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Multi-copy beefy meaty peptide expression gene and carrier, and recombination Pichia pastoris construction

A technology for expressing genes and beef flavor, which is applied in the construction of multi-copy beef flavor peptide expression genes and vectors and recombinant Pichia pastoris, which can solve the problem of excessive linking reagents and acyl carriers, linking reagents affecting products and the environment, and limiting protein hydrolysates Use and other issues to achieve the effect of optimizing selectivity, reducing difficulty, and increasing output

Inactive Publication Date: 2008-03-26
TIANJIN CHUNFA BIO TECH GRP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most flavor peptides are formed by enzymatic hydrolysis of proteins, but a large number of by-products are produced; in addition, bitter peptides are formed during enzymatic hydrolysis of proteins, which severely limits the utilization of protein hydrolysates
Chemical synthesis is also an effective method for synthesizing small molecular peptides, but it has the following disadvantages: ①racemization and side reactions; ②need to protect the genes in the peptide side chain, especially during solid-phase synthesis; ③requires excessive Linking reagents and acyl carriers; ④ Toxic residues of linking reagents affect products and the environment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of 16 copies of BMP

[0034] The self-designed plasmid vector containing 4 copies of BMP expression gene (synthesized by Dalian Bao Biological Company) was digested with Xho I and Sal I, and the digestion system was subjected to agarose gel electrophoresis, and the 106bp size was recovered Small snippet. Then the designed plasmid vector containing 4 copies of BMP expression gene was single-cut with Xho I, and then dephosphorylated with alkaline phosphatase to prevent self-ligation of the plasmid vector, and then the recovered 106bp small fragment was ligated with the linearized vector . The ligation product was transformed into competent E. coli by electroporation, and the bacterial solution was spread on an LB solid plate containing ampicillin. After 16 hours incubation at 37°C, pick the positive transformants that grow on the plate, extract the plasmids, use Xho I and Sal I double enzyme digestion to verify, select transformants that can cut out 20...

Embodiment 2

[0035] Example 2: Construction of expression vector pPIC9

[0036] The selected transformant plasmid into which 16 copies of the BMP expression gene are inserted forward is then digested with EcoR I and Not I, and the cut small fragments are recovered for use. The expression vector pPIC9 was double digested with EcoR I and Not I and then ligated with the above small fragments. The ligated product was transformed into competent E. coli by electroporation through the ligated product, and the bacterial solution was spread on the LB solid plate containing ampicillin . After culturing at 37°C for 16 hours, the positive transformants growing on the plate were picked, and the plasmid was extracted and verified by PCR.

Embodiment 3

[0037] Example 3: Construction of Pichia pastoris engineering strain

[0038]The verified positive transformant plasmid (pPIC9 containing 16 copies of the BMP gene) was digested with Sac I, linearized, and electrotransformed into Pichia pastoris GS115, and the bacterial solution was spread on the MD plate. After culturing at 28°C for 50 hours, the positive transformants were picked, and the host DNA was extracted for PCR verification.

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PUM

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Abstract

The present invention is multicopy beefy meaty peptide (BMP) and its expression gene, vector and construction process in recombinant Pichia pastoris, and relates to molecular biotechnology. Based on the codon partiality expressed and translated with Pichia pastoris, are designed a 4 copy BMP expressing gene sequence and essential limiting cleavage sites, 6His and a termination codon. Through in vitro operation, the copy number is increased to 16, the BMP is inserted to expression vector pPIC9, and after linearization of pPIC9, the 16 copy BMP expressing gene as the target segment is recombined onto the Pichia pastoris genome by means of electric transformation. BMP is obtained in high yield through fermentation and methanol induction of Pichia pastoris, and is separated and purified in 6His affiliated Ni column.

Description

Technical field [0001] The invention relates to a method for constructing recombinant DNA by molecular biology technology to obtain Beefy Meaty Peptide (BMP) in Pichia yeast fermentation broth. Background technique [0002] Beef flavor-enhancing peptide (BMP) was originally isolated from beef papain hydrolysate, and its primary structure is: Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala. BMP, salt and MSG have a good synergistic effect of presenting taste, and has good thermal stability, which is suitable for the heat treatment requirements in the food industry. Therefore, BMP is expected to replace MSG as a new generation of flavor enhancer, with broad market prospects. [0003] At present, most flavor peptides are formed by enzymatic hydrolysis of proteins, but a large number of by-products are produced; in addition, bitter peptides are formed during enzymatic hydrolysis of proteins, which severely limits the utilization of protein hydrolysates. Chemical synthesis is also an effective method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/69C12N1/19C12N15/66C12R1/84
Inventor 王艳萍邢海鹏高文
Owner TIANJIN CHUNFA BIO TECH GRP
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