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Paenibacillus chitin enzyme and preparing method and application thereof

A technology of Paenibacillus and chitinase, which is applied in the biological field, can solve the problems of insufficient specificity and limited use, and achieve the effects of reduced crystallinity, reduced environmental impact, and high crystallinity

Inactive Publication Date: 2019-04-19
SOUTH CHINA UNIV OF TECH
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  • 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

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  • Paenibacillus chitin enzyme and preparing method and application thereof
  • Paenibacillus chitin enzyme and preparing method and application thereof
  • Paenibacillus chitin enzyme and preparing method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0040] Paenibacillus chitinase Pp Cloning, expression and enzymatic characterization of Chi1

[0041] (1) Pp Cloning, expression, isolation and purification of Chi1

[0042] According to the results of genome sequencing, select the gene containing the signal peptide sequence Pp The gene sequence of Chi1 (Genebank No.: KX431050.1) was used as the research object, combined with the signal peptide sequence prediction results, the forward primer Chip1_F: 5'-GAC was designed AAGCTT GCGCATCAAACTACAAGATC-3', reverse primer Chip1_R: 5'-GTGCTCGAGTTTCAGCTTCCAAAGAGC-3', with Paenibacillus pasadenensisThe genome of CS0611 was used as a template and amplified Pp Chi, connect it to the expression vector pET-28a, the restriction sites are HindIII and XhoI, construct the recombinant plasmid pET-28a-Chip1, and sequence it to confirm the sequence correctness.

[0043] The recombinant plasmid pET-28a-Chip1 was transformed into Escherichia coli BL21(DE3) supercompetent cells by heat shock ...

Embodiment 2

[0052] Pp Application of crab shell powder after Chi1 hydrolyzed ionic liquid pretreatment

[0053] (1) Pretreatment of crab shell powder

[0054] Investigate the effect of different ionic liquids on the effect of pretreatment: Add 90mg of crab shell powder passed through a 100 mesh sieve to 3mL of 1-allyl-3-ethylimidazolium chloride, 1-butyl-3-methylimidazolium tetrafluoro Chloride salt, 1-butyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium acetate were pretreated for 1 hour at 100°C, and then added water to precipitate chitin. The solid chitin was separated, and the precipitate was lyophilized, which was the pretreated crab shell powder (treated-CSP1, treated-CSP2, treated-CSP3, treated-CSP4). Take 10 mg of pretreated crab shell powder in 0.8 mL pH5.0 0.2 M citric acid buffer, add 0.2 mL Pp Chi1 (0.2 mg) solution was reacted at 45°C and pH 5.0 for 30 min. The untreated crab shell powder was used as a control to compare the hydrolysis efficiency of crab shell...

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Abstract

The invention discloses a paenibacillus chitin enzyme and a preparing method and application thereof. The paenibacillus chitin enzyme has the amino acid sequence shown in the SEQ ID NO.2. The gene forencoding the paenibacillus chitin enzyme has the nucleotide sequence shown in the SEQ ID NO.1. Through an escherichia coli expression system, the efficient dissoluble expression of the paenibacilluschitin enzyme can be conducted. Then, by preprocessing the crab shell powder through ionic liquid and using the purified paenibacillus chitin enzyme as the catalyst, compared with the unprocessed crabshell powder, the enzymolysis efficiency of the processed crab shell powder is improved by three times. Thus, good industrial application prospects are achieved during the paenibacillus chitin enzymeand the application of the paenibacillus chitin enzyme in the crab shell powder preprocessed by the enzymolysis ionic liquid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Paenibacillus chitinase and its preparation method and application. 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 biomacrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12P19/26C12P19/14
CPCC12N9/2434C12N15/70C12P19/14C12P19/26
Inventor 娄文勇徐培宗敏华程建华杨继国
Owner SOUTH CHINA UNIV OF TECH