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Preparation method and application of 2-phenylethanol by non-cell synthetic biology

A technology of synthetic biology and phenylethanol, applied in multi-enzyme systems, fermentation and other directions, to achieve the effects of high catalytic efficiency, simple operation and excellent repeatability

Active Publication Date: 2019-09-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved in the present invention is aimed at the deficiency of the existing non-cellular synthetic biology preparation technology of 2-phenylethanol. It intends to use the advantage of the high-efficiency catalysis of the multi-enzyme complex to artificially prepare a multi-enzyme reaction system in vitro, which will be highly efficient. The three enzymes and three recombinant enzymes expressing the Ehrlich pathway were placed in a suitable reaction system, and the optimal reaction system for the in vitro co-reaction of the three recombinant enzymes to prepare 2-phenylethanol was constructed. Prepare the target product 2-phenylethanol, and establish a method for the preparation of 2-phenylethanol by non-cell synthetic biology

Method used

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Effect test

Embodiment 1

[0026] Main reagents: recombinant aromatic aminotransferase I, phenylpyruvate decarboxylase and alcohol dehydrogenase, conventional commercially available or constructed with reference to prior art documents or constructed with reference to the method of Example 5 of the present invention. Chemical standards: L-phenylalanine, oxaloacetic acid, and 2-phenylethanol were purchased from Sigma-Aldrich, USA. All other chemical reagents were domestic analytically pure or chromatographically pure. Main instruments: digital display constant temperature water bath HH-2 (Jiangsu Changzhou Guohua Electric Co., Ltd.), constant temperature metal bath CHB-100 (Jiangsu Hangzhou Bioer Technology Co., Ltd.), precision pH meter PHS-3C (Shanghai Precision Scientific Instrument Co., Ltd. ), Mettler-Toledo electronic balance AB204-N (Shanghai Mettler-Toledo Instrument Co., Ltd.), ultrasonic breaker (Ningbo Science and Technology Co., Ltd.), liquid chromatography (Shanghai Tianmei Company), gas chro...

Embodiment 2

[0059] Multi-enzyme co-reaction synthesis of 2-phenylethanol under the optimal conditions of embodiment 2

[0060] Refer to Example 1 for the instruments, reagents and detection methods of this embodiment. Using L-phenylalanine and oxaloacetate as substrates, the optimal synthesis of 2-phenylethanol by recombinant aromatic aminotransferase ⅠARO8, phenylpyruvate decarboxylase ARO10 and alcohol dehydrogenase ADH1 was studied. Conditions, all reaction systems were 0.5 mL, containing 0.01 mM pyridoxal phosphate, 0.2 mM thiamine pyrophosphate and 0.1 mM NADPH.

[0061] Using L-phenylalanine and oxaloacetate in a molar ratio of 0.75:l as substrates, 2-phenylethanol was synthesized according to the aforementioned optimal conditions. Recombinases ARO8, ARO10 and ADH1 were mixed at a ratio of 1:0.25:1 according to the activity of the enzymes, and the amount added was 100 μL. 2+ In the reaction system with a concentration of 1.5mM, after 5min in a water bath at 37°C, the reaction was ...

Embodiment 3 3

[0062] Example 3 Co-reaction test in which the three-enzyme co-immobilization system participates

[0063] Main reagents: recombinant aromatic aminotransferase I, phenylpyruvate decarboxylase and alcohol dehydrogenase are commercially available or constructed by referring to conventional techniques in the field, or constructed by referring to the method of Example 5 of the present invention. Chemical standards: L-phenylalanine, oxaloacetic acid, and 2-phenylethanol were purchased from Sigma-Aldrich, USA. Other reagents were routinely purchased from the market, and the above chemical reagents were all domestic analytical or chromatographically pure. Main instruments: digital display constant temperature water bath HH-2 (Jiangsu Changzhou Guohua Electric Appliance Co., Ltd.), electric blast drying oven 101A-3S (Guangdong Guangzhou Fumin Measurement and Control Technology Co., Ltd.), constant temperature metal bath CHB-100 (Jiangsu Hangzhou Bioer Technology Co., Ltd.), Electrolu...

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Abstract

The invention discloses a noncellular synthetic biology based preparation method of 2-phenylethanol and an application. According to the preparation method and the application, three recombinases including ARO8, ARO10 and ADH1 are utilized to constitute a co-reaction system, and a complicated metabolic process of an Ehrlich pathway requiring multi-step continuous reaction and multi-enzyme participation for synthesis of 2-phenylethanol in microorganisms is simplified to be performed in the same reaction system in vitro, so that the environmental pollution is reduced and the reaction process is shortened. Furthermore, the three recombinases are subjected to carrier-free co-immobilization with glutaraldehyde serving as a cross-linking agent, Combi-CLEAs (Combi-Cross-linked Enzyme Aggregates) are prepared, a target product is synthesized in vitro, accordingly, the enzymes are further recycled on the basis of the beneficial effects, and the production cost is reduced; the preparation method fills up the technical blank of multi-enzyme preparation of 2-phenylethanol in vitro, and important reference is provided for biological preparation of 2-phenylethanol.

Description

technical field [0001] The invention relates to the technical field of biosynthesis, more specifically, to a method for preparing 2-phenylethanol through non-cell synthetic biology and its application. Background technique [0002] 2-Phenylethanol (2-Phenylethanol, 2-PE) is also called β-Phenylethanol (β-Phenylethanol), its chemical name is 2-Phenylethanol (2-Phenylethyl alcohol, 2-PEA), its molecular weight is 122.16, and its structural formula is: [0003] [0004] 2-Phenylethyl alcohol is an aromatic alcohol with a light and delicate smell of roses. It naturally exists in the essential oils of many plants. 2-Phenylethyl alcohol has a soft and sweet aroma and is very popular. It is widely used in the fields of food, cosmetics, tobacco and daily chemical products. It is not only the basic component of all rose fragrances, but also has become a variety of fragrances because of its synergistic and synergistic effects. The components required by the formula are spice subst...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/22C12N11/18
Inventor 林俊芳郭丽琼刘晓蓉云帆
Owner SOUTH CHINA AGRI UNIV
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