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Application of protein and nucleic acid molecule encoding the protein in preparation of phosphohydrolase

A technology of phosphohydrolase and nucleic acid molecules, which is applied in the field of protein and nucleic acid molecules encoding the protein in the preparation of phosphohydrolase, which can solve the problems of many by-products and difficult purification

Active Publication Date: 2019-01-18
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are mainly two methods to produce nucleotides. One is chemical synthesis, which uses bacterial fermentation to produce inosine, and then uses POCl 3 Phosphorylation, this method has many by-products and is difficult to purify; another method is to use Escherichia coli inosine kinase to phosphorylate inosine, this process requires the participation of ATP, and ATP needs to be regenerated by fermentation of Ammonogenes bacteria, which limits the enzymatic method synthetic application

Method used

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  • Application of protein and nucleic acid molecule encoding the protein in preparation of phosphohydrolase
  • Application of protein and nucleic acid molecule encoding the protein in preparation of phosphohydrolase
  • Application of protein and nucleic acid molecule encoding the protein in preparation of phosphohydrolase

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Embodiment 1

[0059] Embodiment 1, preparation and functional verification of acid phosphatase BmacpA

[0060] 1. Cloning of BmacpA gene and BmacpB gene

[0061] Genomic DNA of Bacillus megaterium ACCC10010 and Bacillus megaterium ACCC02970 were extracted respectively, using the genomic DNA of Bacillus megaterium ACCC10010 as a template, using P1 and P2 as primers, PCR amplified the BmacpA gene; using the genomic DNA of Bacillus megaterium ACCC02970 as a template, Using P3 and P4 as primers, PCR amplified BmacpB gene. Among them, the sequences of P1, P2, P3 and P4 are as follows: P1: 5′-ATGTATGTGAAACGATATCG-3′, P2: 5′-CTACTTTTTGTCGAACACATA-3′, P3: 5′-ATGGTAAATCGCACTACAAA-3′, P4: 5′-CTATTTTTGGTTATATAAGC-3 '.

[0062] The obtained BmacpA gene PCR product and the BmacpB gene PCR product are subjected to electrophoresis respectively, and the results show that the BmacpA gene PCR product and the BmacpB gene PCR product are about 600bp bands, and the BmacpA gene PCR product and the BmacpB gene ...

Embodiment 2

[0108] Embodiment 2, the cultivation and functional identification of acid phosphatase phosphate-dissolving engineering bacteria

[0109] 1. Construction of Bacillus megaterium acid phosphatase BmacpA gene shuttle expression vector

[0110] In order to obtain phosphate-soluble engineering bacteria that highly express Bacillus megaterium acid phosphatase BmacpA gene, it is necessary to first construct a shuttle expression vector that can express across hosts. pHT43 (product of German MoBiTec company, purchased from Wuhan Miaoling Biotechnology Co., Ltd.) is a commonly used shuttle expression vector, carrying BamHI and XbaI enzyme recognition sites, with ampicillin and chloramphenicol resistance genes, induced by IPTG, can highly Level secreted expression of target protein. DNAMAN software was used to analyze the gene sequence of Bacillus megaterium acid phosphatase BmacpA, and it was found that there were no restriction sites of BamHI and XbaI, and the recombinant plasmid expr...

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Abstract

The invention discloses the application of a protein and a nucleic acid molecule encoding the protein in the preparation of a phosphohydrolase. The use of the protein of c) or d) as a phosphohydrolase: c) a fusion protein obtained by labeling a carboxyl terminal or / and an amino terminal fusion protein of the protein of a) or b); Said a) being SEQ ID NO. 2 No. 1-203 A protein composed of amino acidsequences at position; Said b) being SEQ ID NO. 2 No. 26-203 A protein composed of amino acid sequences at position; D) substituting and / or deleting and / or adding one or several amino acid residues into the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 6 to obtain a protein with acid phosphatase activity. The present invention can be used for cultivating bioengineered bacteria for efficiently activating phosphorus nutrients in soil.

Description

[0001] This application is a divisional application with the application number 201610051877.2, the application date is January 26, 2016, and the invention name is "Application of acid phosphatase and related biological materials in the construction of phosphorus-dissolving engineering bacteria". technical field [0002] The invention relates to the application of the protein in the biological field and the nucleic acid molecule encoding the protein in the preparation of phosphohydrolase. Background technique [0003] Phosphorus is one of the three essential elements for plant growth and development, and plays an important role in the life process. The phosphorus used by plants mainly comes from the soil. At present, 2 / 3 of the cultivated land in my country is deficient in phosphorus. The main reason for phosphorus deficiency is the insufficient content of available phosphorus in the soil. About 95% of phosphorus is insoluble and ineffective phosphorus, which is difficult fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55
CPCC12N9/16C12N15/70C12N15/74C12Y301/03002
Inventor 孙静文周卫程明芳李书田王玉军
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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