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

A beef flavor and gene expression technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of excessive linking reagents and acyl carriers, linking reagents affecting products and the environment, and limiting the utilization of protein hydrolyzates. Optimizing selectivity, reducing difficulty, and achieving high yields

Inactive Publication Date: 2012-03-14
TIANJIN CHUNFA BIO TECH GRP +1
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  • Abstract
  • Description
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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] Embodiment 1: Construction of 16 copies of BMP

[0034] After the self-designed plasmid vector containing 4 copies of the BMP expression gene (synthesized by Dalian Bao Biology Co., Ltd.) was digested with Xho I and Sal I, the enzyme digestion system was subjected to agarose gel electrophoresis, and a 106bp plasmid was recovered. small snippet. Then, the designed plasmid vector containing 4 copies of the BMP expression gene was single-digested with Xho I, dephosphorylated with alkaline phosphatase to prevent the 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 Escherichia coli by electric shock transformation, and the bacterial solution was spread on the LB solid plate containing ampicillin. After culturing at 37°C for 16 hours, pick the positive transformants grown on the plate, extract the plasmids, use Xho I and Sal I double enzyme digestion ...

Embodiment 2

[0035] Embodiment 2: Construction of expression vector pPIC9

[0036] The selected transformant plasmid with forward insertion of 16 copies of the BMP expression gene was double-digested with EcoR I and Not I, and the excised small fragment was recovered for use. The expression vector pPIC9 was double-digested with EcoR I and Not I and then ligated with the above-mentioned small fragment. The ligated product was transformed into competent E. coli by electric shock, and the bacterial solution was spread on the LB solid plate containing ampicillin . After culturing at 37°C for 16 hours, the positive transformants grown on the plate were picked, the plasmid was extracted, and verified by PCR.

Embodiment 3

[0037] Embodiment 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 and linearized, then electrotransformed into Pichia pastoris GS115, and the bacterial solution was spread on the MD plate. After culturing at 28°C for 50 hours, positive transformants were picked, and their host DNA was extracted for PCR verification.

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Abstract

The present invention provides a multicopy beefy meaty peptide (BMP) expression gene, vector and construction process of recombinant Pichia pastoris, and relates to a method for obtaining the BMP in the Pichia pastoris by using molecular biotechnology, which overcomes the big difficulty, high cost and low yield and so on in the extraction or chemical synthesis of BMP in the prior art. Based on the codon partiality expressed and translated with Pichia pastoris, a 4-copy-containing BMP expressing gene sequence and essential limiting cleavage sites, 6His and a termination codon are designed. Through in vitro operation, the copy number of the BMP is increased to 16, and the BMP is inserted to expression vector pPIC9, after linearization of pPIC9, the 16-copy-containing BMP expressing gene as a 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 by 6His affiliated Ni column.

Description

technical field [0001] The invention relates to a method for constructing recombinant DNA by molecular biology technology to obtain beef flavor peptide (Beefy Meaty Peptide, BMP) in Pichia pastoris fermentation liquid. Background technique [0002] Beef Flavor Enhancement Peptide (BMP) was originally isolated from papain hydrolyzate of beef, and its primary structure is: Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala. BMP has a better synergistic taste effect with salt and MSG, and has good thermal stability, which is suitable for heat treatment requirements in food industry production. Therefore, BMP is expected to replace MSG as a new generation of flavor enhancer and has 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 synt...

Claims

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Application Information

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Patent Type & Authority Patents(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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