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Sucrose phosphorylase mutant and application thereof
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A technology of sucrose phosphorylase and amino acid, applied in the field of genetic engineering, to achieve the effect of improving specificity, significant application value, and reducing yield
Active Publication Date: 2019-03-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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However, the problem in the prior art is that when 2-glycerolglucoside is generated, it is accompanied by the generation of a large amount of by-product 1-glycerolglucoside
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[0046] 2. Using the genomic DNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1, and recover the PCR amplification product.
[0049] 3. Take the PCR amplification product obtained in step 2, perform double digestion with restriction endonucleases XhoI and PstI, and recover the digested product.
[0050] 4. Digest the vector pBAD / HisB with restriction endonucleases XhoI and PstI to recover a vector backbone of about 4000 bp.
[0051] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid pBAD-BaSP.
[0052] According to the sequencing results, the struct...
[0069] The recombinant bacteria BaSP, recombinant bacteria BaSP / L341D, recombinant bacteria BaSP / L341R or recombinant bacteria pBAD were subjected to the following steps:
[0070] 1. Take a single clone of the recombinant bacteria, inoculate it into liquid LB medium, and culture it with shaking at 37°C and 220rpm until OD 600nm =0.7 (in actual application, OD 600nm =0.6-0.8 can be).
[0071] 2. After completing step 1, add L-arabinose to the culture system so that the concentration in the culture system is 0.2g / 100mL, shake and culture at 30°C and 200rpm for 12 hours.
[0072] 3. After completing step 2, take the entire culture system, centrifuge at 4°C and 6000rpm for 15min, and collect the bacterial precipitate.
[0074] The reaction system is composed of the bacterium precipitate obtained in step 3, glycerol, sucrose and phosphate buffer wi...
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Abstract
The present invention discloses a sucrose phosphorylase mutant and application thereof. The protein provided by the invention is obtained by replacing leucine of the amino acid residue at the number 341 site of sucrose phosphorylase with other amino acid residues. The protein can be BaSP / L341D protein obtained by replacing leucine of the amino acid residue at the number 341 site of sucrose phosphorylase with aspartic acid, and can be BaSP / L341R protein obtained by replacing leucine of the amino acid residue at the number 341 site of sucrose phosphorylase with arginine. Through single-point mutation at the amino acid residue at the number 341 site of wild sucrose phosphorylase derived from Bifidobacterium adolescentis with primers, specificity for producing 2-glycosylglycerol from sucrose and glycerin is significantly improved, and the yield of 1-glycosylglycerol that is a byproduct is reduced. The sucrose phosphorylasemutant has great application value in the field of production of the 2-glycosylglycerol.
Description
technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a sucrose phosphorylase mutant and application thereof. Background technique [0002] Sucrose phosphorylase (EC 2.4.1.7) belongs to the 13th family of glycosyltransferases (GH13), which catalyzes the reversible phosphorylation of sucrose molecules in vivo (using the phosphate group as the acceptor of glucose, the product is α-D-glucose- 1 phosphoric acid and D-fructose). Due to the step-by-step reaction mechanism of the enzyme and the special structure of the active site, the glucosyl acceptor in the reaction can be ascorbic acid, hydroquinone, hesperidin, glycerin and other biological substances in addition to the phosphate group. active molecule. The glycosylation of these biologically active molecules can improve their stability, increase their solubility, enhance or even endow them with new physiological activities. The corresponding glycosylated products can...
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