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Carbonyl reductase mutant and application thereof

A reductase and mutant technology, applied in the field of bioengineering, can solve the problems of low atom economy, low activity and poor solubility of ethylsecodione, and achieve the effect of improved stereoselectivity, good activity and remarkable effect

Active Publication Date: 2020-11-27
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

[0003] Chinese patent document CN201810555674.6 reports the use of carbonyl reductase mutants to realize the ethyl condensate ethyl secodione, and its structural standard naming and Chinese standard naming are: (E)-2-ethyl-2-(2-(6-methoxy- 3,4-dihydronaphthalen-1(2H)-ylidene)ethyl)cyclopentane-1,3-dione, Chinese is (cis)-2-ethyl-2-(2-(6-methoxy-3,4- Efficient reduction of dihydronaphthol-1(2H)ethyl)-1,3-cyclopentadione), but the mutant requires additional addition of glucose dehydrogenase (GDH) and a large amount of glucose as a coenzyme for regeneration, atom economy The solubility is not high, and the generation of by-product gluconic acid makes it necessary to continuously adjust the pH value of the reaction during the reaction process. Due to the poor solubility of the ethyl condensate ethylsecodione, it is necessary to use a certain proportion of organic solvent as a co-solvent
Carbonyl reductase TbADH can use isopropanol to regenerate the coenzyme, but because the structure of the target substrate ethyl secodione has greater steric hindrance than isopropanol, and TbADH has the activity of ethyl secodione very low

Method used

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  • Carbonyl reductase mutant and application thereof
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  • Carbonyl reductase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Construction of carbonyl reductase TbADH mutant library

[0027] According to the structure of TbADH, the non-conserved residues in its substrate binding pocket were selected for saturation mutation, and the mutation primers were designed using the degenerate codon NNK, and pET21a-TbADH was used as a template. The obtained monoclonal colonies were picked into 96-well deep-well plates for culture, and the expressed proteins were screened for high-throughput activity. The screening method was to use ethyl condensates as substrates and detect NADP(H )The change. The specific method of the reaction is: the substrate is dissolved in DMSO, the final concentration is 0.8mM, of which DMSO 60μL, NADPH 0.5mg / mL, crude enzyme solution 20μL, potassium phosphate buffer of pH 7.5 to 200μL, microplate reader detection For the decrease of NADPH at 340 nm, if the enzyme activity is relatively high, the consumption of NADPH will be faster, and the slope of the decline curve...

Embodiment 2

[0029] Example 2 Carbonyl reductase TbADH combinatorial mutant library construction

[0030] According to the results of saturation mutation, mutant 3 with improved activity and optimal stereoselectivity was selected as a template to construct combinatorial mutant libraries respectively (Table 1 shows the primer sequences used in the construction of mutant libraries). Experimental determination of the conversion of the ethyl condensate ethyl secodione (the specific operation was carried out as described in Example 4). The mutants obtained by the two-site combined mutation are mutants 4 and 5, and the protein sequences are SEQ ID NO: 5 and 6. Using mutant 4 as a template, the M285 mutation site is introduced, and the better mutant 6 protein obtained by screening The sequence is SEQ ID NO:7. The results are shown in Table 2.

[0031] Table 1 Primer sequences of mutant library

[0032]

[0033] Table 2 The conversion of ethyl secodione and the activity of isopropanol in the...

Embodiment 3

[0035] Example 3: Inducible expression of carbonyl reductase TbADH mutants

[0036] Prepare 50 mL of seed liquid, 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, 37 Cultivate overnight at 200 rpm. The overnight cultured seed solution was transferred to the fermentation medium (LB medium) at an inoculum of 1%, and cultivated to OD at 37°C and 200 rpm 600 0.6-1.0, add 0.1 mM IPTG, and place at 30°C, 200 rpm for 10-12 h. The bacterial cells were collected by centrifugation at 6000 rpm at 4°C and washed once with sodium phosphate buffer (100 mM, pH 7.0). SDS-PAGE electrophoresis pattern shows the induced expression of TbADH and mutants, such as figure 2 As shown, where, M represents Marker, 1 is TbADH wild-type protein expression supernatant, 2 is TbADH wild-type protein expression precipitate, 3 is representative mutant N114G protei...

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Abstract

The invention provides a carbonyl reductase mutant and application thereof in (13R, 17S)-ethyl secol synthesis. Specifically, ethyl condensate ethyl secodione and isopropanol are taken as substrates for directed evolution to obtain the carbonyl reductase mutant, glucose / glucose dehydrogenase or sodium formate / formate dehydrogenase is prevented from being used as coenzyme for regeneration, only isopropanol is used as a co-substrate and a cosolvent, and carbonyl is reduced to prepare (13R, 17S)-ethyl secol with high chiral purity.

Description

technical field [0001] The invention belongs to the field of bioengineering. Specifically, the invention relates to a carbonyl reductase mutant and its application in preparing chiral ethyl secol and structural analogues. Background technique [0002] The desymmetric reduction reaction of 2,2-disubstituted-1,3-cyclopentanedione is a key step in the synthesis of various steroidal compounds (the formation of chiral quaternary carbon at the 13-position) (Chapelon, A.- S.; Moraléda, D.; Rodriguez, R.; Ollivier, C.; Santelli, M., Enantioselective synthesis of steroids. Tetrahedron 2007, 63 , 11511-11616; Sakata, K.; Wang, Y.; Urabe, D.; Inoue,M., Synthesis of the tetracyclic structure of batrachotoxin enabled bybridgehead radical coupling and Pd / Ni-promoted Ullmann reaction. Org. Lett. 2018, 20 , 130-133.). The product after desymmetric monocarbonyl reduction with ethyl secodione as substrate is the key intermediate in the synthesis of steroidal drugs levonorgestrel and gest...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P41/00C12P7/38C12R1/19
CPCC12N9/0006C12N15/70C12P7/38C12P41/002C12Y101/01184
Inventor 陈曦张红榴刘祥涛冯进辉吴洽庆朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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