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Recombinant carrier containing endo-chitinase genes

A chitinase and carrier technology, applied in the field of genetic engineering, can solve the problems of high energy consumption, difficult control of the degree of degradation, and secondary environmental pollution for acid degradation, and achieve the effect of industrialized production.

Inactive Publication Date: 2011-02-09
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Acid degradation consumes a lot of energy, and the degree of degradation is difficult to control. The degradation products are mainly monosaccharides. The waste water containing sulfuric acid or hydrochloric acid generated during the degradation process is discharged directly or neutralized with alkali and then discharged into the environment, causing serious secondary damage to the environment. Pollution

Method used

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  • Recombinant carrier containing endo-chitinase genes
  • Recombinant carrier containing endo-chitinase genes
  • Recombinant carrier containing endo-chitinase genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1. Obtaining of Trichoderma endochitinase cDNA

[0016] Extract total RNA by Trizol method: Grind 100 mg of Trichoderma mycelium into powder in liquid nitrogen, and transfer the powder to a 1.5 mL centrifuge tube when the liquid nitrogen is not yet volatile. Use a 1mL syringe, 26-G (Ф4.5) needle to aspirate the homogenate several times to shear the genomic DNA, and then directly transfer the sample from the syringe to a 1.5mLEppendorf tube. Add 100 μL of chloroform / isoamyl alcohol (24:1), shake vigorously and mix for 30 seconds. Centrifuge at 12,000 rpm on a bench top centrifuge for 5 minutes at room temperature. Carefully transfer the supernatant to a sterile 1.5mL RNase-free centrifuge tube, add 150μL of absolute ethanol, and mix. Transfer all the above solutions to a UNIQ-10 column nested in a 2mL collection tube, leave it at room temperature for 5 minutes, and centrifuge at 8,000 rpm for 1 minute. Take out the column carefully, discard the waste liquid in th...

Embodiment 2

[0024] Example 2. Obtaining of Chitinase DNA from Trichoderma

[0025] Weigh 200mg (fresh weight) of Trichoderma mycelium and grind it into powder in liquid nitrogen. Use 0.5ml of solution I (100mmol / L NaCl; 30mmol / L Tris-HCl, pH7.8; 10mmol / L EDTA, pH8) .0; 10mmol / L β-mercaptoethanol) suspension, the suspension was centrifuged at 12000g for 1min to get a precipitate, and solution I (100mmol / L NaCl; 30mmol / L Tris-HCl, pH7.8; 10mmol / L EDTA, pH8 was added. 0; 10mmol / L β-mercaptoethanol) suspension and centrifuge once. The precipitate was suspended with 0.6 mL of solution II (0.1 mmol / L NaCl; 0.2 mmol / L sucrose; 10 mmol / L EDTA). Add 0.2mL lysate, shake vigorously for 1min, and place the suspension in a 55℃ water bath for 30min. Extract twice with an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) and centrifuge at 12000g for 5min. Take the supernatant and add 1 / 10 volume of 3mol / L NaAc, mix well, add 2 times volume of absolute ethanol, precipitate at room temperature f...

Embodiment 3

[0032] Example 3. Construction of prokaryotic expression vector of Trichoderma endochitinase cDNA

[0033] Such as image 3 Shown is the construction process of a Trichoderma endochitinase cDNA containing signal peptide prokaryotic expression vector.

[0034] The genomic DNA and cDNA sequences were compared to obtain the partial sequence of introns. Two sets of primers were designed to construct a prokaryotic expression vector containing signal peptides (primers P1, P2) and without signal peptides (primers P3, P2). The primer sequence is as follows:

[0035] P1 (SEQ ID NO.4): 5'-GACGAATTCATGTTGGGCTTCCTCGGAAAA-3'

[0036] P2 (SEQ ID NO.5): 5'-AGACTCGAGAGTTGAGACCGCTTCGGATGTT-3'

[0037] P3 (SEQ ID NO.6): 5'-CATGAATTCGCCAGCGGATATGCAAACGCC-3'

[0038] The plasmid pMD-ECH and the E. coli original expression vector pET28a (purchased from Novagen Co., Ltd., USA) were extracted by alkaline method. Using P1 and P2 as primers, using pMD-ECH as template, PCR technology was used to amplify the end...

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Abstract

The invention relates to an endonuclease chitinase, the gene expressing the endonuclease chitinase and the restructuring carrier containing the endonuclease chitinase gene. The endonuclease chitinase gene is cloned from trichoderma using modern biotechnoloty; the microorganism expression vector is constructed, and the expression vector is transformed to an escherichia coli and Pichia pastoris which has the advantages of high expression, easy purification and easy adaptation for large-scale industrial fermentation production; the high secretion type endonuclease chitinase engineering bacteria is constructed, and the chitin is catalyzed and degraded to the chitin oligosaccharide utilizing the engineering bacteria, so as to realize the industrial production and the industrialization demonstration of chitin oligosaccharide.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, in particular to an endochitinase, a gene expressing the enzyme, and a recombinant vector containing the endochitinase gene. Background technique [0002] Chitin is also known as chitin, and its chemical name is (1,4)-2-acetylamino-2-deoxy-β-D-glucose. It is connected by N-acetylglucosamine through β-1,4-glycosidic bonds. The resulting natural polymer amino polysaccharide compounds are widely present in the cells of lower plant fungi, algae, crustaceans, shrimps, crabs, insect shells and the cell walls of higher plants. The annual biosynthesis of chitin in nature is nearly 10 billion tons, of which 10% comes from the ocean. It is the second largest renewable resource on the earth after cellulose. Unfortunately, this resource is not fully utilized. Natural waste as waste, not only caused huge waste, but also caused serious environmental pollution. A large amount of waste chitin resources ex...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/56C12N15/81C12N1/21
Inventor 于平励建荣章慧慧陆海霞朱军莉张蕾唐云平李学鹏
Owner ZHEJIANG GONGSHANG UNIVERSITY
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