CN-121975877-A - Application of alcohol dehydrogenase from Geobacillus thermodenitrificans in catalytic synthesis of alpha, omega-dibasic fatty acid

Abstract

The invention discloses a Geobacillus thermodenitrificans-derived difunctional alcohol dehydrogenase which takes NAD + as a cofactor, can specifically catalyze continuous oxidation reaction of omega-hydroxy fatty acid, and can catalyze omega-hydroxy fatty acid to synthesize alpha, omega-binary fatty acid by single enzyme catalysis without an aldehyde intermediate. The alcohol dehydrogenase has double catalytic functions of alcohol oxidation and aldehyde oxidation, can complete the oxidation process from the hydroxymethyl to the carboxyl in one step, simplifies the reaction steps, avoids the accumulation and separation of intermediate aldehyde, improves the conversion efficiency, provides a concise and efficient biocatalysis path for the biosynthesis of alpha, omega-binary fatty acid, especially lauryldiacid, and has important industrial application value.

Inventors

• WU BIN
• MA XIAO
• MA JIANGFENG
• CAO KUN
• LI YISHU
• HE BINGFANG

Assignees

• 南京工业大学

Dates

Publication Date

20260505

Application Date

20260121

Claims (5)

1. 1. Use of an alcohol dehydrogenase derived from Geobacillus thermodenitrificans for catalyzing the synthesis of an alpha, omega-dibasic fatty acid from an omega-hydroxy fatty acid.
2. 2. The use according to claim 1, wherein the alcohol dehydrogenase has the amino acid sequence shown in SEQ ID NO. 1.
3. 3. The use according to claim 1, characterized in that the alcohol dehydrogenase oxidizes omega-hydroxy fatty acids to alpha, omega-dibasic fatty acids in one step using NAD + as cofactor.
4. 4. The use according to any one of claims 1 to 3, characterized in that the omega-hydroxy fatty acid is a C8 to C16 saturated omega-hydroxy fatty acid.
5. 5. Use according to claim 4, characterized in that the omega-hydroxy fatty acid is 12-hydroxy lauric acid.

Description

Application of alcohol dehydrogenase from Geobacillus thermodenitrificans in catalytic synthesis of alpha, omega-dibasic fatty acid Technical Field The invention relates to the technical field of enzyme catalysis engineering, in particular to application of alcohol dehydrogenase derived from Geobacillus thermodenitrificans Geobacillus thermodenitrificans in catalyzing omega-hydroxy fatty acid to synthesize alpha, omega-binary fatty acid. Background Lauryldiacid has important application in high molecular materials, plasticizers and fine chemical engineering, and market demands steadily increase. The traditional production process takes petroleum-based raw materials as a core, generally requires multi-step chemical oxidation processes, has complex catalytic systems and can generate environmental harmful byproducts in the process. With the increasing demands of industrial greening, the construction of biocatalytic pathways based on fatty acid substrates has become an important technical direction to replace traditional processes. The oxidation of the fatty acid terminal C-H bond is a common reaction catalyzed by heme-dependent cytochrome P450 monooxygenase (CYP) to produce the corresponding 12-hydroxy lauric acid. In the existing biocatalytic route, 12-hydroxy lauric acid is usually converted into the corresponding dibasic acid by a continuous oxidation reaction. The process generally employs a two-step oxidation mechanism, namely, first oxidizing the terminal hydroxyl groups to aldehyde intermediates under the catalysis of an alcohol dehydrogenase (alcohol dehydrogenase, ADH), followed by further oxidation to carboxylic acids under the action of an aldehyde dehydrogenase (aldehyde dehydrogenase, ALDH). The strategy requires sequential reactions of two different catalytic enzymes, the enzyme system is complex in construction, intermediate aldehyde is easy to accumulate in the system and generate side reaction, the reaction efficiency is not improved, and the separation cost of the subsequent process is increased. In order to improve the reaction efficiency, there are studies on attempts to perform combined expression of ADH and ALDH or construct a cascade reaction system, but there are problems such as unbalanced enzyme expression amount, limited catalytic efficiency, and difficult intermediate control. In addition, the aldehyde intermediates themselves are reactive and potentially toxic, and their accumulation can adversely affect cell growth and catalytic stability, further limiting the industrialization potential of this pathway. Therefore, the development of a novel biocatalytic strategy capable of accomplishing the direct oxidation of hydroxyl groups to carboxyl groups in a single enzyme system is an effective approach to solve the above-mentioned problems. Disclosure of Invention The invention obtains the alcohol dehydrogenase (named ADH 2) from Geobacillus thermodenitrificans by screening and researching various sources of alcohol dehydrogenases, and the enzyme has alcohol oxidation and aldehyde oxidation activities at the same time, and can realize the application of synthesizing alpha, omega-binary fatty acid by utilizing NAD + in the continuous oxidation reaction of omega-hydroxy fatty acid. The specific technical scheme of the invention is as follows: Use of an alcohol dehydrogenase derived from geobacillus thermodenitrificans Geobacillus thermodenitrificans (abbreviated as ADH 2) for catalyzing the synthesis of an alpha, omega-dibasic fatty acid from an omega-hydroxy fatty acid. The amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO. 1. The alcohol dehydrogenase of the invention oxidizes omega-hydroxy fatty acid into alpha, omega-binary fatty acid in one step by taking NAD + as a cofactor. Preferably, the omega-hydroxy fatty acid is a C8-C16 saturated omega-hydroxy fatty acid. In a specific example of the present invention, the omega-hydroxy fatty acid is 12-hydroxy lauric acid. A specific example of an application of the invention is described in which the reaction system comprises 2mM substrate (100 mM substrate in DMSO), 0.5 mg/mL purified ADH2 enzyme solution, and NAD + at a final concentration of 0.1 mM is added to activate the enzyme reaction. The reaction was carried out at 45 ℃ under 1000 rpm conditions of 2h. The invention has the advantages that: Aiming at the problems that a two-step oxidation mechanism, alcohol dehydrogenase and aldehyde dehydrogenase are commonly adopted in the prior art and intermediate aldehyde is generated, the novel difunctional alcohol dehydrogenase is screened out, and NAD + is used as a cofactor to complete the one-step oxidation from the hydroxymethylene to the carboxyl, so that the reaction process is simplified, the conversion efficiency is improved, and a more efficient biocatalysis way is provided for the green bio-base production of the dibasic acid. Drawings The accompanying drawings, which are included to provide a further understand