CN103122320A

CN103122320A - Tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate prepared by biological catalysis and strain

Info

Publication number: CN103122320A
Application number: CN2013100243583A
Authority: CN
Inventors: 郑裕国; 王亚军; 盛骏桢; 罗希; 沈寅初
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2013-01-22
Filing date: 2013-01-22
Publication date: 2013-05-29
Anticipated expiration: 2033-01-22
Also published as: CN103122320B

Abstract

The invention provides a microbe strain with high stereoselective carbonyl reduction enzyme activity-Pichia caribbic ZJB-09225 and application thereof in preparing tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate by biological asymmetric reduction of tert-butyl (R)-6-cyano-5-hydroxy-3-carbonylhexanoate. The strain is collected at China Center for Type Culture Collection (Address: Wuhan University, Wuhan City, PRC; Postal Code: 430072), the collection number CCTCC No:M2012411, and the collection date is October 18th, 2012. The invention provides a strain with favorable diastereoselective carbonyl reduction enzyme activity, and provides basis for producing tert-butyl (R)-6-cyano-5-hydroxy-3-carbonylhexanoate by a biological catalysis method.

Description

Biocatalysis prepares 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester and bacterial strain

(1) technical field

The present invention relates to a strain have instead-bacterial strain---Bick pichia spp in card (Pichia caribbic) ZJB-09225 of Prelog cis-selectivity carbonyl reduction enzymic activity, and prepare application in optical purity 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester at biological catalysis.

(2) background technology

Chiral drug is significant in the control of numerous disease, and its study on the synthesis is subject to the great attention of academia, enterprise and government.Chiral alcohol with specific function group is the synthetic crucial chiral building block of many chiral drugs especially, and uses having aspect the fine chemistry industries such as pharmacy, agricultural chemicals, spices very widely.Cardiovascular disorder becomes one of most threatening disease of the mankind, and its M ﹠ M all surpasses malignant neoplastic and leaps to the first.According to World Health Organization's statistics, the whole world has more than 3,000 ten thousand people to die from cardiovascular and cerebrovascular diseases every year.China is annual surpasses 3,000,000 because of cardiovascular and cerebrovascular diseases and complication death.The active treatment hyperlipidemia is the key of prevention cardiovascular and cerebrovascular diseases, and the blood lipid-lowering medicine exploitation has become emphasis and the focus of global drug research.Statins is present main blood lipid-lowering medicine, it is to the rate-limiting enzyme in the Biosynthesis of cholesterol process---and 3-hydroxy-3-methyl coenzyme A (HMG-CoA) reductase enzyme has remarkable restraining effect, reduce the cell free cholesterol levels, feedback raises the cell surface Expression of LDL Receptor, the removing of the residual grain of C-VLDL and low-density lipoprotein in the promotion blood circulation, the level of total cholesterol and low-density lipoprotein in final reduction serum, the effectively generation of atherosclerosis and coronary heart disease.

Global development and in existing more than ten kind of the statins that grinds.First-generation statins comprises Simvastatin, Pravastatin, lovastatin, is a similar fungal metabolite of class formation.His spit of fland of the s-generation comprises the racemize fluvastatin of chemosynthesis.His spit of fland of the third generation is the optical purity statins that synthesizes, as atorvastatin, Rosuvastatin and pitavastatin.Lipid-lowering statins accounts for the 85% above share in lipid lowerers market, the world, and still has room for promotion.

Chiral beta, δ-dihydroxyl caproic acid (ester) structure are pharmacophoric group, the chiral building blocks of synthetic statins, are also the Focal point and difficult points of synthetic statins.The final quality of statins depends on the optical purity of the corresponding isomer of chiral side chain, each traditional Chinese medicines prison department require the e.e. value greater than 99.5%, d.e. value greater than 99%.6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester is the synthetic chiral building block of statins, and synthesis technique comprises chemical asymmetric synthesis and biological asymmetric synthesis.6-cyano group-(3R, there are many defectives in 5R)-dihydroxyl hecanoic acid t-butyl ester chemical synthesis process, synthetic triethyl-boron and-85 ℃ of Cryogenic Conditions that need to use severe toxicity, and non-mapping is induced insufficient, product 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester d.e. value is low, and the boride refuse that this reaction forms is processed and needed through the loaded down with trivial details cancellation of methyl alcohol repeatedly, vacuum distilling etc.Compare with chemical process, biological synthesis process has the following advantages:

(1) solid, zone and chemo-selective are high, and this is mainly because of the strict specific recognition of enzyme to substrate, product; And when using chemical synthesis process, only have could obtain higher stereoselectivity when the group on carbonyl both sides is widely different; (2) security and Environmental compatibility are good, and biocatalysis safety and reaction conditions are gentle, usually with the aqueous solution as reaction system, environmental friendliness.

Have carbonyl reductase (alcoholdehydrogenase) living microorganism and extensively be present in occurring in nature, also meta-rule, product are configured as the S configuration but their asymmetric reduction carbonyl is often followed Prelog; Microorganism with anti--Prelog stereoselectivity carbonyl reduction enzymic activity is very rare at occurring in nature.up to now, asymmetric reduction (the R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester that has of bibliographical information prepares 6-cyano group-(3R, the microbial strains of 5R)-dihydroxyl hecanoic acid t-butyl ester activity has Saccharomyces cerevisiae, Pichia angusta NCYC 495, Pichia angusta NCYCR320, Pichia angusta NCYC R322, Pichia haplophila CBS2028, Beauveria bassiana, Pichia pastoris, Pichia membranefaciens, Pichia angusta, Candida humicola, Candida solani, Candida diddensiae, Candida friedrichii, Kluyveromyces drosophilarum.At present, have no the report that Pichia caribbic has (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester cis-selectivity carbonyl reduction enzymic activity.

(3) summary of the invention

The present invention seeks to overcome existing 6-cyano group-(3R, the deficiency of 5R)-dihydroxyl hecanoic acid t-butyl ester production technology, provide a kind of enrichment from soil, screening to have highly-solid selectively carbonyl reductase living microorganism bacterial classification---Bick Pichia yeast in card (Pichia caribbic) ZJB-09225, and the application in biological asymmetric reduction (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester prepares 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester.

The technical solution used in the present invention is:

One strain has instead-bacterial strain---Bick Pichia yeast in card (Pichia caribbic) ZJB-09225 of Prelog cis-selectivity carbonyl reduction enzymic activity, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number is CCTCC No:M2012411, preservation date is on October 18th, 2012.Through identifying, this bacterial strain belongs to the caribbic kind of Pichia (Pichia), and strain number is ZJB-09225.

The bacterial strain colonial morphology: bacterium colony is creamy white, neat in edge, and circle, opaque, smooth surface, protuberance.

Cellular form: cell is oval, and size is 1.5 μ m * 2.5 μ m approximately, and form is shown in Fig. 1.

Physiological and biochemical property: utilize Vitek2 microorganism automatic identifying system to measure bacterial strain ZJB-09225 to the qualification result of 46 kinds of qualification tests, analyze metabolic index through the Vitek2 readout instrument, determine that bacterial strain ZJB-09225 belongs to the caribbic kind of Pichia (Pichia), homology 98%(sees table 1 for details).

Table 1: utilize Vitek2 microorganism automatic identifying system to analyze bacterial classification ZJB-09225 carbon/nitrogenous source utilization

Result

Experiment	Result	Experiment	Result
				1B arylamine enzyme	-	The assimilation of D-sorbose	+
The L MALIC ACID assimilation	+	The assimilation of L-rhamnosyl	-
				Leucine arylamine enzyme	+	The Xylitol assimilation	+
Arginine GP	+	The D-glucitol assimilation	+
				The erythritol assimilation	+	The sucrose assimilation	+
The glycerol assimilation	-	Urease	-
				Tyrosine arylamine enzyme	-	Alpha-glucosidase	+
β-N-Acetyl-D-glucosamine enzyme	-	The assimilation of D-plumage brown sugar	+
				The myricinic acid assimilation	+	The assimilation of D-trehalose	+
The amygdaloside assimilation	-	Nitrate assimilation	-
				α-semi-lactosi assimilation	+	The L-arabinose assimilation	+
The gentiobiose assimilation	+	The assimilation of D-galacturonic	+
				The D-Glucose assimilation	+	The polychrom hydrolysis	-
The lactose assimilation	-	The assimilation of Pidolidone salt	+
				The methyl glucoside assimilation	+	The wood sugar assimilation	+
The assimilation of D-cellobiose	+	The assimilation of DL-LACTIC ACID salt	+
				Gamma glutamyltransferase	-	The acetate assimilation	+
The D-Maltose assimilation	+	The Citrate trianion assimilation	+
				The assimilation of D-raffinose	+	General uronic acid assimilation	+
PNP-N-acetyl-BD-semi-lactosi ammonia enzyme	-	The L-PROLINE assimilation	+
				The D-MANNOSE assimilation	+	2-ketone group-gluconate assimilation	+
The assimilation of D-melibiose	-	N-acetyl-glucosamine assimilation	+
				The assimilation of D-melizitose	+	The assimilation of D-Glucose hydrochlorate	+

18S rDNA sequential analysis: take the total DNA of bacterial strain ZJB-09225 cell that extracts as template, utilize primer: the 16SrDNA gene of pITS1:5'-TCCGTAGGTGAACCTGCGG-3' and pITS4:5'-TCCTCCGCTTATTGATATGC-3' amplification bacterial strain ZJB-09225, after will being cloned into the T carrier through the fragment of pcr amplification, the recombinant plasmid of the 18S rDNA fragment that contains acquisition after the extracting plasmid, confirm that through order-checking this fragment physical length is 606bp, specific as follows:

TCCGTAGGTG?AACCTGCGGA?AGGATCATTA?CAGTATTCTTTTGCCAGCGC?TTAACTGCGC?GGCGAAAAAC?CTTACACACAGTGTCTTTTT?GATACAGAAC?TCTTGCTTTG?GTTTGGCCTAGAGATAGGTT?GGGCCAGAGG?TTTAACAAAA?CACAATTTAATTATTTTTAT?TGATAGTCAA?ATTTTGAATT?AATCTTCAAAACTTTCAACA?ACGGATCTCT?TGGTTCTCGC?ATCGATGAAGAACGCAGCGA?AATGCGATAA?GTAATATGAA?TTGCAGATTTTCGTGAATCA?TCGAATCTTT?GAACGCACAT?TGCGCCCTCTGGTATTCCAG?AGGGCATGCC?TGTTTGAGCG?TCATTTCTCTCTCAAACCCC?CGGGTTTGGT?ATTGAGTGAT?ACTCTTAGTCGAACTAGGCG?TTTGCTTGAA?AAGTATTGGC?ATGGGTAGTACTGGATAGTG?CTGTCGACCT?CTCAATGTAT?TAGGTTTATCCAACTCGTTG?AATGGTGTGG?CGGGATATTT?CTGGTATTGTTGGCCCGGCC?TTACAACAAC?CAAACAAGTT?TGACCTCAAATCAGGTAGGA?ATACCCGCTG?AACTTAAGCA?TATCAATAAGCGGAGG。

The gene order and the GenBank data that obtain are carried out similarity analysis, bacterial strain ZJB-09225 and Pichiacaribbic(FN428931.1) homology reaches 100%(homology, 100%/606bps, based on18SrDNA).Therefore, determine that bacterial strain ZJB-09225 belongs to the caribbic kind that Pichia belongs to, Chinese is Bick pichia spp in card.

Comprehensive the above results, this bacterial strain belong to through molecular genetics to be identified, bacterial strain ZJB-09225 is defined as Bick Pichia yeast in card, and Latin is called Pichiacaribbic.

The application of described bacterial strain in biological asymmetric reduction (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester prepares 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester.

The reaction formula that this reaction relates to is as follows:

Preferably, described asymmetric reduction reaction carries out under pH7 ~ 9,32 ~ 37 ℃.

Concrete, described application can be as follows: in the damping fluid of pH7 ~ 9, add (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester as substrate, contain the enzyme somatic cells as catalyzer with Bick Pichia yeast (Pichia caribbic) ZJB-09225 in card, react 10 ~ 20h under 32 ~ 37 ℃, the reaction solution separation and purification obtains 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester; Initial substrate concentration is 10 ~ 50g/L damping fluid, and containing enzyme somatic cells addition is 10 ~ 50g DCW/L damping fluid.

Carry out for promoting reaction, also can add the cosubstrate of 10 ~ 50g/L in described damping fluid, described cosubstrate is one of following: glucose, methyl alcohol, ethanol, glycerine, formic acid, Virahol, propyl carbinol, gluconic acid sodium salt, fructose, sorbyl alcohol, seminose, N.F,USP MANNITOL are preferably glucose.

The described enzyme somatic cells that contains can obtain as follows: Bick Pichia yeast in blocking (Pichia caribbic) ZJB-09225 is seeded to the fermention medium of Bick Pichia yeast in suitable card, ℃ lower shaking culture is 2 ~ 3 days in pH7 ~ 9,28 ~ 37, fermented liquid is centrifugal, collect the washing of thalline physiological saline, namely get the described enzyme somatic cells that contains.

Described fermention medium forms can be as follows: Fructus Hordei Germinatus soaks powder 10 ~ 50g/L, glucose 10 ~ 50g/L, CuSO ₄1 ~ 5mg/L, KH ₂PO ₄1 ~ 5g/L, K ₂HPO ₄3H ₂O1 ~ 5g/L, NaCl0.5 ~ 2g/L, solvent are water, pH7 ~ 9.

Beneficial effect of the present invention is mainly reflected in: the invention provides the bacterial strain that a strain has better cis-selectivity carbonyl reduction enzymic activity, produce 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester for biological catalysis and provide the foundation.

(4) description of drawings

Fig. 1 is the cellular form photo of Bick Pichia yeast in card;

Fig. 2 is the impact that the initial pH of fermention medium lives on the bacterial strain enzyme;

Fig. 3 is the impact that the fermentation culture temperature is lived on the bacterial strain enzyme;

Fig. 4 is the impact of the right title reduction reaction of invert point;

Fig. 5 is the impact of the right title reduction reaction of pH value in reaction;

Fig. 6 is the impact of right title reduction reaction of reaction times.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1: screening has catalysis (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester reducing activity microorganism strains

after disperseing according to a certain percentage with physiological saline, soil sample in all parts of the country is seeded to being collected in the enrichment medium (preparation: 50.0g glucose that contains 0.40g/L (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 100.0g soybean sprout boiled 30 minutes, add 0.40g (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, add water and complement to 1.0L, do not regulate the pH value) in, at 30 ℃, under the 150rpm condition, the shaking table shaking culture is until after nutrient solution change muddiness, according to 2.0%(v/v) inoculum size is transferred in the enrichment medium that contains 0.80g/L (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, continuation is at 30 ℃, cultivate under the 150rpm condition.Improve successively (R)-6-cyano group in enrichment medium-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester concentration, until reach 2.0g/L.

With last enrichment culture liquid stepwise dilution, be applied on plate culture medium 30 ℃ and be cultured to and form observable single bacterium colony.Picking list bacterium colony is forwarded to aseptic inclined-plane, and (preparation: 50.0g glucose, 100.0g soybean sprout boiled 30 minutes, and agar 20.0g adds water and complements to 1.0L, does not regulate Medium's PH Value; Sterilized 30 minutes for 115 ℃, cooling bevel), to cultivate 2 days for 30 ℃, the inclined-plane is placed in 4 ℃ of refrigerator preservations.

The bacterial strain that is deposited in the test tube slant is inoculated into fermention medium (preparation: glucose 15.0g/L, peptone 5.0g/L, yeast extract 5.0g/L, KH one by one ₂PO ₄0.50g/L, K ₂HPO ₄0.50g/L, MgSO ₄0.50g/L, NaCl1.00g/L, pH8.0,115 ℃ of sterilizations 30 minutes) in, cultivated 48 hours under 30 ℃, 150rpm condition.Pipette respectively the 20.0mL culture, centrifugal, collect thalline, and wash thalline 3 times with 50.0mM potassium phosphate buffer (pH7.5).Thalline is scattered in the above-mentioned damping fluid of 10.0mL, (the R)-6-cyano group that adds-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, make its final concentration reach 5.0 ~ 20.0g/L, 35 ℃ transform 2 hours, and conversion fluid adopts liquid-phase chromatographic analysis carbonyl reduction enzymic activity after centrifugal, micro-filtration.In bacterium, Pichia caribbic ZJB-09225, i.e. CCTCC No:M2012411, vigor is the strongest, and non-enantiomer selectivity is better.

The carbonyl reduction enzyme activity is defined as: 35 ℃, under the pH7.5 condition, per minute catalysis generates the 1 required enzyme amount of μ mol6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester and is defined as 1 enzyme unit that lives.

Embodiment 2: the impact that the fermention medium initial pH value is lived on P.caribbic ZJB-09225 carbonyl reductase volume enzyme

The P.caribbic ZJB-09225 bacterial classification picking one transfering loop thalline that will be kept on the test tube slant is inoculated into without in bacteria fermentation culture medium, and the component of fermention medium formula is as follows: Fructus Hordei Germinatus soaks powder 30.0g/L, glucose 20.0g/L, CuSO ₄3.0mg/L, KH ₂PO ₄1.36g/L, K ₂HPO ₄3H ₂O2.28g/L, NaCl1.0g/L, solvent are water, 28 ℃ of temperature, the fermention medium initial pH value is respectively 5.0,6.0,7.0,8.0,9.0, cultivates 2 days under the 150rpm condition, collects fermented liquid.

With centrifugal 8 minutes of fermented liquid 12000rpm, abandon supernatant, collect thalline, take the 1g wet thallus with after physiological saline washing 3 times, then use the phosphoric acid buffer (50mM) of 10.0mL, pH7.0 that washed cell is dispersed in to transform in bottle, add 0.14g (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 0.20g glucose, be placed in 30 ℃ of shaking baths, reacted 24 hours, get the 1.0mL conversion fluid, centrifugal 6 minutes of 12000rpm, supernatant liquor adopts 0.45 μ m micro-filtrate membrane filtration.The filtrate of the clarification that obtains is carried out liquid-phase chromatographic analysis 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester concentration, calculates the carbonyl reduction enzyme activity under each condition, the results are shown in Figure 2.Result shows, under pH7.0, bacterial strain carbonyl reduction enzymic activity is the strongest, and biomass is 28.80g/L, and the work of volume enzyme reaches 5.90U/L, d.e. value＞95.0%.

Embodiment 3: the impact that culture temperature is lived on P.caribbic ZJB-09225 carbonyl reductase volume enzyme

The P.caribbic ZJB-09225 bacterial classification picking one transfering loop thalline that will be kept on the test tube slant is inoculated into without in bacteria fermentation culture medium, and fermention medium component formula is as follows: Fructus Hordei Germinatus soaks powder 30.0g/L, glucose 20.0g/L, CuSO ₄3mg/L, KH ₂PO ₄1.36g/L, K ₂HPO ₄3H ₂O2.28g/L, NaCl1.0g/L, solvent are water, pH7.0 is respectively 23 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃ in temperature, cultivates 2 days under the 150rpm condition, collects fermented liquid.

With centrifugal 8 minutes of fermented liquid 12000rpm, abandon supernatant, collect thalline, take the 1g wet thallus with after physiological saline washing 3 times, then use 10.0mL, pH7.0 phosphoric acid buffer (50mM) that washed cell is dispersed in to transform in bottle, add 0.14g (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester and 0.20g glucose, be placed in 30 ℃ of shaking baths, reacted 24 hours, get the 1.0mL conversion fluid, centrifugal 6 minutes of 12000rpm, supernatant liquor adopts 0.45 μ m micro-filtrate membrane filtration.The filtrate of the clarification that obtains is carried out liquid-phase chromatographic analysis 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester concentration, calculates the carbonyl reduction enzyme activity under each condition, the results are shown in Figure 3.Result shows, under 30 ℃ of conditions, bacterial strain carbonyl reduction enzymic activity is the strongest, and biomass reaches 29.90g/L, and the work of volume enzyme reaches 6.62U/L, d.e. value＞95.0%.

Embodiment 4: the preparation of biological catalyst P.caribbic ZJB-09225 cell

The novel carbonyl reductase that obtains from seed selection of the present invention produces picking one ring thalline on the test tube slant of bacterial classification P.caribbic ZJB-09225, be seeded to (the preparation: 50.0g glucose of 30.0mL aseptic seed substratum, soybean sprout 100.0g boiled 30 minutes, filter, water complements to 1.0L, the medium pH nature.121 ℃ of substratum sterilization 20 minutes) in, under 30 ℃, 150rpm condition, the shaking culture base is 24 hours, obtains seed liquor.(preparation: Fructus Hordei Germinatus soaks powder 30.0g/L, glucose 20.0g/L, CuSO 3.0mL seed liquor is seeded to the aseptic fermentation culture of 100.0mL ₄3mg/L, KH ₂PO ₄1.36g/L, K ₂HPO ₄3H ₂O2.28g/L, NaCl1.0g/L, solvent are water, pH7.0), and 30 ℃, cultivated 2 days under the 150rpm condition, collect fermented liquid.After centrifugal 8 minutes of fermented liquid 12000rpm, abandon supernatant and collect thalline, thalline washs 3 times with 0.85% physiological saline, collect and obtain 3.60gP.caribbic ZJB-09225 thalline, this thalline has instead-Prelog cis-selectivity carbonyl reduction enzymic activity (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester.

Embodiment 5: the impact of invert point on Catalytic processes

Select the biological catalyst P.caribbic ZJB-09225 that the present invention prepares to contain the enzyme somatic cells.Transformation system is selected 10.0mL, pH7.0 phosphoric acid buffer (50.0mM), the P.caribbicZJB-09225 final concentration of cells is 20.0g DCW/L, 14g/L (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 20g/L glucose, transform 3 hours respectively under 20 ℃, 23 ℃, 25 ℃, 27 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, conversion fluid adopts the carbonyl reduction enzymic activity of each bacterial classification of liquid-phase chromatographic analysis after centrifugal, micro-filtration, analyze different invert points to the impact of catalytic process enzyme activity, the results are shown in Figure 4.The result demonstration, in the time of 35 ℃, P.caribbic ZJB-09225 catalytic efficiency is the highest, and yield reaches 7.71%, reaches 0.89U/g than enzyme work, d.e. value 95.5%.

Embodiment 6: transform pH to the impact of Catalytic processes

The biological catalyst P.caribbic ZJB-09225 somatic cells of selecting the present invention to prepare.Transformation system selects 10.0mL, pH value to be respectively 3,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0 potassium phosphate buffers (50.0mM), P.caribbic ZJB-09225 final concentration of cells is 20.0g DCW/L, cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester to final concentration is 14.0g/L to add (R)-6-, and 35 ℃ transform 3 hours.Through Liquid Detection 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester concentration and d.e. value, the results are shown in Figure 5.The result demonstration, under the pH7.5 condition, P.caribbic ZJB-09225 catalytic efficiency is the highest, and yield reaches 9.68%, reaches 1.41U/g than enzyme work, d.e. value 96.3%.

Embodiment 7: the impact of cosubstrate on Catalytic processes

The biological catalyst P.caribbic ZJB-09225 somatic cells of selecting the present invention to prepare.transformation system is selected 10.0mL, pH7.0 phosphoric acid buffer (50mM), the ZJB-09225 final concentration of cells is 20.0g DCW/L, (R)-6-cyano group of 14g/L-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, add respectively 20g/L glucose, methyl alcohol, ethanol, glycerine, formic acid, Virahol, propyl carbinol, gluconic acid sodium salt, fructose, sorbyl alcohol, seminose, N.F,USP MANNITOL is as cosubstrate, transform 24h under 30 ℃, conversion fluid is through centrifugal, adopt the carbonyl reduction enzymic activity of each strain bacterial classification of liquid-phase chromatographic analysis after micro-filtration, analyze different cosubstrates to the impact of catalytic process enzyme activity.

The result demonstration, 20g/L gluconic acid sodium salt enzyme is lived the highest, reaches 1.62U/g than enzyme work, and catalysis 24h generates 11.85g/L product, yield 84.6%, d.e. value 96.0%; Other cosubstrate exists as follows on the impact of (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester asymmetric reduction efficient: during without cosubstrate, the ZJB-09225 carbonyl reductase is than enzyme 0.91U/g alive, d.e. value 97.6%; During 20g/L glucose, the ZJB-09225 carbonyl reductase is than enzyme 0.71U/g alive, d.e. value 96.4%; During 20g/L formic acid, the ZJB-09225 carbonyl reductase is than enzyme 0.29U/g alive, d.e. value 90.6%; During 20g/L ethanol, the ZJB-09225 carbonyl reductase is than enzyme 0.53U/g alive, d.e. value 87.5%; During 20g/L methyl alcohol, ZJB-09225 carbonyl reductase volume enzyme 0.65U/g alive, d.e. value 88.9%; During 20g/L glycerine, the ZJB-09225 carbonyl reductase is than enzyme 0.42U/g alive, d.e. value 67.8%; During the 20g/L Virahol, the ZJB-09225 carbonyl reductase is than enzyme 0.68U/g alive, d.e. value 87.4%; During 20g/L fructose, the ZJB-09225 carbonyl reductase is than enzyme 0.70U/g alive, td.e. value 95.8%; During the 20g/L propyl carbinol, the ZJB-09225 carbonyl reductase is than enzyme 0.30U/g alive, d.e. value 84.9%; During the 20g/L seminose, the ZJB-09225 carbonyl reductase is than enzyme 0.54U/g alive, d.e. value 96.8%; During 20g/L N.F,USP MANNITOL, the ZJB-09225 carbonyl reductase is than enzyme 0.35U/g alive, d.e. value 87.8%; During the 20g/L sorbyl alcohol, the ZJB-09225 carbonyl reductase is than enzyme 0.98U/g alive, d.e. value 77.2%.

Embodiment 8:(R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester asymmetric reduction technique

The synthetic biological catalyst P.caribbic ZJB-09225 somatic cells of selecting the present invention to prepare of 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester.Transformation system is selected 100.0mL, pH7.0 phosphoric acid buffer (50.0mM), P.caribbic ZJB-09225 final concentration of cells is 20.0g DCW/L, 50.0g/L (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester, 50.0g/L Sunmorl N 60S, 35 ℃ of lower 300rpm magnetic agitation transform 1 hour.Detect substrate yield 1.47%, 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester d.e. value through HPLC〉99.9%, the ratio enzyme of P.caribbic ZJB-09225 is lived and is 2.84U/g; After 35 ℃ of lower 300rpm magnetic agitation transform 12 hours, detect through HPLC, efficiency of pcr product 19.4%, 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester d.e. value 93.8%(the results are shown in Figure 6).

Claims

1. a strain has instead-bacterial strain---Bick Pichia yeast (Pichiacaribbic) ZJB-09225 in card of Prelog cis-selectivity carbonyl reduction enzymic activity, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number is CCTCC No:M2012411, preservation date is on October 18th, 2012.

2 The strain according to claim 1, characterized in that the gene sequence of 16S rDNA of the strain are as follows：TCCGTAGGTG AACCTGCGGA AGGATCATTA CAGTATTCTTTTGCCAGCGC TTAACTGCGC GGCGAAAAAC CTTACACACAGTGTCTTTTT GATACAGAAC TCTTGCTTTG GTTTGGCCTAGAGATAGGTT GGGCCAGAGG TTTAACAAAA CACAATTTAATTATTTTTAT TGATAGTCAA ATTTTGAATT AATCTTCAAAACTTTCAACA ACGGATCTCT TGGTTCTCGC ATCGATGAAGAACGCAGCGA AATGCGATAA GTAATATGAA TTGCAGATTTTCGTGAATCA TCGAATCTTT GAACGCACAT TGCGCCCTCTGGTATTCCAG AGGGCATGCC TGTTTGAGCG TCATTTCTCTCTCAAACCCC CGGGTTTGGT ATTGAGTGAT ACTCTTAGTCGAACTAGGCG TTTGCTTGAA AAGTATTGGC ATGGGTAGTACTGGATAGTG CTGTCGACCT CTCAATGTAT TAGGTTTATCCAACTCGTTG AATGGTGTGG CGGGATATTT CTGGTATTGTTGGCCCGGCC TTACAACAAC CAAACAAGTT TGACCTCAAATCAGGTAGGA ATACCCGCTG AACTTAAGCA TATCAATAAGCGGAGG。

3. the application of bacterial strain as claimed in claim 1 in biological asymmetric reduction (R)-6-cyano group-5-hydroxyl-3-carbonyl hecanoic acid t-butyl ester prepares 6-cyano group-(3R, 5R)-dihydroxyl hecanoic acid t-butyl ester.

4. application as claimed in claim 3 is characterized in that described asymmetric reduction carries out under pH7 ~ 8,32 ~ 37 ℃.