Oxidation hydrolase gene BtLPMO10B and oxidation hydrolase and application

A technology of hydrolase and gene, which is applied in the field of preparation of oxidative hydrolase, can solve the problems that the crystal structure is difficult to be destroyed, and the catalytic efficiency of glycoside hydrolase is low, so as to achieve the effect of large production potential and economic value, and improve the degradation efficiency

Active Publication Date: 2016-06-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been a large number of reports on these glycoside hydrolases. However, due to the difficulty in destroying the crystal structure of these insoluble natural polysaccharides, the catalytic efficiency of glycoside hydrolases is low.

Method used

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  • Oxidation hydrolase gene BtLPMO10B and oxidation hydrolase and application
  • Oxidation hydrolase gene BtLPMO10B and oxidation hydrolase and application
  • Oxidation hydrolase gene BtLPMO10B and oxidation hydrolase and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 The cultivation of Bacillus thuringiensis subspecies Kustak and the extraction of its genomic DNA

[0025] A single clone of Bacillus thuringiensis subsp. Kustak strain (purchased from the China Agricultural Microorganism Culture Collection and Management Center, No. ACCC10066) was inoculated into 10ml liquid LB medium, and then placed in a temperature of 30°C and a rotation speed of 150rpm Cultivate on a shaker for 16 hours, take 3ml of the bacterial liquid, and centrifuge to collect the bacterial cells for the extraction of genomic DNA. The extraction and purification methods of genomic DNA were completed according to the instructions of the kit.

Embodiment 2B

[0026] Recombinant expression of embodiment 2BtLPMO10B gene in Escherichia coli

[0027] Using the genomic DNA of Bacillus thuringiensis subspecies Kustak ACCC10066 as a template, PCR amplification was performed with the following primer pairs. Primers were designed as follows:

[0028] Forward primer P-F: 5'-ggaattc catatg cacggttttgttgaaaagcccggta-3';

[0029] Reverse primer P-R: 5'-ccg ctcgag cactgttttccataatgataatgca-3'; the underlined forward primer is the restriction endonuclease NedI site, and the reverse primer is underlined the restriction endonuclease XhoI site. TaqDNA polymerase was purchased from Bao Biological Company, and the PCR reaction system was operated according to the product instructions provided by the company. PCR reaction conditions: 94°C pre-denaturation for 5 minutes, then denaturation at 94°C for 30 sec-50°C annealing for 30 sec-72°C extension for 1 min, 30 cycles, and finally 72°C extension for 10 min. The PCR product was double digested wit...

Embodiment 3

[0057] Example 3 Analysis of biochemical properties of oxidohydrolase BtLPMO10B

[0058] (1) Analysis of polysaccharide binding ability

[0059] The binding experiment between oxidohydrolase BtLPMO10B and various polysaccharides was carried out according to the following conditions: 5 mg of various polysaccharides were mixed with 0.5 mg of purified oxidohydrolase BtLPMO10B in a 1.5 ml Eppendorf tube, and the final volume of the reaction was passed through 20 mM, pH 8.0 Make up to 1 ml of Tris-HCl buffer. The combination of enzyme and substrate was carried out at room temperature for 24 hours. In order to make the combination of enzyme and polysaccharide better, a DH-II rotary mixer (Ningbo Xinzhi Company) was used. After the end, the supernatant and the precipitate were collected by centrifugation, and then detected by SDS-PAGE electrophoresis. The result is as figure 2 As shown, the oxidohydrolase BtLPMO10B has a strong binding ability to amorphous chitin (chitin beads an...

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Abstract

The present invention discloses an oxidation hydrolase gene and preparation and application of an oxidization hydrolase encoded by the oxidation hydrolase gene. The oxidation hydrolase BtLPMO10B gene is derived from bacillus thuringiensis subsp. kurstaki (ACCC 10066). The present invention also provides a method for preparation of the oxidization hydrolase BtLPMO10B, to be more specific, by use of a technical method of genetic engineering, the oxidation hydrolase BtLPMO10B gene is cloned to an Escherichia coli expression vector to obtain an escherichia coli recombinant strain capable of expressing the oxidation hydrolase, and the oxidation hydrolase BtLPMO10B prepared by the engineered strain expression has a strong ability to bind insoluble chitin, and is capable of oxidative hydrolysis of a polysaccharide substance to obtain saccharic acids having different degrees of polymerization. The oxidation hydrolase BtLPMO10B may assist polysaccharide hydrolase and can improve the efficiency of hydrolysis of polysaccharides.

Description

technical field [0001] The present invention relates to a gene sequence of a new type of oxidohydrolase BtLPMO10B (derived from Bacillus thuringiensis subspecies Kustak ACCC10066) and a preparation method and application of the encoded oxidohydrolase (Preparation and application of anovellytic polysaccharide monooxygenase). The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the oxidohydrolase. The oxidohydrolase BtLPMO10B involved in the present invention can be applied in the fields of bioenergy, green agriculture, health food, biomedicine and the like. Background technique [0002] Chitin is an insoluble natural polysaccharide mainly derived from marine shrimp and crab shells, insect shells, etc. The efficient biodegradation of chitin is of great significance in the fields of bioenergy, green agriculture, biomedicine, and material chemical engineering. At present, the enzymes that can be used for chitin biodegradation are mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12P19/14A01N63/02A01P3/00
Inventor 赵勇尹恒张卉妍王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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