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

Paenibacillus chitin enzyme and application thereof

A technology of Paenibacillus and chitinase, applied in the direction of enzymes, enzymes, hydrolytic enzymes, etc., can solve the problems of insufficient specificity and limited use, and achieve the effects of reduced crystallinity, improved hydrolysis efficiency, and huge application potential

Inactive Publication Date: 2019-04-19
SOUTH CHINA UNIV OF TECH
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of non-specific enzymes such as protease and cellulase can alleviate the above situation to a certain extent, the lack of specificity limits the use of such enzyme preparations

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
  • Paenibacillus chitin enzyme and application thereof
  • Paenibacillus chitin enzyme and application thereof
  • Paenibacillus chitin enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Paenibacillus chitinase Pp Cloning, expression, isolation and purification of Chi2

[0036] (1) Pp Cloning of Chi2

[0037] According to the results of genome sequencing, select the gene containing the signal peptide sequence Pp The gene sequence of Chi2 (Genebank No.: KX431049.1) was used as the research object, and the forward primer Chip1_F: 5'-GGAATTC was designed based on the prediction results of the signal peptide sequence CATATG GCGGCCTGGACGCCCGGC-3', reverse primer Chip1_R: 5'-GACCG CTCGAG GCCGAAGTAGCCCTTCATCGCGT-3', with Paenibacillus pasadenensis The genome of CS0611 was used as a template and amplified Pp Chi2.

[0038] The PCR reaction system is: 10 μL 5X Q5 reaction buffer, 1 μL 10 mM dNTPs, 2.5 μL 10 μM Chip2_F and Chip2_R, 1 μL genomic DNA, 0.5 μL Q5 high-fidelity DNA polymerase, 10 μL 5X Q5 high GCenhancer, 23 μL ddH2O.

[0039] The PCR reaction conditions were: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, anneali...

Embodiment 2

[0047] Example 2 temperature and pH pair Pp Effect of Chi2 Enzyme Activity

[0048] Determination of chitinase activity: the determination of chitinase activity adopts the method of DNS to determine the reducing sugar in the reaction product, and the substrate is 2 wt% colloidal chitin solution. Add 2mL of DNS reagent to 1mL of the reaction product, place in a boiling water bath for 5min, cool down, add water to 10mL, and measure its absorbance at 540nm. The standard curve was drawn with N-acetylglucosamine as the standard substrate, and the unit of enzyme activity was defined as one unit of 1 μM N-acetylglucosamine produced per minute.

[0049] The influence of temperature: 0.5mL respectively Pp Chi2 solution (0.1 mg / mL, 5mM pH7.0 PBS) and 0.5 mL 2wt% colloidal chitin solution (pH 5.0 0.2 M citric acid buffer) were added to a 10 mL centrifuge tube, and then the reaction system was placed at different temperatures (30 , 35, 40, 45, 50, 55, 60, 65, 70, 80 ℃), magnetic stirri...

Embodiment 3

[0051] Embodiment 3 Different proportions Pp Chi1 and Pp Crab shell powder after Chi2 synergistic hydrolysis pretreatment

[0052] Pp Chi1 is and Pp Another chitinase derived from the same target strain with different Chi2 properties, this example examines different mass ratios Pp Chi1 and Pp Chi2 synergistic hydrolysis of crab shell powder pretreated with ionic liquids.

[0053] Pp Chi1 and Pp Chi2 was prepared by the method in Example 1, Pp The concentration of Chi1 is 3.3mg / mL, Pp The concentration of Chi2 was 2.9 mg / mL. The substrate used is crab shell powder (treated-CSP) pretreated by ionic liquid (1-butyl-3-methylimidazolium acetate). Add the powder into 2mL 1-butyl-3-methylimidazole acetate, incubate at 100°C for 1 hour, add water to precipitate chitin, and separate the precipitate by suction filtration. The resulting precipitate was added to 20 mL of deionized water, ultrasonicated for 5 min, and the precipitate was separated by suction filtration. The abov...

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

No PUM Login to View More

Abstract

The invention discloses a paenibacillus chitin enzyme and application thereof. The paenibacillus chitin enzyme has the amino acid sequence shown in the SEQ ID NO.1. The gene for encoding the paenibacillus chitin enzyme has the nucleotide sequence shown in the SEQ ID NO.2. Through an escherichia coli expression system, the efficient dissoluble expression of the chitin enzyme can be conducted. Then,with a mixed enzyme solution of the chitin enzyme and another chitin enzyme as the catalyst in cooperation with crab shell powder preprocessed by hydrolyzed ionic liquid, compared with the monotonous-enzyme catalysis process, the dual-enzyme cooperative catalysis enzymolysis efficiency is improved by 1.3-3.5 times. Thus, good industrial application prospects are achieved during the application ofthe chitin enzyme and another cooperative chitin enzyme in the crab shell powder enzymolysis process.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Paenibacillus chitinase and an application thereof. Background technique [0002] Chitinase (Chitinase, EC.3.2.1.12) is a kind of hydrolase that can hydrolyze the β-1,4 glycosidic bond in chitin, and it mainly exists in bacteria, fungi and plants. According to its structural characteristics, it can be divided into two families of glycoside hydrolase (Glycosidehydrolase, GH) 18 and 19. Most of the chitinases from bacteria belong to the GH18 family, while the GH19 family chitinases are mainly of plant origin. Chitin oligosaccharides are oligosaccharides obtained by enzymatic hydrolysis of chitin. Its applications in medicine, food health care and plant defense have been discovered, and it has become a hot spot in chitin research. In industry, chemical methods (strong acid, strong alkali) and physical methods are commonly used to hydrolyze chitin biomacromolecules to prep...

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): C12N9/42C12P19/14C12P19/04C12P19/00
CPCC12N9/2434C12P19/00C12P19/04C12P19/14C12Y302/01
Inventor 娄文勇徐培宗敏华程建华杨继国
Owner SOUTH CHINA UNIV OF TECH
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