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A carbonyl reductase resistant to high concentration of alcohol solution and its application

A carbonyl reductase and alcohol solution technology, applied in the field of biological enzyme catalysis, can solve the problems of affecting conversion rate, poor fluidity of conversion system, limiting substrate concentration and dosage, etc.

Active Publication Date: 2020-10-27
CHANGXING PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Chinese patent CN102482648B discloses that selection comes from Novosphingobium aromaticivorans Carbonyl reductase gene construction engineering bacteria catalyze (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, the substrate concentration is 100g / L, the yield is greater than 95%, and the product The diastereomeric excess de is greater than 99.9%, but the patent only provides the technology to prepare the RS configuration, not the SS configuration
For the biological preparation of the SS configuration, Chinese patent CN104745649A discloses the use of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as the substrate, and the substrate dosage concentration It is 50g / L, and the fluidity of the conversion system is promoted through the aid of co-solvents and surfactants, and the contact area between the substrate and the enzyme is increased. If no co-solvent is added, the fluidity of the conversion system will become poor, thereby affecting the conversion rate
However, in order to aid dissolution in this technical solution, additional substances such as toluene and Tween 60 added to the system are not conducive to the final separation and purification of the product, so the product purity is only 99%.
[0008] Chinese patent CN104745649A also discloses a method for preparing biological enzymes in SS configuration, but the conversion system of this method uses glucose as the hydrogen supply system, the substrate concentration is 100g / L, and the reaction system is almost entirely in the water phase. Due to the substrate and SS configuration The products are insoluble in water. With the increase of substrate concentration and no co-solvent, the fluidity of the system will gradually deteriorate, and the conversion rate will gradually decrease, which will limit the dosage of substrate concentration and be difficult to use in industrial production.
[0009] It is worth noting that most of the current art uses (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as a substrate to produce (2S,3S)-N-tert In the butoxycarbonyl-3-amino-1-chloro-2-hydroxy-4-phenylbutane biocatalysis technology, the substrate concentration generally cannot exceed 100g / L, or an additional co-solvent is required to bring the system into Other impurities, thereby affecting the separation, purification and purity of SS configuration products

Method used

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  • A carbonyl reductase resistant to high concentration of alcohol solution and its application
  • A carbonyl reductase resistant to high concentration of alcohol solution and its application
  • A carbonyl reductase resistant to high concentration of alcohol solution and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Carbonyl reductase CXP I, whose amino acid sequence is shown in SEQ ID No.1, contains a polypeptide chain consisting of 253 amino acids, and is obtained by performing site-directed mutation on the gene of CX-LAC II strain.

[0042] The CX-LAC II strain was deposited in the China Type Culture Collection Center (CGMCC) on February 24, 2020. The preservation address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, No. 19432, and it was identified as Lactobacillus furfur ( Lactobacillus farraginis ), facultative anaerobic, in the MRS solid medium, the colony is raised, slightly white, moist, with neat edges, and the colony is round, with a diameter of 3.0 mm±1.0 mm.   Microscopically, Gram-positive, generally short rods arranged in chains, usually immobile. CX-LAC II contains the gene sequence CX II as shown in SEQ ID No.3.

[0043] Conduct site-directed mutation design on the gene sequence of CX II, mutate the 24th base A to T, the 519th base T to A, the ...

Embodiment 2

[0049] This embodiment also provides two amino acid sequences, SEQ ID NO.8 and SEQ ID NO.9, which have no less than 90% homology with the sequence shown in SEQ ID NO.1, which are based on the CX II gene of CX-LAC II For site-directed mutation, the mutation rules should at least include the mutation of K at position 8 to N, the mutation of D at position 173 to E, the mutation of Y at position 190 to P, and the mutation of I at position 226 to N. At the same time, the coenzyme binding domain conservative sequence GATLGIG (position 14-20 in the amino acid sequence of CXP II) and the catalytic conservative sequence YNASK (position 156-160 in the amino acid sequence of CXP II) should be retained during mutation. At the same time, amino acid residues 190-220 need to be reserved. This region forms a loop structure in the protein structure and can be used to bind the loop with the substrate.

Embodiment 3

[0051] With (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as the substrate, the six carbonyl reductase crude enzyme solutions obtained in Example 1 were used for enzyme activity experiments . The names, nucleotide sequences and corresponding mutation sites of the six carbonyl reductases are shown in Table 1:

[0052]

[0053] Prepare the reaction system as follows: 50 mL system: add 1g (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, add different concentrations of isopropanol, stir at 40°C, Add 2.4 g magnesium sulfate, 130 mg NAD + , use potassium phosphate or sodium buffer solution (100 mmol / L, pH8.0) to make up to 50 mL, and add 1 g of crude enzyme solution. After reacting for 24 h, the conversion rate and de value were detected by high performance liquid chromatography (HPLC). The results are shown in Table 2. It can be seen that CXP I can be normally converted to almost exhausted substrate under 60% (m / V) isopropanol concentration, an...

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Abstract

The invention discloses an alcohol solution high-concentration-tolerable carbonyl reductase and application thereof and provides a carbonyl reductase which can still maintain high enzyme activity in ahigh-concentration alcohol solution reaction system. The enzyme is produced by a lactobacillus strain with mutation at a fixed point and is used for reducing a (3S)-4-phenyl-2-butanone derivative into a (2S,3S)-2-hydroxyl-4-phenyl butane derivative. In a reaction system of a substrate, isopropanol and an enzyme liquid, no other cosovelent is added, the concentration of the substrate can be greatly increased, and thus the conversion rate of an enzyme catalysis reaction and the chiral purity of a product can be increased. Therefore, the alcohol solution high-concentration-tolerable carbonyl reductase ensures that the reaction system is simple in separation, and can be applied to preparation of a darunavir intermediate.

Description

technical field [0001] The invention relates to the field of biological enzyme catalysis, in particular to a carbonyl reductase with high alcohol solution tolerance and application thereof. Background technique [0002] The trade name of darunavir is Prezista ® , is a non-peptide HIV protease inhibitor (PI) developed by Tibotec, an Icelandic branch of Johnson & Johnson. Wei, Amprenavir and ABT378 / r) have the highest bioavailability, and work by inhibiting the protease of the virus by blocking the formation of new, mature virions released from the surface of infected host cells. Darunavir has strong antiviral activity in vitro, including HIV strains that are resistant to commonly used PIs. In a randomized clinical trial of the Optimized Background Regimens program, darunavir showed superior virological and immunological responses to the control group. In June 2006, the FDA approved darunavir in combination with other antiretroviral drugs for the treatment of adult patients...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12P13/00
CPCC12N9/0006C12P13/001C12Y101/01184
Inventor 张利坤谈聪杨卫华严燕兵华超关永奎王云
Owner CHANGXING PHARMA
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