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Efficient biological enzyme for synthesizing alcoholic aroma glucosides and preparation method of efficient biological enzyme

A technology for synthesizing alcohol and biological enzymes, applied in the biological field, can solve the problems of easy deformation and deterioration, poor stability, impact, etc., and achieve the effect of strong bactericidal effect

Active Publication Date: 2017-08-18
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research progress of phytoalcohol-based aroma glycosyltransferases is slow, which limits the efficient synthesis of aroma glycosides in vitro and restricts the application of aroma glycosides in food, cosmetics and pharmaceutical industries
[0004] Benzyl alcohol, phenylethyl alcohol, geraniol, linalool, linalool oxide, 4-hydroxycoumarin, geraniol, and cis / trans-3-hexenol, eugenol and other aroma substances exist in many plants Among fruits and fruits, it has pleasant fragrance and floral and fruity aroma, but its own stability is poor, and it is easy to deform and deteriorate in the air, which affects its application in food and medicine, and the glycosylation products of these substances can significantly improve its Stability, water solubility, reduce its toxicity, greatly broaden the application of these aroma substances to new fields, and have great commercial prospects
[0005] Considering that alcoholic aroma plays an important role in production and life, and that glycoside alcoholic compounds can effectively avoid the disadvantages of free aglycon, the chemical synthesis of alcoholic glycoside compounds is inefficient, pollutes the environment, and The product has no selectivity, so this environmentally friendly enzyme method for the synthesis of aroma glycoside compounds has great application value

Method used

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  • Efficient biological enzyme for synthesizing alcoholic aroma glucosides and preparation method of efficient biological enzyme
  • Efficient biological enzyme for synthesizing alcoholic aroma glucosides and preparation method of efficient biological enzyme
  • Efficient biological enzyme for synthesizing alcoholic aroma glucosides and preparation method of efficient biological enzyme

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Experimental program
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preparation example Construction

[0028] For protein preparation, take CsGT1 as an example to illustrate:

[0029] Connect the above gene to pGEX-4T1 vector, transfer it into BL21 or Rosetta(DE3) E. coli, and cultivate to OD at 37°C 600 =0.6-0.8. After adding 1mM IPTG, induce 20-24 hours at 16-18℃ (or at room temperature or 37℃), purify with GST-resin, and detect the purified protein with SDS-Page. figure 1 Shown.

[0030] (3) Activity determination:

[0031] CsGT1 is determined by substrate scanning to benzyl alcohol, phenethyl alcohol, geraniol, linalool, linalool oxide, 4-hydroxycoumarin, geraniol, and cis / trans-3-hexenol alcohols The aromas are very active, and can efficiently synthesize glycosides corresponding to a variety of aromas. CsGT2 has the strongest activity on nerol, and it is worth noting that, unlike CsGT1 and CsGT2, the activity of CsGT3 linalool is much greater than that of linalool oxide. Such as figure 2 Shown.

[0032] (4) Scope of application

[0033] Temperature: such as image 3 As shown, Cs...

Embodiment 1

[0039] Using benzyl alcohol as the substrate and glucose as the donor, react in Tris-HCl as the reaction solution (pH 7.5) for 1 hour at 30 degrees. The product is extracted with ethyl acetate and used directly for LC-MS detection (mobile phase is methanol , Water), the production of benzyl alcohol glycosides can be seen, and the conversion rate is greater than 75%.

Embodiment 2

[0041] Using linalool oxide as the substrate and glucose as the donor, react in phosphoric acid reaction solution (pH 7.0) at 35°C for 0.5 hours. The product is extracted with ethyl acetate and directly used for LC-MS detection (mobile phase is methanol , Water), it can be seen that there is the production of linalool oxide glycosides, the reaction rate is 19.5nmol / min / ug, the efficiency is very high, such as Image 6 Shown.

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Abstract

The invention discloses an efficient biological enzyme for synthesizing alcoholic aroma glucosides and a preparation method of the efficient biological enzyme and belongs to the technical field of biology. The biological enzyme comprises CsGT1, CsGT2 and CsGT3, wherein the nucleotide of the CsGT1 is as shown in SEQ ID No. 1, and the amino acid of the CsGT1 is as shown in SEQ ID No. 2; the nucleotide of the CsGT2 is as shown in SEQ ID No. 3, and the amino acid of the CsGT2 is as shown in SEQ ID No. 4; the nucleotide of the CsGT3 is as shown in SEQ ID No. 5, and the amino acid of the CsGT3 is as shown in SEQ ID No. 6. The efficient biological enzyme has the advantages that by performing prokaryotic expression on protein encoded by the biological enzyme, multiple alcoholic aromas can be used as the substrates to synthesize corresponding glucosides, and the biological enzyme is provided for the industrial efficient production of the aroma glucosides; a synthesizing method using an environment-friendly enzyme system method to synthesize the aroma glucosides is high in application value.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a high-efficiency biological enzyme for synthesizing alcohol-based aroma glycosides and a preparation method thereof. Background technique [0002] Alcoholic aroma has attractive floral and fruity aromas. For example, benzyl alcohol and phenethyl alcohol have biological activities such as nourishing kidney and strengthening yang, liver protection, and anti-oxidation. They also have a pleasant floral and fruity flavor. They are widely used in food, cosmetics, tobacco and daily chemical products. Application; Menthol has a mint aroma, can be used as a flavoring agent for toothpaste, perfume, beverages and candy, etc. It is used as a stimulant in medicine. It acts on the skin or mucous membranes. It has a cooling and antipruritic effect. It can be used for headache and nose when taken orally. Inflammation of the throat, pharynx, and throat; among the most commonly used and the largest amount o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70
CPCC12N9/1048C12N15/70
Inventor 宋传奎荆婷婷高婷张娜董绪平宛晓春
Owner ANHUI AGRICULTURAL UNIVERSITY