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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  • Abstract
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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 t

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
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Example Embodiment

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

[0041] Reference 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 software, and set the size of the probe to Analyze the size and shape of the substrate binding pocket of the enzyme, the results are as follows figure 2 Shown in (A). The outer shape of the substrate binding pockets of the enzymes that have been reported to follow the anti-Prelog rule (PDB: 3CTM, 5ZFM, 4R1S, 5ZED, 5TQW, 3VDR and 6NBR) are all like cubes (six regular entities of equilateral squares), and follow The enzymes of Prelog rule are more like cuboid (PDB: 2WDZ, 4BMV, 5GWT, 3AWD, 4PVC, 4FN4 and 5ZEC). The sequence of 7 anti-Prelog enzymes and the sequence of 7 Prelog enzymes were compared, and the results are shown in Table 1. After comparison, the anti-Prelog enzyme is usually D or F in the ...

Example Embodiment

[0044] Example 2: Using computer to predict the stereoselectivity of 11 undeveloped carbonyl reductases

[0045] According to the rules summarized in Example 1, an undeveloped short-chain dehydrogenase with a sequence similarity of 20-40% and an amino acid sequence length of 320-350 was retrieved in the NCBI database. Carry out homology modeling respectively to observe the size and shape of the enzyme substrate binding pocket and the amino acid residues in key positions. The result is Picture 10 As shown, the shape of the binding pocket of CCR1, CCR2, CCR3, CCR4, HP1, HP2 is similar to a cube, and the shape of the binding pocket of AR1, HP3, AR2, AR3, and HP4 is similar to a cuboid. By comparing the amino acids of key sites (A1, A2, A3, B1, B2), the results are shown in Table 2. Six sequences meet the anti-Prelog law, and 5 sequences meet the Prelog law.

[0046] Table 2 Predict the stereoselectivity of undeveloped carbonyl reductase to tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylh...

Example Embodiment

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

[0050] (1) Target gene cloning

[0051] Take K. marxianus ZJB14056 (Deposit Center Number: ATCC36534) genomic DNA as template, and pass the upstream primer: 5’-TAAGAAGGAGATATA CCATGG ATGACATTTACAGTGGTGACAG-3’, downstream primer 5’-GTGGTGGTGGTGGTG CTCGAG TTACCCACGGTACGCGCCCA-3' is subjected to PCR amplification to obtain amplified products. Take 5μL of PCR amplification product for agarose gel electrophoresis test. See the test results Figure 4 In lanes 1, 2, and 3, the electrophoresis band is clear, and the size is about 1000bp, which is consistent with the theoretical value (1038bp). PCR system (total volume 100μL): 50μL 2×PhantaMax buffer, 40μL double distilled water, 2μL dNTP Mix2, 2μL dNTP mix (10mM each), 2μL each of the upstream and downstream primers, 2μL genomic DNA, Phanta Max Super-Fidelity DNA polymerase 2μL. PCR reaction conditions: 95°C pre-...

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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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