?Ala?Cys?His
275 280 285
Asn?Val?Leu?Phe?Thr?Gly?His?Arg?Ala?Phe?Leu?Thr?Glu?Glu?Ala?Leu
290 295 300
Thr?Ser?Ile?Ser?Gln?Thr?Thr?Leu?Gln?Asn?Ile?Ser?Gln?Leu?Asp?Arg
305 310 315 320
Gly?Glu?Ala?Cys?Pro?Asn?Gln?Leu?Asn?Ala
325 330
<210>3
<211>22
<212>DNA
＜213〉primer
<400>3
cccatatgaa?attggcgata?tc 22
<210>4
<211>19
<212>DNA
＜213〉primer
<400>4
tcaggcgttc?agctggttc 19
Claims (10)
1. isolating D-serum lactic dehydrogenase is characterized in that this enzyme is selected from down group:
(a) polypeptide of aminoacid sequence shown in SEQ ID NO:2;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have function that the catalysis pyruvic acid forms D-lactic acid by (a) polypeptides derived.
2. D-serum lactic dehydrogenase as claimed in claim 1 is characterized in that, the aminoacid sequence of this enzyme is shown in SEQID NO:2, and perhaps this enzyme is the aminoacid sequence that has the SEQ ID NO:2 of 6His label.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:
(a) polynucleotide of D-serum lactic dehydrogenase according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the D-serum lactic dehydrogenase of aminoacid sequence shown in the SEQ ID NO:2.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is be selected from down group a kind of:
(a) sequence of 1-990 position among the SEQ ID NO:1;
(b) sequence of 1-993 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6, perhaps is integrated with the sequence of the described polynucleotide of claim 3 in genome.
8. the preparation method of the described D-serum lactic dehydrogenase of claim 1 is characterized in that, this method comprises:
(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;
(b) from culture, isolate serratia marcescens D-LDH polypeptide.
9. method for preparing D-lactic acid is characterized in that the method comprising the steps of:
In the presence of described D-serum lactic dehydrogenase of claim 1 or the described host cell of claim 7, catalysis pyruvic acid and coenzyme NAD H reaction, thus form D-lactic acid.
10. method as claimed in claim 9 is characterized in that, comprising: in described reaction, the pH in the reaction system is 7.0-8.0, and pyruvic acid concentration is 5-40mM, and/or coenzyme NAD H concentration is 0.3-0.7mM.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667374A (en) * | 2012-09-18 | 2014-03-26 | 华东理工大学 | Method for producing D-lactic acid by taking recombined serratia marcescens as bacterium |
| CN109536467A (en) * | 2018-10-10 | 2019-03-29 | 浙江卓运生物科技有限公司 | The lactic dehydrogenase in lactobacillus panis source and its application and preparation method |
| CN110592057A (en) * | 2019-09-27 | 2019-12-20 | 昆明理工大学 | Chimeric lyase ILTphg and polynucleotides encoding same |
| CN113604493A (en) * | 2021-06-13 | 2021-11-05 | 兰州大学 | LDH, recombinant plasmid for expressing LDH, recombinant probiotic for expressing LDH and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4692173B2 (en) * | 2005-09-13 | 2011-06-01 | 東レ株式会社 | Polypeptide having D-lactic acid dehydrogenase activity, gene encoding the same, and method for producing D-lactic acid |
| CN101302503B (en) * | 2007-05-08 | 2011-04-06 | 上海医药工业研究院 | Mutated plant lactobacillus plantarum D-lactate dehydrogenases, genes, recombinase and use thereof |
| CN101333554B (en) * | 2008-08-05 | 2010-12-15 | 山东大学 | Method for producing D-lactic acid by enzyme resolution of D,L-lactic Acid |
-
2009
- 2009-07-07 CN CN2009100544673A patent/CN101942422B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667374A (en) * | 2012-09-18 | 2014-03-26 | 华东理工大学 | Method for producing D-lactic acid by taking recombined serratia marcescens as bacterium |
| CN109536467A (en) * | 2018-10-10 | 2019-03-29 | 浙江卓运生物科技有限公司 | The lactic dehydrogenase in lactobacillus panis source and its application and preparation method |
| CN110592057A (en) * | 2019-09-27 | 2019-12-20 | 昆明理工大学 | Chimeric lyase ILTphg and polynucleotides encoding same |
| CN113604493A (en) * | 2021-06-13 | 2021-11-05 | 兰州大学 | LDH, recombinant plasmid for expressing LDH, recombinant probiotic for expressing LDH and application |
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| CN101942422B (en) | 2012-10-31 |
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