CN116287028A - A kind of method that bioenzyme utilizes formaldehyde to synthesize acetoin - Google Patents

Abstract

The invention discloses a method for synthesizing acetoin by using formaldehyde through a biological enzyme method, which comprises the following steps in an in-vitro multienzyme reaction system: 1) Formaldehyde is converted into 1, 3-dihydroxyacetone under the action of aldolase; 2) Adding dihydroxyacetone kinase, 3-phosphoacetone isomerase, non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate to the product of step 1)Acid mutases, enolases, pyruvate kinase, NADPH oxidase, alpha-acetolactate synthase, alpha-acetolactate decarboxylase, catalase, ATP, NADP ⁺ 100mM phosphate buffer; and (3) reacting to obtain acetoin. The method of the invention realizes the synthesis of acetoin by using formaldehyde as a substrate through in vitro enzyme catalytic reaction; by controlling the enzyme amount and the substrate concentration, the acetoin synthesis capability is improved, and the growth and production limitation caused by limited tolerance of microorganisms to formaldehyde can be relieved. The acetoin obtained by the method has high yield.

Description

A kind of method that bioenzyme utilizes formaldehyde to synthesize acetoin

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for synthesizing acetoin from formaldehyde by a biological enzymatic method.

Background technique

Acetoin, that is, 3-hydroxybutanone, has a creamy aroma and is often used as a food additive to enhance the aroma. Acetoin is listed by the U.S. Department of Energy as one of the 30 platform compounds worthy of priority development, and is widely used in the fields of medicine, agriculture and chemical industry. Acetoin can be used in the synthesis of important chemicals 2,3-butanediol, tetramethylpyrazine, liquid hydrocarbon fuels, etc.

The current commercial production of acetoin mainly relies on chemical synthesis methods, which mainly include the following: partial hydrogenation reduction of butanedione, selective oxidation of 2,3-butanediol, chlorination of butanone Hydrolysis and direct synthesis of acetaldehyde. These substrates are mainly derived from fossil energy, and the chemical synthesis method consumes a lot of energy, is expensive, and seriously pollutes the environment. Therefore, the use of microbial fermentation to synthesize acetoin has gradually attracted the attention of researchers. The yield of acetoin produced by microorganisms is high, the yield is good, and the safety is high. However, in recent years, the production of acetoin has been difficult to further increase. Therefore, in vitro enzyme-catalyzed production of acetoin has attracted more and more attention from researchers, because in vitro enzyme-catalyzed production has many advantages: high yield, fast reaction rate, high product purity and easy downstream separation.

In recent years, the utilization of one-carbon compounds, including CO ₂ , CO, CH ₄ , HCOOH, CH ₃ OH and HCHO, has attracted much attention and is an attractive long-term feedstock. Among the so-called C1 molecules, formaldehyde is a versatile and particularly reactive compound that holds great promise in the production of value-added chemicals. The microbial fixation of one-carbon compounds mainly enters the central carbon metabolism pathway through the ribulose monophosphate pathway. But the model strain grows slowly by using one carbon compound, and the production efficiency is extremely low. Therefore, there is an urgent need for a method for efficiently utilizing formaldehyde to produce acetoin that can tolerate certain toxicity and has a faster reaction rate.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for synthesizing acetoin by using formaldehyde in a biological enzymatic method.

Technical scheme of the present invention is summarized as follows:

A method for synthesizing acetoin from formaldehyde by a biological enzyme method, in an in vitro multi-enzyme reaction system, the following steps are carried out:

1) catalyzed by aldolase to convert formaldehyde into 1,3-dihydroxyacetone;

2) Catalyzed by dihydroxyacetone kinase to convert 1,3-dihydroxyacetone and ATP into phosphoglyceroketone and ADP; catalyzed by 3-phosphate acetone isomerase to convert phosphoglycerone into 3-phosphate-D-glycerol Aldehydes; 3-phospho-D-glyceraldehyde and NADP ⁺ are converted to 3-phospho-D-glycerate and NADPH, catalyzed by non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase;

Catalyzed by NADPH oxidase, convert NADPH and _O2 to NADP ⁺ and _H2O2 ; Catalyzed by catalase, convert _H2O2 to _H2O and _O2 ; Metathesis by ₃ - _{phosphoglycerate} Enzyme-catalyzed conversion of 3-phospho-D-glycerate to 2-phospho-D-glycerate; enolase catalysis to convert 2-phospho-D-glycerate into phosphoenolpyruvate and H ₂ O ; catalyzed by pyruvate kinase, convert phosphoenolpyruvate and ADP into pyruvate and ATP; catalyzed by α-acetolactate synthase, convert pyruvate and H ₂ O into α-acetolactate and CO ₂ ; α-Acetolactate decarboxylase catalyzes the conversion of α-acetolactate and H ₂ O into acetoin and CO ₂ .

Step 1) is preferably: formaldehyde, aldolase, thiamine pyrophosphate, _MgCl and solvent are mixed to convert formaldehyde into 1,3-dihydroxyacetone; the concentration of the formaldehyde is 53.08-122.1mM, and the formaldehyde The dosage of enzyme is 0.8-3.2U/mL, the concentration of thiamine pyrophosphate is 0.5-1mM, the concentration of _MgCl2 is 10-20mM, and the solvent is 100mM phosphate buffer solution with pH=7.0.

Step 2) is preferably: add 0.135-0.27U/mL dihydroxyacetone kinase, 0.135-0.27U/mL 3-phosphate acetone isomerase, 0.135-0.675U/mL non-phosphorylated Glyceraldehyde-3-phosphate dehydrogenase, 0.135-0.675U/mL 3-phosphoglycerate mutase, 0.135-0.675U/mL enolase, 1-5U/mL pyruvate kinase, 0.405-2U/mL NADPH Oxidase, 0.405-0.81U/mL α-acetolactate synthase, 0.405-0.81U/mL α-acetolactate decarboxylase and 0.405-2U/mL catalase, 3-5mM ATP, 3-5mM NADP ⁺ , the solvent is 100mM phosphate buffer solution with pH=7.0; 35-40°C, react for 18-24h to obtain acetoin.

Beneficial effect

The method of the present invention realizes the synthesis of acetoin by using the enzyme-catalyzed reaction in an in vitro multi-enzyme reaction system with formaldehyde as a substrate; by controlling the amount of enzyme and the concentration of the substrate, its ability to synthesize acetoin is improved, and the The limited tolerance of microorganisms to formaldehyde leads to limited growth and production. The yield of acetoin obtained by the method of the invention is high.

Description of drawings

Fig. 1 is the reaction flowchart of in vitro multi-enzyme catalyzed synthesis of acetoin.

Detailed ways

The present invention will be further described below in conjunction with specific examples. The following examples are intended to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

The source of the original plasmid pET28a is biovector (http://www.biovector.net/);

The competent source of E.coli BL21(DE3) is NEB (http://www.neb-china.com/);

37% formaldehyde was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (https://uat.aladdin-e.com/zh_cn/);

Acetoin standard substance, pyruvate kinase from rabbit muscle cells were purchased from sigma company (http://www.sigmaaldrich.com/sigma-aldrich);

Catalase from bovine liver cells was purchased from Beijing Solarbio Technology Co., Ltd. (https://solarbio.com/index.php);

All other biochemical reagents (such as tryptone, yeast extract, NaCl, TPP, MgCl ₂ , ATP, NADP ⁺ , etc.) were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. (http://www.sangon.com/ ).

Enzymes include enzymes from the following sources:

The aldolase obtained through artificial mutation from Pseudomonas fluorescens biovar I (the amino acid sequence is shown in SEQ ID NO.1);

Dihydroxyacetone kinase (amino acid sequence shown in SEQ ID NO.2) from Citrobacter freundii (Citrobacter freundii);

3-phosphate acetone isomerase (Genbank: 948409), 3-phosphoglycerate mutase (Genbank: 948130), enolase (Genbank: 945032) from Escherichia coli;

Non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase from Thermococcus kodakarensis (the amino acid sequence is shown in SEQ ID NO.3);

α-acetolactate synthase (Genbank: 936852), α-acetolactate decarboxylase (Genbank: 936857) and NADPH oxidase (Genbank: 938267) from Bacillus subtilis;

The disclosure of the sources of the enzymes used above is to enable those skilled in the art to better understand the present invention, but is not limited to the above-mentioned sources. According to the nature of the synthetic pathway, other enzymes with the functions can be used in this invention. invention.

The aldolase was codon-optimized according to its amino acid sequence to obtain the nucleotide sequence of the aldolase (SEQ ID NO.4).

The nucleotide sequence (SEQ ID NO.5) of dihydroxyacetone kinase was obtained by codon optimization according to its amino acid sequence.

The non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase was codon-optimized according to its amino acid sequence to obtain its nucleotide sequence (SEQ ID NO.6).

Aldolase, dihydroxyacetone kinase, 3-phosphate acetone isomerase, 3-phosphoglycerate mutase, enolase, non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase, α-acetolactate synthase The respective genes of α-acetolactate decarboxylase and NADPH oxidase were connected to the pET28a expression vector in turn, transformed into Escherichia.coli BL21 (DE3), and 9 correct recombinant bacteria were obtained, which were named as recombinant bacteria 1, recombinant Bacteria 2, recombinant bacterium 3, recombinant bacterium 4, recombinant bacterium 5, recombinant bacterium 6, recombinant bacterium 7, recombinant bacterium 8 and recombinant bacterium 9.

Example 1

Purification of enzymes related to acetoin synthesis in vitro, the steps are:

1) 9 kinds of recombinant bacteria (recombinant bacteria 1, recombinant bacteria 2, recombinant bacteria 3, recombinant bacteria 4, recombinant bacteria 5, recombinant bacteria 6, recombinant bacteria 7, recombinant bacteria 8, recombinant bacteria 9) were respectively inoculated with 50mg/ In 200mL LB medium containing 1 mL kanamycin, culture in a shaker at 37°C and 220rpm until the _OD600 is 0.6-0.8, and add the inducer IPTG (isopropylthiogalactoside) at a final concentration of 0.5mM , cultivated at 18°C for 16h, centrifuged at 4200rpm for 20min at 4°C, collected the cells, and suspended them with 20mL buffer A.

2) Collect the suspension obtained in step 1), use a high-pressure cell disruptor to break the cells at 4°C, 1200 bar, and an oil pressure of 18Kg/cm ³ , repeat the treatment 3 times, and collect the effluent. Then, centrifuge at 5000 rpm for 40 min at 4° C., and collect the supernatant as 9 crude enzyme solutions.

3) The 9 crude enzyme solutions obtained in step 2) were purified by gravity nickel column respectively. The process is to first flow the crude enzyme solution through the gravity column prepacked with nickel filler at 4°C, and prepare eluents containing different concentrations of imidazole (50mM, 100mM, 150mM, 200mM, 250mM,) from buffer A and buffer B After elution, all the eluted effluents were collected to obtain 9 pure enzyme solutions.

4) The 9 pure enzyme solutions obtained in step 3) were filtered with ultrafiltration tubes with a pore size of 10KD to filter imidazole plasma and concentrate the protein. Then, centrifuge at 4°C, 4500 rpm, and wash with PBS (pH 7.0) buffer 3 times to a remaining volume of about 1 mL. Glycerol with a final concentration of 15% was added to obtain a target protein solution.

The formula of buffer A is: 25mM phosphate buffer, 150mM NaCl, 20mM imidazole, the balance is water, adjust the pH to 7.0.

The buffer B formula is: 25mM phosphate buffer, 150mM NaCl, 500mM imidazole, the balance is water, adjust the pH to 7.0.

Example 2

A method for synthesizing acetoin from formaldehyde by a biological enzyme method, in an in vitro multi-enzyme reaction system, the following steps are carried out:

1) Add 53.08mM formaldehyde, 0.8U/mL aldolase, 0.5mM thiamine pyrophosphate (TPP) and 10mM MgCl ₂ , add 100mM phosphate buffer solution with pH=7.0 to 0.2mL, convert 4h, convert formaldehyde to 1,3-Dihydroxyacetone;

2) Add 0.135U/mL dihydroxyacetone kinase, 0.135U/mL acetone 3-phosphate isomerase, 0.135U/mL non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase, 0.135U/mL 3-phosphoglycerate mutase, 0.135U/mL enolase, 1U/mL pyruvate kinase, 0.405U/mL NADPH oxidase, 0.405U/mLα-acetolactate synthase, 0.405U/mLα -Acetolactate decarboxylase and 0.405U/mL catalase, 3mM ATP, 3mM NADP ⁺ , add 100mM phosphate buffer at pH=7.0 to a total volume of 0.4mL; react at 35°C for 18h to obtain the product acetoin.

After being determined by high performance liquid chromatography, the yield of acetoin was 3.12mM, reaching 34.94% of the theoretical yield.

Example 3

A method for synthesizing acetoin from formaldehyde by a biological enzyme method, in an in vitro multi-enzyme reaction system, the following steps are carried out:

1) Add 109.61mM formaldehyde, 1.6U/mL aldolase, 0.5mM thiamine pyrophosphate (TPP) and 10mM MgCl ₂ , add 100mM phosphate buffer solution with pH=7.0 to 0.2mL, convert 4h, convert formaldehyde to 1,3-Dihydroxyacetone;

2) Add 0.27U/mL dihydroxyacetone kinase, 0.27U/mL acetone 3-phosphate isomerase, 0.27U/mL non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase, 0.27U/mL 3-phosphoglycerate mutase, 0.27U/mL enolase, 2U/mL pyruvate kinase, 0.81U/mL NADPH oxidase, 0.81U/mLα-acetolactate synthase, 0.81U/mLα -Acetolactate decarboxylase and 0.81U/mL catalase, 5mM ATP, 5mM NADP ⁺ , add 100mM phosphate buffer with pH=7.0 to 0.4mL; react at 37°C for 18h to obtain the product acetoin.

After being determined by high performance liquid chromatography, the yield of acetoin was 6.57mM, reaching 36.11% of the theoretical yield.

Example 4

A method for synthesizing acetoin from formaldehyde by a biological enzyme method, in an in vitro multi-enzyme reaction system, the following steps are carried out:

1) 122.1mM formaldehyde, 3.2U/mL aldolase, 1mM thiamine pyrophosphate (TPP) and 20mM MgCl ₂ , add 100mM phosphate buffer solution with pH=7.0 to 0.2mL, transform for 4h, convert formaldehyde to 1, 3-Dihydroxyacetone;

2) Add 0.27U/mL dihydroxyacetone kinase, 0.27U/mL acetone 3-phosphate isomerase, 0.675U/mL non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase, 0.675U/mL 3-phosphoglycerate mutase, 0.675U/mL enolase, 5U/mL pyruvate kinase, 2U/mL NADPH oxidase, 0.81U/mLα-acetolactate synthase, 0.81U/mLα- Acetolactate decarboxylase and 2U/mL catalase, 5mM ATP, 5mM NADP ⁺ , add 100mM phosphate buffer with pH=7.0 to 0.4mL; react at 40°C for 24h to obtain the product acetoin.

After being determined by high performance liquid chromatography, the yield of acetoin was 20.17mM, reaching 99.0% of the theoretical yield.

Claims (3)

1. A kind of method that biological enzyme method utilizes formaldehyde to synthesize acetoin, is characterized in that in the multi-enzyme reaction system in vitro, carries out following steps: 1) catalyzed by aldolase to convert formaldehyde into 1,3-dihydroxyacetone; 2) Catalyzed by dihydroxyacetone kinase to convert 1,3-dihydroxyacetone and ATP into phosphoglyceroketone and ADP; catalyzed by 3-phosphate acetone isomerase to convert phosphoglycerone into 3-phosphate-D-glycerol Aldehydes; catalyzed by non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase, 3-phospho-D-glyceraldehyde and NADP ⁺ are converted to 3-phospho-D-glycerate and NADPH; catalyzed by NADPH oxidase, NADPH and O ₂ into NADP ⁺ and H ₂ O ₂ ; catalyzed by catalase, H ₂ O ₂ is converted into H ₂ O and O ₂ ; catalyzed by 3-phosphoglycerate mutase, 3-phospho- D-glycerate is converted to 2-phospho-D-glycerate; catalyzed by enolase, 2-phospho-D-glycerate is converted to phosphoenolpyruvate and H ₂ O; catalyzed by pyruvate kinase, the Phosphoenolpyruvate and ADP are converted into pyruvate and ATP; catalyzed by α-acetolactate synthase, pyruvate and H ₂ O are converted into α-acetolactate and CO ₂ ; catalyzed by α-acetolactate decarboxylase, Conversion of α-acetolactate and _H2O to acetoin and _CO2 . 2. a kind of biological enzyme method according to claim 1 utilizes the method for formaldehyde to synthesize acetoin, it is characterized in that described step 1) is: formaldehyde, aldolase, thiamine pyrophosphate, _MgCl and solvent Mix to convert formaldehyde into 1,3-dihydroxyacetone; the concentration of formaldehyde is 53.08-122.1mM, the dosage of aldolase is 0.8-3.2U/mL, the concentration of thiamine pyrophosphate is 0.5-1mM, MgCl The concentration of ₂ was 10-20 mM, and the solvent was 100 mM phosphate buffer at pH=7.0. 3. a kind of biological enzyme method according to claim 1 utilizes the method for the synthesis of acetoin from formaldehyde, it is characterized in that: described step 2) is: add 0.135-0.27U/mL in the mixed solution that step 1) obtains Dihydroxyacetone kinase, 0.135-0.27U/mL acetone 3-phosphate isomerase, 0.135-0.675U/mL non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase, 0.135-0.675U/mL 3-phosphoglycerate Bitase, 0.135-0.675U/mL enolase, 1-5U/mL pyruvate kinase, 0.405-2U/mLNADPH oxidase, 0.405-0.81U/mLα-acetolactate synthase, 0.405-0.81U/mLα-acetyl Lactic acid decarboxylase and 0.405-2U/mL catalase, 3-5mM ATP, 3-5mM NADP ⁺ , the solvent is 100mM phosphate buffer solution with pH=7.0; 35-40℃, react for 18-24h to obtain acetoin .

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