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Aldehyde ketone reductase KmAKR mutant and application thereof in catalytic synthesis of chiral alcohol

A reductase and mutant technology, applied in the field of aldehyde-ketone reductase KmAKR mutants, can solve the problems of large amount of catalyst, low asymmetric reduction activity, low activity of non-natural ketoester substrates, etc.

Active Publication Date: 2021-06-04
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The object of the present invention is aimed at the low asymmetric reduction activity of the existing aldehyde and ketone reductase KmAKR to 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester, the large amount of catalyst (cell) and the unnatural ketone For the problem of low activity of ester substrates, provide a stereoselective aldehyde and ketone reductase KmAKR mutant and use the aldehyde and ketone reductase KmAKR mutant gene recombinant bacteria and its enzyme solution as a biocatalyst for asymmetric reduction of carbonyl Compound preparation chiral alcohol, when the substrate is tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, the specific enzyme activity of mutant strain M7 and mutant strain M9 is compared with the starting strain KmAKR-W297H / Y296W / K29H / Y28A / T63M increased by 1.1 times and 0.63 times respectively, and the amount of substrate, catalyst dosage (S / C) and space-time yield all increased significantly

Method used

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  • Aldehyde ketone reductase KmAKR mutant and application thereof in catalytic synthesis of chiral alcohol
  • Aldehyde ketone reductase KmAKR mutant and application thereof in catalytic synthesis of chiral alcohol
  • Aldehyde ketone reductase KmAKR mutant and application thereof in catalytic synthesis of chiral alcohol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction and screening of aldehyde and ketone reductase mutant library

[0034] 1. Starting strain:

[0035] Taking the engineering bacteria E.coli BL21(DE3) / pET28a(+)-kmakr-W297H / Y296W / K29H / Y28A / T63M in the patent application CN201910932502.0 as the starting strain, denoted as strain M5, activated and extracted the plasmid pET28a(+) -kmakr-W297H / Y296W / K29H / Y28A / T63M, wherein the amino acid sequence of aldehyde and ketone reductase kmakr-W297H / Y296W / K29H / Y28A / T63M is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2 Show.

[0036] 2. Single mutation:

[0037] (1) Construction of mutant library

[0038] The preparation of the aldehyde and ketone reductase KmAKR mutant library was achieved by site-directed mutagenesis and random mutagenesis. The vector pET28a(+)-kmakr-W297H / Y296W / K29H / Y28A / T63M in the strain M5 was used as a template and the primers in Table 1 were used for polymerization. Enzyme chain reaction (PCR). Transfer ...

Embodiment 2

[0074] Embodiment 2: Induced expression of starting strain and mutant strain aldehyde ketone reductase and glucose dehydrogenase

[0075] 1. Glucose dehydrogenase genetically engineered bacteria: Insert the glucose dehydrogenase gene esgdh (GenBank No.KM817194.1, nucleotide sequence shown in SEQ ID NO.7) from Exiguobacterium sibirium DSM 17290 into pET28b (+) between the Nco I and Xho I restriction sites, construct a recombinant expression vector; and transfer this expression vector into E.coli BL21(DE3), pick a single colony and inoculate it into LB medium, at 37°C After culturing for 12 hours, it was confirmed by sequencing that the glucose dehydrogenase was successfully constructed, and E. coli BL21(DE3) / pET28b(+)-esgdh was obtained.

[0076] 2. Induced expression: Inoculate the starting strain M5, mutant strain M7, mutant strain M9, and E.coli BL21(DE3) / pET28b(+)-esgdh in Example 1 into 10 mL of kanamycin containing a final concentration of 50 μg / mL, respectively. In LB liq...

Embodiment 3

[0084] Example 3: Purification of aldehyde and ketone reductase in the starting strain of aldehyde and ketone reductase and its mutant strains

[0085] The starting strain M5, the aldehyde and ketone reductase mutant strain M7 obtained in Example 2, and the wet bacterial cell of the mutant strain M9 were washed twice with 0.9 g / mL physiological saline. According to the amount of 100g / L of the total amount of wet bacteria, add pH 7.0, 100mM PBS buffer to resuspend, and ultrasonically break on the ice-water mixture for 6min. Enzyme solution. By centrifuging at 8000rpm at 4°C for 10min, 20mL of the supernatant was collected, and after microfiltration through a 0.45μm membrane, the filtrate was purified with a Ni affinity column to purify the mutant protein.

[0086] The mutant protein was purified using a nickel affinity column (1.6×10 cm, Bio-Rad, USA), and the specific operation was as follows: ① Pre-equilibrated with buffer A (pH 7.0 containing 300 mM NaCl, 20 mM imidazole, 2...

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Abstract

The invention discloses an aldehyde ketone reductase KmAKR mutant and application thereof in catalytic synthesis of chiral alcohol. The aldehyde ketone reductase KmAKR mutant is obtained by performing single-site or multi-site mutation on the 30 site, the 302 site, the 109 site, the 196 site, the 23<rd> site or the 213<rd> site of an amino acid sequence shown in SEQ ID NO. 1. The specific enzyme activities of the constructed aldehyde ketone reductase mutants M7 and M9 are increased by 1.1 times and 0.63 times respectively compared with those of an original strain, and the T5015 is increased by 6.3 DEG C and 4.9 DEG C respectively. For the mutant M9, when the feeding amount of a substrate 6-cyano-(5R)-hydroxy-3-carbonyl tert-butyl hexanoate can reach 350g / L, the reaction can be completed within 3.7h, the substrate conversion rate is greater than 99%, the dep value of the product is always kept at 99.5% or above, and the space-time yield reaches 1.82kg / L.d.

Description

[0001] (1) Technical field [0002] The present invention relates to a kind of aldehyde and ketone reductase KmAKR mutant derived from Kluyveromyces marx, and bichiral diol 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl in the side chain of atorvastatin Applications in chiral biocatalytic synthesis of esters. [0003] (2) Background technology [0004] "Super statins" such as atorvastatin, rosuvastatin, and pitavastatin are important lipid-lowering drugs for the treatment of cardiovascular and cerebrovascular diseases. Disease morbidity and mortality. So far, the cumulative sales of atorvastatin calcium have exceeded 100 billion US dollars, and it is the most successful single drug variety in the history of the human pharmaceutical industry. [0005] Most statin drugs contain 6-substituted-(3R,5R / S)-tert-butyl dihydroxyhexanoate structure, which is not only an important pharmacophore but also a key synthetic precursor. tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N1/21C12P13/00C12R1/19
CPCC12N9/0006C12P13/004C12Y101/01021
Inventor 王亚军程峰邱帅李树芳郑裕国
Owner ZHEJIANG UNIV OF TECH
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