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Ketoreductase mutant and method for producing chiral alcohol

A technique of reducing enzymes and mutants, which is applied in the field of compound synthesis and can solve problems such as low catalytic efficiency

Active Publication Date: 2019-09-20
ASYMCHEM LAB TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although a variety of ketoreductases have been used in commercial production, there are no ketoreductases that can catalyze certain substrates, or the catalytic efficiency is very low

Method used

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  • Ketoreductase mutant and method for producing chiral alcohol
  • Ketoreductase mutant and method for producing chiral alcohol
  • Ketoreductase mutant and method for producing chiral alcohol

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Experimental program
Comparison scheme
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Embodiment approach

[0049] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecules in the above recombinant plasmids are placed in appropriate positions of the recombinant plasmids, so that the above DNA molecules can be replicated, transcribed or expressed correctly and smoothly.

[0050] Although the qualifier used in the present invention is "contains" when limiting the above-mentioned DNA molecule, it does not mean that other sequences irrelevant to its function can be arbitrarily added at both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cutting sites at both ends of the DNA sequence, or additionally add start codons, stop codons, etc., therefore, if using A closed-ended statement will not truly cover these situations.

[0051]The t...

Embodiment 1

[0065] Add 10 mg of 2,6-dichloro-3-fluoroacetophenone to a 10 mL reaction bottle, add 0.1 M PB (phosphate buffer) 7.0, 100 mg isopropanol, and 0.1 mg NAD + , add 10mg of ketoreductase (see Table 1), mix well, the total volume is 1mL, and react at 30°C and 200 rpm for 40 hours. Add 2 mL of ethyl acetate to the reaction sample system, mix well, place in a 5 mL EP tube, and centrifuge at 12,000 rpm for 3 minutes. Take 15 μL of the supernatant in a sample delivery bottle, add 1 mL of ethyl acetate, and detect by HPLC with a detection wavelength of 210 nm.

[0066] Table 1

[0067] mutant active WT - E144S + E144T ++ E144V ++ E144A +++ L152F + L152R + L152A + L152V + E201A + D202A +

[0068] The activity is expressed by the multiple increase compared with the mother parent, + is increased by 1 to 5 times, ++ is increased by 5 to 10 times, and +++ is increased by 10 to 50 times.

[0069] It can be se...

Embodiment 2

[0075] Add 10 mg of 2,6-dichloro-3-fluoroacetophenone to a 10 mL reaction bottle, add 0.1 M phosphate buffer pH 7.0, 100 mg isopropanol, and 0.1 mg NAD + , add 10mg of ketoreductase (see Table 2 for details), mix well, the total volume is 1mL, and react at 30°C and 200 rpm for 40 hours. Add 2 mL of ethyl acetate to the reaction sample system, mix well, place in a 5 mL EP tube, and centrifuge at 12,000 rpm for 3 minutes. Take 15 μL of the supernatant in a sample delivery bottle, add 1 mL of ethyl acetate, and detect by HPLC with a detection wavelength of 210 nm.

[0076] Table 2

[0077]

[0078]

[0079]

[0080] The activity is expressed by the multiple increase compared with the mother parent, + increased by 1 to 5 times, ++ increased by 5 to 10 times, +++ increased by 10 to 50 times, ++++ increased by 50 to 100 times, +++++ Increased by 100 to 1000 times, ++++++ increased by more than 1000 times.

[0081]In industrial production, substrate concentration is very ...

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Abstract

The invention discloses a ketoreductase mutant and a method for producing chiral alcohol. The ketoreductase mutant has a sequence of amino acid mutation as shown in SEQ1 ID NO: 1, and mutation sites at least comprise one of the following sites: a 6th site, a 21st site, a 42nd site, a 58th site, a 61st site, a 76th site, a 87th site, a 94th site, a 96th site, a 108th site, a 113th site, a 117th site, a 144th site, a 146th site, a 147th site, a 149th site, a 151st site, a 152nd site, a 156th site, a 165th site, a 177th site, a 198th site and the like. According to the technical scheme of the invention, ketoreductase takes a ketone compound as a raw material, the chiral alcohol can be efficiently produced through stereoselective reduction, selective resolution can be achieved, production cost and post-treatment difficulty are reduced, and the method is suitable for popularization and application to industrial production of the chiral alcohol.

Description

technical field [0001] The invention relates to the technical field of compound synthesis, in particular to a ketoreductase mutant and a method for producing chiral alcohol. Background technique [0002] Chiral alcohols widely exist in nature and are the structural units of many important biologically active molecules, as well as important intermediates in the synthesis of natural products and chiral drugs. Many chiral drugs contain one or more chiral centers. The pharmacological activity, metabolic process, metabolic rate and toxicity of different chiral drugs are significantly different. Usually one enantiomer is effective, while the other enantiomer are inefficient or ineffective, or even toxic. Therefore, how to efficiently and stereoselectively construct compounds containing chiral centers is of great significance in pharmaceutical research and development. [0003] Ketoreductase (KRED) can also be called carbonyl reductase (Carbony-reductase), the enzyme classificati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12P7/22
CPCC12N9/0006C12N15/70C12P7/22C12Y101/01184
Inventor 洪浩詹姆斯·盖吉肖毅张娜焦学成张克俭杨益明
Owner ASYMCHEM LAB TIANJIN
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