Method for expressing and purifying low temperature chitinase gene chiA in kluyveromyceslactis

A technology of chitinase gene and Kluyveromyces, which is applied in the fields of microbiology, enzyme engineering, fermentation engineering, and genetic engineering, 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

Pending Publication Date: 2019-05-31
DALIAN UNIV
View PDF4 Cites 5 Cited by
  • 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 chiA gene derived from Pseudoalteromonas sp.DL-6 in Escherichia coli. Although the expression level of the target gene is high, the product is mainly concentrated in the cell, making subsequent 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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for expressing and purifying low temperature chitinase gene chiA in kluyveromyceslactis
  • Method for expressing and purifying low temperature chitinase gene chiA in kluyveromyceslactis
  • Method for expressing and purifying low temperature chitinase gene chiA in kluyveromyceslactis

Examples

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 chiA (GenBank No.KF234015) 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 rear opt-chiA gene sequence is shown in SEQ ID NO.1. (GenBank No. MK635352).

[0041] Using the codon-optimized gene sequence of opt-chiA as a template, use Primer5.0 software to optimize the parameters of PCR primers, and design primers with XhoI and EcoRI restriction enzyme sites and protective bases. 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...

Embodiment 2

[0051] Example 2 Secretion and expression of low temperature chitinase gene chiA 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) Take 500 μl of the culture and inoculate it into a 250 mL Erlenmeyer shaker flask containing 50 ml of fresh 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 ...

Embodiment 3

[0089] Example 3 Optimum Temperature and Temperature Stability, Optimum pH and pH Stability Research of Recombinant Chitinase ChiA

[0090] Effect of temperature on the enzyme ChiA

[0091] (1) Optimum reaction temperature: under the condition of pH value 8.0 (50mmol / L Tris-HCl buffer solution), with 4MU-(GlcNAc)2 as the substrate, the enzyme is determined within the temperature range of 4°C to 70°C According to the relative activity of the enzyme at different temperatures, draw a curve to 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 ChiA

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
capacitanceaaaaaaaaaa
electrical resistanceaaaaaaaaaa
particle diameteraaaaaaaaaa
Login to view more

Abstract

The invention relates to the field of genetic engineering, microbiology, enzyme engineering and fermentation engineering, and provides a method for expressing and purifying a low temperature chitinasegene chiA (chitinase A) in kluyveromyceslactis.The method for expressing and purifying the low temperature chitinase gene chiA (chitinase A) in the kluyveromyceslactissuccessfully constructs a recombinant kluyveromyceslactis producing the low temperature chitinasechiA and purifiesthe recombinant kluyveromyceslactisto obtain a high purity recombinant chitinasechiA, and a pure recombinant chitinasechiA is obtained by using a nickel column affinity chromatography. After SDS-PAGE analysis, protein bands of the desired size appear near 110 kDa.According to the method for expressing and purifying the low temperature chitinase gene chiA (chitinase A) in the kluyveromyceslactis, the chitinasechiAexpressed is mostly secreted outside cell, thereby reducing the separation and purification cost and improving the expression efficiency.The protein concentration of purified protein chiAis 1.26 mg/mL and the enzyme activity is 51.45 U/mg.The purified chitinaseChiA has been studied in enzymatic properties such as temperature and temperature stability, pH and pH stability and synergistic degradation, and a foundation for industrial application of such enzyme is laid.

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, purifying, and studying enzymatic properties of a low-temperature chitinase gene chiA (chitinase A) 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 ChiA 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 diseas...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12R1/645
Inventor 陈立功王晓辉迟乃玉张庆芳
Owner DALIAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap