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Biscarbonyl reductase, and coding gene and application thereof

A technology that encodes genes and reductases, which is applied in the field of biocatalytic synthesis, can solve the problems of large enzyme consumption, environmental pollution, and difficulty, and achieve the effects of simplifying synthesis steps, reducing production costs, and reducing production pollution

Active Publication Date: 2014-07-23
ASYMCHEM LAB TIANJIN +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical synthesis of 3R, 5S-dihydroxyl products has the following problems: generally, chiral metal catalysts are required, and the production cost is high; the optical purity of the product is difficult to meet the requirements; a large amount of organic solvents are used, causing serious environmental pollution
It can be seen that there are still some technical problems in the prior art of synthesizing 3R by biocatalytic methods, 5S-dihydroxy products: the nitrilase reaction route is long and difficult, and the cost is high; Introducing another chiral center by other methods; using deoxyribose-5-phosphate aldolase (DERA) synthesis method requires a large amount of enzyme, strong substrate inhibition and initial reaction raw materials are flammable and explosive reagents , industrial production is more difficult

Method used

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  • Biscarbonyl reductase, and coding gene and application thereof
  • Biscarbonyl reductase, and coding gene and application thereof
  • Biscarbonyl reductase, and coding gene and application thereof

Examples

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Effect test

Embodiment 1

[0023] (1) Cloning and expression of dicarbonyl reductase derived from Rhodococcus erythropolis SK121 strain

[0024] In order to facilitate the expression and identification of the double carbonyl reductase gene, compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers. Its primer pair is as follows: upstream primer SEQ ID NO.3: 5'-GGAATTC CATATG ACCGAACTGAAACAAATCACC-3'; downstream primer SEQ ID NO.4: 5'-CCG CTCGAG ACCTTTGTAGTTGTAAAAGCCGTCAC-3'. 扩增得基因序列(SEQ ID NO.2:ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCACGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCGCGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCGTACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAATTCCCGAGGACATCGCCATCAAGCGCGACACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCGATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGGTCAACAACACTGCCGAGGTCATGGGCAC...

Embodiment 2

[0039] The double carbonyl reductase (SEQ ID NO.5) with a sequence homology of more than 80% in Example 1 is derived from Rhodococcus erythropolis PR4.

[0040] In order to facilitate the expression and identification of the double carbonyl reductase gene, compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers. The primer pair is as follows: upstream primer SEQ ID NO.6: 5'-GGAATTC CATATG ACCGAACTGAAACAAATCACC-3'; downstream primer SEQ ID NO.7: 5'-CCG CTCGAG ACCTTTGTAGTTGTAAAAGCCGTCAC-3'. 扩增得基因序列(SEQ ID NO.5:ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCGCAGATCGCCTATCAGACCGCCTATCACGGATTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAGAAGGCCAAGGCTCGGTTCGACTCGTTGGCCGCGGCCTACAAGGCCGAAAACGTCGAGGGCGCCAAGGAAGGCAAGGCCGACGAAGCGCTACAACGTATTACGTACTCGTACGATCTCGCCGAAGCCGTGACCAAGGCCGATCTTGTCATCGAGGCAATTCCCGAGGACTTCGCCATCAAGCGCGACACCTACGAGAAGCTCGCGGCAGTAGCTCCTGAGCACACGGTGTTCGCAACCAACTCCTCGACGCTTCTGCCGAGCGACCTCAAGGAGTTCACCGGCCGCCCCGAGAAGTTCCTCGCACTGCACTTCG...

Embodiment 3

[0048] A dicarbonyl reductase (SEQ ID NO.9) with a sequence homology greater than 80% in Example 1; this gene is a mutant of SEQ NO.1.

[0049] In order to facilitate the expression and identification of the double carbonyl reductase gene, compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers. Its primer pair is as follows: upstream primer SEQ ID NO.10: 5'-GGAATTC CATATG ACCGAACTGAAACAAATCACC-3'; downstream primer SEQ ID NO.11: 5'-CCG CTCGAG ACCTTTGTAGTTGTAAAAGCCGTCAC-3'. 扩增得基因序列(SEQ ID NO.9:ATGACTGAACTGAAGCAGATCACCGTTCTGGGTACCGGAGTTCTCGGCTCACAGATCGCCTATCAGACCGCCTGTCACGGTTTCGACGTCGTCGCGTACGACATCAACGCCGAGGTCATCGAAAAGGCCAAGGCTCGGTTCGACTCGTTGGCCGCGGCCTACAAGGCCGAGAACGTCGAGGGTGCCAAGGAAGGCAAGGCTGACGAAGCGCTGCAACGTATTACGTACTCGTACGATCTAGGCGAAGCCGTCGCCAAGGCCGACCTGGTCATCGAGGCAGTTCCCGAGGACATCGCCATCAAGCGCGACACCTACGAGAAGCTTGCCACGGTTGCTCCTGAGCACACGGTGTTCGCTACCAACTCCTCGACGCTGCTGCCGAGCGATCTCAAGGAGTTCACCGGCCGTCCCGAGAAGTTCCTCGCACTGCACTTCGCAAATCACGTGTGGG...

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Abstract

The invention discloses a biscarbonyl reductase, and a coding gene and application thereof. The biscarbonyl reductase has one of the following amino acid sequences: 1) amino acid sequence disclosed as SEQ NO.1; or 2) SEQ NO.1-derived amino acid sequence subjected to substitution, and / or deletion and / or addition of one or more amino acids with the function of stereoselectively reducing the formula disclosed in the specification into the formula disclosed in the specification, wherein the SEQ NO.1-derived amino acid sequence has more than 80% of homology with SEQ NO.1.R1 is selected from aryl group, alkyl group, cycloalkyl group, alkyl-substituted aryl group, halogen-substituted aryl group, aryl alkyl heterocyclic group, and cycloheteroalkyl or cyclohetero alkylated alkyl group; and R2 is selected from alkyl group, cycloalkyl group, and haloalkyl group or halocycloalkyl group. The biscarbonyl reductase can reduce a diketone substrate in one step to obtain 3R,5S-dihydroxy compounds with single optical purity.

Description

technical field [0001] The invention relates to the technical field of biocatalytic synthesis, in particular to a double carbonyl reductase, its coding gene and its application. Background technique [0002] Chiral alcohols are an important class of intermediate compounds, widely used in the synthesis of chiral drugs and other chiral fine chemicals. 3R, 5S-dihydroxy-6-benzyloxy-tert-butyl hexanoate is a key chiral intermediate of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor statins, which can It is prepared by chemical synthesis and biocatalytic synthesis. However, the chemical synthesis of 3R, 5S-dihydroxyl products has the following problems: chiral metal catalysts are generally required, and the production cost is high; the optical purity of the product is difficult to meet the requirements; a large amount of organic solvents are used, causing serious environmental pollution. [0003] Synthesis of such compounds by biocatalysis is an effective method, that is, the...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P7/62
CPCC12N9/0006C12P7/62C12Y101/01184
Inventor 洪浩詹姆斯·盖吉陈朝勇周炎高峰吕彤郭丽娜
Owner ASYMCHEM LAB TIANJIN
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