Enzyme for high yield of specific functional oligopeptides and derived from Aspergillus niger and engineering bacteria thereof

A technology of engineering bacteria and oligopeptides, applied in the field of genetic engineering, can solve the problems of high purification costs, cumbersome treatment procedures, and large limitations, and achieve the effects of reducing environmental pollution, reducing production costs, and improving efficiency

Active Publication Date: 2020-03-13
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the method of hydrolyzing each enzyme system to prepare high F-value oligopeptides has relatively large limitations and can only be applied to specific protein raw materials
At the same time, during the enzymolysis process, the exposure of the hydrophobic groups of long-chain alkylamino or aromatic amino acids will easily make the enzymolysis solution appear bitter, resulting in cumbersome subsequent separation and purification and debitterization procedures, high purification costs, and high purification and adsorption. During the process, there are many problems such as large loss of amino acids and peptides, difficulty in regeneration, etc.

Method used

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  • Enzyme for high yield of specific functional oligopeptides and derived from Aspergillus niger and engineering bacteria thereof
  • Enzyme for high yield of specific functional oligopeptides and derived from Aspergillus niger and engineering bacteria thereof
  • Enzyme for high yield of specific functional oligopeptides and derived from Aspergillus niger and engineering bacteria thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Construction of M00988 CPA mutant

[0023] 1. Cloning of Aspergillus niger M00988 CPA and construction of expression vector

[0024] According to the carboxypeptidase gene sequence from Aspergillus niger from NCBI, two pairs of primers CPA-F / CPA-R were designed, and the sequences are shown in Table 1.

[0025] Table 1 Sequences of carboxypeptidase gene cloning primers

[0026] The DNA of Aspergillus niger M00988 was extracted as a template, the sequence is shown in SEQ ID NO: 6, and the target gene fragment CPA was obtained by PCR amplification with CPA-F / CPA-R primers. PCR amplification conditions were: pre-denaturation at 94 °C for 5 min, then denaturation at 94 °C for 30 s, annealing at 68 °C for 1 min, extension at 72 °C for 1 min, 35 cycles, and extension at 72 °C for 10 min to obtain a fragment of about 1500 bp in size, which was detected by agarose electrophoresis like figure 1 shown.

[0027] Using the obtained target gene fragment CPA as a temp...

Embodiment 2

[0039] Example 2 Preparation and application of high F value oligopeptide

[0040] 1. Chlorella Protein Extraction

[0041] Accurately weigh a certain amount of Chlorella powder, add deionized water at a ratio of 1:48 (m / V) to the liquid, stir and shake at room temperature for 12 hours to swell. Add 5.37% solid sodium hydroxide to the chlorella suspension, pay attention to slow addition during the addition process, and keep stirring. After completion, sonicate for 60 min at 43 °C. Next, adjust pH=7 with 0.1mol / L sodium hydroxide solution and hydrochloric acid solution, centrifuge at 5000rpm at 25°C for 10min, take the supernatant, add 95% alcohol at 4°C at a ratio of 1:4 (V / V) The solution was stirred for 1 min and mixed well. Centrifuge again at 5000 rpm for 10 minutes at 25°C and discard the supernatant. The obtained precipitate was washed with a small amount of deionized water, and after removing the surface alcohol, the precipitate was dissolved with 0.1 mol / L PBS solu...

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Abstract

The invention belongs to the field of genetic engineering and provides an enzyme for high yield of specific functional oligopeptides and derived from Aspergillus niger M00988 and engineering bacteriathereof. According to the invention, carboxypeptidase capable of high yield of low F-value oligopeptides and engineering bacteria for producing the enzyme are obtained through genetic modification. Through modification of special sites, the efficiency of carboxypeptidase in removing aromatic amino acids is improved, which is good for increasing the F value of products, reducing the production costof the enzymatic method, reducing the energy consumption in the later purification process, reducing environmental pollution and realizing the industrial production application in efficient preparation of high F-value oligopeptides.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a kind of Aspergillus niger ( Aspergillus niger ) high-yielding specific functional oligopeptide enzyme and its engineering bacteria. Background technique [0002] With the continuous improvement of people's living standards, people's consumption concepts, eating habits, nutritional requirements, etc. have undergone tremendous changes, from simply eating well and eating well to nutritional health and even disease prevention and treatment. For a long time, it is generally believed that the protein that mainly plays a nutritional role in food must be broken down into free essential amino acids or non-essential amino acids in the digestive tract before they can be absorbed and utilized by the body. Research in recent years has shown that protein is not only absorbed by the body in the form of amino acids, but also has other absorption methods. Modern nutritional studies have sho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12N15/57C12N15/70C12N1/21C12P21/06C12R1/19
CPCC12N9/485C12N15/70C12P21/06
Inventor 熊科柳佳芸邓蕾裴鹏钢
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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