CN1884502A - Candida parapsilosis CCTCC M203011 (S)- carbonyl reduction enzyme gene sequence and amino acid composition - Google Patents
Candida parapsilosis CCTCC M203011 (S)- carbonyl reduction enzyme gene sequence and amino acid composition Download PDFInfo
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- CN1884502A CN1884502A CN 200610088058 CN200610088058A CN1884502A CN 1884502 A CN1884502 A CN 1884502A CN 200610088058 CN200610088058 CN 200610088058 CN 200610088058 A CN200610088058 A CN 200610088058A CN 1884502 A CN1884502 A CN 1884502A
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Abstract
The invention relates to gene order and amino acid component of (S)-carbonyl reductase of Candida parapsilosis CCTCC M203011, and the (S)-special carbonyl reductase gene can be used in separating recemic compound with biological method, which belongs to biological engineering sphere. The invention is to confirm (S)-special carbonyl reductase of Candida parapsilosis CCTCC M203011, its gene nucleotide sequence is SEQ ID NO:1, and its amino acid component is SEQ ID NO:2. By this gene, the recombination strain can be used in racemic chirality resolution and can arrange conditions for the heterogeneity, conversion approach and reaction mechanism of the microbial stereoselectivity oxidoreductase used in chirality resolution.
Description
Technical field
The gene order of (S)-carbonyl reductase of Candida parapsilosis CCTCC M203011 and amino acid are formed, and should be used for biological process resolution of racemic compound by (S)-specificity carbonyl reductase gene, belong to technical field of bioengineering.
Background technology
The optical purity phenylglycol, i.e. (S)-phenylglycol, can remember work (S)-PED again, be not only indispensable important chiral additives in the liquid crystal material, and become the preparation have the important intermediate of optically active medicine, agricultural chemicals and functional materials, the research of carrying out the phenylglycol method for splitting is extremely meaningful.
Utilize biological process to transform the optically pure chipal compounds of preparation and have the reaction conditions gentleness, product is single, and stereoselectivity, regioselectivity and chemo-selective are higher, and can finish some chemosynthesis and be difficult to the advantages such as reaction of carrying out.The bioconversion reaction of synthesis of optically active material is broadly divided into two classes: a class is racemic modification to be split as two have optically active enantiomorph; Another kind of is from racemize or prochiral precursor, obtains asymmetric optical activity product by catalyzed reaction.
Rise the nineties in the world microorganism and enzyme resolving chiral compound are carried out a large amount of research.Enzyme is made of L-amino acid, and its active centre has constituted an asymmetric environment, helps the identification to raceme, is a kind of catalyzer of height chirality.Its catalytic efficiency height has stronger specificity.The enzymatic resolving racemic is more satisfactory selection.Utilize intact cell that racemic compound is transformed, can obtain the optical purity enantiomorph, in nonaqueous phase and organic-water biphasic reaction system, but also Enzymatic transformation prepares optical pure compound.
At present, part stereoselectivity oxidoreductase gene obtains by genetic engineering means, and for the stereoselectivity oxidoreductase gene that derives from Candida parapsilosis, only be useful on and split 1, the desaturase CpSADH of 3-butyleneglycol and be used for the polyketone reductase enzyme C1 of asymmetric conversion pantoyl lactone and the report of C2, and the homology of these enzymes is not high, there are differences between the gene order.
Summary of the invention
The gene order and the amino acid that the purpose of this invention is to provide (S)-carbonyl reductase of Candida parapsilosis CCTCC M203011 are formed, so that utilize the recombinant bacterial strain of this gene to carry out the racemize chiral separation, and for further studying the diversity of the microorganism stereoselectivity oxydo-reductase that is used for chiral separation, path for transformation and reaction mechanism create conditions.
Technical scheme of the present invention: (S)-specificity carbonyl reductase of Candida parapsilosis (Candida parapsilosis) CCTCCM203011, its gene nucleotide series is SEQ ID NO:1.(S)-specificity carbonyl reductase of this Candida parapsilosis (Candida parapsilosis) CCTCC M203011, its amino acid consists of SEQ ID NO:2.
By three-dimensional selective oxidation reductase enzyme zymologic property being studied, thought the reaction mechanism that the oxydo-reductase that derives from C.parapsilosis CCTCC M203011 will (R)-phenylglycol is asymmetric be converted into (S)-phenylglycol be: at NAD
+Existence under, is the 2-hydroxy acetophenone earlier with (R)-phenylglycol selective oxidation, while NAD
+Be reduced to NADH; In the presence of NADPH, be (S)-phenylglycol with intermediate 2-hydroxy acetophenone asymmetric reduction then, NADPH is oxidized to NADP simultaneously
+
C.parapsilosis CCTCC M203011 alcoholdehydrogenase is active higher to secondary alcohols.The optimum pH of this enzymatic oxidation reaction is pH9.0, and optimum temperuture is 40 ℃.Zine ion has activation to this enzyme, and metal chelator and heavy metal ion can produce restraining effect to the vigor of this enzyme.
The optimum pH of C.parapsilosis CCTCC M203011 carbonyl reductase catalytic reduction reaction is pH4.5, and optimum temperuture is 35 ℃, and heavy metal ion has the obvious suppression effect to the activity of this enzyme.
Candida parapsilosis (Candida parapsilosis CCTCC M203011): colonial morphology is an oyster white, smooth surface, and glossy, bacterium colony is opaque.Thalli morphology is an oval, the cell vegetative propagation, and budding has pseudohypha and septate hypha sometimes.Physio-biochemical characteristics are glucose fermentation, can utilize the D-semi-lactosi, D-wood sugar, L-arabinose, sucrose, maltose, trehalose, methyl-a-D-glycopyranoside, melizitose, glycerine, the D-sorbyl alcohol, D-N.F,USP MANNITOL, D-glyconic acid-1, the 5-lactone, 2-ketone-D-glyconic acid, succsinic acid, citric acid or ethanol can utilize ethamine as sole carbon source, and L-Methionin or cadaverine are as only nitrogen source.Starch forms test: feminine gender, acetic acid produces test: feminine gender, hydrolysis of urea test: feminine gender, the blue B reaction of diazo: feminine gender.30 ℃ of optimum growth temperatures, the suitableeest initiation pH6.5.33 ℃ of the optimum temperutures of the full cell transformation almond of this bacterium alcohol, the suitableeest action pH 6.5.
Beneficial effect of the present invention: the present invention carries out asymmetric conversion racemize phenylglycol and obtains product (S)-phenylglycol with changing effect bacterial strain Candida parapsilosisCCTCC M203011 preferably, and optical purity is more than the 90%e.e..
After substratum composition and culture condition optimization, the asymmetric conversion racemize of bacterial strain uses therefor phenylglycol, the optical purity of product (S)-phenylglycol improves a lot, and has determined substratum composition and the culture condition optimized.
Obtain the gene of (S)-specificity carbonyl reductase of Candida parapsilosis CCTCC M203011, utilize the recombinant bacterial strain of this gene to carry out the racemize chiral separation, and for further studying the diversity of the microorganism stereoselectivity oxydo-reductase that is used for chiral separation, path for transformation and reaction mechanism create conditions.
The biological material specimens explanation
Candida parapsilosis, preservation date on March 1st, 2003, depositary institution: Chinese typical culture collection center C CTCC, strain number: M203011.This bacterial strain is open at Chinese patent CN 1212403 C.
Embodiment
The PCR product of (S)-specificity carbonyl reductase gene of Candida parapsilosis CCTCC M203011 is behind the test kit purifying, utilize BigDye sequencing kit and applying biological system automated DNA sequenator to carry out the order-checking of nucleotide sequence, its nucleotides sequence is classified SEQ ID NO:1 as, and this reductase enzyme amino acid consists of SEQ ID NO:2.
Sequence table
<210>SEQ?ID?NO：1
<211>840
<212>DNA
＜213〉Candida parapsilosis (Candida parapsilosis) CCTCC M203011
<214>
atgggcgaaa?tcgaatctta?ttgtaacaaa?gagttgggac?cattgccaac?aaaagctcca 60
actttgtcaa?agaacgtgct?tgacttgttt?tcccttaagg?gtaaagttgc?ttctgtgact 120
ggatcatctg?gtggtattgg?ttgggctgtt?gctgaagctt?acgctcaagc?tggtgcagat 180
gtagccattt?ggtacaactc?ccatccagct?gatgagaaag?ccgaacactt?gcaaaagaca 240
tatggggtcc?attcgaaagc?ttacaagtgt?aacattagtg?acccaaagag?cgttgaagaa 300
accatctctc?aacaagaaaa?agactttgga?accatcgacg?tgtttgtcgc?taatgctggt 360
gttacttgga?cacaaggacc?agagattgat?gttgacaact?acgattcatg?gaataagata 420
attagtgttg?atttgaatgg?cgtatactac?tgctcacaca?atatcggtaa?gatcttcaaa 480
aaaaacggca?aagggtcttt?gatcataaca?tcatcgatat?ccggcaagat?tgtcaatatc 540
cctcagcttc?aagctccata?taacacggct?aaagctgctt?gtacacattt?ggcaaaatcc 600
ttggccatcg?agtgggcacc?atttgctaga?gtgaacacca?tttcaccagg?ttatattgat 660
actgatatta?cagattttgc?aagcaaagat?atgaaagcta?agtggtggca?attgacacca 720
ttgggaaggg?aggggcttac?tcaagagcta?gttggtggat?atttgtactt?ggcatcgaat 780
gcgtctacat?tcacaactgg?ttctgatgtt?gttattgacg?gtggatacac?gtgtccatag 840
<210>SEQ?ID?NO：2
<211>279
<212>PRT
＜213〉Candida parapsilosis (Candida parapsilosis) CCTCC M203011
<214>
Met?Gly?Glu?Ile?Glu?Ser?Tyr?Cys?Asn?Lys?Glu?Leu?Gly?Pro?Leu
1 5 10 15
Pro?Thr?Lys?Ala?Pro?Thr?Leu?Ser?Lys?Asn?Val?Leu?Asp?Leu?Phe
20 25 30
Ser?Leu?Lys?Gly?Lys?Val?Ala?Ser?Val?Thr?Gly?Ser?Ser?Gly?Gly
35 40 45
Ile?Gly?Trp?Ala?Val?Ala?Glu?Ala?Tyr?Ala?Gln?Ala?Gly?Ala?Asp
50 55 60
Val?Ala?Ile?Trp?Tyr?Asn?Ser?His?Pro?Ala?Asp?Glu?Lys?Ala?Glu
65 70 75
His?Leu?Gln?Lys?Thr?Tyr?Gly?Val?His?Ser?Lys?Ala?Tyr?Lys?Cys
80 85 90
Asn?Ile?Ser?Asp?Pro?Lys?Ser?Val?Glu?Glu?Thr?Ile?Ser?Gln?Gln
95 100 105
Glu?Lys?Asp?Phe?Gly?Thr?Ile?Asp?Val?Phe?Val?Ala?Asn?Ala?Gly
110 115 120
Val?Thr?Trp?Thr?Gln?Gly?Pro?Glu?Ile?Asp?Val?Asp?Asn?Tyr?Asp
125 130 135
Ser?Trp?Asn?Lys?Ile?Ile?Ser?Val?Asp?Leu?Asn?Gly?Val?Tyr?Tyr
140 145 150
Cys?Ser?His?Asn?Ile?Gly?Lys?Ile?Phe?Lys?Lys?Asn?Gly?Lys?Gly
155 160 165
Ser?Leu?Ile?Ile?Thr?Ser?Ser?Ile?Ser?Gly?Lys?Ile?Val?Asn?Ile
170 175 180
Pro?Gln?Leu?Gln?Ala?Pro?Tyr?Asn?Thr?Ala?Lys?Ala?Ala?Cys?Thr
185 190 195
His?Leu?Ala?Lys?Ser?Leu?Ala?Ile?Glu?Trp?Ala?Pro?Phe?Ala?Arg
200 205 210
Val?Asn?Thr?Ile?Ser?Pro?Gly?Tyr?Ile?Asp?Thr?Asp?Ile?Thr?Asp
215 220 225
Phe?Ala?Ser?Lys?Asp?MET?Lys?Ala?Lys?Trp?Trp?Gln?Leu?Thr?Pro
230 235 240
Leu?Gly?Arg?Glu?Gly?Leu?Thr?Gln?Glu?Leu?Val?Gly?Gly?Tyr?Leu
245 250 255
Tyr?Leu?Ala?Ser?Asn?Ala?Ser?Thr?Phe?Thr?Thr?Gly?Ser?Asp?Val
260 265 270
Val?Ile?Asp?Gly?Gly?Tyr?Thr?Cys?Pro
275 279
Claims (2)
1. (S)-specificity carbonyl reductase of Candida parapsilosis (Candida parapsilosis) CCTCC M203011, its gene nucleotide series is SEQ ID NO:1.
2. (S)-specificity carbonyl reductase of Candida parapsilosis as claimed in claim 1 (Candida parapsilosis) CCTCCM203011, its amino acid consists of SEQ ID NO:2.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010151593A1 (en) * | 2009-06-23 | 2010-12-29 | Rutgers, The State University Of New Jersey | Sterospecific carbonyl reductases |
| CN101230363B (en) * | 2007-11-11 | 2012-04-18 | 江南大学 | Method for preparing (R)-styrene glycol by employing asymmetric conversion of recombinant strain |
| CN102492668A (en) * | 2011-11-29 | 2012-06-13 | 华东理工大学 | Carbonyl reductase and gene thereof as well as application of carbonyl reductase in asymmetrical reductive carbonyl compound |
| CN102559520A (en) * | 2011-12-15 | 2012-07-11 | 江南大学 | Method for preparing (S)-(4-chlorphenyl)-(pyridine-2-yl)-methanol by utilizing microbial catalysis |
| CN104152506A (en) * | 2014-08-08 | 2014-11-19 | 江南大学 | Method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system |
| CN104726354A (en) * | 2015-04-09 | 2015-06-24 | 江南大学 | Method of stereoselectively preparing (R)-phenylethanol with spore microcapsule enzyme of (S)-carbonyl reductase II/E228S |
| CN104774778A (en) * | 2015-04-28 | 2015-07-15 | 江南大学 | Method for using recombinant candida parapsilosis strain to efficiently prepare (S)-phenyl glycol |
| WO2016138641A1 (en) * | 2015-03-04 | 2016-09-09 | 华东理工大学 | Generation and use of candida and carbonyl reductase thereof |
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2006
- 2006-06-20 CN CN 200610088058 patent/CN1884502A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101230363B (en) * | 2007-11-11 | 2012-04-18 | 江南大学 | Method for preparing (R)-styrene glycol by employing asymmetric conversion of recombinant strain |
| US9422583B2 (en) | 2009-06-23 | 2016-08-23 | Rutgers, The State University Of New Jersey | Stereospecific carbonyl reductases |
| WO2010151593A1 (en) * | 2009-06-23 | 2010-12-29 | Rutgers, The State University Of New Jersey | Sterospecific carbonyl reductases |
| CN102492668A (en) * | 2011-11-29 | 2012-06-13 | 华东理工大学 | Carbonyl reductase and gene thereof as well as application of carbonyl reductase in asymmetrical reductive carbonyl compound |
| CN102492668B (en) * | 2011-11-29 | 2013-06-26 | 华东理工大学 | Carbonyl reductase and gene thereof as well as application of carbonyl reductase in asymmetrical reductive carbonyl compound |
| CN102559520A (en) * | 2011-12-15 | 2012-07-11 | 江南大学 | Method for preparing (S)-(4-chlorphenyl)-(pyridine-2-yl)-methanol by utilizing microbial catalysis |
| CN102559520B (en) * | 2011-12-15 | 2013-11-20 | 江南大学 | Method for preparing (S)-(4-chlorphenyl)-(pyridine-2-yl)-methanol by utilizing microbial catalysis |
| CN104152506A (en) * | 2014-08-08 | 2014-11-19 | 江南大学 | Method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system |
| CN104152506B (en) * | 2014-08-08 | 2016-10-19 | 江南大学 | The thick enzyme system of recombinant bacterium of aldehyde ketone reductase catalyzes and synthesizes the method for (S)-N, N-dimethyl-3-hydroxyl-3-(2-thiophene)-1-propylamine |
| WO2016138641A1 (en) * | 2015-03-04 | 2016-09-09 | 华东理工大学 | Generation and use of candida and carbonyl reductase thereof |
| CN106164260A (en) * | 2015-03-04 | 2016-11-23 | 华东理工大学 | Candida mycoderma and the generation of carbonyl reductase thereof and application |
| US10294479B2 (en) | 2015-03-04 | 2019-05-21 | East China University Of Science And Technology | Candida carbonyl reductase and method for preparing (R)-lipoic acid precursor |
| CN106164260B (en) * | 2015-03-04 | 2019-10-01 | 华东理工大学 | A kind of Candida carbonyl reductase and the method for being used to prepare (R) -6- hydroxyl -8- chloroctanoic acid ester |
| CN104726354A (en) * | 2015-04-09 | 2015-06-24 | 江南大学 | Method of stereoselectively preparing (R)-phenylethanol with spore microcapsule enzyme of (S)-carbonyl reductase II/E228S |
| CN104774778A (en) * | 2015-04-28 | 2015-07-15 | 江南大学 | Method for using recombinant candida parapsilosis strain to efficiently prepare (S)-phenyl glycol |
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Open date: 20061227 |