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Amylosucrase and method for using same to produce Alpha-arbutin by transformation

A technology of amylosucrase and arbutin, which is applied in the field of food biology, can solve the problems of complicated purification, many steps, expensive conversion rate of substrates, etc., and achieves the effects of low demand, short conversion time and good industrial application potential.

Inactive Publication Date: 2017-10-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional chemical synthesis method to synthesize α-arbutin has the problems of many steps and complicated purification
In recent years, many enzymatic synthesis methods have been reported, such as α-amylase, α-glucosidase, sucrose phosphorylase, tranglucosidase, dextransucrase, but there are problems with relatively expensive substrates or low conversion rates

Method used

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  • Amylosucrase and method for using same to produce Alpha-arbutin by transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Construction of recombinant expression vector pET-CC-AS and acquisition of genetically engineered bacteria BL21(DE3) / pET-CC-AS:

[0029] Synthesized with Nde I / Xho I restriction sites Cellulomonas carboniz The 1935 bp of the amylosucrase gene of T26 was ligated into pET-22b(+) to obtain the pET22-CC-AS plasmid.

[0030] Add 5 μL pET22-CC-AS plasmid (40ng / μL) to 100 μL BL21 competent cell suspension, and mix gently.

[0031] Let it stand in an ice bath for 30 minutes. After the plasmid is transformed, evenly spread it on an LB plate containing 100 μg / mL ampicillin, and culture it upside down at 37°C for 12 hours to obtain a positive clone, which is named recombinant Escherichia coli BL21(DE3) / pET22-CC-AS .

Embodiment 2

[0032] Example 2 Recombination Cellulomonas carboniz Acquisition of T26 enzyme

[0033] Single clones of the genetically engineered strain BL21(DE3) / pET-CC-AS were picked to LB seed medium containing 50 μg / mL ampicillin, and cultured at 37°C with shaking at 200 rpm for 12 hours.

[0034] The seed culture solution was added to the fermentation medium at a ratio of 1:50 and cultivated to OD at 37°C 600 0.6-0.8, add IPTG to a final concentration of 1mM;

[0035] Induce the expression at 28°C and 200rpm for 6h, collect the bacterial liquid; centrifuge at 4°C and 10000rpm for 10min to collect the bacterial cells, discard the supernatant, and wash with normal saline for 3 times;

[0036] After centrifuging to obtain the cells, ultrasonically disrupt them, add the equilibrium buffer at a ratio of 1 g of cells: 20 mL of equilibrium buffer, ultrasonically disrupt in an ice bath, and centrifuge the supernatant to obtain purified amylosucrase by metal affinity chromatography. The low...

Embodiment 3

[0040] Example 3 Efficient preparation of α-arbutin

[0041] The molar concentration ratio of substrate sucrose:hydroquinone is 1:1 to configure the reaction solution, and amylosucrase is added to it for catalytic conversion. The conversion reaction conditions are: the concentration range of the substrate (sucrose, hydroquinone) is 1 %~30%, the dosage of enzyme is 1~3U / mg hydroquinone, pH 5.5~7.0, the conversion reaction temperature is 35~45℃, the conversion reaction time is 3~4h, and the α- The reaction liquid of arbutin.

[0042]After separating and purifying the product in the reaction solution by HPLC, use AVANCE III 400MHz digital

[0043] NMR spectrometer (Broker Biospin International AG) nuclear magnetic instrument, two pictures were obtained by analysis, figure 1 is the hydrogen spectrum, figure 2 It is a carbon spectrogram, the peak position marked in the figure is exactly the same as that of the α-arbutin standard product, and the reaction product is determined ...

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Abstract

The invention relates to an amylosucrase and a method for using same to produce Alpha-arbutin by transformation, which belong to the field of food biotechnology. The invention relates to a recombinant amylosucrase cellulomonas carboniz T26 (registry number: KGM11272.1), the amylosucrase is then added into 1 to 30 percent by mass concentration of sucrose-hydroquinone solution (molar ratio: 1:1) to catalytically synthesize Alpha-arbutin, transformation takes place for 3 to 4 hours, the transformation rate is 60 percent or more, antioxidant Vc is not needed, and the amount of donor demanded is small. Alpha-arbutin produced by the invention is a highly effectively skin-whitening product which has a great market potential, and can be widely applied in industries such as food, cosmetics and pharmacy. The amylosucrase and the method can efficiently produce Alpha-arbutin, and are suitable for mass production.

Description

technical field [0001] The invention relates to amylosucrase and a method for transforming and producing α-arbutin, belonging to the field of food biotechnology. Background technique [0002] Arbutin (arbutin) is a hydroquinone glycoside compound, the chemical name is 4-hydroxyphenyl-D-glucopyranoside, which exists in plants such as bearberry and lingonberry, and is a new non-irritating, non-allergic, Compatible natural whitening active substances. Tyrosinase catalyzes the melanin production reaction of "tyrosine-DOPA-melanin". As a safe and effective tyrosinase inhibitor, arbutin can effectively solve dark spots, age spots, freckles and long-term exposure to sunlight. caused skin damage. There are two isomers of arbutin, namely α-arbutin and β-arbutin. Related studies have shown that α-arbutin has ten times the inhibitory effect on tyrosinase than β-arbutin , and has no inhibitory effect on normal human cells, and its safety is much higher than that of β-arbutin. [000...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P19/44C12P19/18C12R1/19
CPCC12N9/1051C12N15/70C12P19/18C12P19/44C12Y204/01004
Inventor 沐万孟江波王永春张涛
Owner JIANGNAN UNIV
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