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Beta-galactosidase capable of glycosylating resveratrol

A technology of galactosidase and resveratrol is applied in the field of enzyme protein engineering, which can solve the problems of difficult glycosylation modification, low nucleophilicity of phenyl ring hydroxyl group, and difficulty in enzymatic modification of polyhydroxy phenols, etc. The effect of broadening the scope of transglycosylation receptors and having broad application prospects

Active Publication Date: 2014-07-23
SHANDONG UNIV
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  • Abstract
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Problems solved by technology

[0003] Phenolic compounds have very important pharmacological activities, and their glycosylation modification is of great significance. However, due to the low nucleophilicity of the hydroxyl group of the benzene ring of phenolic compounds, it is difficult for natural glycosidases to use phenolic compounds as receptors for glycosylation modification. Enzymatic modification of large, polyhydric phenols is particularly difficult

Method used

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  • Beta-galactosidase capable of glycosylating resveratrol
  • Beta-galactosidase capable of glycosylating resveratrol
  • Beta-galactosidase capable of glycosylating resveratrol

Examples

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Effect test

Embodiment 1

[0031] Example 1: Acquisition of β-galactosidase gene capable of glycosylation of resveratrol and preparation of enzyme protein

[0032] Artificially synthesized β-galactosidase gene sequence with GenBank accession number EU734748.1 (encoded protein GenBank accession number is ACE06986.1), random mutation was performed by error-prone PCR:

[0033] Forward primer:

[0034] 5'-GC ATGAGCAATAAGTTAGTAAAAG-3' (containing NheI restriction site) reverse primer of SEQ ID NO.3:

[0035] 5'-GC TTATTTTAGTAAAAGGGGCTG-3 (with BglⅡ restriction site) SEQ ID NO.4

[0036] PCR reaction system (50 μL): template with a final concentration of 10 ng / μL, primers of 1 μM, 0.2 mM dATP, dGTP, 1 mM dTTP, dCTP, 5 mM MgCl 2 , 2.5U rTaq DNA polymerase, 1× PCR buffer.

[0037] PCR amplification conditions were: pre-denaturation at 95°C for 5 minutes; 30 cycles of reaction (denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 3 minutes); extension at 72°C for 10...

Embodiment 2

[0045] Example 2: Glycosylation of resveratrol by β-galactosidase

[0046]The final concentration of lactose was prepared with pH 7.0 and 50mM potassium phosphate buffer solution to be 0.2M, the final concentration of resveratrol was prepared with acetone to be 0.1M, the amount of enzyme added was 4μg / mL, and the reaction system was 50mL. After reacting at 45°C for 45 minutes, boil at 100°C for 5 minutes to terminate the reaction.

[0047] The boiled reaction solution was centrifuged at 12,000 rpm for 20 minutes, the supernatant was sucked, and the sample was spread on a preparative thin-layer chromatography plate (PLC Silica gel60F254, Merck). After the development, take a 1cm-wide strip plate every 10cm on the thin-layer chromatography plate for color development, and determine the position of the target sugar on the chromatography plate. Then scrape off the silica gel powder containing the target sugar in the non-colored area of ​​the chromatographic plate, redissolve it i...

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Abstract

The invention relates to beta-galactosidase, and in particular relates to beta-galactosidase capable of glycosylating resveratrol, and belongs to the technical field of zymoprotein engineering. The enzyme is a mutant enzyme by directionally evoluting natural beta-galactosidase, has novel substrate specificity of a transglycosylation acceptor, can glycosylate resveratrol molecules which cannot be glycosylated, and can be used as a tool enzyme for glycosylated modification of galactose. The beta-galactosidase has wide application prospect in processing phenolic compound, molecular modification of medicines and the like.

Description

technical field [0001] The invention relates to a beta-galactosidase capable of glycosylating resveratrol, belonging to the technical field of enzyme protein engineering. technical background [0002] Chemical method and enzymatic method are currently the two major methods for glycosylation modification and glycoside synthesis. There are multiple reactive hydroxyl groups on sugar molecules, so that chemical glycosylation must carry out complex group protection and deprotection after glycoside synthesis, in order to specifically synthesize glycosidic bonds in terms of stereo conformation and regional position, such cumbersome steps Greatly limit the practicability of its industrial synthesis of glycosides. In contrast, enzymatic glycosylation has stereoselectivity and regioselectivity, simple steps and environmental friendliness. There are currently two types of enzymes that can be used for glycoside synthesis, namely glycosyltransferase (glycosyltransferase; EC2.4) and gly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12R1/19
Inventor 肖敏卢丽丽郭玉川
Owner SHANDONG UNIV
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