Site-directed mutagenesis-modified agarase mutant with improved thermal stability

A thermostability and site-directed mutation technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of agarase instability and limit the application of agarase, and achieve the effect of increasing half-life

Active Publication Date: 2019-01-15
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, agarase screened from nature is usually unstable in severe industrial

Method used

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  • Site-directed mutagenesis-modified agarase mutant with improved thermal stability
  • Site-directed mutagenesis-modified agarase mutant with improved thermal stability
  • Site-directed mutagenesis-modified agarase mutant with improved thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of agarase mutant D622G

[0026] The gene sequence of the mutant was amplified by overlap extension PCR technique. First design degenerate primers to amplify the source Vibiro The conserved sequence of the agarase rAgaZC-1 gene of sp. was cloned by TAIL-PCR to obtain the agarase gene aga ZC-1 full-length sequence (sequence shown in SEQ ID NO.1); construction of recombinant plasmid pET-22b- aga ZC-1. The recombinant plasmid pET-22b- aga ZC-1 was used as the template for the first round of PCR amplification to aga ZC-1-F, D622G-R as primers to amplify the upstream fragment of the target gene to aga ZC-1-R, D622G-F are primers to amplify the downstream fragment of the target gene; then the purified upstream and downstream fragments are mixed and properly diluted as the template for the second round of PCR to aga ZC-1-F, aga ZC-1-R is the primer for amplifying the target gene. The information of each primer is as follows, where the und...

Embodiment 2

[0036] Example 2 Induced expression and purification of agarase

[0037]The engineered bacteria expressing rAgaZC-1 and mutant enzyme D622G were inoculated into LB medium containing 100 mg / L Amp, cultured overnight at 37°C and 200 rpm, and then transferred to the same medium with 1% inoculum. Continue culturing until OD600 is about 0.6, then add IPTG at a final concentration of 1 mM, and continue culturing at 28°C and 180 rpm for 8 h.

[0038] The fermentation broth was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was concentrated by using a hollow fiber column (MWCO: 10 kDa) and ammonium sulfate precipitation to concentrate the enzyme solution. Ammonium sulfate was removed by dialysis in the buffer solution, and the target protein was purified by NTA-Ni column (BBI, China) after filtration through a 0.22 μm membrane. After the collected enzyme solution was concentrated properly, SDS-PAGE was carried out, and the results were as follows: Figure 2-A As sh...

Embodiment 3

[0039] Example 3 Characterization of agarase

[0040] 1. Agarase Activity Assay

[0041] The activity of agarase was determined by DNS method. Add 100 μL of enzyme solution to 900 μL of 50 mM Tris-HCl (pH 7) solution containing 0.2% agar, and react in a water bath at 40°C for 15 min, then add 1.5 mL of DNS reagent to terminate the reaction. After developing color in boiling water for 5 min, cool to room temperature, shake well and measure OD 540 . The released reducing sugar content was determined according to the glucose standard curve. Definition of enzyme activity unit: Under certain reaction conditions, the amount of enzyme required to hydrolyze agarose to produce 1 μmol reducing sugar per minute is defined as an enzyme activity unit U.

[0042] 2. Catalytic Kinetics of Agarase

[0043] At 38.5°C, the enzyme activity of the pure enzyme at different substrate concentrations (0.1-0.5 g / L) was determined, and the hydrolysis reaction of the pure enzyme catalyzed by the ag...

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Abstract

The invention provides a site-directed mutagenesis-modified agarase mutant with improved thermal stability and belongs to the field of genetic engineering and enzyme engineering. A mutant according tothe invention is obtained in beta;-Agarase rAgaZC-1 to glycine (D622G). The nucleotide sequence of mutant D622G and the corresponding amino acid sequence are shown in SEQ ID NO. 3 and SEQ ID NO. 4. The catalytic efficiency of D622G was higher than that of rAgaZC-1 There was a 17.6% increase. Thermal inactivation kinetics and hydrolysis kinetics analysis showed that the thermal stability of D622Gwas obviously improved, T5010 was increased by 1.5 DEG C, half-life t1/2 at 41 DEG C was prolonged by three times, and more agarose oligosaccharides could be accumulated at 43 DEG C, which enhanced the application potential of agarase in agarose oligosaccharides degradation.

Description

technical field [0001] The invention relates to an agarase mutant with improved thermostability through site-directed mutation transformation, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Agarose (Agarose) is the main component of agarose, which is a chain structure formed by the alternating connection of β-D-galactose (Gal) and α-3,6-endo-L-galactose (ALGal). Agarase is a type of glycoside hydrolase (Glucoside Hydrolase, GH) that exclusively degrades agarose to produce agarose oligosaccharides, most of which are derived from marine microorganisms, mainly Gram-negative bacteria, including alternate unit cells Bacteria, Pseudoalteromonas, Microcormosa, Agarophage, etc. [0003] When the agarose is above the sol-gel transition temperature, it is in a sol state. Since the agarose in the sol state has a smaller specific surface area and flow restriction than the gel state, it has a better affinity for agarase, that is, th...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12P19/14C12P19/00C12P19/04
CPCC12N9/2468C12P19/00C12P19/04C12P19/14C12Y302/01081
Inventor 林娟苏冰梅许鑫琦鄢仁祥
Owner FUZHOU UNIV
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