Application of carbonyl reductase and carbonyl reductase mutants in Indacaterol drug intermediates

A carbonyl reductase and enzymatic activity technology, which is applied in the field of bioengineering, can solve the problems of large steric hindrance of substrate II and no conversion activity of carbonyl reductase, and achieves the effects of improved conversion activity, simple and convenient preparation method, and environmental friendliness.

Active Publication Date: 2020-01-21
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In the currently known reports, there is no report on the biocatalytic preparation of chiral alcohols using substrate II or its analogues. The main reason is that substrate II has a large steric hindrance, and most carbonyl reductases do not convert it. Vitality, and as an intermediate of the drug indacaterol, the configuration of the product should be the R configuration, and the optical purity should be >99%

Method used

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  • Application of carbonyl reductase and carbonyl reductase mutants in Indacaterol drug intermediates
  • Application of carbonyl reductase and carbonyl reductase mutants in Indacaterol drug intermediates
  • Application of carbonyl reductase and carbonyl reductase mutants in Indacaterol drug intermediates

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Experimental program
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Effect test

Embodiment 1

[0018] Embodiment 1: RasADH and mutant gene obtained

[0019] 1.1 Synthesis of RasADH gene and acquisition of its mutant gene

[0020] The RasADH gene is derived from Ralstonia sp.DSM 6428, and has a crystal structure (PDB: 4BMS). According to the codon preference of Escherichia coli, the gene is optimized and sent to the company for synthesis. The codon-optimized genes are respectively A NdeI (CATATG) restriction site was added at the 5' end, and an XhoI (CTCGAG) restriction site was added at the 3' end, and cloned into the PET21a vector. Since the crystal structure of RasADH is known, Discovery Studio 4.1 software was used to dock the substrate II with its three-dimensional structure to obtain the interaction relationship between the substrate and the protein. According to the docking results, the amino acids 189, 191, 202, and 205 near the active center were selected for site-directed mutagenesis, and the PET21a plasmid with the RasADH gene was used as a template and prime...

Embodiment 2

[0035] Embodiment 2: the construction of RasADH and its mutant expression bacteria

[0036] After the digested and purified PCR product was transformed into Escherichia coli BL21(DE3) competent cells by chemical transformation, single clones were picked and placed in 4 mL of LB medium containing ampicillin (10 mg / mL), and fresh bacterial liquid was taken Sent to a sequencing company for sequencing, and the sequencing results showed that the correct mutant was the strain expressing the RasADH mutant.

Embodiment 3

[0037] Example 3: Induced expression of RasADH and mutants thereof

[0038] Prepare 50 mL of seed liquid, and the medium is LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L), pick a single colony of genetically engineered bacteria with an inoculation loop and insert it into the medium, at 37 °C, Incubate overnight at 200 rpm. The seed solution cultivated overnight was transferred to the fermentation medium with an inoculum size of 1%, and cultivated at 25°C and 200rpm for 20h. After 5 mL of the fermentation broth was concentrated and ultrasonicated, the activity of RasADH and its mutants was detected. Enzyme activity definition of RasADH and its mutants: the amount of enzyme needed to consume 1 μmol NADPH per minute is 1 enzyme activity unit (U).

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Abstract

The invention discloses application of carbonyl reductase (RasADH) derived from Ralstonia sp. DSM 6428 and RasADH mutants as biocatalysts for catalyzing 5-(alpha-haloacetyl)-8-substituted oxygen radical-(1H)-quinolin-2-ketone to synthesize Indacaterol chiral intermediates. Being used for reducing the substrate, namely the 5-(alpha-haloacetyl)-8-substituted oxygen radical-(1H)-quinolin-2-ketone, the RasADH is relatively low in enzyme activity, so that addition of a large amount of thallus or protein during transformation is required. By adopting a protein engineering means, seven mutants of theRasADH, namely RasADH E189A, RasADH Q191A, RasADH R202F, RasADH F205A, RasADH Q191A / F205A, RasADH Q191A / R202F / F205A and RasADH E189A / Q191A / R202F / F205A, can be obtained; and the seven RasADH mutants have significantly improved enzyme activity on the substrate. Therefore, highly efficient transformation can be realized during transformation of the substrate; and moreover, when product configurationis R, stereo-selectivity is higher than 99%.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to carbonyl reductase RasADH and mutants from Ralstonia sp.DSM 6428, sequences encoding the enzyme, and asymmetric reduction of carbonyl reductases and mutants in indacaterol pharmaceutical intermediates, Application in the preparation of highly optically pure chiral alcohols. Background technique [0002] Indacaterol is a β-selective adrenoceptor agonist with potent bronchodilatory activity and is useful in the treatment of asthma and chronic obstructive pulmonary disease (COPD). Since 2009, it has been listed in more than 70 countries and regions around the world. In June 2012, it was approved by the State Food and Drug Administration for listing in China. It is the first long-acting β2 receptor agonist approved for the treatment of COPD in China (Seth, H.D.; Sultan, S.; Gotfried, M.H. Role of Indacaterol, a Once-Daily Bronchodilator, in Chronic Obstructive Pulm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P17/12
CPCC12N9/0006C12Y101/01184C12P17/12
Inventor 张红榴陈曦李娟冯进辉吴洽庆朱敦明
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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