Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Marine streptomyces niveus chitosanase gene and application thereof

A technology of chitosanase and streptomyces, applied in application, glycosylase, genetic engineering and other directions, can solve the problems of uncontrollable range of polymerization degree and poor specificity of products

Active Publication Date: 2020-05-15
INST OF PROCESS ENG CHINESE ACAD OF SCI
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, most of the current chitosanases have poor specificity, resulting in an uncontrollable range of product polymerization degrees.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Marine streptomyces niveus chitosanase gene and application thereof
  • Marine streptomyces niveus chitosanase gene and application thereof
  • Marine streptomyces niveus chitosanase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The construction of embodiment 1 chitosan hydrolase SnCSN expression bacterial strain

[0035] With reference to the gene encoding chitosan hydrolase SnCSN (GenBank No: AQU65829.1), the present invention entrusts Huada Gene Co., Ltd. to synthesize the gene encoding chitosan hydrolase (excluding signal peptide) through codon optimization. 765 bases, the nucleotide sequence is shown in SEQ ID NO.1, the cloning vector is pET22b, the cloning sites BamHI and XhoI, the carrier resistance is ampicillin (Amp), and the optimized species E.coli ( figure 1 ). Escherichia coli DH5α carrying the expression plasmid pET22b-SnCSN was cultured, the plasmid was extracted using the Plasmid Mini Kit I kit, and then the expression plasmid was introduced into competent Escherichia coli BL21(DE3) to obtain a recombinant strain. The amino acid sequence is shown as SEQ ID NO.2, which contains 255 amino acids, and the predicted protein molecular weight is 29.8kDa.

[0036] SEQ ID NO.1:

[003...

Embodiment 2

[0057] Expression and detection of embodiment 2 chitosan hydrolase SnCSN

[0058] (1) Prepare LB medium containing Amp resistance: 5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride, sterilize at 120°C for 20min, cool to room temperature and add Amp to make the final concentration 100μg / L mL.

[0059] (2) Inoculate the recombinant strain obtained in Example 1 onto solid LB medium containing Amp resistance, culture overnight at 37°C, pick a single colony and inoculate it into 10 mL of liquid LB culture containing Amp resistance, at 37°C, After culturing on a shaker at 200rmp for 24 hours, inoculate the above-mentioned bacterial solution into 600ml liquid LB culture containing Amp resistance, and cultivate to OD at 37°C and 200rmp bed 600nm When =0.6, add 0.5mM IPTG, induce expression at 16°C, 200rmp for 12 hours, centrifuge at 4000rmp, and collect the bacteria.

[0060] (3) A small amount of bacterial cells were taken for SDS-PAGE analysis, and the expression of the ta...

Embodiment 3

[0061] Expression, purification and detection of embodiment 3 chitosan hydrolase SnCSN

[0062] (1) Example 2 was collected into thalli, suspended in buffer solution A (50mM Tri-HCl, pH 7.9, 500mMNaCl) and carried out sonication, 12,000rmp was centrifuged to collect the supernatant, and carried out SDS-PAGE detection ( figure 2 ) predicted protein molecular weight of 29.8kDa.

[0063] (2) Purify the above protein using a nickel column:

[0064] 1. Equilibrate the column with buffer solution A (50mM Tris / HCl, pH 7.9, 0.5M NaCl) at a flow rate of 1mL / min.

[0065] 2. Load the sample at a flow rate of 1 mL / min, and collect the breakthrough.

[0066] 3. Wash the column with buffer solution A, flow rate 1mL / min, wash 40mL,

[0067] 4. Elution with buffer solution A+20mM imidazole, flow rate 1mL / min, wash 40ml, collect one tube every 4min.

[0068] 5. Collect samples for G250 detection to see if there is any protein eluted in the last tube. If there is no protein, proceed to th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention provides a marine streptomyces niveus chitosanase gene and application thereof. According to the preference of escherichia coli codon, a chitosanase coded gene(GenBank accession number:AQU65829) of the marine streptomyces niveus is optimized, the homology of an optimized nucleic acid sequence with an original nucleic acid sequence is 73.39%, the chitosanase SnCSN is successfully expressed in escherichia coli BL21 (DE3), and the obtained chitosanase SnCSN can efficiently degrade chitosan to obtain chitosan oligosaccharide with chitobiose as a main product; and the chitosanase SnCSN has higher hydrolytic activity on chitosan substrates with different degrees of deacetylation, and complex chitosan oligosaccharide with different degrees of deacetylation is obtained. The chitosanase can be used for large-scale preparation of chitosan oligosaccharide and chitin oligosaccharide, and the industrial application prospects are good.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, in particular, the invention relates to a chitosanase gene of marine Streptomyces snow-white and application thereof. The chitosanase of the invention can be used to catalyze chitosan to prepare chitobiose. Background technique [0002] Chitosanase (Chitosanases, EC.3.2.1.132) is a glycoside hydrolase mainly derived from archaea, bacteria, fungi, and plants, which catalyzes the hydrolysis of β-1 in partially acetylated chitosan in an endo-cutting manner, 4-glucosamine bond to generate chitosan oligosaccharide. Because chitosanase can be used to prepare chitosan oligosaccharide, a substance with unique biological activity, it has a wide range of applications in the fields of medicine, food, cosmetics, etc., and more and more researchers have paid attention to the enzyme. According to the carbohydrate-active enzyme database (Carbohydrate-Active enZYmes, CAZY), chitosanase is distribut...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/56C12N9/24C12N15/70C12N1/21C12P19/26C12P19/14C12P19/12C12R1/19
CPCC12N9/2402C12N15/70C12P19/26C12P19/14C12P19/12C12Y302/01132
Inventor 杜昱光李建军陈彤程功焦思明任立世
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com