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A chitinase and its gene for preparation of chitooligosaccharides

A chitinase gene and chitinase technology are applied in the fields of glycosylase, genetic engineering, and plant gene improvement, which can solve the problems of enzyme inactivation, long growth cycle, and reduction of enzyme activity, and achieve high activity and stability, maintaining activity and stability, and efficient degradation

Active Publication Date: 2021-08-31
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of intracellular enzymes requires mechanical disruption for release from cells, which may result in inactivation or reduced enzyme activity, limiting the large-scale production and application of chitinases
In addition, the operation of Pichia pastoris is complicated, and its growth cycle is long, but it has the advantages of high copy, high-density fermentation, and the expression gene is stable in the host genome, so choosing a suitable host is also very important

Method used

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  • A chitinase and its gene for preparation of chitooligosaccharides
  • A chitinase and its gene for preparation of chitooligosaccharides
  • A chitinase and its gene for preparation of chitooligosaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Chitinase gene chi1602 the acquisition

[0041] (1) Microbulbifer sp . Separation and extraction of genomic DNA:

[0042] Take 1.5 mL bacterial culture Microbulbifer sp . (purchased from China Marine Microorganism Culture Collection Management Center) in a sterilized Ep tube, centrifuged at 12000 rpm for 1 min, discarded the supernatant, and collected the bacteria.

[0043] Add 400 μL of lysate solution (40 mM Tris-acetic acid, 20 mM sodium acetate, 1 mM EDTA, 1% SDS, pH 7.8), mix well, and place in a 37°C water bath for 1 h.

[0044] Then add 200 μL of 15 mol / L sodium chloride solution, mix well and centrifuge at 13000 rpm for 15 min.

[0045] Take the supernatant, extract twice with phenol and once with chloroform.

[0046] Add twice the volume of absolute ethanol and 1 / 10 volume of potassium acetate (3 M, pH 8.0), store at -20°C for 1 h, centrifuge at 13,000 rpm for 15 min, discard the supernatant, and wash the precipitate twice with 70% ethanol;...

Embodiment 2

[0057] Example 2 Expression and amplification of chitinase-encoding gene chi1602 in Escherichia coli

[0058] The result obtained in embodiment 1-(4) Sec I and not I double-enzyme-digested target gene, and Sec I and not The pET-28a (+) plasmid of I double digestion is connected, obtains recombinant plasmid pET-28 (a)- chi1602 (Such as figure 1 shown).

[0059] Take 10 uL of the constructed plasmid DNA and add it to 100 uL of prepared Escherichia coli BL21(DE3) competent cells, shake well and place on ice, ice bath for 30 min; place in a 42°C water bath for 45 s heat shock; put Quickly move the centrifuge tube to ice-water mixture for 2 min; add 800 uL LB liquid medium to each tube, and recover on a shaker at 37°C for 1 h (80 rpm to 200 rpm); centrifuge at 4000 rpm for 5 min, and discard 800 uL Clear, and mix the rest; smear on a plate (LB-agar plate, containing 100 ug / mLAmp), culture overnight at 37°C, pick the recombinants into LB liquid medium, wait until the bact...

Embodiment 3

[0061] Example 3 Construction of Yeast Recombinant Expression Vector

[0062] The result obtained in embodiment 1-(4) SnaB I and Avr ⅡThe target gene of double enzyme digestion, and after SnaB I and Avr Ⅱ The double-digested pPIC9k plasmid was ligated to obtain the recombinant plasmid pPIC9k- chi1602 (Such as figure 1 shown).

[0063] Transfer the recombinant plasmid into E. coliTop10 competent cells were spread on the LB screening plate containing 100 mg / mL Amp and cultured overnight, and the recombinants were picked and identified by bacterial liquid PCR. If they were positive recombinants, they were sent for sequencing, and the sequencing results further proved the sequence of the target gene and the correctness of the insertion site. Select the bacterial solution with the correct inserted gene, follow the extraction steps of the OMEGA PlasmidMini Kit, extract the recombinant plasmid, and store it at -20°C for later use.

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Abstract

The present invention provides a chitinase and its gene for the preparation of chitin oligosaccharides, and a method for cloning and expressing it in Escherichia coli and yeast cells. The nucleotide sequence of the chitinase gene is as shown in SEQ ID Shown in NO.1; The amino acid sequence of chitinase is shown in SEQ ID NO.2; The recombinant vector containing said gene is that chitinase gene is connected to Escherichia coli plasmid or yeast plasmid; The cells of said gene are composed of The recombinant vector is transformed; the cell containing the chitinase gene is Escherichia coli that contains the nucleotide molecule or is transformed with the recombinant vector or contains the nucleotide molecule or is transformed with the recombinant vector Transformed Pichia pastoris.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chitinase and a gene thereof for preparing chitooligosaccharides. [0002] technical background [0003] The marine environment can provide new compounds with great application potential, such as enzymes with potential market value, which are rarely found in marine sediments despite the continuous production of crustacean waste in the marine environment. These phenomena indicate that there are abundant chitinase-producing microorganisms widely distributed in the marine environment. The chitinase produced by these microorganisms from the marine environment can hydrolyze chitin and complete the processing of chitin in nature. It has been reported that marine bacteria Alteromonassp. strain 0-7, Micrococcus sp. AG84, Pseudoalteromonas sp. DL-6, Vibriofurnissii, Aeromonas caviae and Pseudoalteromonas sp. DXK012 can degrade chitin into chitooligosaccharides for their metabolis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N15/81C12N1/21C12N1/19C12P19/26C12R1/19C12R1/84
CPCC12N9/2442C12N15/70C12N15/815C12P19/26C12Y302/01014
Inventor 李仁宽叶秀云胡亚娟何雨洁
Owner FUZHOU UNIV
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