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Novel chitinase P1724 and application thereof

A technology of P1724 and chitinase, which is applied in the field of transgenic engineering, can solve problems such as unreported, energy consumption, and CHI-X is not suitable, and achieve good application prospects, energy saving, and short processing time.

Active Publication Date: 2017-12-15
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To maintain the higher reaction temperature required by CHI-X, more energy will be consumed
Therefore, CHI-X is not suitable for food, biological control and other fields where the ambient temperature is lower than 30°C
[0008] At present, chitinases with two catalytic domains and high activity at low temperature have hardly been reported.

Method used

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  • Novel chitinase P1724 and application thereof
  • Novel chitinase P1724 and application thereof
  • Novel chitinase P1724 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Configure microbial culture medium

[0029] (1) according to table 1, table 2, table 3, table 4, configure microbial culture medium, wherein, solution B described in table 1 is the K of 8g / L 2 HPO 4 .

[0030] Table 1 Medium composition list

[0031] serial number

composition

volume and weight

1

Solution A

50ml / L

2

Solution B

50ml / L

3

trace metal solution

10ml / L

4

Wolf's Vitamin Solution

10ml / L

5

Yeast extract

1g

6

Resazurin (0.1%)

1ml / L

[0032] Table 2 Component list of solution A

[0033]

[0034]

[0035] Table 3 Composition list of trace metal solution (100×)

[0036] serial number

composition

weight(g / L)

1

nitrilotriacetic acid

12.8

2

FeSO 4 ·7H 2 o

0.42

3

MnCl 2 4H 2 o

0.1

4

CoCl 2 ·6H 2 o

0.17

5

CuCl 2 2H 2 o

0.02

6

ZnSO 4 ·7H 2 o

0.21...

Embodiment 2

[0048] Enrichment of microbial communities

[0049] (1) Using the microbial culture medium to adjust the soil collected from the Qinghai-Tibet Plateau to a homogenate sample with a water content of 90%.

[0050] (2) Put the homogenized soil sample in an anaerobic bottle with pure N 2 Replace the air in the bottle to create an anaerobic environment.

[0051] (3) Place the sample in an environment at 4°C for 24 hours to balance the gas phase and residual oxygen.

[0052] (4) In the anaerobic box, about 50ml of soil homogenate samples mixed uniformly and under anaerobic conditions are mixed with flake chitin (0.8%, w / v) in a 100ml serum bottle, filled with an appropriate amount of nitrogen, Incubate at 4°C.

[0053] (5) When the methane production in the anaerobic bottle reaches about 35%, take out 5 ml of culture solution and mix it with 50 ml of fresh 0.8% chitin flakes, and continue to cultivate at 4°C.

[0054] (6) Passaging in this way for 3 times, after a total of 2 years...

Embodiment 3

[0056] Analyzing microbial community metagenomes

[0057] (1) Use PowerSoil from MOBIO TM The DNA Isolation Kit extracts the total DNA of the sample. The metagenomic sequencing mode mainly uses Hiseq X-ten and 150P. The analysis process is to perform sequence filtering, assembly and gene and species classification annotation after obtaining the raw data of off-machine. The databases for functional annotation of gene sequences mainly include COG, Uniprot, KEGG, eggNOG, EC, etc. CAZy annotation is the most important analysis involved in the present invention. The main carbohydrate-degrading enzymes (such as cellulase, chitinase) belong to the GHs class.

[0058] (2) Nucleotide sequence of P1724 gene: SEQ ID NO 1.

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Abstract

The invention belongs to the technical field of transgenic engineering and aims to provide chitinase provided with 2 catalytic domains and having higher activity under the low temperature condition. According to the adopted technical scheme, microbial communities are enriched and cultured in a low temperature environment, metagenomes of the microbial communities are analyzed, a chitinase P1724 gene is cloned, recombinant bacteria are established, P1724 protein is expressed, HIS-TAG is purified, and the properties of recombinant protein serving as the chitinase are identified. The chitinase provided with the 2 catalytic domains is directly obtained for the first time from unseparated microbial strains in the environment through a macroscopic gene method, has better activity under the low temperature condition, has the advantages that the treating time is shorter than the treating time required for medium-high-temperature enzyme, the production cost is low, more energy is saved in application process, is more suitable for industrial production and can have a better application prospect in engineering application.

Description

technical field [0001] The invention belongs to the technical field of transgenic engineering, and in particular relates to a low-temperature chitinase P1724 gene obtained from the Qinghai-Tibet plateau wetland environmental microbial genome and a potential application method thereof. Background technique [0002] Chitin, also known as chitin and chitin, is a long-chain polymer connected by N-acetyl-D-glucosamine with β-1,4-glycosidic bonds, and is also the second most abundant polysaccharide in nature. polysaccharides. The amount of chitin produced in nature every year is extremely large. As an important source of organic carbon and nitrogen, chitin and its degradation products participate in the material exchange and energy flow of the ecosystem. Chitinase can catalyze the decomposition of chitin to produce chitooligosaccharides and chitobiose. Oligosaccharides, the hydrolyzed products of chitinase, have antibacterial effects, can regulate the immunity of organisms, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70C12N1/21C12P19/26C12P19/14
CPCC12N9/2442C12N15/70C12P19/14C12P19/26C12Y302/01014
Inventor 代玉梅李艳杨凤张玲利刘晓风
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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