Aldo-keto reductase and application thereof in synthesis of (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate

An aldehyde-ketone reductase and reductase technology, applied in the field of bioengineering, can solve the problems of cumbersome operation, low recovery rate and the like

Active Publication Date: 2016-06-01
弈柯莱生物科技(集团)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the above-mentioned reported methods, the main configuration of the product obtained by the asymmetric reduction of the carbonyl group of baker’s yeast is (2S,3S)-configuration products, which need to undergo the step of configuration reversal by chemical methods, the operation is cumbersome, and the recovery low rate

Method used

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  • Aldo-keto reductase and application thereof in synthesis of (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate
  • Aldo-keto reductase and application thereof in synthesis of (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate
  • Aldo-keto reductase and application thereof in synthesis of (2S,3R)-2-benzoylaminomethyl-3-hydroxybutyrate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 random mutation

[0050] Using the aldehyde and ketone reductase gene BYKY-KRED derived from Chinese patent application 201410541899.8 (its nucleotide sequence is shown in SEQ ID NO: 1 of this application) as a template, with SEQ ID NO: 5 and SEQ ID NO: 6 as primers, BYKY-KRED was subjected to random mutation. The random mutation PCR program is as follows: 10×MutazymeII reaction buffer 5 μL, 40 mMdNTP mix 1 μL (final concentration 200 μM), forward primer and reverse primer 250 ng / μL, MutazymeII DNA polymerase 2.5 U / μL, DNA template 1 ng, ddH 2 O to make up 50 μL. PCR amplification steps are: (1) 95°C, pre-denaturation for 2min; (2) 95°C, denaturation for 30s; (3) 56°C annealing for 30s; (4) 72°C extension for 1min; steps (2) to (4) repeated 30 times; (5) Continue extending at 72°C for 10 minutes, then cool to 4°C. The PCR product was purified by agarose gel electrophoresis, and the target band in the range of 800 to 900 bp was recovered using an agarose gel...

Embodiment 2

[0051] Embodiment 2 aldehyde and ketone reductase gene isolation

[0052]Select positive colonies and add them to a deep-well 96-well plate containing 1mL LB medium (containing 100 μg / mL ampicillin) for culture to obtain BYKY-KRED mutant genetically engineered Escherichia coli, which is inoculated into 100mL liquid LB medium for cultivation. Transfer the overnight culture to 1L of fresh LB liquid medium and grow to OD 600 When the temperature reached 0.6-0.8, IPTG was added to a final concentration of 100 μM to induce the expression of the recombinant protein, and the temperature was lowered to 30°C to continue culturing for 24 hours. Centrifuge at 5000rpm to collect the bacteria, wash once with 0.2M sodium phosphate buffer (pH7.0), and resuspend the bacteria in 500 μL of PBS lysis buffer (pH7.2) containing 1mg / mL lysozyme and 1U / mL Benzonase nuclease, Freeze and thaw 3 times at -80°C to break the bacteria, centrifuge at 10,000rpm for 10min, take 50μL of the supernatant of th...

Embodiment 3

[0053] Embodiment 3 high-density fermentation

[0054] The BYKY-KRED mutant genetically engineered Escherichia coli obtained according to Example 2 was inoculated into a 1L shake flask filled with 200 mL of LB medium, and cultured at 37° C., 180-220 rpm for 10-16 hours. The above-mentioned cultured seed culture solution was inoculated in a 3L upper tank fermentation medium (M9 medium, containing glucose 4g / L, disodium hydrogen phosphate 12.8g / L, potassium dihydrogen phosphate in a ratio of 10% (v / v). 3g / L, ammonium chloride 1g / L, sodium sulfate 0.5g / L, calcium chloride 0.0152g / L, magnesium chloride hexahydrate 0.41g / L), at 20~30℃, 300~800rpm, air flow 2~ Cultured under the condition of 6L / min. After culturing for 6-10 hours, feed medium containing 50% glycerol was fed at a rate of 5-20 mL / h until the end of fermentation. Fed feed medium for several hours to OD 600 When reaching 20-40, add 0.1-1mM IPTG to start induction. After 10 to 20 hours of induction, put the tank into...

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Abstract

The invention provides a new aldo-keto reductase mutant and a method using the recombinant aldo-keto reductase mutant or a lyophilized powder thereof to carry out asymmetric reduction. When the new aldo-keto reductase is used to catalyze a substrate with the concentration reaching up to 1000g / L, the optical purity of products still reaches up to 98% or above, and extra addition of an expensive coenzyme is avoided. Compared with other asymmetric reduction preparation methods, a method using the aldo-keto reductase has the advantages of high concentration of the products, high optical purity of the products, mild reaction conditions, environmental protection, simple operation and easy industrial amplification, so the aldo-keto reductase has very good industrial application prospect.

Description

technical field [0001] The present invention relates to the technical field of bioengineering, in particular to an aldehyde and ketone reductase mutant, the construction of a genetically engineered bacterium for producing the aldehyde and ketone reductase mutant, a production method of the aldehyde and ketone reductase mutant and its application in biocatalysis Application in the preparation of (2S,3R)-2-benzamidomethyl-3-hydroxybutyrate. Background technique [0002] Enzymes belonging to the class of ketoreductases (KRED) or carbonyl reductases are useful for the synthesis of optically active alcohols from the corresponding precursor stereoisomeric ketone substrates or the corresponding racemic aldehyde substrates. Typically, KRED converts ketone and aldehyde substrates to the corresponding alcohol products, but can also catalyze the reverse reaction, oxidizing alcohol substrates to the corresponding ketone / aldehyde products. The reduction of ketones and aldehydes and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/21C12P13/02C12P13/00C12R1/19
Inventor 罗煜丁时澄瞿旭东李辉王海涛
Owner 弈柯莱生物科技(集团)股份有限公司
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