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(+)gamma-lactamase with racemate gamma-lactam resolution activity and its application

A technology for encoding lactamase and lactamase, which is applied in the field of enzyme engineering to achieve high purity and high catalytic efficiency

Inactive Publication Date: 2012-11-28
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the current scientific research and practice, the (+) γ-amidase and its coding gene in Bradyrhizobium japonicum have not been cloned and expressed and applied to obtain high-purity (-) γ-lactam

Method used

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  • (+)gamma-lactamase with racemate gamma-lactam resolution activity and its application
  • (+)gamma-lactamase with racemate gamma-lactam resolution activity and its application
  • (+)gamma-lactamase with racemate gamma-lactam resolution activity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] This embodiment is a (+) γ-lactamase in soybean bradyrhizobium that has the activity of resolving racemate γ-lactam.

[0047] The (+) γ-lactamase (BJA) is the following (a) or (b) protein:

[0048] (a) Its amino acid sequence composition is: SEQ ID No: 1 in the sequence listing;

[0049] (b) The amino acid sequence of SEQ ID No: 1 in the sequence listing has undergone one or more amino acid substitutions, and / or deletions, and / or additions, and has the activity of splitting racemate γ-lactam from SEQ ID No:1 derived protein.

[0050] A (+) gamma-lactamase coding gene with the activity of splitting racemic gamma-lactam is the gene of (a) or (b):

[0051] (a), its nucleotide sequence composition is: SEQ ID No: 2 in the sequence listing;

[0052] (b), the nucleotide sequence encoding the amino acid sequence of SEQ ID No: 2 in the sequence listing.

[0053]A recombinant expression vector contains the (+) gamma-lactamase coding gene.

[0054] A transgenic cell line cont...

Embodiment 2

[0058] This example is a method for preparing (+) γ-lactamase by cloning and expressing the (+) γ-lactamase (BJA) gene in Example 1; and the (+) γ-lactamase Applied method in the resolution of racemic γ-lactams.

[0059] The cloning and expression methods of the (+) γ-lactamase (BJA) gene are as follows:

[0060] A. Genome extraction:

[0061] a. Take out Bradyrhizobium japonicum (CGMCC: 1.2550) bacteria stored at -80°C, inoculate them into the rhizobium fermentation medium at a volume ratio of 0.2%, and carry out shaking culture to obtain the bacterial liquid; The temperature of the shaking culture described above is 30° C., the time is 120 hours, and the rotation speed is 220 rpm;

[0062] The raw materials of the rhizobia fermentation medium include: yeast extract 1g, mannitol 10g, soil extract 20ml, distilled water 800ml;

[0063] The raw materials of the soil extract include: 50g of soil, 200ml of deionized water; the preparation method of the soil extract is as follow...

Embodiment 3

[0099] This example is a method for producing (-) γ-lactam using Bradyrhizobium soybean as a strain.

[0100] The method comprises the steps of:

[0101] A. Strain selection: Bradyrhizobium japonicum was selected as the strain, and the strain preservation number was CGMCC No.1.2550; the Bradyrhizobium japonicum strain was stored at -80°C;

[0102] B. Oscillating culture: Inoculate the Bradyrhizobium soybean species into the Rhizobium culture medium at a volume ratio of 0.2%, and carry out shaking culture to obtain a fermented liquid; the temperature of the shaking culture is 20° C. to 30° C. ℃, the time is 4~7 days, and the rotation speed is 200-250rpm;

[0103] The raw materials included in the rhizobia culture medium are: 0.1% nitrogen source, 1% carbon source, 20% soil extract, and sufficient deionized water;

[0104]The raw materials included in the soil extract are: 25% soil, 75% deionized water; the preparation method of the soil extract is: mix the soil with deionized...

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Abstract

The invention provides a (+)gamma-lactamase with racemate gamma-lactam resolution activity and application of its coding gene in chiral compounds, as well as a method for production of (-)gamma-lactam with bradyrhizobium japonicum. The method comprises: extracting the genomic DNA of the bradyrhizobium japonicum, constructing a recombinant thallus for recombinant protein extraction, then conducting expression to obtain the (+)gamma-lactamase; and adding a gamma-lactam substrate into a phosphate buffer solution in which the recombinant thallus for recombinant protein extraction or the (+)gamma-lactamase suspends, and then carrying out reaction and extraction so as to obtain the (-)gamma-lactam. Or the method can be performed as the following: extracting the genomic DNA of the bradyrhizobium japonicum, constructing the recombinant thallus for recombinant protein extraction, then conducting expression to obtain the (+)gamma-lactamase, adding the substrate, and implementing reaction and extraction, thus obtaining the (-)gamma-lactam. The (+)gamma-lactamase provided in the invention derives from the bradyrhizobium japonicum and has high catalytic efficiency. The obtained (-)gamma-lactam has high purity.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and specifically relates to a (+) gamma-amidase with the activity of splitting racemate gamma-lactam and the application of its coding gene in chiral compounds. Background technique [0002] (-) 2-Azabicyclo[2,2,1]hept-5-en-3-one (abbreviated as (-)γ-lactam) is an important intermediate in the synthesis of antiviral drugs such as abacavir. The (+ / -) γ-lactam of high optical purity is one of the main factors limiting the production cost reduction and yield increase of the above-mentioned various antiviral drugs and metabolic regulation drugs. At present, γ-lactamase is a new type of amidase, which can catalyze the hydrolysis of the amide bond of amide compounds. (+)γ-lactamase can efficiently resolve racemic γ-lactam to obtain optically pure (-)γ-lactam. Optically pure (-)γ-lactam is an important intermediate raw material for the preparation of antiviral drug abacavir ((-)abacavir), which has g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/86C12N15/55C12N15/63C12N5/10C12N1/21C12P41/00C12P17/10C12R1/01C12R1/19
CPCY02P20/582
Inventor 郑国钧朱绍洲宋大伟宫萃宇
Owner BEIJING UNIV OF CHEM TECH
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