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A kind of method for preparing chiral amine by multi-enzyme coupling system

A chiral amine and system technology, applied in the field of biocatalytic asymmetric transformation, to reduce production costs, solve the problem of coenzyme regeneration, and promote continuous effects

Active Publication Date: 2019-01-08
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of amine dehydrogenase and other NADH-dependent oxidoreductases to construct a multi-enzyme coupling system to catalyze the preparation of chiral amines and achieve coenzyme regeneration has not been reported.

Method used

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  • A kind of method for preparing chiral amine by multi-enzyme coupling system
  • A kind of method for preparing chiral amine by multi-enzyme coupling system
  • A kind of method for preparing chiral amine by multi-enzyme coupling system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1) Preparation of amine dehydrogenase:

[0031] Strain construction, cultivation and collection: construct the amine dehydrogenase gene shown in SEQ ID NO.1, the amine dehydrogenase gene sequence is derived from Bacillusbadius, and use the above amine dehydrogenase gene to prepare engineering bacteria expressing amine dehydrogenase Recombinant Escherichia coli E. coli BL21(DE3)-adh; the above-mentioned engineered bacteria were inserted into 200 mL LB medium at an inoculum size of 1%, and kanamycin was added before inoculation to make the final concentration 40 μg / mL. The incubation time was 6 hours at 37° C. with a rotating speed of 200 rpm. The inducer IPTG was added so that the final concentration was 10 mg / ml, and culture was continued for 4 hours at 30° C. and 200 rpm.

[0032] Preparation of crude enzyme solution: centrifuge the fermented solution obtained at the end of the culture in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain cells, discard the sup...

Embodiment 2

[0043] 1) Preparation of amine dehydrogenase: same as step 1) of Example 1.

[0044] 2) Preparation of coenzyme regeneration enzyme:

[0045] Formate dehydrogenase:

[0046] Strain construction, cultivation and collection: construct the formate dehydrogenase gene shown in SEQ ID NO.3, the formate dehydrogenase gene sequence is derived from Candida boidinii, and use the above formate dehydrogenase gene to prepare a project that can express formate dehydrogenase Recombinant Escherichia coli E. coli BL21(DE3)-fdh; Inoculate the strain into 200 mL LB medium with 1% inoculum size, and add kanamycin before inoculation to make the final concentration 40 μg / mL. The incubation time was 6 hours at 37° C. with a rotating speed of 200 rpm. The inducer IPTG was added so that the final concentration was 10 mg / ml, and culture was continued for 4 hours at 30° C. and 200 rpm.

[0047] The preparation steps of the crude enzyme solution and the pure enzyme are the same as those of the amine d...

Embodiment 3

[0053] 1) Preparation of amine dehydrogenase: same as step 1) of Example 1.

[0054] 2) Preparation of coenzyme regeneration enzyme: refer to step 2) of Example 1 to obtain glycerol dehydrogenase; refer to step 2) of Example 2 to obtain formate dehydrogenase.

[0055] 3) Using amine dehydrogenase and coenzyme regeneration enzyme to construct a multi-enzyme coupling system, in this example, the amine dehydrogenase-formate dehydrogenase-glycerol dehydrogenase coupling system:

[0056] glycerol, sodium formate and NAD + Soluble in the ammonium chloride-ammonia buffer solution with a concentration of 500mM and a pH value of 8, the addition of glycerol is 2M, the addition of sodium formate is 300mM, NAD + The amount of amine dehydrogenase added is 0.2mM, add amine dehydrogenase enzyme solution, formate dehydrogenase enzyme solution, glycerol dehydrogenase enzyme solution, and make the final concentration of amine dehydrogenase 100U / L, formate dehydrogenase and glycerol dehydrogena...

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Abstract

The invention discloses a method for preparing chiral amine from multi-enzyme coupled systems and belongs to the technical field of biological catalysis asymmetric conversion. An amine dehydrogenase and glycerol dehydrogenase coupled system, an amine dehydrogenase and formate dehydrogenase coupled system as well as an amine dehydrogenase, glycerol dehydrogenase and formate dehydrogenase coupled system are constructed, and asymmetric preparation of the chiral amine and regeneration of coenzyme nicotinamide adenine dinucleotide are realized. The multi-enzyme coupled systems and the method for preparing the chiral amine from the multi-enzyme coupled systems have advantages that the product conversion rate is high, the reaction condition is mild, operation is simple, the coenzyme can be regenerated and the like, the cost is low, the enzyme utilization rate is high, and the method is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of biocatalytic asymmetric transformation, and relates to a method for preparing chiral amines by a multi-enzyme coupling system. Background technique [0002] Chiral amines are the building blocks of many bioactive molecules and important intermediates in the synthesis of natural products and chiral drugs. About 40% of optically active drugs contain chiral amine units. At the same time, chiral amines can be used as resolving agents, chiral auxiliary agents and chiral compounds, and are widely used in fine chemicals, pharmaceuticals and agricultural chemicals. At present, biocatalytic preparation of chiral compounds is widely used in the chemical and pharmaceutical industries and has broad prospects. The asymmetric reductive amination of ketones and amino donors to prepare chiral amines catalyzed by amine dehydrogenase is a simple and efficient reaction route. [0003] Amine dehydrogenases require coenzym...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/00C12N9/06C12N9/04
Inventor 王世珍方柏山任红林鹏
Owner XIAMEN UNIV
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