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Recombinant bacillus amyloliquefaciens as well as construction method and application thereof

A technology of amylolytic spores and construction methods, applied in the field of recombinant Bacillus amyloliquefaciens and its construction, can solve the problems of low price and high cost of polyglutamic acid degrading enzymes, achieve great application advantages and save production costs

Active Publication Date: 2018-10-09
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Purpose of the invention: In order to solve the problem that the polyglutamic acid degrading enzyme used in the preparation of small molecule γ-PGA by the existing biological enzymatic method is less and expensive, the first aspect of the present invention provides a recombinant Bacillus amyloliquefaciens, which can secrete polyglutamic acid Glutamic acid degrading enzyme is used to produce low-molecular-weight polyglutamic acid; the second aspect of the present invention provides the construction method of recombinant Bacillus amyloliquefaciens, and the third aspect of the present invention provides the application of recombinant Bacillus amyloliquefaciens

Method used

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  • Recombinant bacillus amyloliquefaciens as well as construction method and application thereof
  • Recombinant bacillus amyloliquefaciens as well as construction method and application thereof
  • Recombinant bacillus amyloliquefaciens as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Construction of embodiment 1 plasmid pNX01

[0060] (1) Construction of vector skeleton Frag1

[0061] Using the shuttle plasmid pHY300PLK (TaKaRa Code No.3060) as a template, the origin of replication ori-177 of Escherichia coli and the ampicillin selection marker (such as SEQ ID NO: 1) were obtained, and the primer ori-F: 5'--3'( Such as SEQ ID NO: 10) and Amp-R: 5'--3' (such as SEQ ID NO: 11) for PCR amplification, the reaction conditions are shown in Table 1, using the DNA gel recovery kit (AP -MN-P-250G) recover the fragment to obtain the vector backbone Frag1.

[0062] Table 1 PCR reaction system and reaction conditions of Frag1

[0063]

[0064] (2) Construction of vector skeleton Frag2

[0065] According to the replication protein sequence (as shown in SEQ ID NO: 2) of the endogenous plasmid of B. amyloliquefaciens NX-2S (Bacillus amyloliquefaciens CCTCC NO: M 2016346) that has been determined, design primer rep-F respectively: (as shown in SEQ ID NO :12)...

Embodiment 2

[0091] Construction of embodiment 2 recombinant plasmid pNX01-pgdS

[0092] The polyglutamic acid degrading enzyme gene PgdS is derived from Bacillus subtilis B. subtilis NX-2, and the primers PgdS- F: (such as SEQ ID NO: 20) and PgdS-R: (such as SEQ ID NO: 21), use the bacterial genomic DNA extraction kit (TIANamp Bacteria DNA Kit) provided by Tiangen Biochemical Technology Co., Ltd. to extract the genome to extract The genome of pgdS was used as a template, and a standard PCR amplification system and program was used (see Table 9 for PCR reaction conditions) to obtain a pgdS gene fragment, the nucleotide sequence of which was shown in SEQID NO:9.

[0093] Table 9 PCR reaction system and reaction conditions of polyglutamic acid degrading enzyme gene PgdS

[0094]

[0095] The plasmid pNX01 was double-digested with Spe I and Not I, and the enzyme digestion system and reaction conditions are shown in Table 10;

[0096] Table 10 Plasmid pNX01 digestion reaction system and r...

Embodiment 3

[0101] Example 3 Pretreatment of recombinant plasmid pNX01-pgdS--demethylation

[0102] The recombinant plasmid pNX01-pgdS and E.coli GM2163 (Shanghai Haoran Biotechnology Co., Ltd. No. M0099) (competent mixing, and the same heat shock transformation method as in step (4) of Example 1 were used for the sequencing comparison. The plasmid was transformed into GM2163, and the transformants were picked for enzyme digestion verification again. The verified correct plasmid could be directly used as the plasmid to be electroporated because it did not have a BamH I site and avoided the recognition and cutting of the Bacillus amylobacter restriction repair system.

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Abstract

The invention discloses a construction method of recombinant bacillus amyloliquefaciens. The method is characterized in that bacillus amyloliquefaciens in which indigenous plasmids are removed are used as host bacteria; a polyglutamate degrading enzyme gene pgdS is inserted between Spe I and Not I restriction enzyme cutting sites of a pNX01 carrier to obtain recombinant plasmids, and the recombinant plasmids are imported into the host bacteria to obtain the product. The invention further discloses the recombinant bacillus amyloliquefaciens constructed through the abovementioned construction method and application thereof in fermenting and producing of gamma-polyglutamic acid; the process of fermenting and producing gamma-PGA and the polyglutamic acid degrading enzyme process are coupled torealize the effect of directly obtaining two products of low-molecular weight gamma-PGA and polyglutamic acid degrading enzyme by one-step fermenting; the gamma-PGA is degraded through the polyglutamic acid degrading enzyme which is secreted to the outside of a cell during being the production of high polymer gamma-PGA. Therefore, the cumbersome operation of adding degrading enzyme from outside is avoided; the production technology is simplified; the low-molecule polyglutamic acid is synthesized by one-step fermenting.

Description

technical field [0001] The invention relates to bacillus amyloliquefaciens, in particular to a recombinant bacillus amyloliquefaciens and its construction method and application. Background technique [0002] γ-polyglutamic acid (γ-PGA) is a kind of homopolyamino acid polymerized by D / L-glutamic acid monomer through γ-glutamine bond. Its unique molecular structure endows it with excellent physical and chemical properties , such as water solubility, biocompatibility, biodegradability and edibility, etc., have been widely used in food, agriculture, cosmetics, medicine, water treatment and other fields. As a glutamic acid polymer, γ-PGA has a molecular weight that is an important parameter affecting its biological performance. Different uses have different requirements for the molecular weight of γ-PGA. In recent years, studies have found that low-molecular-weight polyglutamic acid (Mw<500kDa) has unique physiological properties, especially in the fields of cosmetics and me...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/55C12N15/75C12N15/65C12N15/66C12P13/02C12R1/07
CPCC12N9/80C12N15/65C12N15/66C12N15/75C12P13/02
Inventor 徐虹沙媛媛李莎许宗奇冯小海邱益彬张亚涛
Owner NANJING UNIV OF TECH
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