Glucose oxidase-expressing fructooligosaccharide-synthesizing engineered strain, and its construction method and application

Abstract

The invention discloses a glucose oxidase-expressing fructooligosaccharide-synthesizing engineered strain. The genome of the engineered strain contains the fusion gene gox-fopA-C; the starting strain of the engineered strain is Aspergillus niger ATCC 20611; the fusion gene gox-fopA-C is prepared through fusion of the gox gene coding glucose oxidase GOX and the fopA-C gene coding the FopA-C terminal domain of beta-fructofuranosidase; and the fusion gene has a nucleotide sequence as shown in SEQ ID No. 1, the upstream of the nucleotide sequence is a glyceraldehyde-3-phosphate dehydrogenase promoter PgpdA of Aspergillus nidulans, and the downstream of the nucleotide sequence is a tryptophan terminator TtrpC of Aspergillus nidulans. The invention also discloses application of the engineered strain to preparation of fructooligosaccharide. Experimental results show that the activity of glucose oxidase in the engineered strain reaches 90.0 U / g, the activity of beta-fructofuranosidase reaches 380 U / g, mycelia are directly used for preparation of fructooligosaccharide, and the content of prepared fructooligosaccharide reaches 71.2%. The method has the advantages of simple and convenient operation, capacity of realizing one-step enzymatic preparation of high-purity fructooligosaccharide and good industrial application value.

Description

technical field [0001] The invention relates to a fructo-oligosaccharide synthesis engineering strain expressing glucose oxidase, a construction method thereof and an application in preparing fructo-oligosaccharide, belonging to the technical field of genetic engineering. Background technique [0002] Fructooligosaccharides (FOS for short), also known as functional fructooligosaccharides, is a sucrose triose (GF) formed by combining several D-fructose with β-1,2 glycosidic bonds on the sucrose molecule. 2 ), Kestose (GF 3 ) and fructopentose (GF 4 ) and their mixtures. Fructose-oligosaccharides have physiological effects such as regulating the balance of intestinal flora, reducing blood lipids, and promoting calcium and magnesium absorption, and are widely used in the fields of food, beverages, and medicine. In industry, microbial enzymatic methods are usually used to produce large amounts of fructo-oligosaccharides. High-concentration sucrose is used as raw material, and...

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Problems solved by technology

[0003] At present, the preparation methods of high-purity fructo-oligosaccharides can be divided into two types: one is a two-step method, that is, firstly, β-fructofuranosidase is used to convert sucrose into about 55% fructo-oligosaccharides, and then the glucose is filtered out by chromatography, etc. and sucrose to obtain high-purity fructo-oligosaccharide products, but this method has the disadvantages of high cost of separation equipment, cumbersome and complicated operation and control, and high maintenance costs, making the production cost of high-purity fructo-oligosaccharides high; the other is the mixed enzyme method , that is, glucose oxidase is added to the system of β-fructofuranosidase reaction to synthesize fructo-oligosaccharides, and the by-product glucose is oxidized to gluconic acid to relieve the inhibitory effect and promote the further synthesis of fructo-oligosaccharides, thereby obtaining high-purity fructo-oligosaccharides Polyfructose, but this method requires the preparation of glucose oxidase in vitro, which not only increases the cost, but also easily causes product pollution, and is not suitable for large-scale industrial production

[0004] So far, there is no report on the expression of glucose oxidase in fructooligosaccharide-synthesizing strains at home and abroad, and there is no use of glucose oxidase and β-fructofuranosidase (FopA) C-terminal domain for fusion expression to construct engineering bacteria and apply A report on the preparation of fructooligosaccharides

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