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CN-122012542-A - Short-chain dehydrogenase gene for synthesizing medium-long chain aldehyde, recombinant genetic engineering bacteria and application

CN122012542ACN 122012542 ACN122012542 ACN 122012542ACN-122012542-A

Abstract

The invention discloses a short-chain dehydrogenase gene for synthesizing medium-long chain aldehyde, recombinant genetic engineering bacteria and application thereof, wherein the enzyme can efficiently catalyze the medium-long chain olefine aldehyde to generate medium-long chain aldehyde through selective hydrogenation reduction under mild conditions, and has good substrate adaptability and catalytic activity. By introducing glucose dehydrogenase and glucose to construct an NAD (P) H coenzyme cyclic regeneration system, a stable and efficient double-enzyme coupling biocatalysis reaction system is successfully established, and the coenzyme consumption is obviously reduced while the reaction efficiency is ensured. Compared with the traditional chemical synthesis method, the biocatalysis process has the advantages of mild reaction conditions, high selectivity, few byproducts, environmental friendliness and the like, provides a new technical approach for green and sustainable preparation of long-chain aldehydes in perfume grade dodecanal and the like, and has good industrial application prospect.

Inventors

  • WU SHIJIN
  • CHEN XIAOMIN
  • LI TONGTONG
  • QIU LEQUAN

Assignees

  • 浙江工业大学

Dates

Publication Date
20260512
Application Date
20260325

Claims (10)

  1. 1. A short-chain dehydrogenase IbFabI gene for synthesizing medium-long chain aldehyde is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
  2. 2. A protein encoded by the short-chain dehydrogenase IbFabI gene as claimed in claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.
  3. 3. A recombinant genetically engineered bacterium expressing the gene of claim 1.
  4. 4. Use of the short-chain dehydrogenase IbFabI gene of claim 1 in the preparation of medium-long-chain aldehydes by biocatalytic reduction of medium-long-chain olefines.
  5. 5. The use according to claim 4, wherein the use is the catalysis of dodecenal to form dodecanal or the catalysis of decenal to form decanal.
  6. 6. The method according to claim 5, wherein the method comprises the steps of performing ultrasonic disruption on recombinant genetically engineered bacteria containing a short-chain dehydrogenase IbFabI gene, taking a supernatant or a pure enzyme solution extracted from the supernatant after centrifugation of the disrupted mixture as a catalyst, taking medium-long-chain enal as a substrate, NAD (P) +as a cofactor, glucose dehydrogenase as a coenzyme, glucose as an auxiliary substrate, isopropanol as a cosolvent, taking a buffer solution with pH of 7-9 as a reaction medium, performing reaction in a shaker at a rotation speed of 25-35 ℃ and 100-300rpm, extracting the reaction solution with ethyl acetate after the reaction is completed, and dehydrating with anhydrous magnesium sulfate to obtain the long-chain aldehyde in the product.
  7. 7. The use according to claim 6, wherein the amount of catalyst in the reaction system is 1-5mg/mL in terms of protein content, the substrate is added after 1M of mother liquor is prepared with isopropanol, the final concentration is 10-50 mM, the NAD (P) + is added at a final concentration of 10-15mM, the coenzyme is added at a final concentration of 1-3U/mL, and the glucose is added at a final concentration of 30-70mM.
  8. 8. The use according to claim 6, wherein the reaction medium is a potassium phosphate buffer solution having a pH of 7, 50 mM.
  9. 9. The method according to claim 6, wherein the supernatant is prepared by suspending wet cells in PBS buffer solution of 50mM and pH 7.0, ultrasonic-crushing cells 10min under 4 ℃ low-temperature ice-water bath under conditions of power 400W, 3 s on and 4 s off, centrifuging the ultrasonic-crushed mixture at 12,000 rpm for 10min, centrifuging the supernatant at 12000 rpm for 20min, and sucking the supernatant to obtain a crude enzyme solution containing the target short-chain dehydrogenase.
  10. 10. The method according to claim 6, wherein the pure enzyme solution is prepared by transferring the supernatant to an equilibrated Ni 2+ column by chromatography, eluting a large amount of the hetero protein with an elution buffer containing 10mM imidazole after loading, then gradient eluting the remained hetero protein and a part of the target protein with an elution buffer containing 25 mM and 40mM imidazole, eluting with an elution buffer containing 250 mM imidazole, eluting a large amount of the target protein, collecting the effluent containing the target protein, eluting all the residual proteins in the column with an elution buffer containing 250 mM imidazole, desalting and concentrating the effluent containing the target protein with a ultrafiltration tube with a cut-off molecular weight of 10 kDa at 4 ℃ and 7000 rpm for 30min, and collecting the retentate as the pure enzyme solution, wherein the elution buffer comprises 300mM sodium chloride, and the solvent is 50mM PBS buffer, pH7.0.

Description

Short-chain dehydrogenase gene for synthesizing medium-long chain aldehyde, recombinant genetic engineering bacteria and application Field of the art The invention belongs to the technical field of biochemical engineering, and particularly relates to a short-chain dehydrogenase IbFabI gene, a recombinant expression vector and recombinant genetic engineering bacteria for synthesizing medium-long chain aldehyde, and application thereof, in particular to application thereof in biocatalysis preparation of perfume compound dodecanal. (II) background art Fatty aldehydes and derivatives thereof are important basic chemicals and intermediates in the fields of medicine, food, flavors and fragrances, and fine chemistry. The medium-long chain fatty aldehyde has unique and soft odor characteristics, and has important application value in the flavor industry. Dodecanal, which is a typical medium-long chain fatty aldehyde, has the characteristics of fatty wax fragrance, citrus fragrance and floral fragrance, is a key component in a variety of essence formulations, and has stable and continuous industrial requirements. Currently, dodecanal is mainly prepared by chemical synthetic methods, such as chemical oxidation of fatty alcohols or multi-step chemical reaction routes. However, the conventional chemical method generally has the problems of harsh reaction conditions, insufficient selectivity, more byproducts, heavy environmental burden and the like, and is difficult to meet the development requirements of green manufacturing and sustainable development. The biocatalysis technology gradually becomes an important development direction for green preparation of fatty aldehyde compounds due to the advantages of mild reaction conditions, high regioselectivity, environmental friendliness and the like. The dehydrogenase/reductase biocatalysts, especially the short chain dehydrogenase/reductase family with NAD (P) H as cofactor, are receiving attention because of their stable structure and strong substrate adaptability. However, in the prior report, enzyme resources capable of catalyzing medium-long chain olefine aldehyde substrates with high efficiency and meeting industrial application requirements are still limited, and especially aiming at biocatalytic conversion of dodecene aldehyde to dodecanal, enzyme preparations with definite sources, stable performance and good application prospect are still lacking. Therefore, a short-chain dehydrogenase capable of catalyzing reduction of the dodecenal to generate the dodecanal with high efficiency is developed, and a corresponding recombinant expression system is constructed, so that the method has important technical significance and application value for realizing green and sustainable preparation of the dodecanal. (III) summary of the invention The invention aims to provide a short-chain dehydrogenase IbFabI gene with definite sources and excellent catalytic performance for synthesizing medium-long chain aldehyde, and constructs a corresponding recombinant expression vector and recombinant genetic engineering bacteria, which are used for preparing the medium-long chain aldehyde by high-efficiency biocatalysis reduction of the medium-long chain olefine aldehyde, in particular to preparing the dodecanal by reduction of the dodecene aldehyde, thereby realizing the green preparation of the dodecanal. The technical scheme adopted by the invention is as follows: The invention provides a short-chain dehydrogenase IbFabI gene for synthesizing medium-long-chain aldehyde, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The invention also provides a coded protein of the short-chain dehydrogenase IbFabI gene, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2. The invention also provides an expression vector containing the gene and recombinant genetic engineering bacteria constructed by the expression vector, wherein the expression vector is a prokaryotic expression vector, and the host cell is escherichia coli E.coli BL21 (DE 3) as an optimal choice. The invention provides an application of a short-chain dehydrogenase IbFabI gene in preparation of medium-chain aldehyde by biocatalysis reduction of medium-chain olefine aldehyde. Further, the application is the catalysis of dodecenal to make dodecanal or the catalysis of decenal to make decanal. Further, the application is that wet bacterial body obtained by fermenting and culturing recombinant genetic engineering bacteria containing short-chain dehydrogenase IbFabI gene is subjected to ultrasonic crushing, supernatant (i.e. crude enzyme liquid) obtained by centrifuging the crushing mixed liquid or pure enzyme liquid extracted from the supernatant is taken as a catalyst, medium-long-chain enal is taken as a substrate, NAD (P) +is taken as a cofactor, glucose Dehydrogenase (GDH) is taken as coenzyme, glucose is taken as an auxiliary substrate, isopropanol is taken as a cosolvent, buffer solution with pH