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Expression and purification method of low-temperature chitinase gene chiC in Kluyveromyces lactis

A technology of chitinase gene and Kluyveromyces, which is applied in the fields of fermentation engineering, enzyme engineering, genetic engineering, and microbiology. It can solve the problems of poor stability of chitinase and high cost of separation and purification, and reduce the separation Purification cost, effect of improving expression efficiency

Inactive Publication Date: 2019-07-05
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there have been reports on the expression of the chitinase chiC gene from Pseudoalteromonas sp.DL-6 in Escherichia coli. The cost of separation and purification increases, and the growth temperature of Escherichia coli is 37°C, so the stability of low-temperature chitinase is relatively poor

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  • Expression and purification method of low-temperature chitinase gene chiC in Kluyveromyces lactis
  • Expression and purification method of low-temperature chitinase gene chiC in Kluyveromyces lactis
  • Expression and purification method of low-temperature chitinase gene chiC in Kluyveromyces lactis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of Kluyveromyces lactis expression plasmid pKLAC2-chiA

[0039] 1.1 Primer design and amplification of target gene

[0040] According to the low-temperature chitinase chiC (GenBank No.KF234017) gene sequence and the codon preference of Kluyveromyces lactis, the signal peptide sequence was removed, and the gene mature peptide nucleotide sequence was codon-optimized using GeneOptimizer software, codon optimization The latter opt-chiC is shown in SEQ ID NO.1 (GenBank No. MK635353).

[0041] Using the codon-optimized gene sequence of opt-chiC as a template, the PCR primer parameters were optimized using Primer 5.0 software, and primers with XhoI and EcoRI restriction sites and protective bases were designed. by TaKaRa LA Taq TM DNA Polymerase PCR amplifies the mature target fragment without signal peptide, and introduces a 6×His affinity purification tag at the C-terminus. The PCR reaction conditions were: 98°C for 1 min, 1 cycle; 98°C for 10 s, 5...

Embodiment 2

[0051] Example 2 Secretion and expression of low temperature chitinase gene chiC in Kluyveromyces lactis GG799

[0052] 2.1 Preparation of Kluyveromyces lactis GG799 (preserved in the laboratory) electroporation competent cells and their electroporation transformation

[0053] (1) pick fresh single colony of yeast in 5ml YPD liquid culture medium composed of 1% yeast powder, 2% peptone, and 2% glucose, and cultivate overnight at 30°C at 200rpm;

[0054] (2) Inoculate 500 μl of the culture into a 250 mL Erlenmeyer shaker flask containing 50 ml of YPD liquid medium, culture at 30°C and 200 rpm overnight, until the OD600 reaches 1.3-1.5;

[0055] (3) Centrifuge the cell culture at 1500g for 5min at 4°C, and resuspend the cell pellet with 20mL of ice-cold sterile water;

[0056] (4) Centrifuge according to step 3, and resuspend the bacterial cell pellet with 20ml of ice-cold sterile water;

[0057] (5) Centrifuge according to step 3, and resuspend the bacterial cell pellet with ...

Embodiment 3

[0089] Example 3 Optimum Temperature and Temperature Stability, Optimum pH and pH Stability of Recombinant Chitinase Gene chiC

[0090] The effect of temperature on the enzyme ChiC

[0091] (1) Optimum reaction temperature: under the condition of pH value 8.0 (50mmol / L Tris-HCl buffer solution), with 4MU-(GlcNAc) 2 As a substrate, measure the enzyme activity in the temperature range of 4°C to 70°C, draw a curve according to the relative activity of the enzyme at different temperatures, and determine the optimum reaction temperature of the enzyme.

[0092] (2) Thermal stability of the enzyme: the enzyme solution was incubated at different temperatures (4°C to 70°C) for 1 hour, and then the residual enzyme activity was detected at the optimum reaction pH and temperature, and compared with untreated Enzyme activity was compared to calculate relative activity.

[0093] Effect of pH on Enzyme ChiC

[0094] (1) Optimum reaction pH value: the enzyme activity reaction temperature i...

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Abstract

The invention relates to the field of genetic engineering, microbiology, enzyme engineering and fermenting engineering and provides a cloning, expression and purification method of low-temperature chitinase gene chiC in Kluyveromyces lactis. Recombinant Kluyveromyces lactis capable of producing low-temperature chitinase chiC is constructed successfully herein; high-purity recombinant chitinase chiC is acquired by purifying; pure recombinant chitinase chiC is acquired by nickel column affinity chromatography purification; SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis shows that a protein band of expected size is present near 90 kDa. Most of the expressed chitinase chiC is secreted out of cells, so that separation and purification cost is reduced, and expression efficiency is improved. The protein concentration of the purified chiC is 1.48 mg / mL, and enzymic activity is 108.56 U / mg. The enzymatic properties of the purified chitinase ChiC, such as temperature, temperature stability, pH, pH stability and coordinative degradation, are studied, and basis is laid for the industrial application of the chitinase.

Description

technical field [0001] The invention relates to the fields of genetic engineering, microbiology, enzyme engineering and fermentation engineering, and provides a method for cloning, expressing and purifying a low-temperature chitinase gene chiC (chitinase C) in Kluyveromyces lactis. Background technique [0002] Chitin is a polysaccharide second only to cellulose in nature, and is the most abundant renewable resource in the marine environment. Its degradation is mainly completed by the chitinase degradation system secreted by marine microorganisms. Among them, chitinase ChiC is one of the key enzymes, which can degrade the backbone of polysaccharides into short-chain oligosaccharides. The high value-added chitooligosaccharides, chitin monosaccharides and their derivatives produced by the degradation of chitin by chitinase have good tissue compatibility, biodegradability, and regulation of immunity, antibacterial, induction of plant disease resistance, It can promote biologic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/80
CPCC12N9/2442C12Y302/01014C12N15/80
Inventor 陈立功王文毫王晓辉迟乃玉张庆芳
Owner DALIAN UNIV