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Method for modifying carbonyl reductase stereoselectivity, carbonyl reductase mutant and application

A stereoselective and reductase technology, applied in the direction of microbial-based methods, oxidoreductases, applications, etc., can solve the problems of carbonyl reductase structural differences, low protein structure similarity, and weak reaction selectivity

Active Publication Date: 2020-10-16
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein structures of the three families have low similarity, so despite overlapping substrate specificities, there are large differences in the structures of the different carbonyl reductases
The chemical method has defects such as harsh reaction conditions, poor reaction selectivity, and low optical purity of the product.

Method used

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  • Method for modifying carbonyl reductase stereoselectivity, carbonyl reductase mutant and application
  • Method for modifying carbonyl reductase stereoselectivity, carbonyl reductase mutant and application
  • Method for modifying carbonyl reductase stereoselectivity, carbonyl reductase mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Establishing a method for predicting stereoselectivity of carbonyl reductase

[0041] refer to figure 1 , search the crystal structure of carbonyl reductase in the PDB database, import its three-dimensional model into CaverAnalyst 2.0, select the pocket analysis of the software, and set the size of the probe to The size and shape of the substrate binding pocket of the enzyme were analyzed, and the results were as follows: figure 2 Shown in (A). The outer shape of the substrate-binding pockets of the reported enzymes following the anti-Prelog rule (PDB: 3CTM, 5ZFM, 4R1S, 5ZED, 5TQW, 3VDR, and 6NBR) is like a cube (six equilateral square regular entities), while following Prelog-ruled enzymes are more cuboid-like (PDB: 2WDZ, 4BMV, 5GWT, 3AWD, 4PVC, 4FN4 and 5ZEC). The sequences of the 7 anti-Prelog enzymes were compared with the sequences of the 7 Prelog enzymes, and the results are shown in Table 1. After comparison, it is concluded that anti-Prelog enzy...

Embodiment 2

[0044] Example 2: Computer Prediction of Stereoselectivity of Eleven Unexplored Carbonyl Reductases

[0045] According to the rules summarized in Example 1, undeveloped short-chain dehydrogenases with a sequence similarity of 20-40% to KmCR and an amino acid sequence length of 320-350 were searched in the NCBI database. Homology modeling was carried out on them respectively, and the size, shape and amino acid residues at key sites of the enzyme substrate binding pocket were observed. The result is as Figure 10 As shown, the shape of the binding pocket of CCR1, CCR2, CCR3, CCR4, HP1, HP2 is similar to a cube, while the shape of the binding pocket of AR1, HP3, AR2, AR3 and HP4 is similar to a cuboid. By comparing the amino acids at the key sites (A1, A2, A3, B1, B2), the results are shown in Table 2, 6 sequences satisfy the law of inverse Prelog, and 5 sequences satisfy the law of Prelog.

[0046] Table 2 Predicted stereoselectivity of an unexplored carbonyl reductase towards...

Embodiment 3

[0049] Example 3: Cloning of wild-type carbonyl reductase KmCR gene and construction of its recombinant expression vector

[0050] (1) Target gene cloning

[0051] Using Kluyveromyces marxianus (K.marxianus) ZJB14056 (Depository Number: ATCC36534) genomic DNA as a template, through the upstream primer: 5'-TAAGAAGGAGATATA CCATGG ATGACATTTACAGTGGTGACAG-3', downstream primer 5'-GTGGTGGTGGTGGTG CTCGAG TTACCCACGGTACGCGCCCA-3' was amplified by PCR to obtain the amplified product. Take 5 μL of PCR amplification products for agarose gel electrophoresis test. See the test results Figure 4 Swimming lanes 1, 2, and 3 have clear electrophoresis bands with a size of about 1000bp, which is consistent with the theoretical value (1038bp). PCR system (total volume 100 μL): 50 μL 2×PhantaMax buffer, 40 μL double distilled water, 2 μL dNTP Mix2, 2 μL dNTP mixture (10 mM each), 2 μL each of upstream and downstream primers, 2 μL genomic DNA, Phanta Max Super-Fidelity DNA polymerase 2 μL. ...

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Abstract

The invention discloses a method for modifying carbonyl reductase stereoselectivity, a carbonyl reductase mutant and application. The carbonyl reductase stereoselectivity is identified by combining the size and shape of an enzyme with a pocket, the external shape of the pocket of a reverse Prelog enzyme is close to a cube, the external shape of the pocket of the Prelog enzyme is more similar to acuboid, and the enzyme stereoselectivity can be reversed or improved by regulating and controlling key site amino acid. The stereoselectivity (99.5% dep, R) of the carbonyl reductase mutant KmCR-E87D / A129C / V239N prepared by the method is reversed compared with that (99.5% dep, S) of a wild KmCR, and 35.4mM of 6-cyano (3R, 5R)-dihydroxy tert-butyl caproate can be asymmetrically synthesized within10 hours.

Description

technical field [0001] The invention relates to a stereoselective transformation method of carbonyl reductase, using the method to construct a stereoselective carbonyl reductase mutant, and its application in the preparation of optically pure chiral alcohol compounds, in particular to the use of computer-aided prediction of carbonyl reductase Stereoselectivity and key sites, carbonyl reductase was screened and applied to the chiral synthesis of atorvastatin 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester and (S)-4-chloro- Chiral biosynthesis of ethyl 3-hydroxybutyrate. Background technique [0002] Carbonyl reductase (Carbonyl reductase, EC 1.1.1.184) belongs to oxidoreductase, which can catalyze the asymmetric reduction of latent chiral carbonyl compounds such as aliphatic ketones, aromatic ketones, aldehydes and quinones to generate corresponding chiral alcohols, and has a wide range of substrates spectrum, good catalytic activity and stereoselectivity. NAD(H) (n...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12P13/00C12P7/62C12R1/19
CPCC12N9/0006C12P13/004C12P7/62C12Y101/01184
Inventor 王亚军程峰陈祎郑裕国
Owner ZHEJIANG UNIV OF TECH
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