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Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid

A hydantoinase and amino acid technology, which is applied in the field of preparing a series of optically pure L-amino acids, can solve the problems of hydantoinase activity and poor thermal stability, and achieve scale production, good substrate tolerance, and avoid inhibition The effect of action

Inactive Publication Date: 2011-05-25
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a bacterial strain that produces hydantoinase and L-N-carbamoylase simultaneously, and the hydantoinase and L-N-carbamoylase produced by the bacterial strain can overcome the present biotransformation DL-5-substituted seawater Due to the disadvantages of hydantoinase and L-N-carbamyl hydrolase activity and poor thermal stability in the process of preparing L-amino acid from N-carbamoyl amino acid, the strain was successfully applied to the efficient production of L-amino acid

Method used

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  • Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid
  • Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid
  • Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] This experiment illustrates the source, screening and identification procedures of strains.

[0030] Screening medium (g L -1 ): DL-5-substituted hydantoin 20, sucrose 20, potassium dihydrogen phosphate 3, sodium chloride 1, magnesium sulfate 0.25, yeast extract 0.1, pH 7.5;

[0031] Phenol red plate medium (g L -1 ): hydantoin or methyl hydantoin 3, peptone 5, yeast extract 3, sodium chloride 3, phenol red 0.05, agar 20, pH 7.7;

[0032] Fermentation medium (g L -1 ): methylhydantoin 3, sucrose 20, yeast extract 1, sodium chloride 3, corn steep liquor 20mL (dry weight about 4g), potassium dihydrogen phosphate 3, magnesium sulfate 0.25, pH 7.5;

[0033] Broth plate medium (g L -1 ): peptone 10, beef extract 5, sodium chloride 5, agar 20, pH 7.5.

[0034] Primary screening:

[0035] About 1,500 soil samples were collected from nature, and about 1 g of each of 3 to 4 kinds of soil samples was added to a test tube filled with 10 mL of screening medium. -1 After three ...

Embodiment 2

[0041] This experiment illustrates the biological properties of Bacillus fordii MH602.

[0042] The present invention identifies the biological characteristics of Bacillus fordii MH602, which is Gram-positive bacteria with spore formation. After growing in the broth plate medium for 16 hours, the colony size is 2-2.5mm, the growth temperature range is 40-55°C, the optimum temperature is 45°C, the growth pH is 6.5-8.5, and the optimum pH is 7.5. Physiological and biochemical manifestations include positive peroxidase test and growth under oxygen conditions.

Embodiment 3

[0044] This experiment illustrates the isolation and cloning procedures of hydantoinase and L-N-carbamoylase genes.

[0045] Total DNA was extracted by the phenol-chloroform method. According to the sequence homology of hydantoinase gene, degenerate primers were designed to amplify the partial sequence of hydantoinase gene, and its flanking sequence was further amplified by genome walking method. The coding sequences of key enzymes hydantoinase and L-N-carbamyl hydrolase were obtained. The PCR fragment containing the mature enzyme coding sequence was recovered by electrophoresis and cloned into pMD18-T vector for sequence analysis. The results showed that this fragment has a full-length reading frame of 1428bp and 1242bp, and the number of amino acids encoding mature hydantoinase and carbamoylase is 474 and 412, respectively. Its amplification process is as figure 1 shown.

[0046] Amplification primers for partial hydantoinase gene fragment H1

[0047] According to the h...

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Abstract

The invention relates to a new microbe Bacillus fordii MH602 strain which can simultaneously produce hydantoinase and L-N-carbamyl hydrolase, the hydantoinase and L-N-carbamyl hydrolase produced by the strain, and an application for preparing serial optical pure L-amino acid through the strain. The hydantoinase and L-N-carbamyl hydrolase that are produced by Bacillus fordii MH602 strain can overcome the defect of worse activity and heat stability of the hydantoinase and L-N-carbamyl hydrolase in the L-amino acid preparation process by prior biologically inverting DL-5 substituted hydantoin or N-hydantoinamino acid, which has wide substrate spectrum, and can catalyze a plurality of substrates and have better substrate selectivity for aromatic hydantoin. For the natural amino acid and non-protein amino acid which are difficult to produce by a fermentation method or a chemosynthesis method, the preparation of L-amino acid by using the Bacillus fordii MH602 is simple and stable in operation, good in repeatability, which realizes stable and cheap preparation of L-amino acid and facilitates to scale production.

Description

technical field [0001] The invention belongs to the technical field of applied microorganisms, and in particular relates to a new microorganism Bacillus fordii MH602 strain producing hydantoinase and L-N: carbamoylase at the same time, and hydantoinase and L-N-carbamoylase genes produced by the strain , and the application of using the strain to prepare a series of optically pure L-amino acids. Background technique [0002] Natural L-amino acids and unnatural L-amino acids and their derivatives are widely used in food, medicine, agriculture and other industries. Hydantoinase and L-N-carbamoylase are one of the key biocatalysts for the production of natural and non-natural L-amino acids and their derivatives, especially suitable for essential amino acids and non-natural amino acids that are difficult to produce by conventional fermentation methods. In recent years, non-natural L-amino acids can be used as components of functional protein inhibitors to prevent HIV infection, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/78C12N15/55C12P13/04C12R1/01
Inventor 何冰芳欧阳平凯梅艳珍柏中中姜岷周华韦萍
Owner NANJING TECH UNIV
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