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Site-directed mutation modified algin lyase mutant and application thereof

A technology of alginate lyase and site-directed mutation, which is applied in the field of alginate lyase mutants to achieve the effects of improving catalytic efficiency, improving enzyme production capacity, and increasing yield

Active Publication Date: 2020-07-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the performance of the existing alginate lyase needs to be further improved to meet the needs of industrial applications

Method used

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  • Site-directed mutation modified algin lyase mutant and application thereof
  • Site-directed mutation modified algin lyase mutant and application thereof
  • Site-directed mutation modified algin lyase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Simulation of crystal structure of alginate lyase derived from Escherichia coli

[0035] Using the reported alginate lyase AlyA5 alginate lyase (PDB code: 4BE3) as a template (crystal structure of the exolytic PL7 alginate lyase AlyA5 from Zobelligalactanivorans, published in 2013) (the amino acid similarity between the two is 38.2%), using molecular simulation Docking software that simulates the crystal structure of E. coli-derived alginate lyase. The 212th glutamic acid and the 222nd arginine near the flower-activating domain of the alginate lyase were selected as the active sites of the alginate lyase in this study after the 3D structure simulation docking of the protein in Example 1.

Embodiment 2

[0036] Example 2: The effect of the active site mutation of alginate lyase on the expression of the enzyme activity of alginate lyase

[0037] Using the site-directed mutagenesis kit (TaKaRa), design primers 212A-F, 212A-R, 222A-F, 222A-R (as shown in Table 1), and use the constructed pET-28a (+) as a template to perform PCR. The 212th glutamic acid and the 222nd arginine of alginate lyase were replaced with alanine respectively. The PCR reaction conditions were: 98°C for 5min, 34 cycles (98°C for 30S, 57.6°C for 60S, 72°C for 1.5min) , 72°C for 10 min. PCR amplification system: template 1 μL, upstream and downstream primers 1 μL, Prime Star (Premix) DNA 20 μL, ddH 2 O 17 μL. The gel recovery kit was used to purify and recover the PCR product, and the concentration of the recovered product was checked by electrophoresis. Carry out enzyme digestion under the action of Bam HI and Mlu I fast-cutting enzymes, try to use T4 ligase method to ligate the PCR recovered product, tran...

Embodiment 3

[0040] Example 3: The effect of a single point mutation in the active site of alginate lyase on the expression of the enzyme activity of alginate lyase

[0041] Using the site-directed mutagenesis kit (TaKaRa), design primers 212H-F, 212H-R and 222K-F, 222K-R (as shown in Table 1), and use the constructed pET-28a(+)-Alg212A as a template for PCR , to replace glutamic acid at position 212 or arginine at position 222 by mutation, and the PCR reaction conditions were: 98°C for 5min, 34 cycles (98°C for 30S, 57.8°C for 60S, 72°C for 1.5min), and 72°C for 10min. PCR amplification system: template 1 μL, upstream and downstream primers 1 μL each, Prime Star (Premix) DNA 20 μL, ddH 2 O17 μL. The gel recovery kit was used to purify and recover the PCR product, and the concentration of the recovered product was checked by electrophoresis. Carry out enzyme digestion under the action of Bam HI and Mlu I fast-cutting enzymes, try to use T4 ligase method to ligate the PCR recovered product,...

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Abstract

The invention discloses a site-directed mutation modified algin lyase mutant and application thereof, and belongs to the technical field of enzyme engineering. The algin lyase mutant provided by the invention is obtained by replacing glutamic acid at position 212 of the an algin lyase with an amino acid sequence as shown in SEQ ID NO.1 by histidine and / or replacing arginine at position 222 by lysine. According to the invention, the active center amino acid of the algin lyase is determined through molecular docking simulation, and site-directed saturated mutation is carried out to change aminoacid residues near active sites of protein molecules, so that catalytic efficiency of the algin lyase is improved, and the yield of the algin lyase is further improved. According to the invention, constructed recombinant escherichia coli with enhanced secretion capacity of algin lyase can improve enzyme activity of the algin lyase by 2.83 times compared with an original strain. The improved genetic engineering bacteria have obviously improved enzyme production capacity, and activity of algin lyase produced by shake flask fermentation reaches 15000U / mL or above, so that the improved genetic engineering bacteria are more suitable for industrial application, production cost can be reduced, and production efficiency is improved.

Description

technical field [0001] The invention relates to a mutant of alginate lyase modified by site-directed mutation and application thereof, belonging to the technical field of enzyme engineering. Background technique [0002] Brown algae is one of the three major algae, and its main components are alginate, mannitol and laminarin. Among them, the research on the utilization of mannitol and laminarin is relatively mature. Therefore, how to efficiently degrade alginate and realize industrial application is a hot research topic at present. Alginate is a linear polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G), and its degradation product, fucoidan oligosaccharide, has important physiological activities, such as anti-oxidation, anti- Therefore, it has received extensive attention. [0003] Alginate lyase (Aly) can degrade alginate through β-elimination reaction mechanism, and form unsaturated carbon-carbon double bond at the non-reducing end of the degra...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12R1/19
CPCC12N9/88C12Y402/02011C12N15/70
Inventor 史劲松李恒李星霖吴雯龚劲松
Owner JIANGNAN UNIV
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