阅读说明：本技术 一种源于黑曲霉菌的高产特异性功能低聚肽的酶及其工程菌 (High-yield specific functional oligopeptide enzyme derived from aspergillus niger and engineering bacterium thereof ) 是由 熊科 柳佳芸 邓蕾 裴鹏钢 于 2019-12-09 设计创作，主要内容包括：本发明属于基因工程领域,提供了一种源于黑曲霉菌M00988的高产特异性功能低聚肽的酶及其工程菌。本发明通过基因改造获得能够高产低F值寡肽的羧肽酶及生产该酶的工程菌。通过对特殊位点的改造,提高了羧肽酶切除芳香族氨基酸的效率,能够有利于提高产品F值,降低酶法生产成本,减少后期纯化过程中的能耗,降低环境污染,实现高效制备高F值低聚肽的工业化生产应用。(The invention belongs to the field of genetic engineering, and provides a high-yield specific functional oligopeptide enzyme derived from aspergillus niger M00988 and engineering bacteria thereof. The carboxypeptidase capable of producing oligopeptide with high yield and low F value and the engineering bacteria for producing the carboxypeptidase are obtained through gene modification. By modifying the special sites, the efficiency of removing aromatic amino acids by carboxypeptidase is improved, the F value of the product can be improved, the production cost of an enzyme method is reduced, the energy consumption in the later purification process is reduced, the environmental pollution is reduced, and the industrial production application of efficiently preparing the high-F-value oligopeptide is realized.)

1. A carboxypeptidase, characterized in that the amino acid sequence is any one of SEQ ID NO 1-3.

2. A nucleotide sequence of carboxypeptidase with an amino acid sequence shown in any one of SEQ ID NO 1-3.

3. The nucleotide sequence of claim 2, as set forth in SEQ ID NOS 4-6.

4. Use of the carboxypeptidase of claim 1 for the preparation of an oligopeptide with a high F-number.

5. A plasmid or engineered bacterium comprising the carboxypeptidase nucleotide sequence of claim 2.

6. Use of the engineered bacterium of claim 5 in the preparation of a carboxypeptidase.

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to aspergillus nigerAspergillus niger) The high-yield specific functional oligopeptide enzyme and the engineering bacteria thereof.

Background

With the continuous improvement of living standard of people, the consumption concept, eating habit, nutritional requirements and the like of people are greatly changed from simple full eating to nutrition and health, even preventing and treating diseases. It has long been generally accepted that proteins, which are essential nutrients in food, must be broken down in the digestive tract into free essential or nonessential amino acids for absorption and utilization by the body. Recent studies have shown that proteins are not only absorbed by the human body in the form of amino acids, but that there are other ways of absorption. Modern nutritional studies have shown that, besides providing human nutrients like proteins and amino acids, many peptides with unique sequences or amino acid compositions also have specific physiological functions, and especially some low molecular weight active oligopeptides have become one of the most popular subjects in the current food science community. Wherein the oligopeptide with high F value is a small peptide system consisting of high branched chain amino acid content and low aromatic amino acid content. It has received a high degree of attention from the food and medical community because of its unique amino acid composition and physiological function.

The high F value oligopeptide is a mixed small peptide system mainly composed of 2 to 10 amino acids, and the F value (the molar ratio of branched chain amino acid to aromatic amino acid content) of the high F value oligopeptide is generally more than 20. Due to the unique amino acid composition, the high F value oligopeptide has various physiological effects. It has the physiological functions of resisting oxidation, preventing senility, protecting liver, treating hepatic encephalopathy and treating phenylketonuria. Has good conditioning effect on sub-health state of chronic fatigue syndrome. At present, the method for preparing the bioactive peptide by the enzymolysis method has the advantages of mild production conditions, high safety of the obtained bioactive peptide, good solubility, stability, functional specificity and the like, and becomes a method commonly adopted for producing the bioactive peptide. When the high F value oligopeptide is prepared by an enzyme method, analysis and judgment are needed based on the amino acid sequence of the protein in the raw material and the enzyme cutting site thereof, and the conditions of the preparation process are researched and optimized. Therefore, the method for preparing the oligopeptide with high F value by hydrolyzing each enzyme system is more limited and can only be applied to specific protein raw materials. Meanwhile, in the enzymolysis process, the long-chain alkylamino or the hydrophobic group of the aromatic amino acid is exposed, so that the enzymolysis liquid is easy to have bitter taste, and the subsequent treatment procedures of separation, purification, debitterizing and the like are complicated, the purification cost is high, the loss of amino acid and polypeptide in the purification and adsorption process is large, the regeneration is difficult and the like. Therefore, the key problem to be solved urgently is to improve the enzyme digestion specificity and effectively remove aromatic amino acids in the preparation of high-F-value oligopeptide by using the enzyme method. The enzyme digestion specificity in the process of producing the high F value oligopeptide by the enzyme method is improved, so that the F value of the product is improved, the production cost of the enzyme method is reduced, the energy consumption in the later purification process is reduced, the environmental pollution is reduced, and the industrial production application of efficiently preparing the high F value oligopeptide is realized.

Disclosure of Invention

Aiming at the problems existing in the preparation of high F value oligopeptide by an enzyme method, the invention provides an engineering bacterium of high-yield specific functional oligopeptide from aspergillus niger, and the high F value oligopeptide can be efficiently obtained.

In order to achieve the purpose, the invention adopts the following technical scheme.

A carboxypeptidase has an amino acid sequence shown in any one of SEQ ID NO 1-3. Wherein, SEQ ID NO. 2 is that tyrosine at the position 206 of the sequence shown in SEQ ID NO.1 is mutated into serine, and SEQ ID NO. 3 is that serine at the position 135 of the sequence shown in SEQ ID NO.1 is mutated into glycine.

The carboxypeptidase is derived from Aspergillus nigerAspergillus niger) M00988, the preservation number is CGMCC No. 18828.

A nucleotide sequence of carboxypeptidase with an amino acid sequence shown in any one of SEQ ID NO 1-3. Preferably, the amino acid sequences corresponding to SEQ ID NO. 1-3 are shown in SEQ ID NO. 4-6.

A plasmid or engineering bacterium containing the nucleotide sequence of the carboxypeptidase.

An application of the engineering bacteria in preparing carboxypeptidase.

An application of the carboxypeptidase in preparing the oligopeptide with high F value.

The invention has the following advantages:

the invention provides a genetically modified carboxypeptidase and an engineering bacterium for producing the same. By modifying the special sites, the efficiency of removing aromatic amino acids in the polypeptide substrate by the carboxypeptidase is improved, the F value of the product can be improved, the production cost of an enzyme method is reduced, the energy consumption in the later purification process is reduced, the environmental pollution is reduced, and the industrial production application of efficiently preparing the high-F-value oligopeptide is realized.

Biological preservation information

Aspergillus nigerAspergillus niger) And the strain is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 06 days in 2019, the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, China, and the preservation number is CGMCC number 18828.

Drawings

FIG. 1 is a gel electrophoresis picture of PCR products of CPA gene;

FIG. 2 is an agarose gel electrophoresis image of the procedure for constructing the Y206S and S135G mutants;

FIG. 3 is a SDS-PAGE electrophoresis of a crude recombinase enzyme solution;

FIG. 4 is the amino acid content of the oligopeptide product;

FIG. 5 shows the values of F for the oligopeptides obtained by different enzymes.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.