Recombinant expression plasmid vector and recombinant strain to be used in producing oxalate decarboxylase, and method of producing recombinant oxalate decarboxylase

A technology of oxalate decarboxylase and expression plasmid, which is applied in the field of oxalate decarboxylase and can solve the problems of insufficient practical or commercial level

Inactive Publication Date: 2010-12-15
AMANO ENZYME INC
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  • Claims
  • Application Information

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Problems solved by technology

However, this productivity, even if adequate at a research level, is not sufficient at a practical or commercial level

Method used

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  • Recombinant expression plasmid vector and recombinant strain to be used in producing oxalate decarboxylase, and method of producing recombinant oxalate decarboxylase
  • Recombinant expression plasmid vector and recombinant strain to be used in producing oxalate decarboxylase, and method of producing recombinant oxalate decarboxylase
  • Recombinant expression plasmid vector and recombinant strain to be used in producing oxalate decarboxylase, and method of producing recombinant oxalate decarboxylase

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preparation example Construction

[0069] (Method for preparing oxalate decarboxylase-producing bacteria and oxalate decarboxylase-producing bacteria)

[0070] If an appropriate host bacterium is transformed with the expression vector of the present invention, a recombinant bacterium producing OXDC can be obtained. Therefore, as a second aspect of the present invention, there are provided (1) a method for producing an OXDC-producing bacterium characterized by transforming a host bacterium with the expression vector of the present invention, and (2) a recombinant bacterium obtained by the production method. The recombinant bacterium of the present invention holds an expression plasmid vector containing a microorganism-derived oxdc gene under the transcriptional regulation of a Bacillus α-amylase promoter. The copy number of the expression plasmid vector is not particularly limited, and is, for example, 1-700.

[0071] The host bacteria here are not particularly limited, and are preferably Escherichia coli or ba...

Embodiment

[0085] 1. Selection of oxdc gene expression system

[0086] To increase productivity, combinations of hosts and promoters were investigated. As hosts, (1) Bacillus subtilis 168 strain (ATCC (American Type Culture Collection)) known as an OXDC producer and (2) Escherichia coli (E. coli JM109 strain (TAKARA BIO company)). In accordance with these hosts, a promoter in which high expression of the introduced gene is expected to be selected ( figure 1 ). In addition, the amy promoter is the promoter of the α-amylase gene. The α-amylase gene is a gene possessed by various Bacillus subtilis (Bacillus genus), and several genes cloned using an α-amylase promoter have been reported so far. The other lac promoter is a general promoter used when Escherichia coli is used as a host, and is known to express a large amount.

[0087] 2. Acquisition of oxdc gene

[0088]The oxdc gene (yvrK) to be cloned was obtained from the chromosome of Bacillus subtilis 168 strain. Specifically, first...

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Abstract

It is intended to provide a means of highly producing oxalate decarboxylase originating in a microorganism. A recombinant expression plasmid vector, which contains a-amylase promoter of a microorganism belonging to the strain Bacillus and an oxalate decarboxylase gene originating in a microorganism that is provided under the control of the promoter, is constructed. A host bacterium is transformed by this vector to give an oxalate decarboxylase-producing bacterium. A recombinant oxalate decarboxylase is produced by culturing the producing bacterium as described above and then harvesting the oxalate decarboxylase thus produced.

Description

technical field [0001] The present invention relates to oxalate decarboxylases of microbial origin. Specifically, it relates to a recombinant expression plasmid vector and recombinant bacteria used in the production of oxalate decarboxylase, a preparation method of oxalate decarboxylase producing bacteria, and a production method of recombinant oxalate decarboxylase. Background technique [0002] Oxalic acid is contained in many foods (especially spinach or other green vegetables, green tea, cocoa beans, etc.), or produced in the human body (generated during metabolism, cannot be further decomposed in the organism, and is excreted together with urine). compound. Oxalic acid is known to be a substance that causes stones by binding to calcium in the human body. In addition, an increase in urinary oxalic acid concentration (increased risk of stone formation) can also be seen due to excessive intake of oxalic acid or excessive production of oxalic acid in the body. In additio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21C12N9/00
CPCC12N9/88C12Y401/01002
Inventor 小山贵史小嶋裕三儿岛宪二箕田正史
Owner AMANO ENZYME INC
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