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Oxidation hydrolase gene BtLPMO10A 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 BtLPMO10A and oxidation hydrolase and application
  • Oxidation hydrolase gene BtLPMO10A and oxidation hydrolase and application
  • Oxidation hydrolase gene BtLPMO10A 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 2BtLPMO10A 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'-cgctgcccagccggcgatggcccatggatatgtagaatcaccagcg-3';

[0029] Reverse primer P-R: 5'-gctttgttagcagccggatctcaaaatagtgtaggagtttgcatacg-3'; polymerase PrimeSTAR was purchased from Treasure Biotech, 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. After the PCR reaction, the PCR product was used as a primer, and the pET22b plasmid was used as a carrier to carry out secondary PCR amplification according to the above PCR ampli...

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 BtLPMO10A 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 BtLPMO10A 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 BtLPMO10A has a strong binding ability to crystalline chitin (α-chitin and ...

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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 BtLPMO10A gene is derived from bacillus thuringiensis subsp. kurstaki (ACCC 10066). The present invention also provides a method for preparation of the oxidization hydrolase BtLPMO10A, to be more specific, by use of a technical method of genetic engineering, the oxidation hydrolase BtLPMO10A 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 BtLPMO10A 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 BtLPMO10A 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 novel oxidohydrolase BtLPMO10A (derived from Bacillus thuringiensis subspecies Kustack 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 BtLPMO10A involved in the present invention can be applied in the fields of bioenergy, green agriculture, healthy 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 mainly ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/70C12P19/14A01N63/02A01P3/00
Inventor 赵勇尹恒王文霞张卉妍
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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