Beta-mannase mutant food-grade bacillus subtilis expression vector, expression system, construction method and application

A technology of Bacillus subtilis and mannanase, which is applied in the field of food-grade Bacillus subtilis expression vectors for β-mannanase mutants, and can solve problems such as limited application and antibiotic hazards

Pending Publication Date: 2022-07-05
HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology

[0004] At present, antibiotics are often used as screening markers in genetic engineering. Due to the potential harm of antibiotics, the application of exogenous

Method used

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  • Beta-mannase mutant food-grade bacillus subtilis expression vector, expression system, construction method and application
  • Beta-mannase mutant food-grade bacillus subtilis expression vector, expression system, construction method and application
  • Beta-mannase mutant food-grade bacillus subtilis expression vector, expression system, construction method and application

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[0046] The technical solutions of the present invention will be described in detail below with reference to the embodiments. The following examples are only used to illustrate and explain the present invention, but not to limit the technical solutions of the present invention.

[0047] 1 Materials and methods

[0048] 1.1 Materials and Reagents

[0049] The plasmids, strains and primers used in the present invention are shown in Table 1, Table 2 and Table 3, respectively.

[0050] Table 1 Plasmids used in the present invention

[0051]

[0052] ①: Wu, Y.; Liu, Y.; Lv, X.; Li, J.; Du, G.; Liu, L. CAMERS-B: CRISPR / Cpf1 AssistedMultiple-Genes Editing and Regulation System for Bacillus Subtilis.BiotechnolBioeng 2020, 117(6), 1817–1825.

[0053] Table 2 bacterial strains used in the present invention

[0054]

[0055]

[0056] ②: Kawamura, F.; Doi, R.H. Construction of a Bacillus Subtilis DoubleMutant Deficient in Extracellular Alkaline and Neutral Proteases. J Bacterio...

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Abstract

According to the invention, a bacillus licheniformis beta-mannase mutant is subjected to food-grade expression in bacillus subtilis, a bacillus subtilis d-alanine racemase gene dal is knocked out through a CRISPR/Cpf1 genome editing technology, and a bacillus subtilis food-grade expression host is constructed; replacing an antibiotic resistance gene segment on a bacillus subtilis expression vector with a dal gene to construct a food-grade expression vector of the bacillus licheniformis beta-mannase mutant; the vector is introduced into a food-grade expression host, so that food-grade expression of beta-mannase is realized, and the enzyme activity is 17601.3 U/mL; when the 0.5 U/mL food-grade beta-mannase is applied, 0.5% of konjac glucomannan is degraded at 60 DEG C for 1 h, the content of mannan oligosaccharides in degradation products accounts for 85% of the total hydrolysis products, and main components in the mannan oligosaccharides are mannan oligosaccharides DP6 and DP3 with the polymerization degrees of six and three.

Description

technical field [0001] The present invention relates to a β-mannanase mutant food-grade Bacillus subtilis expression vector, expression system, construction method and application. Background technique [0002] Bacillus subtilis has many advantages such as good biosafety (GRAS), convenient molecular manipulation, high genetic stability, strong protein secretion ability, robust culture, and friendly fermentation, and is widely used in the industry. In 1997, the complete genome of B. subtilis was published, and then its transcriptome, secretome, metabolism, and genetic regulation network were further studied and published. With the support of these basic research work, B. subtilis became the first cell of excellent chassis cells. The basic conditions are becoming more mature. [0003] β-Mannanase (EC 3.2.1.78) is an endohydrolase capable of hydrolyzing β-1,4 mannoside bonds, and its hydrolyzed product is mannose oligosaccharide (MOS). MOS can promote intestinal probiotics, in...

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/75C12N15/66C12N1/21C12P19/00C12R1/125
CPCC12N9/2491C12N15/75C12Y302/01025C12P19/00
Inventor 张春晓王丽丽陈赟刘金龙梁琪
Owner HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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