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Endochitinase and coding gene and application thereof in production of chitobiose

A technology of chitinase and coding gene, which is applied in the field of bioengineering, can solve the problems of chitinase research starting late, not realizing industrialization, and low yield of chitobiose, so as to achieve less energy consumption and promote development , the effect of high conversion rate

Active Publication Date: 2016-06-08
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have reported that not only the yield of chitobiose is low, but also its content is low, which brings great difficulty to the subsequent separation and purification, so the preparation technology needs to be further improved
[0006] Domestic research on chitinase started relatively late, and has not yet achieved industrialization

Method used

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  • Endochitinase and coding gene and application thereof in production of chitobiose
  • Endochitinase and coding gene and application thereof in production of chitobiose
  • Endochitinase and coding gene and application thereof in production of chitobiose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 Discovery of chitinase gene

[0054] 1. Design of degenerate primers for PCR amplification of the conserved region of chitinase genome

[0055] Using Paenibacillus barengoltzii CAU904 as the genome template, according to the chitinase amino acid sequence reported in the GenBank database, the online software BlockMaker (http: / / blocks.fhcrc.org / blocks / blockmkr / make_blocks. html) to search for conserved regions. According to the conserved amino acid sequence of chitinase, the online design software CODHOP (http: / / blocks.fhcrc.org / codehop.html) was used to select the amino acid sequence of chitinase from Paenibacillus GH18 family reported in GenBank. For homologous alignment, select the sequence conservation regions THINYAFA and DLDWEYPV to design degenerate primers Chi2DF and Chi2DR in Table 2.

[0056] PCR reaction conditions: pre-denaturation at 94°C for 5 minutes; 20 cycles of denaturation at 94°C for 30s, annealing at 62-52°C for 30s, and extension at 72...

Embodiment 2

[0062] Embodiment 2 Expression of endochitinase gene

[0063] 1. Construction of recombinant expression vector

[0064] Genomic DNA of Paenibacillus barengerzia was used as a template to amplify the chitinase PbChi70 gene with the removal of the signal peptide using the upstream and downstream primers Chi2EF and Chi2ER. The agarose gel electrophoresis of the PCR amplification product is shown in figure 2 . The PCR amplification product was recovered, connected to the pMD18-T vector, and transformed into Escherichia coli DH5α by heat shock method. The positive recombinant plasmid was named pMD18-T-PbChi70 and sent for sequencing. Double-digest the plasmid pMD18-T-PbChi70, recover the target gene fragment, connect it with the expression vector pET-28a(+) that has been cut by the same double enzyme, transform the ligated product into Escherichia coli DH5α, extract the plasmid and identify the positive recombinant. The correct recombinant plasmid was named pET28a-PbChi70.

[0...

Embodiment 3

[0067] Example 3 Purification and property determination of endochitinase

[0068] 1. Purification of Chitinase

[0069] The positive clones were picked and cultured in 50 mL LB liquid medium (50 μg / mL) at 37° C. on a shaker until logarithmic phase, and used as seed solution. Inoculate the seed liquid in 100mL LB liquid medium, use a 500mL Erlenmeyer flask containing 50μg / mL kanamycin, culture on a shaker at 37°C until the OD600 reaches 0.6-0.8, add IPTG with a final concentration of 1mM, and induce overnight.

[0070] After the culture was completed, the bacterial liquid was collected by centrifugation at 10,000 rpm for 2 minutes. The bacterial liquid was suspended in 20 mL of pH7.4 phosphate buffer, and the cells were disrupted by ultrasonication at 200 W for 3 s, intermittently for 4 s, and 90 times. After the cells are disrupted, centrifuge at 10,000 rpm for 10 minutes to collect the supernatant, namely the crude enzyme solution. The pellet was resuspended in phosphate ...

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Abstract

The present invention discloses endochitinase and a coding gene and application thereof in production of chitobiose. The protein is as follows: (a) or (b) or (c), wherein (a) is a protein comprising 40th-site to 696th-site amino acid residues from N-terminal of a sequence 2; (b) is a protein comprising an amino acid sequence shown as the sequence 2 in a sequence table; and (c) is a chitinase-activity protein which is derived from a sequence 1 and obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence of the (a) or (b). When the endochitinase is used for production of the chitobiose, conversion rate is high, energy consumption is less, no pollutant is produced, and the endochitinase has important economic value. The protein has a large potential for industrial application.

Description

technical field [0001] The present invention relates to a chitinase gene. More specifically, it relates to an endochitinase gene and its application in preparing chitobiose. Belongs to the field of bioengineering. Background technique [0002] Chitin (Chitin) is a water-insoluble linear polymer formed by N-acetylglucosamine linked by β-1,4-glucosidic bonds (Bendtetal. , 5:119-126), is the renewable biomass second only to cellulose in nature, and the biosynthesis of chitin is as high as tens of billion tons every year. Chitin is widely found in the carapaces of shrimps, crabs, and insects, as well as in the cell walls of fungi and plants. Chitin and its derivatives, such as chitooligosaccharides and chitosan, have received more and more attention in recent years due to their high utilization value in antibacterial and antitumor aspects. The production of traditional chitin-related products is mainly obtained through chemical acid hydrolysis, and the production process has...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/26C12P19/14C12R1/01
Inventor 闫巧娟付星杨绍青江正强郭禹杨鑫斌
Owner CHINA AGRI UNIV
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