A method for preparing chiral amines catalyzed by a marine strain and its amine dehydrogenase

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...

Examples

Embodiment 1

[0047] 1) Cultivation of strains: Pseudomonas balearica (Pseudomonas balearica) cultivation: Pseudomonas balearica (P.balearica) with 1% (ml / ml) inoculum size, bacterial classification is inserted into 50mL 216L culture medium. 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 / L NH 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 (pH 7.6 for precipitation) ) resuspended, fully washed and then centrifuged, repeating the above precipitation resuspension-washing-centrifugation...

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 2500mg / L cell fluid obtained in step 2), prepare a whole-cell catalytic system: 0.01mol / L 5-decanone, 0.01mol / L 2-aminopropane, 0.02mol / L sec-butyl Alcohol, 0.2mol / L potassium phosphate buffer, 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 reduction Amination affords 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-hydrochloride buffer solution of pH 7.5 to prepare 25 mg / L cell fluid;

[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 to obtain The product R-phenethylamine.

[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%.

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...

Examples

Embodiment 1

[0047] 1) Cultivation of strains: Pseudomonas balearica (Pseudomonas balearica) cultivation: Pseudomonas balearica (P.balearica) with 1% (ml / ml) inoculum size, bacterial classification is inserted into 50mL 216L culture medium. 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 / L NH 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 (pH 7.6 for precipitation) ) resuspended, fully washed and then centrifuged, repeating the above precipitation resuspension-washing-centrifugation...

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 2500mg / L cell fluid obtained in step 2), prepare a whole-cell catalytic system: 0.01mol / L 5-decanone, 0.01mol / L 2-aminopropane, 0.02mol / L sec-butyl Alcohol, 0.2mol / L potassium phosphate buffer, 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 reduction Amination affords 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-hydrochloride buffer solution of pH 7.5 to prepare 25 mg / L cell fluid;

[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 to obtain The product R-phenethylamine.

[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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