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A kind of biocatalyst and its preparation method and application

A biocatalyst and functionalization technology, applied in the field of bioengineering, can solve problems such as easy leakage of enzymes, hindrance of substrate binding, enzyme inactivation, etc., and achieve the effects of mild process conditions, low cost, and simple method operation

Active Publication Date: 2020-09-18
CHONGQING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The physical method is simple, fast, and not easy to inactivate the enzyme, but the enzyme is easy to leak, and the carrier itself is easy to cover the active site of the enzyme and hinder the substrate binding
Chemical methods are beneficial to increase enzyme stability, but are prone to enzyme inactivation (Suwan Myung, Chun You and Y.-H. Percival Zhang, Recyclable cellulose-containing magnetic nanoparticles: immobilization of cellulose-binding module-tagged proteins and a synthetic metabolism featuring substrate channeling, J. Mater. Chem. B, 2013, 1, 4419-4427; Wen Wang, Daniel I.C. Wang and Zhi Li, Facile fabrication of recyclable and active nanobiocatalyst: purification and immobilization of enzyme in one pot with Ni-NTA functionalized magnetic nanoparticle ,Chem.Commun.,2011,47,8115–8117; FuhuaZhao, Hui Li, Yijun Jiang, Xicheng Wang and Xindong Mu, Co-immobilization ofmulti-enzyme on control-reduced graphene oxide by non-covalent bonds: anartificial biocatalytic system for the one-pot production of gluconic acid from starch, Green Chem., 2014, 16, 2558-2565)

Method used

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  • A kind of biocatalyst and its preparation method and application
  • A kind of biocatalyst and its preparation method and application
  • A kind of biocatalyst and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Enzyme activity assay

[0058] QNR enzyme activity assay:

[0059] Standard reaction mixture system: buffer solution B (PBS, pH7.2-7.4), 3 μmol 3-quininone, 0.3 μmol NADH, appropriate amount of enzyme QNR, total volume 1 mL. 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.

[0060] GDH enzyme activity assay:

[0061] Standard reaction mixture system: buffer solution B (PBS, pH7.2-7.4), 10 μmol glucose, 1 μmol NAD + , an appropriate amount of enzyme GDH, in a total volume of 1 mL. Changes in absorbance values ​​were measured at λ = 340 nm. Enzyme activity unit definition: 1 μmol NAD converted within 1 min at 25°C + The amount of enzyme required.

Embodiment 2

[0063] Co-immobilization of His-QNR and His-GDH

[0064] Take 50mg Ni 2+ Functionalized agarose microspheres, equilibrated with buffer solution A (containing 50mM sodium dihydrogen phosphate and 300mM sodium chloride, pH value 7.8-8.2); then add 2mL containing quinine reductase (His-QNR) and glucose Hydrogenase (His-GDH) cell lysate mixed supernatant, diluted to 5 mL with buffer solution A containing 10 mM imidazole. At a temperature of 25° C., pH 8.0, and a rotational speed of 50 rpm, the above suspension was continuously stirred for 2 h. The above suspension was centrifuged to collect the enzyme-loaded agarose microspheres, which were washed twice with buffer solution A. The washed enzyme-loaded agarose microspheres were redispersed in buffer solution A, and incubated in a refrigerator at 4°C for 4 days.

[0065] The biocatalyst prepared in embodiment 2 is carried out polyacrylamide gel electrophoresis (SDS-PAGE) analysis

[0066] SDS-PAGE analysis results are shown in t...

Embodiment 3

[0078] Biocatalyst catalyzed asymmetric synthesis of (R)-3-quinine alcohol:

[0079] Reaction system: 50mg biocatalyst, 5mM 3-quinine, 9mM glucose, 0.05mM NAD + and 0.05mM NADH, buffer solution B (10mM phosphate buffered saline, PBS), containing NaCl 137mM, KCl 2.7mM, NaCl 2 HPO 4 10mM, KH 2 PO 4 2mM, pH 7.2-7.4), total volume 10mL. The reaction temperature is 25°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 biotransformation is complete, the reaction mixture is centrifuged to separate the biocatalyst. 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 three times, collect the organic phase, concentrate under reduced pressure and evaporate to dryness. At 90°C, dissolve the solid wit...

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Abstract

The invention provides a biocatalyst which contains NADH-dependent specific 3-quinuclidone reductase (QNR), glucose dehydrogenase (GDH) and Ni<2+>functionalized agarose microspheres. The biocatalyst has diffraction peaks when diffraction angles 2 theta are 27.5+ / -0.2 degrees, 31.7+ / -0.2 degrees, 45.5+ / -0.2 degrees, 52.1+ / -0.2 degrees, 66.4+ / -0.2 degrees and 75.8+ / -0.2 degrees respectively. The biocatalyst provided by the invention has the benefits that a preparation method is simple to operate, does not need special equipment and is mild in process conditions, low in cost and suitable for industrialization. The biocatalyst provided by the invention is used for bio-catalytic synthesis of (R)-3-quinuclidone, can realize carbonyl reduction and co-enzyme in-situ regeneration at the same time, and does not need to add expensive coenzymes NADH. Meanwhile, the biocatalyst provided by the invention can be recycled and has long-term operation stability.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a 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 drugs such as aclidinium bromide, solifenacin, ravatropate and tasalidine. At present, the industry mainly uses chiral catalysts to asymmetrically reduce 3-quinine to synthesize (R)-3-quinine alcohol, such as: XylSkewphos / PICA-Ruthenium(II) complex or BINAP / IPHAN-Ru(II) complex things etc. However, this chemical synthesis method needs to screen chiral ligands; the transition metals used are expensive, highly toxic, and difficult to remove from the product; and the prepared product has low optical purity and needs further purification. Another method is racemic resolution, which has the disadvantage that its theoretical yield is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12P41/00C12P17/18
CPCC12N9/0006C12P17/182C12Y101/01184C12Y101/9901
Inventor 李伟陈倩刘笃强李晶于明安
Owner CHONGQING MEDICAL UNIVERSITY
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