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Bacillus subtilis for efficient secretory expression of diacetylchitobiose deacetylase and application thereof

A technology of Bacillus subtilis and deacetylase, applied in the field of metabolic engineering, can solve the problems of insufficient enzyme activity, limited catalytic efficiency of chitobiose deacetylase Dac, etc., and achieve improved enzyme activity, convenient promotion and application, and mild conditions Effect

Active Publication Date: 2021-06-15
SHANDONG RUNDE BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem that the catalytic efficiency of chitobiose deacetylase Dac is limited and the enzyme activity is not high enough, the present invention provides a method for improving the catalytic efficiency of chitobiose deacetylase Dac, thereby increasing the extracellular enzyme activity

Method used

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  • Bacillus subtilis for efficient secretory expression of diacetylchitobiose deacetylase and application thereof
  • Bacillus subtilis for efficient secretory expression of diacetylchitobiose deacetylase and application thereof
  • Bacillus subtilis for efficient secretory expression of diacetylchitobiose deacetylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Molecular docking and selection of saturation mutation sites

[0043] The CDOCKER module of the software Discovery Studio was used for molecular docking, and according to the operation manual of the DiscoveryStudio software, the three-dimensional protein structure file of the chitobiose deacetylase Dac model and the three-dimensional structure file of the ligand N-acetylglucosamine GlcNAc were used for molecular docking ,Such as figure 1 shown.

[0044] When the substrate molecule and the protein molecule are successfully docked, the amino acids that interact with GlcNAc around the binding position of the substrate molecule GlcNAc and the protein molecule Dac are the key sites in the protein molecule Dac that interact with the substrate molecule GlcNAc, such as figure 2 shown.

[0045] According to literature reports, the key sites affecting the catalytic effect of chitobiose deacetylase are the 152-position histidine H152 and the 120-position tyrosine Y12...

Embodiment 2

[0046] Example 2: Establishment of Site-Directed Saturation Mutation Library

[0047] According to the base sequence of chitobiose deacetylase Dac, degenerate primers were designed, as shown in Table 1. Using pP43mut-yncM-Dac (see application number CN201910193636.5 for the construction method) as a template, perform PCR amplification, and transform Escherichia coli DH5α (such as image 3 ), all the large intestine transformants were prepared into a mixed bacterial solution and the plasmid was extracted, and then transformed into the expression host Bacillus subtilis WB600.

[0048] Table 1

[0049]

[0050]

Embodiment 3

[0051] Example 3: High-throughput screening and amplification verification of site-directed saturation mutation library

[0052] (1) High-throughput screening:

[0053] Use a sterilized toothpick to pick about 100 single clones from each saturated mutation point library and transfer them to a 48-well plate for seed culture. Each well contains 600 μl of liquid LB medium supplemented with kana antibiotics (10 mg / mL), (the composition of LB medium is as follows: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L), shake the plate at 37 ° C The bed was cultured for 12 hours to obtain the seed solution, and then in a new 48-well plate, 200 μl of the seed solution was transferred to each well containing 600 μl of liquid TB medium supplemented with kana antibiotics (10 mg / mL) for fermentation and culture, (TB medium composition As follows: glycerol 4g / L, tryptone 12g / L, yeast powder 24g / L, dipotassium hydrogen phosphate 12.54g / L, potassium dihydrogen phosphate 2.31g / L), at 37°C, 700rp...

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Abstract

The invention discloses a bacillus subtilis for efficient secretory expression of diacetylchitobiose deacetylase and application thereof. The molecular structure of the diacetylchitobiose deacetylase Dac and the RBS sequence of an expression vector pP43mut-N1-yncM-Dac are modified, so that the catalytic rate and translation expression rate of the diacetylchitobiose deacetylase Dac are increased, the enzyme activity is improved, and the bacillus subtilis capable of producing glucosamine GlcN at high yield is constructed. Compared with the prior art, the extracellular enzyme activity of the diacetylchitobiose deacetylase can reach 3769.5 U / mL. The conversion method is mild in condition, small in environmental pollution, high in substrate selectivity, specific in target product, high in reaction rate and high in product yield. The method is beneficial to solving the problems of serious pollution, tedious steps and the like in a chemical synthesis method, and is simple in process, easy to control and convenient to popularize and apply.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering, and in particular relates to a bacillus subtilis capable of efficiently secreting and expressing chitobiose deacetylase and an application thereof. Background technique [0002] As a natural water-soluble monosaccharide, glucosamine GlcN has important applications in the fields of medicine and health care, skin beauty, and agricultural production. At present, in many countries, glucosamine GlcN has been listed as a medicine. In addition, by combining glucosamine GlcN with ferulic acid, a new type of cosmetic material with good water solubility can be obtained, which has the effect of whitening and moisturizing. On the other hand, glucosamine GlcN can be used as the main component of a detoxification agent for crops, and when used during the plant growth period, it can discharge harmful substances such as heavy metals and pesticide pollutants in plants. [0003] In recent years, th...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N1/21C12N15/75C12N15/62C12P19/26C12R1/125
CPCC12N9/80C12N15/75C12P19/26C07K2319/02
Inventor 刘龙陈坚吕雪芹卢伟堵国成李江华张弘治刘延峰卢健行毛馨竹
Owner SHANDONG RUNDE BIOTECH CO LTD
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