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Marine bacterial strain and method for preparing chiral amine from catalyzing of amine dehydrogenase of marine bacterial strain

A technology for catalytic preparation of chiral amines, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of no gene and protein structure, low reaction selectivity, etc., and achieve simple equipment and high yield. , good catalytic activity and selectivity

Inactive Publication Date: 2016-05-11
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reaction selectivity of the enzyme is still low, and there is no report on gene and protein structure information.

Method used

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  • Marine bacterial strain and method for preparing chiral amine from catalyzing of amine dehydrogenase of marine bacterial strain
  • Marine bacterial strain and method for preparing chiral amine from catalyzing of amine dehydrogenase of marine bacterial strain
  • Marine bacterial strain and method for preparing chiral amine from catalyzing of amine dehydrogenase of marine bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1) Cultivation of bacterial strains: cultivation of Pseudomonas balearica (Pseudomonas balearica): Pseudomonas balearica (P.balearica) is cultured in 50mL216L with the inoculum size of 1% (ml / ml) Base. The composition of 216L medium is 10.0g / L peptone, 5.0g / L yeast powder, 0.5g / L beef extract, 0.5g / L sodium citrate, 0.2g / LNH 4 NO 3 , 1.0g / L sodium acetate, configured with sea water. The culture conditions are as follows: the initial pH is 7.0, the volume fraction of the filling volume is 10%, the culture temperature is 30° C., the shaking table rotates at 150 rpm, and the culture time is 72 hours.

[0048] 2) Preparation of whole cells: centrifuge the fermented liquid obtained in step 1) in a refrigerated centrifuge (4°C, 10000rpm, 15min) to obtain cells, discard the supernatant, and use Tris-HCl buffer (pH7. 6) resuspend, centrifuge after sufficient washing, repeat the above-mentioned precipitation resuspension-washing-centrifugation operation 3 times to obtain cell...

Embodiment 2

[0053] 1)-2) The experimental steps are as in the steps 1)-2) of Example 1, and in step 2), the cells are prepared with 0.2 mol / L potassium phosphate buffer solution of pH 10 to prepare 2500 mg / L cell fluid;

[0054] 3) Whole-cell catalysis: Based on the 2500 mg / L cell fluid obtained in step 2), a whole-cell catalytic system was prepared: 0.01mol / L 5-decanone, 0.01mol / L 2-aminopropane, 0.02mol / L sec-butanol, 0.2mol / L potassium phosphate buffer solution, pH 10, 2500mg / L cells, reaction volume 500mL, reaction system pH 10, reaction temperature 50°C, 300rpm shaking reaction for 70 hours, using the whole cell to catalyze asymmetric reductive amination to obtain R -5-decylamine.

[0055] 4) Separation and detection: Separation was carried out according to step 4) in Example 1, and detected by high performance liquid chromatography, the yield of product amine was 60.1%, and the e.e. value was 95.7%.

Embodiment 3

[0057] 1)-2) The experimental steps are as in the steps 1)-2) of Example 1, and in step 2), the cells are prepared with 0.2 mol / L Tris-hydrochloric acid buffer solution of pH 7.5 to prepare a 25 mg / L cell solution;

[0058] 3) Whole-cell catalysis: Based on the 25mg / L cell fluid obtained in step 2), prepare a whole-cell catalytic system: 0.01mol / L acetophenone, 0.02mol / L ammonia water, 0.01mol / L isopropanol, 0.2mol / L Tris-HCl buffer solution, 25mg / L cells, reaction system pH 7.5, reaction volume 5L, reaction temperature 10°C, 300rpm shaking reaction, reaction time 180 hours, using the whole cell to catalyze asymmetric reductive amination The product R-phenethylamine is obtained.

[0059] 4) Separation and detection: Separation was carried out according to step 4) in Example 1, and detected by high performance liquid chromatography, the yield of product amine was 91.7%, and the e.e. value was 93.2%.

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Abstract

The invention discloses a marine bacterial strain and a method for preparing chiral amine from catalyzing of amine dehydrogenase of the marine bacterial strain and belongs to the technical field of asymmetric synthesis chiral compounds obtained through a biological method. Psedomonas balearica from the sea is obtained through screening, and under the condition that ketone serving as a substrate, an amino donor and an auxiliary substrate are added, the chiral amine is prepared by the utilization of asymmetric reductive amination of microorganism and crude enzyme catalyzed ketone of the bacterial strain. A reaction system and reaction conditions of asymmetric reductive amination of microorganism catalyzing and crude enzyme catalyzing of the strain are determined. The method has good industrial application prospects in the field of preparation of chiral amine through biological catalysis and has important meaning for research on development of marine novel biocatalyst and a green synthesis method for chiral amine in the future.

Description

technical field [0001] The invention relates to a marine bacterial strain and a method for preparing chiral amines catalyzed by amine dehydrogenase thereof. The whole cell and crude enzyme solution are used to catalyze asymmetric reductive amination to obtain chiral amines. Background technique [0002] The ocean is a complex, open, and changing biological organic system, and its complex structural characteristics have created the diversity of biological groups. Marine bacteria have properties not found in other ecosystem members and are an important source of new and unique biocatalysts. The uniqueness of the marine environment and the diversity of bacterial enzymes produce great differences between marine proteins and terrestrial biological proteins. The unique marine environment creates extreme enzymes with characteristics such as salt resistance, heat resistance, cold resistance, pressure resistance, acid resistance and alkali resistance. In recent years, researchers a...

Claims

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Application Information

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IPC IPC(8): C12P13/02C12R1/38
CPCC12P13/001
Inventor 王世珍方柏山任红林鹏
Owner XIAMEN UNIV
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