CN111925949B

CN111925949B - Curvularia lunata B-36 and application thereof in synthesis of chiral alcohol

Info

Publication number: CN111925949B
Application number: CN202010834132.XA
Authority: CN
Inventors: 李军; 杜文婷; 施菁; 樊淼樑
Original assignee: Hangzhou Medical College
Current assignee: Hangzhou Medical College
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-03-04
Anticipated expiration: 2040-08-18
Also published as: CN111925949A

Abstract

The invention discloses Curvularia lunata (Curvularia hominis) B-36 and application thereof in synthesis of chiral alcohol. The strain B-36 of the curvularia lunata is preserved in the China center for type culture Collection in 2017, 11 and 03, and the preservation number is CCTCC NO: m2017654. The invention discovers for the first time that curvularia lunata B-36 has the capability of catalyzing and reducing 1-chloro-2-heptanone, can biologically convert 1-chloro-2-heptanone into (S) -1-chloro-2-heptanol, and has the advantages of simple reaction and high product yield.

Description

Curvularia lunata B-36 and application thereof in synthesis of chiral alcohol

Technical Field

The invention relates to the technical field of microorganisms and biocatalysis, in particular to Curvularia lunata (Curvularia hominis) B-36 and application thereof in synthesizing chiral alcohol.

Background

Remodelin (treprostinil) is a prostacyclin-based targeted drug approved for marketing at 5 months CFDA in 2014 for the treatment of pulmonary hypertension. Pulmonary hypertension is a rare disease with low survival rate, called "cancer in cardiovascular disease". Prior to targeted drug therapy, the 3-year survival rate of chinese pulmonary hypertension patients was only 39%. The (S) -1-chloro-2-heptanol is an important chiral intermediate for synthesizing the Remoduline, and the preparation technology is the key for efficiently synthesizing the Remoduline.

At present, the chiral drug intermediate is mainly synthesized by a chemical method and a biological method. When chiral drugs are synthesized by a chemical method, the stereoselectivity is low, the reaction steps are multiple, the product yield is low, a heavy metal catalyst needs to be added, the environment is polluted, and the control standard of the drugs on heavy metals is difficult to achieve. Biological rules utilize enzymes or whole cells of microorganisms to catalyze reactions.

The biological method has the characteristics of good specificity and low pollution. The microbial whole cell not only contains oxidoreductase, but also contains coenzyme, and the coenzyme in-situ regeneration is realized by adding a hydrogen donor. Thus, whole-cell catalysis by microorganisms is a common technique for asymmetric reduction of chiral compounds.

Although some microorganisms have been used for biotransformation, the number of strains that can be successfully and effectively used for industrial production is very limited. Therefore, the screening and development of microorganisms capable of catalyzing the preparation of the chiral intermediate (S) -1-chloro-2-heptanol of Remadulin are of great significance.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide Curvularia lunata (Curvularia hominis) B-36, and the Curvularia lunata B-36 can be used for preparing optically pure (S) -1-chloro-2-heptanol by asymmetrically reducing 1-chloro-2-heptanone.

The technical scheme adopted by the invention is as follows:

curvularia lunata (Curvularia hominis) B-36, which is preserved in China center for type culture Collection; the preservation number is: CCTCC NO: m2017654; the preservation time is as follows: 11/03/2017.

The biological characteristics of the strain are as follows:

colony morphology: the colony on the PDA plate is compact and round, spreads to the periphery, and the hypha is white at the initial stage, then turns into brown green, and finally turns into black green. Cell morphology: the hyphae have septa and branches, and have a diameter of about 2.8-3.5 μm. Conidia are crescent-shaped, and the size of the conidia is 20-30 x 10-14 mu m.

The application of the Curvularia lunata B-36 in the synthesis of chiral alcohol.

In one embodiment, the curvularia lunata B-36 is applied to the synthesis of chiral alcohol, wet somatic cells of the curvularia lunata B-36 after fermentation are used as a catalyst, 1-chloro-2-heptanone is used as a substrate, a biocatalytic reaction is carried out in a buffer solution, and after the reaction is finished, a reaction solution is separated and purified to obtain (S) -1-chloro-2-heptanol;

the initial concentration of the substrate is 10-1000 mM, preferably 30-70 mM.

In one embodiment of the invention, the initial concentration of the substrate may include, but is not limited to: 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 100mM, 200mM, 300mM, 500mM, 600mM, 800mM or 1000 mM.

In an embodiment of the present invention, the biocatalytic preparation of (S) -1-chloro-2-heptanol using the Curvularia lunata B-36 is represented as follows:

in one embodiment of the present invention, in the biocatalytic reaction, the amount of the wet somatic cells is 20 to 1000g/L buffer solution, preferably 30 to 50g/L buffer solution, on a dry weight basis, including but not limited to: 30g/L, 40g/L or 50g/L buffer.

In one embodiment of the invention, the biocatalytic reaction is carried out at 25-40 ℃ for 24-60 hours in a buffer solution with pH of 5.0-7.0.

In one embodiment of the invention, the biocatalytic reaction is carried out in a phosphate buffer or a citrate buffer, including but not limited to K₂HPO₄-KH₂PO₄、Na₂HPO₄-citric acid or Na₂HPO₄-KH₂PO₄. Preferably, the biocatalytic reaction is at K₂HPO₄-KH₂PO₄In a buffer.

Coenzyme regeneration efficiency is the rate-limiting step of the biological catalytic oxidation-reduction reaction, and adding an auxiliary substrate is an effective method for improving the whole-cell catalytic efficiency. In another embodiment of the invention, a co-substrate is also included in the biocatalytic reaction.

In an embodiment of the invention, the co-substrate is a saccharide and/or an alcohol; preferably, the co-substrate is selected from one or more of the following: glucose, fructose, sucrose, maltose, methanol, ethanol, isopropanol or glycerol; when the co-substrate is a saccharide, the co-substrate is preferably glucose; when the substrate is an alcohol, the co-substrate is preferably isopropanol; when the co-substrate is a saccharide or an alcohol, the substrate is preferably isopropanol or glucose.

In an embodiment of the invention, when the cosubstrate is a saccharide, the addition amount of the cosubstrate is 10 to 200g/L buffer solution by mass, preferably 50 to 100g/L buffer solution; including but not limited to: 50g/L, 60g/L, 70g/L, 80g/L, 90g/L or 100g/L buffer, most preferably 50g/L buffer. When the auxiliary substrate is alcohol, the addition amount of the auxiliary substrate is 5-30% of the volume of the biocatalytic reaction buffer solution by volume, preferably 10-20% of the volume of the biocatalytic reaction buffer solution by volume, and most preferably 10% of the biocatalytic reaction buffer solution by volume.

The optical purity and yield of the obtained product are highest when 50g/L glucose is used as an auxiliary substrate in the biocatalytic reaction.

In one embodiment of the invention, the screening method of the curvularia lunata B-36 comprises the following steps:

collecting a soil sample from a poppy area in Shaoxing city in Shaoxing province of Zhejiang, adding the collected soil sample into a 250mL conical flask filled with 70mL of enrichment medium, carrying out shaking culture at 30 ℃ and 220rpm for 6-7 days, taking 1mL of culture solution after the culture solution becomes turbid, transferring the culture solution into a fresh enrichment medium, continuing culturing for 6-7 days, and repeating the enrichment culture for 3-4 times. 1-chloro-2-heptanone is used as a unique carbon source in the enrichment medium. The enriched culture solution is diluted in a gradient way, coated on a separation flat plate, and separated and cultured for multiple times to obtain a single colony. And selecting a single colony, inoculating the single colony to a seed culture medium, culturing for 24 hours, and transferring to an enzyme production culture medium for culturing for 48 hours. 1-chloro-2-heptanone is used as a substrate, whole cells of the screened microorganism are used as a catalyst, after biotransformation is carried out for 24 hours in a phosphate buffer solution, an enantiomeric excess value (ee value) of a target product (S) -1-chloro-2-heptanol in a transformation solution is detected by adopting a chiral gas chromatography, and the new microorganism strain Curvularia lunata B-36(Curvularia hominis B-36) is screened from the enantiomeric excess value.

In one embodiment of the present invention, the method for preparing curvularia lunata B-36 wet somatic cells comprises:

inoculating Curvularia hominis B-36 into fermentation medium, and shake culturing at 30 deg.C and 200rpmCulturing for 24h, centrifuging the fermentation liquor, washing and precipitating, and collecting to obtain wet thallus cells; the final concentration of the fermentation medium is as follows: 20-30 g/L glucose, 25-35 g/L peptone and K₂HPO₄ 2.0～4.0g/L，(NH₄)₂SO₄ 4.0～6.0g/L，NaCl 1.0～2.0g/L，pH 5.5～7.0。

In one embodiment of the present invention, wet bacterial cells obtained by the above method for producing wet bacterial cells are added to a phosphate buffer solution having a pH of 6.0 at a concentration of 40g/L on a dry basis, and a substrate of 1-chloro-2-heptanone (50 mM) and glucose (10% (v/v) as an auxiliary substrate for coenzyme regeneration is added thereto at 30 ℃ and 200rpm for 24 hours. And after the reaction is finished, centrifuging the reaction liquid, taking supernatant, adding isovolumetric ethyl acetate for extraction for three times, taking ethyl acetate layer liquid after extraction, distilling and concentrating by a rotary evaporator for 5 times of volume, then adding silica gel, uniformly mixing, transferring to a chromatographic column, carrying out silica gel column chromatography separation by using petroleum ether/ethyl acetate (7: 3 (v/v)) as an eluent, collecting eluent containing a product, concentrating and evaporating by a rotary evaporator to dryness to obtain a pure product of (S) -1-chloro-2-heptanol, and detecting the content of the (S) -1-chloro-2-heptanol in a gas phase.

The invention has the following beneficial effects:

the novel microbial strain Curvularia hominis B-36 provided by the invention can be used for preparing (S) -1-chloro-2-heptanol by biological asymmetric reduction of 1-chloro-2-heptanone, and the optically pure (S) -1-chloro-2-heptanol prepared by catalysis of the novel strain has the advantages of high stereoselectivity, high yield and the like.

By adopting chiral biological catalysis of Curvularia hominis B-36 cells, when the concentration of a substrate is 50mM, the ee value of the product (S) -1-chloro-2-heptanol reaches 99.9%, and the yield reaches over 90.0%. The new microbial strain obtained by screening from the soil sample can be used for preparing chiral alcohol by asymmetric reduction, so that reference is provided for researching the key chiral intermediate (S) -1-chloro-2-heptanol of the drug for treating pulmonary hypertension by biosynthesis.

The strain is obtained by separating and screening soil samples collected from the corn poppy area of Shaoxing city in Zhejiang province.

ITS sequence characteristics of the strain: using the extracted total DNA of the cells as a template, the ITS region of the strain was amplified using the universal primers ITS1 and ITS4, and the PCR product was subjected to 1% agarose gel electrophoresis. The fungal ribosome ITS (internal Transcribed spacer) gene sequence of the colony of the Curvularia lunata B-36 is confirmed to be shown in SEQ ID NO.1 by sequencing.

This sequence was submitted to GenBank (GenBank accession No. MH656705), and the ITS sequence of strain B-36 was compared for homology (BLAST) on the NCBI website (http:// www.ncbi.nlm.nih.gov), indicating that: the B-36 strain has 100% sequence homology with Curvularia lunata UTHSC 08-3737(Curvularia hominis UTHSC 08-3737) (GenBank accession No. HG779010.1). The strain was identified as Curvularia lunata (Curvularia hominis) based on colony and cell morphology combined with molecular biology identification.

The strain Curvularia lunata (Curvularia hominis) B-36 is preserved in the China center for type culture Collection; the preservation number is: CCTCC NO: m2017654; the preservation time is as follows: 11/03/2017. The strain was detected as a viable strain by the depository at 11/20 in 2017 and deposited.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a gas phase detection spectrum of a standard substance of 1-chloro-2-heptanone as a substrate and 1-chloro-2-heptanol as a product, provided in an example of the present invention;

FIG. 2 is a gas chromatogram of an extract from a bioreduction reaction of Curvularia hominis B-36.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Optimized culture medium of Curvularia hominis B-36 strain

Through the design of a response surface experiment, the composition of the obtained optimized culture medium of the Curvularia hominis B-36 strain is as follows: 15-30 g/L glucose, 20-35 g/L peptone and K₂HPO₄ 1.0～4.0g/L，(NH₄)₂SO₄2.5-6.0 g/L NaCl 1.0-2.5 g/L. The culture conditions of Curvularia hominis B-36 are as follows: the initial pH is 4.0-7.0, the liquid amount of the conical flask is 100-200 mL/500mL, the culture temperature is 20-40 ℃, the rotation speed of a shaking table is 180-240 rpm, the inoculation amount is 1-10%, and the culture time is 12-36 h.

Screening of Curvularia hominis B-36 species:

(1) enrichment culture: soil samples collected from different sampling points in the corn poppy area of Shaoxing city are inoculated into an enrichment medium, the culture medium is placed on a shaking table at 30 ℃ and 200rpm for 7 days, 1mL of culture solution is taken to be transferred into a fresh enrichment medium after the culture solution becomes turbid, the culture is continued for 7 days, and the circulation culture is carried out for 3 times. The enrichment medium formula is as follows: 30mM of 1-chloro-2-heptanone, (NH)₄)₂SO₄5g/L，K₂HPO₄ 4g/L，NaCl 2.5g/L，pH 6.0。

(2) Plate culture: and diluting the final enrichment culture solution, and then coating the diluted final enrichment culture solution on a plate culture medium, wherein the plate culture medium consists of the enrichment culture medium and 20g/L agar.

(3) Slant culture: slant culture medium (PDA medium) formulation: 200g/L of potato, 20g/L of glucose and 20g/L of agar. Culturing the inclined plane at 30 ℃ for 2-3 d, picking single bacterial colony in the plate culture medium to the inclined plane, and storing in a refrigerator at 4 ℃.

(4) Seed culture: a ring of thallus is picked from a mature culture slant and inoculated into a 500mL shake flask containing 200mL seed culture medium, and cultured for 24h at 30 ℃ and 200 rpm. The seed culture medium comprises the following components: glucose 25g/L, peptone 27g/L, K₂HPO₄3g/L，(NH₄)₂SO₄ 4.5g/L，NaCl 2g/L，pH 6.0。

(5) Fermentation culture: the fermentation medium has the same formula as seed culture medium. And (3) transferring the seed solution into a 500mL shake flask filled with 200mL fermentation medium by an inoculation amount of 5-10%, and culturing at 30 ℃ and 200rpm for 24 h.

Identification of Curvularia hominis B-36 species:

Preparation of wet somatic cells

The culture medium formula of the seed liquid and the fermentation liquid is as follows: glucose 25g/L, peptone 27g/L, K₂HPO₄ 3g/L，(NH₄)₂SO₄4.5g/L，NaCl 2g/L，pH 7.0。

Inoculating mature slant strain into 500mL conical flask containing 200mL seed culture medium, performing shake culture at 30 deg.C and 200rpm for 24h to obtain seed solution, transferring the seed solution into 500mL conical flask containing 200mL fermentation culture medium at 5% (v/v) inoculum size, at 30 deg.C and 200rpm for 24 h. And after the culture is finished, centrifuging the fermentation liquor, washing the fermentation liquor by using a phosphate buffer solution, and collecting wet thalli for later use.

Example 2

Biological catalytic reaction: preparation of (S) -1-chloro-2-heptanol by catalytic reduction of 1-chloro-2-heptanone

The wet bacterial cells obtained in example 1 were suspended in 20mL phosphate buffer solution K₂HPO₄-KH₂PO₄(pH 6.0) wherein the wet cell density is 40g/L on a dry basis; 50mM 1-chloro-2-heptanone was added as a substrate, and 50g/L glucose was added as an auxiliary substrate for coenzyme regeneration, and the reaction was carried out at 30 ℃ and 200rpm for 24 hours. And (3) after the reaction is finished, centrifuging the reaction solution, taking supernate, adding equal volume of ethyl acetate for extraction, and detecting the content of (S) -1-chloro-2-heptanol in a gas phase.

The method for detecting the concentration of the product and separating and purifying the product comprises the following steps:

(1) and (3) concentration detection: after the biocatalytic reaction is finished, the reaction solution is extracted by using ethyl acetate with the same volume. Unreacted substrate and product in the extract can be analyzed by gas chromatography (see FIG. 2), and FIG. 1 shows gas detection patterns of the substrate 1-chloro-2-heptanone and the product 1-chloro-2-heptanol as standard substances. The unreacted substrate and the product in the extract were quantified by internal standard method. The internal standard was n-dodecane. 1ml of the extract was added to 2. mu.l of dodecane and mixed well before gas phase analysis. Gas chromatography conditions: agilent gas chromatograph (Agilent GC-7890A); warian CP-chiralsil-Dex CB chiral capillary gas chromatography column (25m × 0.25mm × 0.25 μm, df ═ 0.25) in usa. The carrier gas is high-purity nitrogen, and the flow rate is 2 mL/min; the sample injection amount is 1 mu L, and the split ratio is 20: 1; the temperature of the detector and the injection port is 250 ℃; keeping the column temperature at 80 deg.C for 1min, heating to 150 deg.C at 8 deg.C/min, and maintaining for 2 min; the detector is FID. And (4) calculating the concentration and ee value of the product in the reaction solution by using a relative correction factor method according to a gas chromatography detection spectrogram.

(2) Separation and purification: and after extraction, distilling and concentrating an ethyl acetate layer by a rotary evaporator for 5 times of volume, adding silica gel, uniformly mixing, transferring to a chromatographic column, performing silica gel column chromatography separation by using petroleum ether-ethyl acetate-7: 3(v/v) as an eluent, collecting eluent containing a product, and concentrating by the rotary evaporator to obtain a pure (S) -1-chloro-2-heptanol product.

The yield calculation method comprises the following steps:

an internal standard method: taking n-dodecane as an internal standard substance, and measuring a product concentration standard curve. During the determination, a certain amount of dodecane is added into a sample as an internal standard substance, and the concentration of a product is calculated according to the concentration of the internal standard substance. The yield calculation formula is as follows:

c in formula (1)_pIs (S) -1-chloro-2-heptanol concentration, C₀Is the initial concentration of 1-chloro-2-heptanone.

The optical purity of the product is characterized by enantiomeric excess (ee). The calculation formula is as follows:

c in formula (2)_SAnd C_RThe molar concentrations of S-type and R-type 1-chloro-2-heptanol, respectively.

Example 2 the concentration of the product (S) -1-chloro-2-heptanol was 48.9 mM; the ee value is: 99.9 percent. The yield was: 97.8 percent.

Examples 3 to 5:

the cells obtained in example 1 were suspended in 20mL of phosphate buffer K at different pH values₂HPO₄-KH₂PO₄Wherein the concentration of cells is 40g/L on a dry weight basis; 50mM 1-chloro-2-heptanone was added as a substrate, and 50g/L glucose was added as an auxiliary substrate, and the reaction was carried out at 30 ℃ and 200rpm for 24 hours. The concentration and ee of the product (S) -1-chloro-2-heptanol using the method for measuring the concentration of the product of example 2 are shown in the following Table. The yields of examples 3 to 5 were 85%, respectively; 97.8 percent; 80.6 percent.

Examples 6 to 8:

the cells obtained in example 1 were suspended in 20mL of different phosphate buffers (pH 6.0) at a concentration of 40g/L on a dry weight basis; 50mM 1-chloro-2-heptanone was added as a substrate, and 50g/L glucose was added as an auxiliary substrate, and the reaction was carried out at 30 ℃ and 200rpm for 24 hours. The concentration and ee of the product (S) -1-chloro-2-heptanol using the method for measuring the concentration of the product of example 2 are shown in the following Table. The yields of examples 6 to 8 were 97.8%, respectively; 75 percent; 81.4 percent.

Examples 9 to 11:

the cells obtained in example 1 were suspended in 20mL of K₂HPO₄-KH₂PO₄(pH 6.0) at a concentration of 40g/L cells on a dry weight basis; 50mM 1-chloro-2-heptanone was added as a substrate, and 50g/L glucose was added as an auxiliary substrate, and the reaction was carried out at 200rpm for 24 hours at different temperatures. The concentration and ee of the product (S) -1-chloro-2-heptanol using the method for measuring the concentration of the product of example 2 are shown in the following Table. The yields of examples 9 to 11 were 85.6%, respectively; 97.8 percent; 77 percent.

Examples 12 to 14:

the different weights (dry weight basis) of cells obtained in example 1 were suspended in 20mL of K₂HPO₄-KH₂PO₄(pH 6.0) 50mM 1-chloro-2-heptanone was added as a substrate, and 50g/L glucose was added as an auxiliary substrate, and the reaction was carried out at 30 ℃ and 200rpm for 24 hours. The concentration and ee of the product (S) -1-chloro-2-heptanol using the method for measuring the concentration of the product of example 2 are shown in the following Table. The yields of examples 12 to 14 were 74.4%, respectively; 97.8 percent; 93 percent.

Examples 15 to 23:

the cells obtained in example 1 were suspended in 20mL of K₂HPO₄-KH₂PO₄(pH 6.0) at a concentration of 40g/L cells on a dry weight basis; adding 50mM 1-chloro-2-heptanone as substrate, adding saccharides with different mass concentrations or alcohols with different volume ratios as auxiliary substrate, and adding 3The reaction was carried out at 0 ℃ for 24h in a shaker at 200 rpm. The concentration and ee of the product (S) -1-chloro-2-heptanol using the method for measuring the concentration of the product of example 2 are shown in the following Table. The yields of examples 15 to 23 were 97.8%, respectively; 73.4 percent; 63.4 percent; 55.6 percent; 59.2 percent; 29.6 percent; 44.6 percent; 63.6 percent; 16.8 percent.

Examples 24 to 26:

the cells obtained in example 1 were suspended in 20mL of K₂HPO₄-KH₂PO₄(pH 6.0) at a concentration of 40g/L cells on a dry weight basis; adding 1-chloro-2-heptanone with different concentrations as a substrate, adding 50g/L glucose as an auxiliary substrate, reacting at 30 ℃ and 200rpm for 24 h. The concentration and ee of the product (S) -1-chloro-2-heptanol using the method for measuring the concentration of the product of example 2 are shown in the following Table. The yields of examples 24 to 26 were 57.4%, respectively; 97.8 percent; 85.2 percent.

Example 27:

the cells obtained in example 1 were suspended in 20mL of K₂HPO₄-KH₂PO₄(pH 6.0) at a concentration of 40g/L cells on a dry weight basis; 1000mM 1-chloro-2-heptanone was added as a substrate, and 50g/L glucose was added as an auxiliary substrate, and the reaction was carried out at 30 ℃ and 200rpm for 60 hours. The concentration and ee of the product (S) -1-chloro-2-heptanol using the method for measuring the concentration of the product of example 2 are shown in the following Table. The yield of example 27 was 76.3%.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Hangzhou college of medicine

<120> curvularia lunata B-36 and application thereof in synthesis of chiral alcohol

<130> PA20018699

<160> 1

<170> PatentIn version 3.3

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<211> 575

<212> DNA

<213> Curvularia lunata (Curvularia hominis) B-36

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gggcgggttc gcccgccacc aggaccacat cataaacctt ttttatgcag ttgcaatcag 180

cgtcagtata acaaatgtaa atcatttaca actttcaaca acggatctct tggttctggc 240

atcgatgaag aacgcagcga aatgcgatac gtagtgtgaa ttgcagaatt cagtgaatca 300

tcgaatcttt gaacgcacat tgcgcccttt ggtattccaa agggcatgcc tgttcgagcg 360

tcatttgtac cctcaagctt tgcttggtgt tgggcgtttt ttgtctttgg ttgccaaaga 420

ctcgccttaa aaggattggc agccggccta ctggtttcgc agcgcagcac atttttgcgc 480

ttgcaatcag caaaagagga cggcaatcca tcaagactcc ttctcacgtt tgacctcgga 540

tcaggtaggg atacccgctg aacttaagca tatca 575

Claims

1. Curvularia lunata (A) and (B)Curvularia hominis) B-36, preserved in China center for type culture Collection; the preservation number is: CCTCC NO: m2017654; the preservation time is as follows: 11/03/2017.

2. Synthesis of Curvularia lunata B-36 according to claim 1 (A)S) -1-chloro-2-heptanol.

3. The application of claim 2, wherein the application comprises taking fermented wet bacterial cells of the Curvularia lunata B-36 as a catalyst, taking 1-chloro-2-heptanone as a substrate, carrying out a biocatalytic reaction in a buffer solution, and separating and purifying a reaction solution after the reaction is finished to obtain the (A)S) -1-chloro-2-heptanol;

the initial concentration of the substrate is 10-1000 mM.

4. Use according to claim 3, wherein the initial concentration of the substrate is between 30 and 70 mM.

5. The use of claim 3, wherein the catalysis of the biological reaction is followed by extraction with ethyl acetate of equal volume, extraction is followed by taking the ethyl acetate layer liquid, concentration by distillation in a rotary evaporator is 4.5-5.5 times the volume, then silica gel is added and mixed uniformly, the mixture is transferred to a chromatographic column, silica gel column chromatography separation is carried out with petroleum ether/ethyl acetate volume ratio of 7: 3 as an eluent, the eluent containing the product is collected, and the concentrated and evaporated to dryness in a rotary evaporator is carried out, thus obtaining (1)S) Pure product of (E) -1-chloro-2-heptanol.

6. The use according to claim 3, wherein the wet bacterial cells are used in an amount of 20 to 100g/L buffer solution on a dry basis.

7. The use according to claim 3, wherein the wet bacterial cells are used in an amount of 30 to 50g/L buffer solution on a dry basis.

8. The use according to claim 7, wherein the wet somatic cells are prepared by the following method:

slant culture:

culturing the inclined plane at 30 ℃ for 2-3 d, picking single bacterial colony in the plate culture medium to the inclined plane, and storing in a refrigerator at 4 ℃; the formula of the slant culture medium is as follows: 200g/L of potato, 20g/L of glucose and 20g/L of agar;

seed culture:

selecting a ring of thalli from a mature culture inclined plane, inoculating the selected ring of thalli into a 500mL shake flask filled with 200mL seed culture medium, and culturing for 24h at 30 ℃ and 200 rpm; the seed culture medium comprises the following components: glucose 25g/L, peptone 27g/L, K₂HPO₄ 3 g/L，(NH₄)₂SO₄4.5 g/L，NaCl 2 g/L，pH 6.0；

Fermentation culture:

transferring the seed solution into a 500mL shake flask filled with 200mL fermentation medium by an inoculation amount of 5-10%, and culturing at 30 ℃ and 200rpm for 48 h; the fermentation medium has the same formula as seed culture medium.

9. The use according to claim 3, wherein the biocatalytic reaction is carried out at 25-40 ℃ in a buffer solution having a pH of 5.0-7.0 for 24-60 h.

10. Use according to claim 3, wherein the biocatalytic reaction is carried out in phosphate buffer or citrate buffer or Tris-HCl buffer.

11. Use according to claim 3, wherein the biocatalytic reaction is at K₂HPO₄-KH₂PO₄In a buffer.

12. Use according to any one of claims 3 to 11, wherein a co-substrate is included in the biocatalytic reaction.

13. Use according to claim 12, wherein the co-substrate is a saccharide and/or an alcohol.

14. Use according to claim 12, wherein the co-substrate is selected from one or more of: glucose, fructose, sucrose, maltose, methanol, ethanol, isopropanol or glycerol.

15. Use according to claim 12, wherein the co-substrate is glucose.

16. The use according to claim 12, wherein when the co-substrate is a saccharide, the amount of the co-substrate added is 10 to 200g/L by mass of the buffer; when the auxiliary substrate is alcohol, the addition amount of the auxiliary substrate is 5-30% of the volume of the biocatalytic reaction buffer solution by volume.

17. The use according to claim 12, wherein when the co-substrate is a saccharide, the amount of the co-substrate added is 50 to 100g/L by mass of the buffer; when the auxiliary substrate is alcohol, the addition amount of the auxiliary substrate is 10-20% of the volume of the biocatalytic reaction buffer solution by volume.