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Sucrose phosphorylase mutant and its application in the production of glycerol glucoside

A phosphorylase mutant, sucrose phosphorylase technology, applied in the field of genetic engineering to achieve the effects of improving specificity, major application value and reducing yield

Active Publication Date: 2021-07-16
NANJING HUASHI NEW MATERIAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem in the prior art is that when 2-glycerol glucoside is generated, it is accompanied by the generation of a large amount of by-product 1-glycerol glucoside

Method used

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  • Sucrose phosphorylase mutant and its application in the production of glycerol glucoside
  • Sucrose phosphorylase mutant and its application in the production of glycerol glucoside
  • Sucrose phosphorylase mutant and its application in the production of glycerol glucoside

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, construct recombinant bacteria

[0034] 1. Construction of recombinant bacteria BaSP

[0035] 1. Extract the genomic DNA of Bifidobacterium adolescentis.

[0036] 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.

[0037] F1: 5'-GCCTGGTGCCGCGCGGCAGC CTCGAG atgaaaaacaaggtgcag-3';

[0038] R1: 5'-CAGCTGCAGACCGAGCTCACC CTGCAG tcaggcgacgacaggcggattg-3'.

[0039] 3. Take the PCR amplification product obtained in step 2, perform double digestion with restriction endonucleases XhoI and PstI, and recover the digested product.

[0040] 4. Digest the vector pBAD / HisB with restriction endonucleases XhoI and PstI to recover a vector backbone of about 4000 bp.

[0041] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid pBAD-BaSP.

[0042] According to the sequencing results, the struc...

Embodiment 2

[0049] Embodiment 2, application of recombinant bacteria to prepare 2-glycerol glucoside

[0050] The recombinant bacteria BaSP, recombinant bacteria BaSP / L341W, or recombinant bacteria pBAD were subjected to the following steps:

[0051] 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).

[0052] 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.

[0053] 3. After completing step 2, take the entire culture system, centrifuge at 4°C and 6000rpm for 15min, and collect the bacterial precipitate.

[0054] 4. Prepare the reaction system.

[0055] The reaction system is composed of the bacterium precipitate obtained in step 3, glycerol, sucrose and phosphate buffer with pH 6.0. In the reaction syst...

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Abstract

The invention discloses that the purpose of the invention is to provide a sucrose phosphorylase mutant and its application. A protein provided by the present invention is obtained by replacing the 341st amino acid residue of sucrose phosphorylase with leucine; the protein provided by the present invention can be the 341st amino acid residue of sucrose phosphorylase BaSP / L341W protein obtained by substituting leucine for tryptophan; the present invention significantly improves its Specificity in the production of 2‑glycerol glucoside from sucrose and glycerol as feedstock reduces the yield of the by-product 1‑glycerol glucoside. It has great application value for the production field of 2-glycerol glucoside.

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, and the product is α-D-glucose -1 phosphate 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 b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/44
CPCC12N9/1051C12P19/44C12Y204/01007
Inventor 苏桂珍汪昌国薛虹宇史鲁秋其他发明人请求不公开姓名
Owner NANJING HUASHI NEW MATERIAL CO LTD
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