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Self-feeding type dual-function biocatalyst and preparation method and application thereof

A biocatalyst, self-sufficient technology, applied in the field of bioengineering, can solve the problems of long production cycle, time-consuming, low catalytic efficiency, etc., and achieve the effects of mild process conditions, large economic benefits, and high catalytic activity

Inactive Publication Date: 2020-07-28
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main defects of the chemical synthesis method: (1). The transition metal used is expensive, highly toxic, and difficult to remove from the product; (2). Time-consuming screening and synthesis of chiral ligands are required; (3). The prepared product Optical purity is low, need further purification
The biocatalytic process of enzyme-coupled asymmetric synthesis of (R)-3-quinine alcohol as figure 1 As shown, the enzyme coupling is to complete the two processes of carbonyl reduction and coenzyme regeneration in two independent bacteria respectively. The transfer between two bacteria is blocked, mass transfer resistance leads to long biotransformation time, low catalytic efficiency, long production cycle and increased cost, which is not conducive to industrialization

Method used

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  • Self-feeding type dual-function biocatalyst and preparation method and application thereof
  • Self-feeding type dual-function biocatalyst and preparation method and application thereof
  • Self-feeding type dual-function biocatalyst and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Enzyme Activity Determination

[0047] Assay of Carbonyl Reductase Activity in a Self-Contained Bifunctional Fusion Enzyme:

[0048] Standard reaction mixture system: buffer solution (PBS, pH7.2-7.4), 3 μmol 3-quinine, 0.3 μmol NADH, appropriate amount of self-contained bifunctional biocatalyst, total volume 1mL. Changes in absorbance values ​​were measured at λ = 340 nm. Definition of enzyme activity unit: the amount of enzyme required to convert 1 μmol NADH within 1 min at 25°C.

[0049] Determination of Coenzyme Regenerating Enzyme Activity in a Self-Contained Bifunctional Fusion Enzyme:

[0050] Standard reaction mixture system: buffer solution (PBS, pH7.2-7.4), 10 μmol glucose, 1 μmol NAD + , an appropriate amount of self-contained bifunctional biocatalyst, with a total volume of 1 mL. Changes in absorbance values ​​were measured at ? = 340 nm. Enzyme activity unit definition: 1 μmol NAD converted within 1 min at 25°C + amount of enzyme required.

Embodiment 2

[0052] Expression of a self-sufficient bifunctional fusion enzyme

[0053] Take the expression plasmid pET28a-mlg to transform competent Escherichia coli E.coli BL21(DE3), inoculate LB solid medium (1% peptone, 0.5% yeast extract, 1% sodium chloride and 1.5 % agarose), cultured overnight at 37°C. Pick a single clone colony and inoculate it into LB liquid culture medium (1% peptone, 0.5% yeast extract and 1% sodium chloride), cultivate overnight at 37°C with a rotational speed of 150rpm. Inoculate the LB liquid medium with the culture solution at a ratio of 1:50, cultivate to OD at 37°C with a rotational speed of 150rpm 600 About 0.6-1.0. Add 0.2mM isopropyl-β-D-thiogalactoside to induce the expression of self-sufficient bifunctional fusion enzyme. The culture temperature is 25°C, the rotation speed is 150rpm, and the culture time is 36 hours. 4°C, 10,000rpm centrifuge to collect the bacteria, wash with buffer solution, and obtain self-supporting bifunctional enzyme whole-ce...

Embodiment 3

[0067] Biocatalyst catalyzed asymmetric synthesis of (R)-3-quinine alcohol:

[0068] Reaction system: 80mg biocatalyst, 5mM 3-quinine, 9mM glucose, 0.2mM NAD + , buffer solution A (10mM phosphate buffered saline, PBS, containing NaCl 137mM, KCl 2.7mM, Na 2 HPO 4 10mM, KH 2 PO 4 2mM, pH 7.2-7.4), total volume 10mL. The reaction temperature is 30°C, stirring continuously at 100rpm, and the pH is controlled at 7.5-8.0 during the reaction. The reaction is monitored by TLC, and the mobile phase is V 二氯甲烷 / V 甲醇 =9 / 1. After the biotransformation was completed, 0.1 g of trifluoroacetic acid was added to remove the protein, and the reaction mixture was centrifuged. The supernatant was adjusted to a pH greater than 12 with high-concentration NaOH, and then concentrated under reduced pressure at 80°C to 1 / 4 of the total volume. Add an equal volume of n-butanol for extraction 3 times, collect the organic phase, concentrate under reduced pressure and evaporate to dryness. Dissol...

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Abstract

The invention provides a self-feeding type dual-function biocatalyst. The self-feeding type dual-function biocatalyst comprises NADH-dependent special 3-quininone reductase(MlQR), glucose dehydrogenase(GDH) and a flexible connector. The biocatalyst has carbonyl reduction and coenzyme in situ regeneration capacity, the carbonyl reduction activity is 3678U / g, the coenzyme regeneration activity is 5070U / g, cyclic utilization of a coenzyme is guaranteed, biotransformation can be smoothly performed, the maximum substrate consumption is 486g / L, and the space time yield is 1505.5 g.L<-1>d<-1>. The biocatalyst is used for biocatalysis synthesis of (R)-3-quinuclidinol, can realize carbonyl reduction and coenzyme in situ regeneration at the same time, and is free from additional addition of costly coenzyme NADH. The preparation method of the biocatalyst is simple to operate, is free from special equipment, and is mild in technology condition, low in cost and suitable for industrialization.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a self-supporting bifunctional biocatalyst and its preparation method and application. Background technique [0002] (R)-3-quinine alcohol (molecular formula C 7 h 13 NO, molecular weight 127.18, CAS number: 25333-42-0) is a key intermediate for the synthesis of marketed drugs such as aclidinium bromide, ravatorate, solifenacin and penhyclidine. At present, the industry mainly uses chiral catalysts to asymmetrically hydrogenate 3-quinine to synthesize (R)-3-quinine alcohol, such as: XylSkewphos / PICA-Ruthenium(II) complex or BINAP / IPHAN-Ru(II) complex things etc. The main defects of the chemical synthesis method: (1). The transition metal used is expensive, highly toxic, and difficult to remove from the product; (2). Time-consuming screening and synthesis of chiral ligands are required; (3). The prepared product The optical purity is low and further purifica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/70C12N15/62C12P17/18C12R1/19
CPCC12N9/0006C12N15/70C12P17/182C12Y101/9901C07K2319/00
Inventor 李伟陈倩李珊珊陈宇涵石山李阳于明安
Owner CHONGQING MEDICAL UNIVERSITY
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