A high-yielding specific functional oligopeptide enzyme derived from Aspergillus niger and its engineering bacteria

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

Active Publication Date: 2020-05-26
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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  • A high-yielding specific functional oligopeptide enzyme derived from Aspergillus niger and its engineering bacteria
  • A high-yielding specific functional oligopeptide enzyme derived from Aspergillus niger and its engineering bacteria
  • A high-yielding specific functional oligopeptide enzyme derived from Aspergillus niger and its engineering bacteria

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 of NCBI Aspergillus niger, two pairs of primers CPA-F / CPA-R were designed, and the sequences are shown in Table 1.

[0025] Table 1 The sequences of primers for carboxypeptidase gene cloning

[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. The PCR amplification conditions are: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 68°C for 1 minute, extension at 72°C for 1 minute, 35 cycles, and extension at 72°C for 10 minutes to obtain a fragment with a size of about 1500 bp, which was detected by agarose electrophoresis Such as figure 1 shown.

[0027] Use the obtained target gen...

Embodiment 2

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

[0040] 1. Chlorella protein extraction

[0041] Accurately weigh a certain amount of chlorella powder, add deionized water at a solid-liquid ratio of 1:48 (m / V), stir and swell at room temperature for 12 hours. Add 5.37% sodium hydroxide solid to the chlorella suspension, pay attention to adding slowly during the addition process, and keep stirring. After completion, sonicate for 60 min at 43 °C. Next, use 0.1mol / L sodium hydroxide solution and hydrochloric acid solution to adjust the pH=7, centrifuge at 5000rpm 25°C for 10min, take the supernatant, add 95% alcohol at 4°C at a ratio of 1:4 (V / V) solution, stirred for 1 min, and mixed thoroughly. Centrifuge again at 5000 rpm at 25° C. for 10 minutes, and discard the supernatant. Wash the obtained precipitate with a small amount of deionized water, remove the surface alcohol, and then dissolve the precipitate with 0.1mol / L PBS solution with pH=6.

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Abstract

The invention belongs to the field of genetic engineering and provides an enzyme derived from Aspergillus niger M00988 with high yield of specific functional oligopeptides and an engineering bacterium thereof. The invention obtains the carboxypeptidase capable of high-yield low F value oligopeptide and the engineering bacterium producing the enzyme through genetic modification. Through the modification of special sites, the efficiency of carboxypeptidase to remove aromatic amino acids is improved, which can help improve the product F value, reduce the cost of enzymatic production, reduce energy consumption in the later purification process, reduce environmental pollution, and achieve high efficiency. Industrial production application for 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-yield specific functional oligopeptide enzyme and its engineering bacteria. Background technique [0002] With the continuous improvement of people's living standards, great changes have taken place in people's consumption concepts, eating habits, and nutritional requirements, from simple eating well to nutrition and health, and even disease prevention and treatment. For a long time, it has been generally believed that protein in food, which mainly plays a nutritional role, must be broken down into free essential amino acids or non-essential amino acids in the digestive tract before it can be absorbed and utilized by the body. Recent studies have shown that protein is not only absorbed by the body in the form of amino acids, but also in other ways. Modern nutritional research shows that, in addition to providing human nut...

Claims

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Application Information

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Patent Type & Authority Patents(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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