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Chitinase derived from marine environment and gene thereof

A chitinase gene, chitinase technology, applied in genetic engineering, plant genetic improvement, introduction of foreign genetic material using vectors, etc., can solve the problem of uncultivable microorganisms, etc. The effect of lasting stability

Active Publication Date: 2019-03-01
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under standard laboratory conditions, the vast majority of environmental microorganisms are not culturable

Method used

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  • Chitinase derived from marine environment and gene thereof
  • Chitinase derived from marine environment and gene thereof
  • Chitinase derived from marine environment and gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 The acquisition of Chitinase gene

[0033] Operate according to the genome walking kit.

[0034] (1) Extraction of metagenomic DNA from mangrove tidal flat soil samples: OMEGA Soil DNA kit was used to extract metagenomic DNA from soil samples.

[0035] (2) Acquisition of chitinase conserved sequence

[0036] The Chitinase gene of subfamily A of the 18 families already available at NCBI was analyzed, and degenerate primers were designed to amplify the conserved sequence of the Chitinase gene. The degenerate primer sequences are as follows:

[0037] ChiA F: 5' GGTGGACATCGACTGGGARTWYCC 3';

[0038] ChiA R: 5' CCCAGGCGCCGTAGARRRTCRTARSWCA 3'.

[0039] The PCR program was: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min, cycle amplification 35 times; final extension at 72°C for 10 min. After the amplification, the PCR product was taken for electrophoresis detection, and the target ...

Embodiment 2

[0055] Example 2 Expression and amplification of Chitinase coding gene in Escherichia coli

[0056] With the target gene obtained in embodiment 1-(5), and through to Sac I and xho The pET28a (+) plasmid of 1 double digestion is connected, obtains the recombinant plasmid pET28a (+)-Chitinase (such as figure 1 shown).

[0057] Take 10uL of the constructed plasmid DNA, add it to 100uL of the prepared competent Escherichia coli BL21(DE3), shake well and place it on ice for 30min; place it in a water bath at 42°C for 45s; place the centrifuge tube quickly Transfer to ice-water mixture for ice bath for 2min; add 800uL LB medium to each tube, lightly aspirate and disperse with a pipette, then recover on a shaker at 37°C for 1h (80rpm-200rpm); centrifuge, 4000rpm×5min, remove 700uL supernatant , and mix the remaining part; smear the plate (LB-agar plate, containing 100ug / mLAmp), place it upright at 37°C for 1 hour, and culture it upside down overnight. The positive clones contain...

Embodiment 3

[0059] Example 3 Purification of recombinant Chitinase

[0060] The fermentation broth prepared in Example 2 was centrifuged at 12,000 rpm for 10 min, and then the cells were resuspended in 20 mM Tris-HCl buffer (pH 7.4) and ultrasonically disrupted. The cells were broken and centrifuged at 12000rpm for 10min, and the supernatant was collected. Dilute the crude enzyme solution 5 times with buffer A (20mM Tris-HCl, 20mM imidazole, 500mM NaCl, pH 7.4), load it on a nickel column at a flow rate of 1mL / min, and use buffer A to elute weakly bound proteins to Equilibrate the column, then use buffer B (20mM Tris-HCl, 200mM imidazole, 500mM NaCl, pH 7.4) to elute the target protein at a flow rate of 1mL / min, collect the eluate, and measure the purity of the target protein by SDS-PAGE.

[0061] After the purification was completed, the specific activity of the obtained chitinase increased from 0.14 U / mg of the crude enzyme solution to 0.63 U / mg of the pure enzyme, the purification fac...

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Abstract

The invention provides a chitinase derived from a marine environment and a gene thereof and a method for cloning and expressing the chitinase in escherichia coli and yeast cells. The nucleotide sequence of the chitinase gene is shown as SEQ ID NO. 1; the amino acid sequence of chitinase is shown as SEQ ID NO. 2; a carrier containing the gene is an escherichia coli plasmid or a yeast plasmid; a cell containing the gene is transformed from the carrier; the cell of the gene of the chitinase is escherichia coli which comprises the nucleotide molecule or is transformed by the carrier or pichia pastoris which comprises nucleic acid molecule or is transformed by the carrier.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chitinase derived from a marine environment and a gene thereof. technical background [0002] The development of microbes based on metagenomic studies of samples allows us to unlock a lot of functional diversity, resulting in new genes and useful biomolecules. Chitin-encoding gene resources are very rich, but 99% of microorganisms are not cultivable, which brings great difficulties to obtain chitin genes. The metagenomic method can bypass the cultivation of microorganisms and directly study the genetic information of all microorganisms. In 2014, Karin Hjort et al. used the library constructed by soil metagenomics to target the chitinase of uncultivated bacterial communities and discovered a new type of bacterial chitinase Chi18H8. Chi18H8 was the first obtained from the metagenomic library. A purified enzyme with potential as an agent for the control of plant fungal d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N15/81C12N1/21C12N1/19C12R1/19C12R1/645
CPCC12N9/2442C12N15/70C12N15/81C12Y302/01014
Inventor 叶秀云胡亚娟李仁宽
Owner FUZHOU UNIV
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