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Alpha-galactosidase mutant and application thereof

A technology for galactosidase and mutants, applied in the field of α-galactosidase mutants, can solve problems such as low efficiency and complex extraction process, and achieve the effects of reducing cost, high catalytic efficiency and improving transformation efficiency

Active Publication Date: 2022-07-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no chemical synthesis method for isoerythroside, which is mainly obtained from red algae by alcohol extraction process, but this method has problems such as complicated extraction process and low efficiency

Method used

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  • Alpha-galactosidase mutant and application thereof
  • Alpha-galactosidase mutant and application thereof
  • Alpha-galactosidase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Determination of key amino acids in the framework of α-galactosidase AgaV enzyme activity

[0020] The α-galactosidase AgaV selected in the present invention has certain synthetic activity, and catalyzes the reverse hydrolysis reaction of D-galactose and glycerol to synthesize isokainate. Studies have shown that α-galactosidase, as a glycoside hydrolase, mainly catalyzes the hydrolysis reaction of glycoside compounds, and only a small part can catalyze the synthesis reaction. The present invention finds that α-galactosidase AgaB derived from Geobacillus stearothermophilus has high sequence similarity (75%) with AgaV through BLAST search, and obtains the crystal structure of AgaB in the PDB database (PDB: 4fnq), which is used as template, the three-dimensional structural model of α-galactosidase AgaV was constructed ( figure 1 ).

[0021] Through sequence alignment, it was found that the α-galactosidase AgaA and AgaB derived from Geobacillus stearothermophilu...

Embodiment 2

[0022] Example 2 Construction of α-galactosidase AgaV G355 mutant

[0023] As described in Example 1, the amino acid at position 355 of alpha-galactosidase AgaV was saturated mutated. The construction method is as follows, and the primers used are shown in the following table:

[0024] Primer Sequence(5'to 3') G355A-F CAGATCGCCGAAGCCGCAAAAGAACTGGGCATC SEQ ID NO: 3 G355C-F CAGATCGCCGAAGCCTGTAAAGAACTGGGCATC SEQ ID NO: 4 G355D-F CAGATCGCCGAAGCCGATAAAGAACTGGGCATC SEQ ID NO: 5 G355E-F CAGATCGCCGAAGCCGAAAAAGAACTGGGCATC SEQ ID NO: 6 G355F-F CAGATCGCCGAAGCCTTTAAAGAACTGGGCATC SEQ ID NO: 7 G355H-F CAGATCGCCGAAGCCCATAAAGAACTGGGCATC SEQ ID NO: 8 G355I-F CAGATCGCCGAAGCCATTAAAGAACTGGGCATC SEQ ID NO: 9 G355K-F CAGATCGCCGAAGCCAAAAAAGAACTGGGCATC SEQ ID NO: 10 G355L-F CAGATCGCCGAAGCCCTGAAAGAACTGGGCATC SEQ ID NO: 11 G355M-F CAGATCGCCGAAGCCATGAAAGAACTGGGCATC SEQ ID NO: 12 G355N-F CAGATCGC...

Embodiment 3

[0026] Example 3 Fermentation expression of recombinant α-galactosidase mutants in Escherichia coli

[0027] The specific expression method is as follows:

[0028] (1) Inoculate each G355 mutant constructed in Example 2 and the original enzyme α-galactosidase AgaV into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate, and culture at 37° C. and 180 rpm overnight , to prepare the seed solution.

[0029] (2) The seed liquid was inoculated into fresh 50 mL LB liquid medium with 2% inoculation amount, and cultivated to OD at 37°C and 180 rpm 600 When it is 0.6-1.0, take it out and cool it in an ice-water bath for 5 min, add the inducer IPTG (isopropyl-β-D thiogalactoside) (final concentration 0.1 mmol / L), and induce expression for 20 h at 20 °C and 180 rpm.

[0030] (3) Take the fermentation broth of induced expression, centrifuge at 12000rpm for 20min, discard the supernatant, and then use 50mM Na 2 HPO 4 -KH 2 PO 4 (pH 7.0) buffer, resuspend and wash the cel...

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Abstract

The invention discloses an alpha-galactosidase mutant with high specific activity and an application of the alpha-galactosidase mutant. According to the present invention, alpha-galactosidase AgaV derived from Anxybacillus vitaminiphilus is adopted as a template, amino acid mutation is performed by using a molecular biological technology, such that mutants with improved catalytic efficiency, such as G355D, G355E, G355F, G355H, G355I, G355K, G355L, G355N, G355Q, G355R, G355T, G355V and G355Y, are obtained; the mutant disclosed by the invention has the excellent property of high catalytic efficiency, the efficiency of catalytically synthesizing the isored alga glycoside by taking D-galactose and glycerol as substrates is improved by 1.3-3 times, the cost of high synthesis of the isored alga is favorably reduced, the conversion efficiency of producing the isored alga glycoside by using the enzyme is improved, and the mutant has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of functional gene modification, in particular to a class of alpha-galactosidase mutants and applications thereof. Background technique [0002] Galactosyl-glycerol is an alcohol-soluble galactoside composed of D-galactosyl and glycerol and is present in many red and seaweed species. α-D-galactosyl-glycerol and β-D-galactosyl-glycerol are widely used in cosmetics, health products, food and medicine. In general, α-D-galactosyl-glycerol has two enantiomeric forms: kaicoside and isokainate, which mainly play the role of anti-osmotic pressure in algal cells, and have chemical antioxidant, anti-inflammatory, immune Regulates and scavenges free radicals. Studies have shown that isokaicoside is the main photosynthetic product of red algae, and red algae (sea dill and seaweed) have been confirmed to contain high concentrations of isokaicoside. [0003] At present, there is no chemical synthesis method for isokain...

Claims

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Application Information

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IPC IPC(8): C12N9/40C12N15/56C12P19/44
CPCC12N9/2465C12Y302/01022C12P19/44Y02A50/30
Inventor 吴斌王佳玲韦利军高振储建林何冰芳
Owner NANJING UNIV OF TECH
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