CN1293194C - D-Pantothe internal ester hydrolase c DNA and its clone expression and application - Google Patents
D-Pantothe internal ester hydrolase c DNA and its clone expression and application Download PDFInfo
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Abstract
The present invention relates to a D-pantothenic acid external ester hydrolase cDNA and the cloning expression and application thereof, particularly to the technical field of a D-pantothenic acid external ester hydrolase cDNA structure gene. In the D-pantothenic acid external ester hydrolase cDNA, a reverse transcriptional PCR technology is used and total RNA of a Fusariummoniliforme (GGMCC 0536) strain is used as a template to synthesize a first chain of cDNA, a segment with the length of 1.5 kb is obtained by the enlargement under the action of a primer 1 and a primer 2, the segment is connected with a T carrier after glue cutting recovery and purification treatment to obtain a cloning carrier recombined plasmid pMD18-T-LAC, the cloning carrier is used as a template to be enlarged under the action of a primer 3 and a primer 4, and a structure gene with the length of 1146 bp is obtained to code the D-pantothenic acid external ester hydrolase cDNA. An expression carrier recombined plasmid pTrc 99 ca-LAC or pYX210-LAC and a corresponding recombined bacterium containing the recombined plasmid are constructed, and D-pantothenic acid is prepared by using the recombined bacterium to biologically converting D-pantothenic acid external ester or D, L-pantothenic acid external ester.
Description
Technical field
The present invention relates to derive from D-pantoic acid lactone hydrolase cDNA structure gene and cloning vector thereof in beading fusarium (Fusarium moniliforme) CGMCC 0536 bacterial strain, the expression vector recombinant plasmid, electricity transforms and makes up the reorganization bacterium and transform preparation D-pantoic acid, relates to D-pantoic acid lactone hydrolase cDNA structure gene technical field.
Background technology
Pantoic acid, Pantothenic acid, D (+)-N (α, γ-dihydroxyl-β, the beta-dimethyl-butyryl)-beta Alanine [D (+)-N (alpha, gamma-dihydroxy-β, β-dimethylbutyl)-β-alanine], claiming pantothenic acid, vitamin B5 or B3 again, is a kind of important medicine, foodstuff additive and fodder additives.It is again the integral part of coenzyme A, involved in sugar, fat, protein metabolism, and its product form is generally its dextrorotatory form calcium salt D-calcium pantothenate.The preparation method of D-pantoic acid mainly contains: chemical resolution method, and the physics Split Method, biological Split Method has only the utilization in the biological Split Method to produce optionally asymmetric hydrolysis D-pantoyl lactone of D-pantoic acid lactone hydrolase bacterial strain at present, obtains the D-pantoic acid.
The bacterial strain that can produce the D-pantoic acid lactone hydrolase has fusarium Pseudomonas Fusarium, and gibberella belongs to Gibberella, and gliocladium germ belongs to Gliocladium, aspergillus niger Pseudomonas Aspergillus, and column disk spore Pseudomonas Cylindrocarpon, Volutella etc.
People such as Sakamoto [the Shimi zu S of Japan in 1994, Kataoka M, Shimizu K, HirakataM, Sakamoto K, Yamada H.Purification and characterization of a novellactonohydrolase, catalyzing the hydrolysis of aldonate lactones andaromatic lactones, from Fusarium oxysporum.Eur J Biochem, 1992,209:383-390.] but stereospecificity in the Fusarium Oxysporm IF05942 bacterial strain of Genera genus is split D, the enzyme of L-pantoyl lactone has carried out separation and purification, and the reaction conditions of enzyme has been carried out exploratory study, has determined optimum reaction condition [the Kataoka M of enzymatic conversion, Shimizu K, Sakamoto K, Yamada H, Shimizu S.Appl.Microbiol.biotechnol, 1995,43:974～977].Simultaneously they form the molecular weight of enzyme and subunit and also measure, and find that this enzyme is made up of two subunits, and molecular weight is about 12500.Inhibitor to this enzyme is also studied, and the factor that can suppress this enzyme has: Zn
2+, Cd
2+, Cu
2+, Hg
2+And EDTA.
[the Kobayashi M such as Michihiko Kobayashi of Japan in 1998, Shinohara M, SakohC, Kataoka M, Shimizu S.Lactone-ring-cleaving enzyme:Genetic analysis, novel RNAediting, and evolutionary implications.Proc.Natl.Acad.Sci.USA.1998,95:12787～12792.] the D-pantoic acid lactone hydrolase gene in Fusarium Oxysporm AKU 3702 bacterial strains is studied, find about the about 2.2kb of D-pantoic acid lactone hydrolase genome total length, contain 6 intension, by the construction cDNA library, utilize gene probe to screen encoding D-pantoic acid lactone hydrolase cDNA gene, its total length is about about 1.2kb, with this enzyme cDNA gene with change in the intestinal bacteria after carrier is connected, and carried out activity expression, detect through SDS-PAGE, discovery has the gene expression product of two kinds of different molecular weights (49kDa and 51kDa), the two all has the D-pantoic acid lactone hydrolase, suspects that the enzyme of 49kDa is the product of 51kDa enzyme under the intracellular protease effect.
The gene order that the people such as Sakamoto of Japan in 2002 have delivered to come from Eukaryotic D-pantoic acid lactone hydrolase also will go in the prokaryote behind this genetic modification, carried out overexpression, this expression product has stereospecificity to split D after measured, and the vigor of L-pantoyl lactone is also analyzed its sequence.It is better to utilize this expression product to carry out the suitability for industrialized production effect.This has been researched and solved and has utilized somatic cells directly to transform the difficulty of being brought, and has also improved the vigor of enzyme and the yield of product.
Summary of the invention
The object of the invention provides a kind of D-pantoic acid lactone hydrolase cDNA and clone, expression and application.Deliver and derive from beading fusarium (Fusarium moniliforme) CGMCC 0536 bacterial strain [Chinese patent .01104070.X, 2001.] middle D-pantoic acid lactone hydrolase cDNA gene nucleotide series and amino acid sequence coded thereof, structure contains the cloning vector of this sequence, expression vector and the reorganization bacterium that has transformed expression vector, and utilize and to contain expression vector reorganization bacterium with D-pantoyl lactone or D, the L-pantoyl lactone prepares the D-pantoic acid for the substrate bio-transformation.
Technical scheme of the present invention is: utilize reverse transcription PCR technology (RT-PCR technology) and be synthetic cDNA first chain of template to derive from total RNA in beading fusarium (Fusarium moniliforme) CGMCC 0536 bacterial strain.Under the effect of primer 1, primer 2, utilize cDNA first chain for template amplification the segment of about 1500bp, this fragment is connected on the pMD18-T carrier recombination bacillus coli that obtains cloning vector pMD18-T-LAC and transformed pMD18-T-LAC.Recombinant plasmid is checked order, and sequencing result is utilized software analysis, this sequence contains an open reading frame.According to sequencing result design primer 3, primer 4, utilizing primer 3, primer 4 is template with cloning vector pMD18-T-LAC, has obtained the long encoding D of the 1146bp-pantoic acid lactone hydrolase cDNA structure gene that is.This gene nucleotide series is:
ATGGCTAAGC TTCCTTCTAC GGCCCAGATT ATTGACCAGA AGTCCTTTAA TGTCTTGAAG 60
GATGTGCCGC CTCCCGCAGT GGCCAATGAC TCTCTGGTGT TCACTTGGCC TGGTGTGACT 120
GAGGAGTCTC TTGTTGAGAA GCCTTTTCAT GTCTACGATG AAGAGTTTTA CGACGTCATC 180
GGAAAGGACC CCTCTTTGAC CCTCATCGCA ACATCGGACA CCGACCCAAT CTTCCATGAG 240
GCTGTCGTAT GGTATCCTCC TACTGAAGAG GTCTTCTTTG TCCAGAATGC TGGCGCTCCC 300
GCTGCTGGCA CTGGCTTGAA CAAGTCTTCC ATCATTCAGA AGATTTCCCT CAAGGAGGCC 360
GACGAGGTCC GCAAGGGCAA GAAGGATGAG GTCAAGGTCG CGGTTGTTGA CTCAAACCCT 420
CAGGTCATCA ACCCCAATGG TGGCACTTAC TACAAGGGCA ACATCATCTT TGCTGGTGAG 480
GGCCAAGGCG ACGATGTTCC CTCCGCCCTG TACCTGATGA ACCCTCTCCC TCCTTACAAC 540
ACCACCACCC TCCTCAACAA CTACTTTGGT CGCCAGTTCA ACTCCCTCAA CGACGTCGGT 600
ATCAACCCCA GGAACGGTGA CTTGTACTTC ACCGATACCC TCTATGGATA CCTCCAGGAC 660
TTCCGTCCTG TTCCTGGTCT GCGAAACCAA GTCTATCGTT ACAACTTTGA CACCGGCGCC 720
GTCACTGTCG TCGCTGATGA CTTTACCCTC CCTAACGGTA TTGGCTTTGG CCCCGACGGC 780
AAGAAGGTCT ATGTCACCGA CACTGGTATC GCTCTTGGCT TTTACGGCCG CAACCTCTCT 840
TCACCCGCCT CTGTTTACTC CTTCGATGTA AACCAGGACG GTACTCTCCA GAACCGCAAG 900
ACCTTTGCTT ACGTCGCGTC TTTCATCCCC GATGGTGTTC ATACCGACTC CAAGGGCCGT 960
GTTTATGCCG GTTGCGGCGA TGGTGTCCAC GTCTGGAACC CTTCGGGCAA GCTAATCGGC 1020
AAGATCTACA CCGGTACTGT TGCTGCTAAC TTCCAGTTTG CCGGCAAGGG AAGGATGATT 1080
ATTACTGGAC AGACCAAGTT GTTCTATGTT ACTTTAGGGG CTTCGGGTCC CAAGCTCTAT 1140
GATTAG 1146
Utilize software that this gene order is analyzed, and know its amino acid sequence coded by inference and be:
MAKLPSTAQI IDQKSFNVLK 20
DVPPPAVAND SLVFTWPGVT 40
EESLVEKPFH VYDEEFYDVI 60
GKDPSLTLIA TSDTDPIFHE 80
AVVWYPPTEE VFFVQNAGAP 100
AAGTGLNKSS IIQKISLKEA 120
DEVRKGKKDE VKVAVVDSNP 140
QVINPNGGTY YKGNIIFAGE 160
GQGDDVPSAL YLMNPLPPYN 180
TTTLLNNYFG RQFNSLNDVG 200
INPRNGDLYF TDTLYGYLQD 220
FRPVPGLRNQ VYRYNFDTGA 240
VTVVADDFTL PNGIGFGPDG 260
KKVYVTDTGI ALGFYGRNLS 280
SPASVYSFDV NQDGTLQNRK 300
TFAYVASFIP DGVHTDSKGR 320
VYAGCGDGVH VWNPSGKLIG 340
KIYTGTVAAN FQFAGKGRMI 360
ITGQTKLFYV TLGASGPKLY 380
D* 381
The present invention with D-pantoic acid lactone hydrolase cDNA structure gene with expression vector pTrc99a (PharmaciaBiotech, Inc., Piscataway) or shuttle expression carrier pYX212 (Novogen company) connect and to have made up recombinant expression pTrc99a-LAC or the pYX212-LAC that contains D-pantoic acid lactone hydrolase cDNA structure gene.
Recombinant plasmid pTrc99a-LAC is converted into the recombination bacillus coli that intestinal bacteria obtain to contain recombinant plasmid pTrc99a-LAC, or recombinant plasmid pYX212-LAC is converted into yeast saccharomyces cerevisiae, obtain to contain the recombinant Saccharomyces cerevisiae of shuttle expression carrier pYX212-LAC.With the reorganization bacterium is the enzyme source, utilizes D-pantoyl lactone or D, and the L-pantoyl lactone is that substrate conversion prepares the D-pantoic acid.
Beneficial effect of the present invention:
The present invention has delivered a kind of D-pantoic acid lactone hydrolase cDNA structure gene nucleotide sequence that derives among beading Fusarium (Fusarium moniliforme) CGMCC 0536; And this gene is connected with expression vector to make up obtains containing this expression of gene recombinant plasmid pTrc99a-LAC or pYX212-LAC, correspondence is converted into intestinal bacteria or yeast saccharomyces cerevisiae respectively again, acquisition contains the recombination bacillus coli of recombinant plasmid pTrc99a-LAC or contains the recombinant Saccharomyces cerevisiae of shuttle expression carrier pYX212-LAC, the reorganization bacterium that contains this gene after measured has stereospecificity to split D-pantoyl lactone or D, the vigor of L-pantoyl lactone can utilize this reorganization bacterium biological process to split preparation D-pantoic acid.The recombination bacillus coli enzyme activity reaches 37-41U/g, and the recombinant Saccharomyces cerevisiae enzyme activity reaches 60-64U/g.
Description of drawings
Fig. 1 cloning vector pMD18-T-LAC physical map.
Fig. 2 pTrc99a-LAC recombinant plasmid physical map.
Fig. 3 pYX212-LAC recombinant plasmid physical map.
Fig. 4 D-pantoic acid lactone hydrolase cDNA gene PCR amplification argrose electrophorogram.
1. the cDNA fragment of utilizing the amplification of primer 1 and primer 2 to obtain;
2.250bp DNA Marker；
3. the cDNA fragment of utilizing primer 3 and primer 4 amplifications to obtain.
The enzyme of the positive recombinant plasmid pTrc99a-LAC of Fig. 5 is cut structure iron.
1.DL2000DNA Marker；
2.D-pantoic acid lactone hydrolase cDNA structure gene fragment;
3.pTrc-LAC/EcoRI；
4.pTrc-LAC/SalI；
5.pTrc-LAC/EcoR/I and SalI；
6.λDNA/HindIII DNA Marker。
The enzyme of the positive recombinant plasmid pYX212-LAC of Fig. 6 is cut structure iron.
1.λDNA/HindIII DNA Marker；
2.D-pantoic acid lactone hydrolase cDNA structure gene fragment;
3.pYX212-LAC/EooRI；
4.pYX212-LAC/SalI；
5.pYX212-LAC/EcoRI and SalI；
6.DL2000DNA Marker。
Embodiment
Reference literature [Chomczynski P., Sacchi N., Single Step Method of RNAIsolation by Acid Guanidinium Thiocyanate Phenol Chloroform Extraction.Anal.Biochem.1987,162:156-159] method and it is improved, in order to extract total RNA of Fusariummoniliforme CGMCC 0536 thalline, concrete operations are as follows: get the Fusariummoniliforme CGMCC 0536 mycelium 0.4g that handled, liquid nitrogen flash freezer also grinds (adding a small amount of quartz sand) powdered, powder is moved in the 50mL centrifuge tube fast, add 12mL sex change liquid (different sulphur hydracid guanidine 6mol/L, citric acid 37.5mol/L, dodecyl creatine sodium 0.75mol/L, beta-mercaptoethanol 0.15mol/L), fully shake up.In centrifuge tube, add 1.2mL sodium acetate (2M pH 4.0) mixing, add the saturated phenol mixing of 12mL, add the 12mL chloroform: primary isoamyl alcohol (volume ratio is 24: 1) mixing.Concussion 10s, ice bath 15min.The centrifugal 20min of 10000g.Supernatant liquor moves to new pipe, adds and the isopyknic mixed solvent of supernatant liquor (phenol: chloroform: the primary isoamyl alcohol volume ratio is 25: 24: 1), and concussion is complete, ice bath 15min.4 ℃ of centrifugal 20min of 10000g, supernatant liquor moves to new pipe, adds the equal-volume Virahol, mixing ,-20 ℃ of precipitation 1h.4 ℃ of centrifugal 20min of 10000g collect RNA, topple over supernatant (carefully), and the RNA resolution of precipitate in 3.5mL sex change liquid, is dissolved mixing.With several pipes of lysate packing, 700 μ L/ pipe adds the equal-volume Virahol ,-20 precipitation 1h.Behind 4 ℃ of centrifugal 10min of 10000g, 75% ethanol is washed 2 times, each 500 μ L/ pipe, will precipitate hanged after, the static 10min of room temperature, the centrifugal 5min of room temperature 10000g, the supernatant that inclines, air-dry on the operator's console.Every pipe adds the water (RNase-free water) of 100 μ L deoxyribonucleases, after dissolving is good, surveys the OD value, and gets an amount of sample and carry out the denaturing formaldehyde electrophoresis.
Utilize commercial mRNA separating kit to separate separating mRNA from total RNA that embodiment 1 obtains.Isolating principle is: have only mRNA at 3 ' end a PolyA structure to be arranged in total RNA, when total RNA when containing the medium of Oligo (dT), mRNA with 3 ' terminal PolyA structure is adsorbed, after utilizing washing lotion with impurity and other RNA flush awaies, utilize elutriant that mRNA is eluted again, can obtain the higher mRNA of purity.Present embodiment utilizes the mRNA separating kit of Promega company, and concrete operations are as follows:
1.0.1 the RNA of～1mg adds the water (RNase-free water) of deoxyribonuclease to 500 μ L (1 μ g/ml), 65 ℃ of water-bath 10min, add the Biotinglated-Oligo (dT) that 3 μ L are provided by test kit, add 20 * SSC that 13 μ L are provided by test kit, room temperature is placed to cooling (being no more than 10min storage period) fully in the soft back of mixing.
2. SA-PMP (magnetic bead that contains Oligo (dT) that provides by test kit) is provided, flick test tube SA-PMP is hanged, be positioned on the magnet stand, carefully remove supernatant, use the 0.5 * SSC that provides by test kit to wash (300 μ L/ time) 3 times, SA-PMP is resuspended among 100 μ l0.5 * SSC.
3. product in 1 is put into 2, room temperature is placed 10min, softly reverses 1～2min, and SA is positioned on the magnet stand, carefully removes supernatant, washes (300 μ L/ time) 4 times with 0.1 * SSC, at every turn soft thorough mixing.
4. with RNase-free water (100 μ L) resuspended SA, the soft mixing, be positioned on the magnet stand, supernatant is moved in the clean centrifuge tube, repeat to wash SA-PMP (as if precipitation is arranged, but 4 ℃ of centrifugal 1min of 12000g) several times with 150 μ LRNase-free water.
Under the effect of ThermoScript II, be synthetic cDNA first chain of template with mRNA, the quantity of T base is between 12～18 in the Oligo of design (dT) primer.Carry out reverse transcription reaction at 50 ℃ of following constant temperature, the reaction times is 1～2h, and cDNA first chain of acquisition is as the cDNA second chain synthetic template.
At first, encoding D-pantoic acid lactone hydrolase gene order of announcing in the gene database is analyzed, we have designed a pair of primer according to analytical results.
The genome of encoding D-pantoic acid lactone hydrolase and cDNA design primer 1 in the fusarium bacterial strain of announcing in the reference gene database: sequence is 5 '-GCTAAGCTTCCTTCTACGGC-3 ', primer 2: Oligo (dT15), giving birth to worker bio-engineering corporation by Shanghai provides, and designs according to mRNA 3 ' end structure.
Utilizing high-fidelity Pyrobest archaeal dna polymerase is template with D-pantoic acid lactone hydrolase cDNA first chain, under the initiation of primer 1, primer 2, and synthetic cDNA second chain.Detect the discovery amplification through agarose gel electrophoresis and obtain the fragment that a length is 1.5kb, agarose gel electrophoresis the results are shown in Figure 4.
Cut glue and reclaim this fragment and purifying, utilize the Taq archaeal dna polymerase to introduce base A to fragment 5 ' end.Under the effect of T4DNA ligase enzyme, this fragment is connected with the T carrier, obtain cloning recombinant plasmid pMD18-T-LAC and see Fig. 1.
This recombinant plasmid electricity is converted in the intestinal bacteria, utilizes the white screening system of basket to screen, picking white cloning and sequencing utilizes the software analysis sequencing result at random, designs following primer according to analytical results:
Primer 3:5 '-CCGGAATTCATGGCTAAGCTTCCTTCTACGGC-3 '
Primer 4:5 '-ATTCCGGTCGACCTAATCATAGAGCTTGGGAC-3 '
EcoRI and SalI restriction enzyme site in primer 3 and primer 4, have been introduced respectively.
With the clone's recombinant plasmid pMD18-T-LAC among the embodiment 3 is template, under the primer 3 in embodiment 3 and the initiation of primer 4, utilize high-fidelity Pyrobest archaeal dna polymerase to increase, obtain long structure gene for the encoding D of 1146bp-pantoic acid lactone hydrolase cDNA, utilize EcoRI and SalI restriction enzyme that amplified fragments is handled after the order-checking, and utilize the T4DNA ligase enzyme that this fragment is connected construction of expression vector pTrc99a-LAC or shuttle expression carrier pYX212-LAC with the commercialization carrier pTrc99a or the shuttle expression carrier pYX212 that handle with identical restriction enzyme.The expression vector pTrc99a-LAC electricity that makes up is converted into intestinal bacteria, be coated with dull and stereotyped 37 ℃ and carry out overnight incubation, or shuttle expression carrier pYX212-LAC electricity is converted into yeast saccharomyces cerevisiae, being coated with flat board cultivated 2-3 days at 30 ℃, picking clone extracting plasmid carries out enzyme and cuts evaluation at random, and qualification result is seen Fig. 5 and Fig. 6.
Use enzyme with recombination bacillus coli that contains expression vector pTrc99a-LAC that obtains among the embodiment 4 or the recombinant Saccharomyces cerevisiae wet thallus that contains shuttle expression carrier pYX212-LAC as transforming, with D-pantoyl lactone or D, the L-pantoyl lactone is a substrate, carries out conversion reaction and prepares the D-pantoic acid.Transformation system composition and conversion operation are as follows: adding 0.5g wet thallus and concentration of substrate are 2% 50mL reaction solution in the 250mL triangular flask, react 60min under 30 ℃ of shaking table 150r/min conditions, and centrifugal removal thalline, clear liquid are the aqueous solution that contains the D-pantoic acid.With HPLC assay determination D-pantoic acid growing amount, know reorganization bacterium enzyme by inference and live.Measurement result sees Table 1 and table 2.
The D-pantoic acid lactone hydrolase enzyme activity determination result that table 1. is measured for the enzyme source with the recombination bacillus coli
| Bacterial strain/plasmid | Enzyme (U/g (wet cells)) alive |
| E. coli jm109 | 0 |
| E. coli jm109/pTrc99a | 0 |
| E. coli jm109/pTrc99a-LAC-1 | 37 |
| E. coli jm109/pTrc99a-LAC-2 | 41 |
The D-pantoic acid lactone hydrolase enzyme activity determination result that table 2. is measured for the enzyme source with the recombinant Saccharomyces cerevisiae
| Bacterial strain/plasmid | Enzyme (U/g (wet cells)) alive |
| Yeast saccharomyces cerevisiae W303-1A | 0 |
| Yeast saccharomyces cerevisiae W303-1A/pYX212-LAC-1 | 60 |
| Yeast saccharomyces cerevisiae W303-1A/pYX212-LAC-2 | 64 |
Claims (5)
1. D-pantoic acid lactone hydrolase cDNA who derives from beading fusarium (Fusarium moniliforme) CGMCC 0536 bacterial strain, its nucleotides sequence is classified as:
atggctaagc ttccttctac ggcccagatt attgaccaga agtcctttaa tgtcttgaag 60
gatgtgccgc ctcccgcagt ggccaatgac tctctggtgt tcacttggcc tggtgtgact 120
gaggagtctc ttgttgagaa gccttttcat gtctacgatg aagagtttta cgacgtcatc 180
ggaaaggacc cctctttgac cctcatcgca acatcggaca ccgacccaat cttccatgag 240
gctgtcgtat ggtatcctcc tactgaagag gtcttctttg tccagaatgc tggcgctccc 300
gctgctggca ctggcttgaa caagtcttcc atcattcaga agatttccct caaggaggcc 360
gacgaggtcc gcaagggcaa gaaggatgag gtcaaggtcg cggttgttga ctcaaaccct 420
caggtcatca accccaatgg tggcacttac tacaagggca acatcatctt tgctggtgag 480
ggccaaggcg acgatgttcc ctccgccctg tacctgatga accctctccc tccttacaac 540
accaccaccc tcctcaacaa ctactttggt cgccagttca actccctcaa cgacgtcggt 600
atcaacccca ggaacggtga cttgtacttc accgataccc tctatggata cctccaggac 660
ttccgtcctg ttcctggtct gcgaaaccaa gtctatcgtt acaactttga caccggcgcc 720
gtcactgtcg tcgctgatga ctttaccctc cctaacggta ttggctttgg ccccgacggc 780
aagaaggtct atgtcaccga cactggtatc gctcttggct tttacggccg caacctctct 840
tcacccgcct ctgtttactc cttcgatgta aaccaggacg gtactctcca gaaccgcaag 900
acctttgctt acgtcgcgtc tttcatcccc gatggtgttc ataccgactc caagggccgt 960
gtttatgccg gttgcggcga tggtgtccac gtctggaacc cttcgggcaa gctaatcggc 1020
aagatctaca ccggtactgt tgctgctaac ttccagtttg ccggcaaggg aaggatgatt 1080
attactggac agaccaagtt gttctatgtt actttagggg cttcgggtcc caagctctat 1140
gattag 1146
2. by the described D-pantoic acid lactone hydrolase of claim 1 cDNA amino acid sequence coded, it consists of:
MAKLPSTAQI IDQKSFNVLK 20
DVPPPAVAND SLVFTWPGVT 40
EESLVEKPFH VYDEEFYDVI 60
GKDPSLTLIA TSDTDPIFHE 80
AVVWYPPTEE VFFVQNAGAP 100
AAGTGLNKSS IIQKISLKEA 120
DEVRKGKKDE VKVAVVDSNP 140
QVINPNGGTY YKGNIIFAGE 160
GQGDDVPSAL YLMNPLPPYN 180
TTTLLNNYFG RQFNSLNDVG 200
INPRNGDLYF TDTLYGYLQD 220
FRPVPGLRNQ VYRYNFDTGA 240
VTVVADDFTL PNGIGFGPDG 260
KKVYVTDTGI ALGFYGRNLS 280
SPASVYSFDV NQDGTLQNRK 300
TFAYVASFIP DGVHTDSKGR 320
VYAGCGDGVH VWNPSGKLIG 340
KIYTGTVAAN FQFAGKGRMI 360
ITGQTKLFYV TLGASGPKLY 380
D* 381。
3. clone the method for the described D-pantoic acid lactone hydrolase of claim 1 cDNA, it is characterized in that:
A) utilizing the reverse transcription PCR technology, is that template has been synthesized cDNA first chain with the total RNA in beading fusarium (Fusarium moniliforme) CGMCC 0536 bacterial strain;
B) primer 1:5 '-GCTAAGCTTCCTTCTACGGC-3 '
Primer 2: Oligo (dT15),
Under primer 1, primer 2 effect, be template with cDNA first chain, utilize high-fidelity Pyrobest archaeal dna polymerase to increase and obtain the fragment of length for 1.5kb;
C) reclaim this fragment and purifying through cutting glue, utilize the Taq archaeal dna polymerase to introduce base A to fragment 5 ' end, under the effect of T4 dna ligase, this fragment is connected with the T carrier, obtain cloning vector recombinant plasmid pMD18-T-LAC and the intestinal bacteria that transformed pMD18-T-LAC, and recombinant plasmid is checked order;
D) design primer 3:5 '-CCGGAATTCATGGCTAAGCTTCCTTCTACGGC-3 '
Primer 4:5 '-ATTCCGGTCGACCTAATCATAGAGCTTGGGAC-3 '
Under primer 3, primer 4 effects, be template with pMD18-T-LAC, utilize high-fidelity Pyrobest archaeal dna polymerase to increase and obtain the structure gene of length for encoding D-pantoic acid lactone hydrolase cDNA of 1146bp.
4. express the method for the described D-pantoic acid lactone hydrolase of claim 1 cDNA, it is characterized in that utilizing EcoRI and SalI restriction enzyme that length is handled for the amplified fragments of 1146bp, and utilize the T4DNA ligase enzyme that this fragment is connected with the commercialization carrier pTrc99a or the shuttle expression carrier pYX212 that handle with identical restriction enzyme, construction of expression vector recombinant plasmid pTrc99a-LAC, and electricity is converted into intestinal bacteria, be coated with flat board and carry out overnight incubation at 37 ℃, or structure shuttle expression carrier recombinant plasmid pYX212-LAC, and electricity is converted in the yeast saccharomyces cerevisiae, is coated with dull and stereotypedly to cultivate 2-3 days at 30 ℃; Picking clone extracting plasmid carries out enzyme and cuts evaluation at random.
5. application rights requires 1 described D-pantoic acid lactone hydrolase cDNA conversion reaction to prepare the method for D-pantoic acid, it is characterized in that recombination bacillus coli that contains expression vector pTrc99a-LAC that obtains or the recombinant Saccharomyces cerevisiae wet thallus that contains shuttle expression carrier pYX212-LAC are as the conversion enzyme, with D-pantoyl lactone or D, the L-pantoyl lactone is a substrate, carry out conversion reaction and prepare the D-pantoic acid, transformation system composition and conversion operation are as follows: adding 0.5g wet thallus and concentration of substrate are 2% 50mL reaction solution in the 250mL triangular flask, react 60min under 30 ℃ of shaking table 150r/min conditions, centrifugal removal thalline, clear liquid are the aqueous solution that contains the D-pantoic acid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100351369C (en) * | 2005-11-22 | 2007-11-28 | 浙江杭州鑫富药业股份有限公司 | Microorganism of producing D-pantothenic acid enternal ester hydrolase and process for preparing D-pantothenic acid thereof |
| CN109456908A (en) * | 2018-11-15 | 2019-03-12 | 江南大学 | A kind of genetic engineering bacterium producing D-pantoyl lactone hydrolase and its construction method and application |
| CN110004135A (en) * | 2019-03-18 | 2019-07-12 | 合肥华恒生物工程有限公司 | A kind of method of ball shape fixation hydrolase containing D-pantoyl lactone cell and preparation method thereof and hydrolysis D-pantoyl lactone |
| CN111455013B (en) * | 2020-05-14 | 2022-06-07 | 安徽泰格生物科技有限公司 | Method for auxiliary resolution of pantolactone by weak base salt |
| CN113106129A (en) * | 2020-09-29 | 2021-07-13 | 安徽华恒生物科技股份有限公司 | Preparation method of D pantolactone with high conversion rate |
| CN112175839B (en) * | 2020-09-30 | 2022-09-13 | 遵义医科大学 | D-pantoic acid lactone hydrolase, producing strain, genetic engineering strain and application thereof |
| CN113046337B (en) * | 2021-03-18 | 2023-04-07 | 赤峰制药股份有限公司 | Pantolactone hydrolase mutant strain and application thereof |
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