Chitosanase and application thereof

Abstract

The invention discloses chitosanase having an amino acid sequence as shown in SEQ ID NO.1. A gene encoding the chitosanase has a nucleotide sequence as shown in SEQ ID NO.2. The chitosanase is derivedfrom the GH5 family of glucoside hydrolase, can degrade chitosan to generate GlcN-(GlcN)4 and can be used for preparing an enzymatic COS mixture of a new component. The chitosanase has excellent biocatalytic activity, can hydrolyze chitosan to generate glucosamine, chitobiose, chitotriose and chitotetraose hydrochloride, and is relatively high in product purity; and when the pH is 10 and temperature is 50 DEG C, the chitosanase has relatively enzymatic activity of 898.6U / mg which is higher than those of most other known chitosanase, can effectively degrade chitosan, has mild reaction condition, easy control, high efficiency, environmental friendliness. The chitosanase has an industrial application value for producing novel chitosanase mixture.

Description

technical field

[0001] The present invention relates to a chitosan enzyme and application thereof, in particular to a chitosan enzyme capable of degrading chitosan to generate GlcN-(GlcN) 4 The chitosanase and its application in the preparation of chitosan oligosaccharides belong to the technical field of functional gene discovery, cloning and characterization. Background technique

[0002] Chitosan is derived from the complete or partial deacetylation of chitin, and is randomly composed of D-glucosamine (GlcN, D) and N-acetyl-D-glucosamine (GlcNAc, A) through β-1,4 glycosidic bonds. connected. Although chitosan has unique and excellent polymer structure diversity, its high molecular weight and low bioavailability greatly limit its application. However, the degradation product of chitosan, chitooligosaccharide (COS), is widely used in food industry, medicine, agriculture and cosmetic industry due to its superior solubility and biological function.

[0003] Chitosanase (EC...

Examples

Embodiment 1

[0023] Cloning of embodiment 1 chitosanase gene Csn-gly

[0024] In order to mine a GlcN-(GlcN) 4 Chitosanase, the inventors passed to be able to generate (GlcN) 2 -(GlcN) 4 The chitosanase derived from Streptomyces griseus HUT 6037 was used as a template, and a chitosanase with an amino acid sequence similarity of 88.28% to the template was discovered by means of molecular biology. The chitosanase was derived from Streptomyces bacillaris SDUM420012 (purchased from Weihai Marine Microorganism Resource Center of Shandong University), by cloning the gene expressing the enzyme, thereby contributing to the present invention. Its nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.1. Blast results showed that Csn-gly is a new member of glycoside hydrolase class 5 family.

[0025] Using the genome of Streptomyces bacillaris SDUM420012 as a template, primers for seamless connection were designed on the upstream and...

Embodiment 2

[0032] Embodiment 2 Contains the expression vector construction of chitosanase gene

[0033] The gene fragment was connected with the PET-28a cloning vector using seamless cloning technology, and the connection product was transferred into E.coli DH5α competent cells. The LB plates containing kanamycin sulfate resistance were screened for positive transformants, the clones were verified by colony PCR using T7 universal primers, and positive clones were picked for sequencing.

Embodiment 3

[0034] Embodiment 3 Contains the recombinant plasmid of chitosanase gene and the construction of engineering bacterium

[0035] The recombinant plasmids with correct sequencing were extracted and transformed into host E.coli BL21 competent cells, and the constructed engineering bacteria were grown on kanamycin sulfate-resistant plates.

technical field

[0001] The present invention relates to a chitosan enzyme and application thereof, in particular to a chitosan enzyme capable of degrading chitosan to generate GlcN-(GlcN) 4 The chitosanase and its application in the preparation of chitosan oligosaccharides belong to the technical field of functional gene discovery, cloning and characterization. Background technique

[0002] Chitosan is derived from the complete or partial deacetylation of chitin, and is randomly composed of D-glucosamine (GlcN, D) and N-acetyl-D-glucosamine (GlcNAc, A) through β-1,4 glycosidic bonds. connected. Although chitosan has unique and excellent polymer structure diversity, its high molecular weight and low bioavailability greatly limit its application. However, the degradation product of chitosan, chitooligosaccharide (COS), is widely used in food industry, medicine, agriculture and cosmetic industry due to its superior solubility and biological function.

[0003] Chitosanase (EC...

Examples

Embodiment 1

[0023] Cloning of embodiment 1 chitosanase gene Csn-gly

[0024] In order to mine a GlcN-(GlcN) 4 Chitosanase, the inventors passed to be able to generate (GlcN) 2 -(GlcN) 4 The chitosanase derived from Streptomyces griseus HUT 6037 was used as a template, and a chitosanase with an amino acid sequence similarity of 88.28% to the template was discovered by means of molecular biology. The chitosanase was derived from Streptomyces bacillaris SDUM420012 (purchased from Weihai Marine Microorganism Resource Center of Shandong University), by cloning the gene expressing the enzyme, thereby contributing to the present invention. Its nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.1. Blast results showed that Csn-gly is a new member of glycoside hydrolase class 5 family.

[0025] Using the genome of Streptomyces bacillaris SDUM420012 as a template, primers for seamless connection were designed on the upstream and...

Embodiment 2

[0032] Embodiment 2 Contains the expression vector construction of chitosanase gene

[0033] The gene fragment was connected with the PET-28a cloning vector using seamless cloning technology, and the connection product was transferred into E.coli DH5α competent cells. The LB plates containing kanamycin sulfate resistance were screened for positive transformants, the clones were verified by colony PCR using T7 universal primers, and positive clones were picked for sequencing.

Embodiment 3

[0034] Embodiment 3 Contains the recombinant plasmid of chitosanase gene and the construction of engineering bacterium

[0035] The recombinant plasmids with correct sequencing were extracted and transformed into host E.coli BL21 competent cells, and the constructed engineering bacteria were grown on kanamycin sulfate-resistant plates.

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