CN109913428B - 7 beta-hydroxysteroid dehydrogenase, coding gene, vector, engineering bacteria and application - Google Patents

Abstract

The invention discloses 7 beta-hydroxysteroid dehydrogenase derived from clostridium mosaic (Clostridium Marseille), a coding gene, a vector and application thereof in preparing ursodeoxycholic acid intermediate 3, 12-diketone-7 beta-cholanic acid by biological catalysis, wherein the amino acid sequence of the 7 beta-hydroxysteroid dehydrogenase is shown as SED ID NO 1; the invention provides a novel 7 beta-hydroxysteroid dehydrogenase for stereoselectively preparing 3, 12-diketone-7 beta-cholanic acid; the 7 beta-hydroxysteroid dehydrogenase can be used for preparing ursodeoxycholic acid intermediate 3, 12-diketone-7 beta-cholanic acid; the preparation method of the 3, 12-diketone-7 beta-cholanic acid has the advantages of high optical purity, mild condition, environmental friendliness and the like, and has higher industrialized application potential.

Description

7 beta-hydroxysteroid dehydrogenase, coding gene, vector, engineering bacteria and application

Technical Field

The invention relates to 7 beta-hydroxysteroid dehydrogenase and application thereof, in particular to recombinant 7 beta-hydroxysteroid dehydrogenase, a coding gene, a recombinant expression vector and a recombinant expression transformant containing the gene, and application of the enzyme or recombinant cells containing the enzyme as a catalyst in preparing ursodeoxycholic acid intermediate 3, 12-diketone-7 beta-cholanic acid.

Background

Ursodeoxycholic acid (UDCA) is a main effective component contained in rare Chinese medicine bear gall powder, is a secondary bile acid from bear gall, is generated by bacterial metabolism of primary bile acid, is mainly used for treating gall-stone diseases and various acute and chronic liver diseases clinically, and has good treatment effect. The traditional Chinese medicine bear gall powder is mainly extracted from artificially cultured bear in a mode of 'living bear drainage gall taking', so that the method has a protection law of wild animals, and the yield of UDCA is low and cannot meet the market demand, so that the artificial synthesis of the UDCA has important significance. The following table provides the structural formulas, chemical names and abbreviations of the important compounds in the synthesis step.

The classical chemical process for preparing UDCA uses Cholic Acid (CA) as raw material, and 7 steps of reaction are needed to prepare UDCA. Since the chemical oxidation is non-selective, the carboxyl groups and 3a, 7 a-hydroxyl groups on CA must be protected and deprotected repeatedly, and the catalytic hydrogenation step of 7-keto-lithocholic acid (7-keto-LCA) is low in selectivity, and the process route is long, resulting in lower yield. In addition, the reduction of the intermediate 7-keto-LCA adopts metallic sodium or Pd/C catalytic hydrogenation, so that the selectivity is low, and the industrial scale-up production is not easy to control and unsafe.

The technology based on the enzymatic preparation of UDCA mainly comprises 3 technologies, as follows:

sun B et al (Sun B, kantzow C, bresch S.Biotechnology and bioengineering,2013, 110:68-77) describe a process for chemically enzymatic preparation of UCDA (scheme a). The final product was obtained by first preparing DHCA by chemical oxidation of CA, then reducing DHCA to 12-keto-UDCA by 3α -HSDH of Comamonas testosteroni (Comanomonas testosteroni) and 7β -HSDH of Klebsiella aerogenes (Collinsella aerofaciens), and finally by wolff-kishner reduction. In a 1L reaction system, the two enzymes are used for catalyzing 0.1mol/L DHCA to prepare 12-keto-UDCA, and the final reaction yield reaches 99.5 percent. Monti D et al (Monti D, ferrandi EE, zanella to I, huan L, polentini F, carrea G, riva S.advanced Synthesis & Catalysis,2009, 351:1303-1311) describe another chemoenzymatic process for preparing UDCA starting from CA (scheme b). 7,12-dione-3 a-cholanic acid (7, 12-dione-3 a-CA) was first prepared by oxidation of CA using 7a-HSDH and commercial 12a-HSDH (Genzyme Biochemicals ltd.) of bacteroides fragilis (Bacteroides fragilis), followed by reduction of 7,12-dione-3 a-CA to 12-keto-ursodeoxycholic acid (12-keto-UDCA) by 7β -HSDH of Clostridium absonum, and finally by a wolff-kishner reduction reaction to obtain the final product. The control of 3 reversible reactions is involved in the route, the difficulty is great, and the literature reports that the highest yield of the 12-keto-UDCA prepared by taking CA as a starting substrate through the route b is only 73 percent.

Route c is a complete bio-enzymatic catalysis route, and both patent CN105368828A and CN105861613a use this process to prepare UDCA. CN105368828A uses 7. Alpha. -HSDH of Escherichia coli to oxidize CDCA first, and then 7-keto-LCA is reduced to UDCA using 7. Beta. -HSDH of the genus Living ruminococcus (Ruminococcus gnavus), which reports a catalytic level of 100g/L CDCA with a final yield of 88-94%.

In summary, the 3 chemical enzymatic routes described above all require the use of 7β -HSDH as a biocatalyst in the preparation of UDCA. However, the 7. Beta. -HSDH sequences reported so far are very limited and viability has been published and verified as key enzymes for the UDCA production process, and only 5 7. Beta. -HSDH sequences derived from Clostridium sardiniense (GenBank: JN191345.1, ferrandi EE, bertolesi GM, polentini F, negri A, riva S and Monti D.applied microbiology and biotechnology,2012,95,1221-1233.), collinsella aerofaciens (PDB: 5FYD,Liu L,Aigner A and Schmid RD.Applied microbiology and biotechnology,2011,90,127-135.), ruminococcus gnavus (GenBank: KF052988, lee JY, arai H, nakamura Y, fukiya S, wada M and Yokota A. Journal of lipid research,2013,54,3062-3069.), ruminococcus torques (GenBank: CBL26204, zheng MM, waRF, li CX and Xu JH. Process Biochemistry,2015,50,598-604.), and Turneriella parva (GenBank: WP 014_1, CNC) are greatly restricted. Therefore, the method has important significance in mining and screening the novel 7 beta-HSDH sequence with good catalytic performance.

Disclosure of Invention

The invention aims to provide a novel 7beta-HSDH derived from clostridium Marseillensis (Clostridium Marseille), which can reduce carbonyl at C-7 position of dehydrocholic acid to generate 3, 12-diketone-7beta-cholanic acid in a stereoselective manner, and provides more enzyme source selections for a chemical enzyme method of ursodeoxycholic acid.

The technical scheme adopted by the invention is as follows:

the invention provides a 7 beta-HSDH derived from clostridium mosaic (Clostridium Marseille), wherein the amino acid sequence of the 7 beta-HSDH is shown as SEQ ID NO: 1.

The 7 beta-hydroxysteroid dehydrogenase is obtained by a gene database mining method, the selected 7 beta-HSDH is subjected to total gene synthesis, recombinant escherichia coli cells are constructed, and the activity of the 7 beta-HSDH is verified.

Due to the specificity of the amino acid sequence, any polypeptide comprising SEQ NO:1 or a variant thereof, such as a conservative variant, a biologically active fragment or a derivative thereof, as long as the fragment or variant thereof has a homology of 90% or more with the aforementioned amino acid sequence. The alteration may comprise a deletion, insertion or substitution of an amino acid in the amino acid sequence; wherein, for conservative changes of the variant, the substituted amino acid has similar structure or chemical properties as the original amino acid, such as replacement of isoleucine with leucine, the variant may also have non-conservative changes, such as replacement of glycine with tryptophan.

The invention also designs a coding gene of the 7beta-HSDH. The nucleotide sequence of the gene is shown in SEQ ID NO: 2.

Due to the specificity of the nucleotide sequence, any of SEQ ID NOs: 2, and all variants of the polynucleotide having a homology of 90% or more to the polynucleotide are within the scope of the present invention. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotides may be made as either a living variant or a non-living variant, including substitution, deletion and insertion variants. As known in the art, an allelic variant is an alternative to a polynucleotide, which may be a substitution, deletion or insertion of a polynucleotide, without substantially altering the function of the polypeptide it encodes.

The invention also relates to a recombinant vector containing the coding gene and recombinant genetic engineering bacteria obtained by utilizing the recombinant vector to transform.

The recombinant vector is constructed by connecting the nucleotide sequence of the encoding gene of the 7beta-HSDH of the invention to various vectors by a conventional method. The vector may be any of a variety of vectors conventional in the art, such as various plasmids, phage or viral vectors, and the like, preferably pET28a. Preferably, the recombinant expression vector of the present invention can be obtained by the following method: the 7β -hsdh gene product obtained by PCR amplification was ligated with pMD-18T to form a cloning vector. The cloning vector is subjected to restriction enzyme NcoI/BamHI double digestion, and after gel cutting recovery, is connected with pET28a recovered by enzyme cutting treatment, so as to construct the 7 beta-HSDH gene recombinant expression plasmid pET28a-7 beta-HSDH.

The invention also provides a genetically engineered bacterium containing the coding gene or the recombinant vector. The genetically engineered bacterium can be obtained by transforming the recombinant expression vector of the invention into a host microorganism. The host microorganism may be various host microorganisms conventional in the art as long as it is satisfied that the recombinant expression vector can stably self-replicate and that the carried 7β -hsdh gene of the present invention can be efficiently expressed. The invention selects escherichia coli, preferably escherichia coli E.coli BL21 (DE 3). And (3) converting the recombinant plasmid pET28a-7 beta-hsdh into E.coli BL21 (DE 3) to obtain recombinant E.coli BL21 (DE 3)/pET 28a-7 beta-hsdh. The recombinant bacteria are used as enzyme sources to carry out biological catalysis.

The invention also relates to application of the 7β -HSDH in preparing ursodeoxycholic acid intermediate 3, 12-diketone-7β -cholanic acid by biocatalysis, wherein the application is as follows: the wet bacterial crushed liquid obtained by fermenting and culturing recombinant genetic engineering bacteria containing 7 beta-HSDH coding gene is used as a biological catalyst, and a substrate, an auxiliary substrate and NADP are added into a buffer solution with pH value of 5.0-9.0 ⁺ Reacting at 20-50deg.C and 50-250rpm (preferably 30 deg.C and 200rpm for 0.5-5 hr), separating and purifying the reaction solution to obtain 3, 12-diketone-7β -cholanic acid; the substrate is dehydrocholic acid, the auxiliary substrate is glucose, and glucose dehydrogenase is added to form a coenzyme circulation system; the dosage of the catalyst is 5-50g/L based on the weight of wet bacteria, the initial concentration of the substrate is 0.05-1.0mol/L, the dosage of the auxiliary substrate is 5-100g/L, and the dosage of the glucose dehydrogenase is 0.5-50g/L based on the weight of wet bacteria obtained by fermenting and culturing engineering bacteria containing glucose dehydrogenase genes.

The reaction formula is as follows:

the Glucose Dehydrogenase (GDH) wet cell of the present invention is prepared as follows: recombinant strain BL21 (DE 3)/pET 28a-GDH containing GDH (preferably GDH derived from Bacillus subtilis, genBank: WP_ 003246720.1) was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, cultured at 37℃for 12 hours, inoculated into fresh LB liquid medium containing 50. Mu.g/mL kanamycin at a 2% by volume of the inoculated amount, and cultured at 37℃to a cell concentration OD ₆₀₀ And (3) adding IPTG with a final concentration of 0.1-1.0mmol/L into the LB liquid culture medium, performing induced culture at 28 ℃ for overnight, centrifuging the culture solution at 4 ℃ for 5min at 12000rpm, discarding the supernatant, and collecting wet thalli containing recombinant GDH.

Further, the preparation method of the biocatalyst comprises the following steps: inoculating recombinant genetically engineered strain containing 7 beta-HSDH coding gene into LB liquid culture medium containing 50 μg/ml kanamycin, culturing at 37deg.C for 12 hr, inoculating into fresh LB liquid culture medium containing 50 μg/ml kanamycin at 2% of the volume concentration, culturing at 37deg.C to thallus concentration OD ₆₀₀ And (3) adding IPTG with the final concentration of 0.1-1.0mmol/L into the LB liquid culture medium with the value of 0.4-0.6, performing induced culture at 28 ℃ for overnight, centrifuging the culture solution at 4 ℃ and 12000rpm for 5min, discarding the supernatant, and collecting wet thalli containing recombinant 7β -HSDH.

The post-treatment and crystallization of 3, 12-diketone-7 beta-cholanic acid in the conversion reaction liquid: adding 1-10% diatomite into the reaction system and stirring for 1-2h; slowly dripping hydrochloric acid solution into the reaction system until the pH value is about 2.0, continuously stirring for 1h, and filtering to collect a filter cake; adding 0.5-5 times of organic solvent such as acetone into the filter cake, stirring for 1-2 hr, and filtering to remove solid; distilling the filtrate under reduced pressure, and collecting solid matters; adding a proper amount of distilled water into the solid, and dripping NaOH solution until the solution is dissolved and clarified; dropwise adding a hydrochloric acid solution into the system until the pH value is about 2.0, stirring for about 2-10h, and filtering to collect a filter cake; and drying to constant weight to obtain the product 3, 12-diketone-7 beta-cholanic acid.

The beneficial effects of the invention are mainly as follows: the invention provides a novel 7beta-HSDH derived from clostridium mosaic (Clostridium Marseille), which is an enzyme with the activity of 7beta-HSDH which is found in a strain for the first time; the 7 beta-HSDH has lower similarity with the known 7 beta-HSDH, higher activity on dehydrocholic acid and excellent stereoselectivity. The 7 beta-HSDH can be used for preparing ursodeoxycholic acid intermediate 3, 12-diketone-7 beta-cholanic acid, the substrate concentration can reach more than 40g/L, and the reaction yield is maintained at more than 99%; the preparation method of the 3, 12-diketone-7 beta-cholanic acid has the advantages of high optical purity, mild condition, environmental protection and the like, and has great industrialized application potential.

Drawings

FIG. 1 is a physical map of the expression vector pET28a-7β -hsdh;

FIG. 2 is an amino acid sequence alignment of C.Marseille 7β -HSDH and reported 7β -HSDH;

FIG. 3 is a SDS-PAGE map of the induced expression of engineering bacteria; lane 1 is protein molecular weight Marker, lane 2 is e.coll BL21 (DE 3), lane 3 is uninduced e.coll BL21 (DE 3)/pET 28a-7β -hsdh, lane 4 is IPTG-induced e.coll BL21 (DE 3)/pET 28a-7β -hsdh;

FIG. 4 is a graph showing the effect of pH on 7. Beta. -HSDH viability; the buffer system is as follows: citric acid-sodium citrate (pH 5.0-6.0), na ₂ HPO ₄ -NaH ₂ PO ₄ (pH 6.0-7.5)，Tris-HCl(pH 7.5-9.0)；

FIG. 5 is a graph showing the effect of temperature on 7. Beta. -HSDH activity.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1: acquisition of 7 beta-hydroxysteroid dehydrogenase Gene

The 7 beta-hydroxysteroid dehydrogenase is obtained by a gene database mining method, and the sequence is screened by utilizing protein PDB and NCBI databases to obtain a 7 beta-HSDH sequence (NCBI Reference Sequence:WP_ 066892209.1). The sequence has homology with 7 beta-HSDH sequences which have been published and verified for activity, and the homology is as follows: turneriella parva (GenBank: WP_ 014801383.1), 20%; clostridium sardiniense (GenBank: JN 191345.1), 44%; collinsella aerofaciens (PDB: 5 FYD), 73%; ruminococcus gnavus (GenBank: KF 052988), 75%; ruminococcus torques (GenBank: CBL 26204), 81%. The enzymes according to the invention belong to the family of short-chain dehydrogenases (SDRs), and the sequences described above are subjected to homology alignment (FIG. 2), clearly showing the conserved domains in the primary structure of SDRs. The N-terminal sequences G-X-X-X-G-X-G (corresponding to G-15, G-19 and G-21, numbering corresponding to the numbering of the alignment in FIG. 2) correspond to the characteristic coenzyme binding sites of the SDR family. Furthermore, three completely conserved amino acid residues S-143, Y-156 and K-160 (numbered according to the alignment) can be identified, corresponding to the catalytic triplet structure of the SDR family.

The 7 beta-HSDH is derived from clostridium mosaic (Clostridium Marseille), is optimized according to the amino acid sequence of the enzyme and according to the codon preference of escherichia coli, ncoI and BamHI are designed according to the characteristics of an expression vector pET28a, the 7 beta-hydroxysteroid dehydrogenase gene 7 beta-HSDH (shown as SEQ ID NO: 2) is synthesized by a total gene synthesis method through the conventional operation of genetic engineering, the length of the nucleotide sequence is 792bp, the sequence codes for a complete open reading frame, and the amino acid sequence of the coded enzyme is shown as SEQ ID NO: 1.

Example 2: construction of recombinant expression vector pET28a-7 beta-hsdh and construction of recombinant engineering bacteria

The 7. Beta. -hsdh gene fragment synthesized in example 1 was subjected to double cleavage and recovery treatment using NcoI and BamHI restriction enzymes, and ligated overnight at 16℃with a commercial vector pET28a treated with the same restriction enzymes using T4DNA ligase, thereby constructing a recombinant expression vector pET28 a-7. Beta. -hsdh. Transforming the constructed recombinant expression vector pET28a-7 beta-hsdh into E.coli BL21 (DE 3) competent cells, coating the competent cells on LB plates containing 50 mug/mL kanamycin at a final concentration, and culturing overnight at 37 ℃; colony PCR identification is carried out by randomly picking clones from colonies growing on a flat plate, and positive clone sequencing verification shows that the recombinant expression vector pET28a-7β -hsdh is successfully transformed into an expression host E.coli BL21 (DE 3), and the 7β -hsdh gene is successfully cloned to NcoI and BamHI sites of pET-28 a.

Example 3: preparation of recombinant 7β -HSDH-containing somatic cells

Inoculating the genetically engineered bacterium pET28a-7 beta-hsdh constructed in example 2 into LB culture medium containing 50 mug/mL kanamycin, and culturing at 37 ℃ until the concentration of thalli OD ₆₀₀ And (3) adding IPTG with the final concentration of 0.1mmol/L into the LB liquid medium with the value of 0.4-0.6, performing induction culture at 28 ℃ for overnight, centrifuging the culture solution at 4 ℃ and 12000rpm for 5min, discarding the supernatant, and collecting wet thalli containing the recombinant 7β -HSDH. 1g of wet cells were weighed, suspended in 10mL of phosphate buffer (pH 7.0), sonicated, and the size and expression level of the soluble recombinant protein were verified by SDS-PAGE (see FIG. 3).

Example 4: activity measurement of recombinant genetically engineered bacterium E.coli BL21 (DE 3)/pET 28a-7 beta-hsdh

The bacterial cell disruption solution obtained in the method of example 3 is used as a catalyst to catalyze the substrate dehydrocholic acid.

The catalytic system comprises the following components and catalytic conditions: 10mL of phosphate buffer (50 mmol/L, pH 7.0) was added with a wet cell disruption solution (final concentration 1.0 g/L) of recombinant 7β -HSDH, dehydrocholic acid (final concentration 75 mmol/L) and NADPH (final concentration 60 mg/L) to construct a reaction system. The reaction temperature is 30 ℃, and the enzyme activity is detected by sampling after 10 minutes of reaction under the condition of 200 rpm. Under the same conditions, E.coli BL21 (DE 3) and E.coli BL21 (DE 3)/pET 28a cell disruption solution were used as controls.

Definition of enzyme activity unit (U): the amount of enzyme required to consume 1. Mu. Mol of NADPH in 1min at 30℃and pH 7.0 was defined as 1U. The consumption of NADPH was determined by means of a spectrophotometer at 340 nm. The enzyme activity of recombinant 7β -HSDH was calculated from the consumption of NADPH in the system. The measurement results are shown in Table 1.

TABLE 1 enzyme Activity assay for recombinant 7β -HSDH

Example 5: recombinant 7β -HSDH coenzyme type preference assay

The bacterial cell disruption solution obtained in the method of example 3 was used as a catalyst, and dehydrocholic acid was used as a substrate.

The catalytic system comprises the following components and catalytic conditions: 10mL of phosphate buffer (50 mmol/L, pH 7.0) was added with a wet cell disruption solution (final concentration 1.0 g/L) of recombinant 7β -HSDH, dehydrocholic acid (final concentration 75 mmol/L), and NADH or NADPH (final concentration 60 mg/L) to constitute a reaction system. The reaction temperature was 30℃and the rotation speed was 200rpm for 10 minutes, and the sample was taken to detect the enzyme activity (the method was the same as in example 4). Under the same conditions, a reaction solution without adding a coenzyme was used as a control. The measurement results are shown in Table 2.

TABLE 2 recombinant 7β -HSDH coenzyme preference

Coenzyme type	Enzyme activity (U/g) Wet cell )
		Control	0
NADH	21.6
		NADPH	529.3

The results show that the enzyme activity of the 7β -HSDH is significantly higher when NADPH is used as a coenzyme than when NADH is used as a coenzyme, so that the enzyme is an NADPH-coenzyme-dependent hydroxysteroid dehydrogenase.

Example 6: determination of glucose dehydrogenase concentration in coenzyme circulation System

Inoculating recombinant BL21 (DE 3)/pET 28a-gdh into LB liquid medium containing 50 μg/mL kanamycin, culturing at 37deg.C for 12 hr, inoculating into fresh LB liquid medium containing 50 μg/mL kanamycin at 2% by volume, culturing at 37deg.C to thallus concentration OD ₆₀₀ And adding IPTG with a final concentration of 0.1mmol/L into the LB liquid culture medium with a value of 0.4-0.6, performing induced culture at 28 ℃ for overnight, centrifuging the culture solution at 4 ℃ and 12000rpm for 5min, discarding the supernatant, and collecting wet thalli containing recombinant GDH. The thallus broken liquid can be applied to coenzyme circulation.

Conversion system: 10mL of phosphate buffer (50 mmol/L, pH 7.0) was added with recombinant 7β -HSDH wet cell disruption solution (final concentration 20g/L, prepared by the method of example 3), dehydrocholic acid (final concentration 75 mmol/L), NADP ⁺ (final concentration 60 mg/L), glucose 60g/L, the final concentration of the prepared GDH wet bacterial body crushing liquid is 2g/L respectively, the reaction temperature is 30 ℃, the stirring rotation speed is 200rpm, the acetonitrile is used for quenching reaction after the reaction is finished, and the conversion rate of the substrate dehydrocholic acid is detected by HPLC. HPLC detection conditions are described in the reference (Liu L, aigner A and Schmid RD.applied microbiology and biotechnology,2011,90,127-135.). Under the same conditions, the reaction solution without adding GDH wet cell disruption solution was used as a control. The results show that when the final concentration of GDH cells is 2g/L, namely when the weight ratio of the 7 beta-HSDH cells to the GDH cells is 10:1, the coenzyme circulation system can meet the requirement of 7 beta-HSDH on NADPH, and the reaction conversion rate is high>99%. As a control, the experimental group without GDH was free of productionAnd (5) generating a product.

Example 7: determination of glucose dehydrogenase concentration in coenzyme circulation System

Conversion system: 10mL of phosphate buffer (50 mmol/L, pH 7.0) was added with recombinant 7β -HSDH wet cell disruption solution (final concentration 20g/L, prepared by the method of example 3), dehydrocholic acid (final concentration 75 mmol/L), glucose dehydrogenase cell disruption solution (final concentration 2g/L, prepared by the method of example 6), NADP ⁺ (final concentration 60 mg/L), glucose concentrations were 10g/L and 60g/L, respectively, at 30℃and stirring speed 200rpm, and after the reaction was completed, the reaction was quenched with acetonitrile and the conversion of the substrate dehydrocholic acid was measured by HPLC. Under the same conditions, a reaction solution without glucose was used as a control. The results showed that the reaction conversion was 41.0% and 60% respectively when the final concentration of glucose was 10g/L and 60g/L>99% and no product was produced in the control group without glucose. Thus, the coenzyme cycle system was able to meet the demand for NADPH by 7β -HSDH at a glucose concentration of 60 g/L.

Example 8: NADP in coenzyme circulation System ⁺ Concentration determination

Conversion system: 10mL of phosphate buffer (50 mmol/L, pH 7.0) was added with recombinant 7β -HSDH wet cell disruption solution (final concentration 20g/L, prepared by the method of example 3), dehydrocholic acid (final concentration 75 mmol/L), glucose dehydrogenase cell disruption solution (final concentration 2g/L, prepared by the method of example 6), glucose concentration 60g/L, NADP ⁺ The concentration is 40mg/L, the reaction temperature is 30 ℃, the stirring rotation speed is 200rpm, after the reaction is finished, acetonitrile is used for quenching the reaction, and the conversion rate of the substrate dehydrocholic acid is detected by HPLC. Under the same conditions, without addition of NADP ⁺ As a control. The results show that when NADP ⁺ At a concentration of 40mg/L, the reaction conversion rate>99% of the system does not add NADP ⁺ The reaction conversion rate of the control group of (C) can reach 46.6%, probably because of intracellular NADP after the bacterial cells are broken ⁺ The release into the reaction system can be caused by coenzyme circulation under the action of GDH. Thus, when NADP ⁺ At a concentration of 20mg/L, the coenzyme circulation system is capable of meeting the demand of 7β -HSDH for NADPH.

Example 9: reaction pH optimization

The bacterial cell disruption solution obtained in the method of example 3 was used as a catalyst, and dehydrocholic acid was used as a substrate.

Conversion system: 10mL of buffer solutions (50 mmol/L, pH 5.0-9.0) with different pH values are added with recombinant 7β -HSDH wet cell disruption solution (final concentration 1g/L, prepared by the method of example 3), dehydrocholic acid (final concentration 75 mmol/L), GDH cell disruption solution (final concentration 2 g/L), glucose concentration 60g/L, NADP ⁺ The concentration is 40mg/L, the reaction temperature is 30 ℃, the stirring rotation speed is 200rpm, the reaction is carried out for 10min, after the reaction is finished, the acetonitrile is used for quenching the reaction, and the activity of the 7beta-HSDH is detected by HPLC. The results are shown in FIG. 4, where the 7β -HSDH showed the highest enzyme activity in phosphate buffer pH 6.0.

Example 10: reaction temperature optimization

The bacterial cell disruption solution obtained in the method of example 3 was used as a catalyst, and dehydrocholic acid was used as a substrate.

Conversion system: 10mL of phosphate buffer (50 mmol/L, pH 6.0) was added with recombinant 7β -HSDH wet cell disruption solution (final concentration 1g/L, prepared by the method of example 3), dehydrocholic acid (final concentration 75 mmol/L), glucose dehydrogenase cell disruption solution (final concentration 2 g/L), glucose concentration 60g/L, NADP ⁺ The concentration is 40mg/L, the reaction temperature is 25 ℃,30 ℃,35 ℃, 40 ℃, 45 ℃,50 ℃ and the stirring rotation speed is 200rpm for reaction for 10min, after the reaction is finished, the acetonitrile is used for quenching the reaction, and the activity of the 7beta-HSDH is detected by HPLC. The experimental results are shown in FIG. 5, and the 7β -HSDH exhibits the highest enzyme activity at 40 ℃. In view of the temperature stability of the enzyme, 30℃was still used as the reaction temperature in the subsequent 3, 12-dione-7β -cholanic acid preparation experiments.

Example 11: application of recombinant genetic engineering bacteria E.coli BL21 (DE 3)/pET 28a-7 beta-hsdh in preparation of 3, 12-diketone-7 beta-cholanic acid

The bacterial cell disruption solution obtained in the method of example 3 was used as a catalyst, and dehydrocholic acid was used as a substrate.

The catalytic system comprises the following components and reaction conditions: 250mL of phosphate buffer (50 mmol/L, pH 6.0) was added with recombinant 7β -HSDH wet cell disruption solution (final concentration 20g/L, prepared in the method of example 3), GDH wet cell (final concentration 2g/L, prepared in the method of example 6), dehydrocholic acid (final concentration 40g/L, about 100 mmol/L), NADP ⁺ 40mg/L, dextranThe glucose concentration is 60g/L to form a reaction system, the rotating speed is 200rpm at 30 ℃, the acetonitrile is used for quenching reaction after the reaction is finished, and the conversion rate of the substrate dehydrocholic acid is detected by HPLC. After the reaction is finished, the conversion rate of dehydrocholic acid is more than 99 percent.

Example 12: separation and refining in preparation of 3, 12-diketone-7 beta-cholanic acid

Adding 10% diatomite into the conversion solution obtained in the example 11, dropwise adding 3mol/L HCl into the system while stirring, adjusting the pH to about 2.0, and continuously stirring for 2 hours; filtering the conversion solution, adding 500mL of acetone into a filter cake, stirring for 2h, and filtering to remove solids; collecting acetone solution, and performing reduced pressure distillation until no outflow occurs; 250mL of distilled water is added into a distillation flask, stirred, and 5mol/L NaOH solution is added dropwise until dissolution and clarification are achieved; 3mol/L HCl is dripped into the system, the pH is regulated to about 2, the mixture is stirred for about 5 hours, a filter cake is collected by filtration, the mixture is dried to constant weight at 80 ℃, 9.54g of refined 3, 12-diketone-7 beta-cholanic acid is collected, and the molar yield of the product is 94.9%.

Sequence listing

<110> Shanghai Oobo biomedical technology Co., ltd

<120> a 7 beta-hydroxysteroid dehydrogenase, coding gene, vector, engineering bacterium and application

<130> 2017.11.6

<141> 2017-12-13

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 792

<212> DNA

<213> Clostridium Marseille

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gttgaagtta tcaccctggg taccaccatc accccgtctc tgctgaaaaa cctgccgggt 600

ggtccggctg gtgaagctgt tatgaaagct gctctgaccc cggaagcttg cgttgaagaa 660

gctttcgaaa acctgggtaa aaaattctct atcatcgctg gtgaacacaa caaagcttct 720

atccacgact ggaaagctaa ccacaccgaa gacgaattca tctcttacat gggttctttc 780

tacgaacgtt aa 792

<210> 2

<211> 263

<212> PRT

<213> Clostridium Marseille

<400> 2

Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala

1 5 10 15

Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly

20 25 30

Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu

35 40 45

Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Lys

50 55 60

Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr

65 70 75 80

Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr

85 90 95

Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu

100 105 110

Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys

115 120 125

Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu

130 135 140

Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys

145 150 155 160

Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys

165 170 175

Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro

180 185 190

Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Val Met

195 200 205

Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn

210 215 220

Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser

225 230 235 240

Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr

245 250 255

Met Gly Ser Phe Tyr Glu Arg

260

Claims (3)

1. A preparation method of a composition containing clostridium marseisClostridium Marseille) The application of the 7 beta-hydroxysteroid dehydrogenase in preparing ursodeoxycholic acid intermediate by biological catalysis is characterized in that the substrate is dehydrocholic acid, and the ursodeoxycholic acid intermediate is 3, 12-diketone-7 beta-cholanic acid; the amino acid sequence of the 7 beta-hydroxysteroid dehydrogenase is shown as SEQ ID NO. 2.

2. The application of claim 1, wherein the application is: adding glucose dehydrogenase, glucose and NADP to the buffer System ⁺ The optical pure 3, 12-diketone-7 beta-cholanic acid is prepared by catalyzing dehydrocholic acid to perform asymmetric reduction reaction by using 7 beta-hydroxysteroid dehydrogenase recombinant bacteria as a catalyst.

3. The use according to claim 1, wherein the crude 3, 12-diketone-7 beta-cholanic acid is acidified and separated in a reaction system, the organic reagent is dissolved, the solid is removed by filtration, the reflux and the alkali are added for dissolution, and the refined product is finally obtained by acidification and separation.