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Diketoreductase mutant and application thereof

A technology of reductase and dicarbonyl, which is applied in the field of mutants of dicarbonyl reductase, can solve the problems of large amount of enzyme solution, increased total volume of reaction system, low enzyme catalytic activity, etc., and achieves the improvement of enzyme activity and enzyme specificity. Effect

Active Publication Date: 2015-04-29
ASYMCHEM LAB TIANJIN +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application of industrial production, there are still some problems that need to be further solved, such as the low catalytic activity of the enzyme, which leads to a large amount of enzyme solution, and increases the total volume of the reaction system, resulting in an increase in production batches and production costs.

Method used

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  • Diketoreductase mutant and application thereof
  • Diketoreductase mutant and application thereof
  • Diketoreductase mutant and application thereof

Examples

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Effect test

Embodiment 1

[0056] Site-directed saturation mutation was performed on a double carbonyl reductase (DKR) mutant derived from Rhodococcus erythropolis SK121 strain (the amino acid sequence of which is shown in SEQ ID NO: 6).

[0057] The amino acid sequence of double carbonyl reductase (DKR) was simulated on the Swiss-model website to simulate the three-dimensional structure of the protein, and then the binding simulation between the substrate and the protein was carried out through Docking. Finally, through Pymol analysis, the selection may be related to the binding of the substrate and NAD. Amino acids related to NAD proton transport were used as mutant amino acids ( Figure 4 ).

[0058] According to the mutated amino acid and the base sequence on both sides (see the mutation site in Table 1 for the mutated amino acid), use Primmer 5.0 to design the corresponding mutant primers (Table 1). Using the pET22b(+) expression vector containing the double carbonyl reductase gene (purchased from...

Embodiment 2

[0062] Example 2: Preliminary screening of double carbonyl reductase mutants

[0063] According to the content described in Example 1, pick a single colony on the above-mentioned solid medium and inoculate it in a 96-deep well plate, add 1 ml of LB liquid medium containing 50 μg / ml ampicillin in advance to each well, and culture at 37°C with shaking at 220 rpm After 3 hours, add IPTG at a final concentration of 0.1 mM, induce culture at 18°C, 220 rpm for 16 hours, and centrifuge at 4000 g for 15 minutes to collect the cells, and the cells are broken by an ultrasonic breaker (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), 4 Centrifuge at 12000rpm for 5min to obtain the supernatant, that is, the crude enzyme solution of the mutant, which is used for primary activity screening with a microplate reader. Add 30 μL DMSO, 1.5 μL main material tert-butyl 6-benzyloxy-3,5-dioxo-hexanoate (30 mg / mL dissolved in DMSO), 2.5 μL NADH (20 mg / mL) to the 96-well plate, 216 μL of phosphate bu...

Embodiment 3

[0070] Example 3: Re-screening of double carbonyl reductase mutants

[0071] Inoculate the mutant whose enzyme activity is higher than that of the parent in Example 2 into 500 ml of LB liquid medium containing 50 μg / ml ampicillin, culture with shaking at 37° C. until OD600=0.6, add IPTG to a final concentration of 0.1 mM, and Expression was induced at 18°C. After 16 hours of induction, the cells were collected by centrifugation at 6000g for 10 minutes. The cells were disrupted with an ultrasonic disruptor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), and centrifuged at 10,000 g for 20 min at 4°C to obtain the supernatant, which was used for activity detection. Add 0.05g of the main material (tert-butyl 6-benzyloxy-3,5-dioxo-hexanoate or neopentyl 6-benzyloxy-3,5-dioxo-hexanoate to a 10ml reaction flask ), 0.5ml polyethylene glycol PEG-400, after the raw materials are dissolved, add 4.0ml phosphate buffer (100mM, pH=6.0), the main raw material is evenly dispersed in the b...

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Abstract

The invention discloses a diketoreductase mutant and an application thereof. An amino acid sequence of the diketoreductase mutant is an amino acid sequence which is mutated from an amino acid sequence coded by a sequence shown in SEQ ID NO:9, the mutated amino acid sequence has at least two of the following mutation sites: a 94th bit, a 151st bit, a 231st bit, a 236th bit and a 251st bit, and I of the 94th bit is mutated into V, A or G; V of the 151st bit is mutated into Q, N or S; F of the 231st bit is mutated into W, Y or P; I of the 236th bit is mutated into L, V or A; and Q of the 251st bit is mutated into H, R or K; or the amino acid sequence of the diketoreductase mutant has mutation sites in the mutated amino acid sequence and more than 90% homogenous with the mutated amino acid sequence; and activity of the diketoreductase mutant with the mutation sites is greatly improved.

Description

technical field [0001] The invention relates to the field of enzymes and enzyme engineering, in particular to a double carbonyl reductase mutant and its application. Background technique [0002] As a biocatalyst, enzymes can give full play to their high efficiency and high specificity in living organisms. However, in industrial applications, there are generally problems such as inability to adapt to industrial production conditions and low catalytic ability to unnatural substrates. Enzyme molecules must be modified by means of protein engineering methods to adapt to different application requirements. Protein engineering methods can be summarized into three types: rational design, irrational design and semi-rational design. [0003] Rational design refers to changing individual amino acids in protein molecules through site-directed mutation (Site-directed Mutagenesis) or other methods on the basis of understanding the spatial structure of proteins, thereby producing prote...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/62
CPCC12N9/0006C12N15/70C12P7/62
Inventor 洪浩詹姆斯·盖吉高峰刘立辉刘芳于文燕郭莉娜崔瑜霞唐芳荣张娜
Owner ASYMCHEM LAB TIANJIN
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