阅读说明：本技术 一种多酶级联生产(r/s)-羟基蛋氨酸的方法 (Method for producing (R/S) -hydroxymethionine by multienzyme cascades ) 是由 刘立明 刘佳 张灿 宋伟 罗秋玲 陈修来 高聪 叶超 于 2019-11-18 设计创作，主要内容包括：本发明公开了一种多酶级联生产(R/S)-羟基蛋氨酸的方法,属于生物工程技术领域。本发明利用一锅法同步反应催化蛋氨酸生产(R)-HMTBA,通过分批补料解除底物抑制,转化周期由23h缩短至15h,(R)-HMTBA产量可达到97.0g/L,ee>99％,蛋氨酸摩尔转化率达到96.3％；利用一锅法同步反应催化蛋氨酸生产(S)-HMTBA,通过分批补料解除底物抑制,转化周期由23h缩短至18h,(S)-HMTBA产量可达到98.0g/L,ee>99％,蛋氨酸摩尔转化率达到98.6％。(The invention discloses a method for producing (R/S) -hydroxymethionine by multienzyme cascades, which belongs to the technical field of bioengineering and utilizes pot method synchronous reaction to catalyze methionine to produce (R) -HMTBA, substrate inhibition is removed by batch feeding, the conversion period is shortened from 23h to 15h, the yield of the (R) -HMTBA can reach 97.0g/L, ee is more than 99 percent, and the mol conversion rate of the methionine reaches 96.3 percent, utilizes pot method synchronous reaction to catalyze the methionine to produce (S) -HMTBA, substrate inhibition is removed by batch feeding, the conversion period is shortened from 23h to 18h, the yield of the (S) -HMTBA can reach 98.0g/L, ee is more than 99 percent, and the mol conversion rate of the methionine reaches 98.6 percent.)

The method for producing hydroxymethionine by conversion of , which is characterized in that methionine is used as a substrate, L-amino acid deaminase, formate dehydrogenase and stereoselective dehydrogenase are used for catalyzing methionine to generate ketomethionine by synchronous cascade, the stereoselective dehydrogenase comprises R-stereoselective dehydrogenase or S-stereoselective dehydrogenase, and the conversion system comprises (1) or (2):

(1) methionine, formate, whole cell expressing L-amino acid deaminase, co-expressing R-stereoselectivityWhole cell and NAD of dehydrogenases and formate dehydrogenases⁺(ii) a The concentration of the methionine is 5-30 g/L, the mol ratio of the methionine to the formate is 1: 1.5-2.5, the concentration of the whole cell expressing the L-amino acid deaminase is 10-20 g/L, the concentration of the whole cell co-expressing the R-stereoselective dehydrogenase and the formate dehydrogenase is 10-20 g/L, and the NAD⁺The concentration of methionine is 0.4-0.6 mmol/L, methionine and formate are fed intermittently into a conversion system according to a molar ratio of 1: 1.5-2.5 in the conversion process, and the concentration of methionine is maintained to be 5-30 g/L;

(2) methionine, formate, whole cell expressing L-amino acid deaminase, whole cell co-expressing S-stereoselective dehydrogenase and formate dehydrogenase and NAD⁺(ii) a The concentration of the methionine is 10-30 g/L, the mol ratio of the methionine to the formate is 1: 1.5-2.5, the concentration of the whole cell expressing the L-amino acid deaminase is 15-20 g/L, the concentration of the whole cell co-expressing the S-stereoselective dehydrogenase and the formate dehydrogenase is 15-20 g/L, and the NAD⁺The concentration of methionine is 0.4-0.8 mmol/L, methionine and formate are fed intermittently into a conversion system according to a molar ratio of 1: 1.5-2.5 in the conversion process, and the concentration of methionine is maintained to be 5-35 g/L.

2. The method of claim 1, wherein the temperature of the conversion is 25 to 32 ℃, the pH is 7.0 to 8.0, the rotation speed is 500 to 600rpm, and the aeration rate is 1 to 2 vvm.

3. The method of claim 1, wherein the conversion system further comprises a Tris-HCl solution at a pH of 7.0 to 8.0.

4. The method of claim 1, wherein the amino acid sequence of the L-amino acid deaminase is as set forth in SEQ id No. 6.

5. The method of claim 1, wherein the formate dehydrogenase has the amino acid sequence set forth in SEQ ID NO 8.

6. The method of claim 1, wherein the R-stereoselective dehydrogenase is a D-lactate dehydrogenase having an amino acid sequence shown in SEQ ID NO. 7.

7. The method according to claim 1, wherein the S-stereoselective dehydrogenase is an L-lactate dehydrogenase having an amino acid sequence shown in SEQ ID NO. 5.

8. The method of claim 1, wherein the formate salt comprises sodium formate or ammonium formate.

9. The method of claim 1, wherein the R-stereoselective dehydrogenase or the S-stereoselective dehydrogenase and the formate dehydrogenase are expressed in the same E.coli (Escherichia coli).

10. The process as claimed in claim 9, wherein the E.coli is E.coli BL21(DE 3).

Technical Field

The invention relates to a method for producing (R/S) -hydroxymethionine by kinds of multienzyme cascade, belonging to the technical field of biological engineering.

Background

Hydroxy-methionine (2-Hydroxy-4- (methythio) butanoic acid, HMTBA), known as 2-Hydroxy-4-methylthiobutanoic acid, also known as methionine Hydroxy analogue, with molecular formula C₅H₁₀O₃S, the relative molecular mass is 150.196, because α carbon is chiral carbon, two different stereoisomers exist, (R) -hydroxymethionine, (S) -hydroxymethionine (figure 1), the calcium salt form of hydroxymethionine is of the components forming the compound α -keto acid tablet, is used for treating renal failure, and can be converted into L-methionine in vivo, so is widely applied to feed additives of livestock and poultry.

The method for synthesizing hydroxymethionine mainly includes chemical synthesis method and enzyme conversion method. The chemical method for synthesizing HMTBA comprises hydrolysis of cyanohydrin and ester, butadiene oxidation method and the like: (1) the cyanohydrin hydrolysis method is to synthesize 3-methylthiopropanal by the reaction of methyl mercaptan and acrolein, then react with sodium cyanide to produce 2-hydroxy-4-methylthiobutyronitrile, and finally generate HMTBA by two-step hydrolysis reaction of 65-70% hydrochloric acid or sulfuric acid; (2) the ester hydrolysis method comprises the steps of catalyzing 2-hydroxy-4-methylthiobutyronitrile obtained in the step (1) at 98 ℃ by manganese oxide or sodium tetraborate to generate 2-hydroxy-4-methylthiobutanamide, reacting with methyl formate to obtain 2-hydroxy-4-methylthiobutyric acid methyl ester, and finally using H at 95 DEG₂SO₄Hydrolyzing for 5h to prepare hydroxy methionine; the method can avoid the generation of ammonium sulfate, but the synthesis process is complex; (3) the butadiene oxidation method takes butadiene as a raw material, obtains 2-hydroxy-4-methylthiobutanol through multi-step chemical oxidation, and obtains the hydroxymethionine through oxidation of Gordon bacteria or Rhodococcus. The chemical synthesis reaction is carried out at a high temperature and uses a strong acid such as sulfuric acid, thereby generating energyThe high demand causes equipment corrosion, and toxic acrolein, methyl mercaptan and the like are used as raw materials, so that the application of HMTBA in the food and medicine industries is severely limited.

Compared with the chemical synthesis method, the enzyme conversion has many advantages, such as operation under the conditions of normal temperature and pressure and less use of toxic chemicals, the utilization of L-methionine with simple structure as a substrate is safer and nontoxic, the absence of enzymes directly catalyzing amino acid synthesis of hydroxy acid in nature requires two conversion processes, (1) conversion of L-methionine into ketomethionine, (2) conversion of ketomethionine into hydroxymethionine, Busto et al combined with two conversion processes designs a multi-enzyme cascade reaction in which L-amino acid deaminase derived from protein myxofaciens is expressed to convert L-methionine into ketomethionine, D-isocaproate reductase derived from Lactobacillus paracasei and FDH catalyze ketomethionine synthesis (R) -HMTBA are expressed in Escherichia coli, L-isocaproate reductase derived from Lactobacillus fuses is expressed to convert ketomethionine into (S) -HMTBA, and L-isocaproate dehydrogenase derived from Lactobacillus paracasei is expressed to convert ketomethionine into (S) -HMD-TBA, and the coenzyme circulation system is used to simultaneously catalyze the cascade reaction to convert L-methionine dehydrogenase to produce HME-96-methionine dehydrogenase, and finally the conversion reaction is expressed to achieve the bottleneck of the conversion reaction of MTG-96-methionine-96-methionine conversion and the final conversion reactions.

Disclosure of Invention

The invention aims to optimize the reaction conditions of transformation, shorten the transformation period, reduce the addition amount of wet cells and further reduce the production cost of HMTBA so as to be suitable for industrial production of (R/S) -HMTBA.

The th object of the invention is to provide methods for producing HMTBA by using methionine as a substrate and using L-amino acid deaminase, formate dehydrogenase and stereoselective dehydrogenase to synchronously cascade and catalyze methionine to generate ketomethionine, wherein the stereoselective dehydrogenase comprises R-stereoselective dehydrogenase or S-stereoselective dehydrogenase, and the system for conversion comprises (1) or (2):

(1) methionine, formate, whole cell expressing L-amino acid deaminase, whole cell co-expressing R-stereoselective dehydrogenase and formate dehydrogenase and NAD⁺(ii) a The concentration of the methionine is 5-30 g/L, the mol ratio of the methionine to the formate is 1: 1.5-2.5, the concentration of the whole cell expressing the L-amino acid deaminase is 10-20 g/L, the concentration of the whole cell co-expressing the R-stereoselective dehydrogenase and the formate dehydrogenase is 10-20 g/L, and the NAD⁺The concentration of the methionine is 0.3-0.6 mmol/L, in the conversion process, methionine and formate are fed intermittently into a conversion system in batches according to the molar ratio of 1: 1.5-2.5, and the concentration of the methionine is maintained to be 5-30 g/L;

(2) methionine, formate, whole cell expressing L-amino acid deaminase, whole cell co-expressing S-stereoselective dehydrogenase and formate dehydrogenase and NAD⁺(ii) a The concentration of the methionine is 10-30 g/L, the mol ratio of the methionine to the formate is 1: 1.5-2.5, the concentration of the whole cell expressing the L-amino acid deaminase is 15-20 g/L, the concentration of the whole cell co-expressing the S-stereoselective dehydrogenase and the formate dehydrogenase is 15-20 g/L, and the NAD⁺The concentration of the methionine is 0.4-0.8 mmol/L, in the conversion process, methionine and formate are fed intermittently into a conversion system in batches according to the molar ratio of 1: 1.5-2.5, and the concentration of the methionine is maintained to be 5-35 g/L.

, the transformation system also includes Tris-HCl solution with pH value of 7.0-8.0.

, the temperature of the conversion in the step (1) is 25-32 ℃, the pH value is 7.0-8.0, the conversion time is 15-18 h, the rotating speed is 500-600 rpm, and the ventilation volume is 1-2 vvm.

, the temperature of the conversion in the step (2) is 25-32 ℃, the pH value is 7.0-8.0, the conversion time is 18-20 h, the rotating speed is 500-600 rpm, and the ventilation volume is 1-2 vvm.

, the conversion process utilizes coenzyme regeneration system which uses formate as substrate to make NAD through formate dehydrogenase⁺Coenzyme regeneration system for conversion to NADH.

Further , the formate salt includes sodium formate or ammonium formate.

Further , the source of the L-amino acid deaminase includes, but is not limited to, Proteusvulgaris.

, the amino acid sequence of the L-lactate dehydrogenase is shown in SEQ ID NO. 6, and the nucleotide sequence of the gene coding the L-amino acid deaminase is shown in SEQ ID NO. 1.

Further , the R-stereoselective dehydrogenase is a D-lactate dehydrogenase.

Further , the source of the D-lactate dehydrogenase includes, but is not limited to, Pediococcus acidilactici (Pediococcus acidilactici).

, the amino acid sequence of the D-lactate dehydrogenase is shown as SEQ ID NO. 7, and the nucleotide sequence of the gene encoding the D-lactate dehydrogenase is shown as SEQ ID NO. 2.

Further , the S-stereoselective dehydrogenase is an L-lactate dehydrogenase.

Further , the source of the L-lactate dehydrogenase includes, but is not limited to, Bacillus coagulans (Bacillus coagulans).

, the amino acid sequence of the L-lactate dehydrogenase is shown as SEQ ID NO. 5, and the nucleotide sequence of the gene encoding the L-lactate dehydrogenase is shown as SEQ ID NO. 4.

Further , the source of formate dehydrogenase includes, but is not limited to, Candida (Candidabineii).

, the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO. 8, and the nucleotide sequence of the gene encoding the formate dehydrogenase is shown in SEQ ID NO. 3.

Further , the R-stereoselective dehydrogenase or S-stereoselective dehydrogenase and formate dehydrogenase are expressed in E.coli .

Further , the Escherichia coli is Escherichia coli BL21(DE 3).

The invention has the beneficial effects that:

(1) the method utilizes pot method to synchronously react and catalyze methionine to produce (R) -HMTBA, removes substrate inhibition by batch feeding, shortens the conversion period from 23h to 15h, shortens the conversion period from 23h to 18h, achieves the yield of S-HMTBA of 98.0g/L, ee of 99% and the mol conversion rate of methionine of 96.3%, and utilizes pot method to synchronously react and catalyze methionine to produce (S) -HMTBA, removes substrate inhibition by batch feeding, and shortens the conversion period from 23h to 18h, achieves the yield of S-HMTBA of 98.0g/L, ee of 99% and the mol conversion rate of methionine of 97.3%.

(2) The conversion substrate is methionine with low price, formate dehydrogenase, L-amino acid deaminase, R-stereoselective dehydrogenase or S-stereoselective dehydrogenase required by conversion are prepared in large quantity by high-density fermentation, and the whole cell is utilized for conversion, so that the operation is simple and convenient, the downstream purification is simple and easy, and processes for synthesizing HMTBA by the biological enzyme method which are efficient, energy-saving and easy for industrial amplification production can be established.

Drawings

FIG. 1: (R/S) -Hydroxymethionine.

FIG. 2: SDS-PAGE verifies the L-amino acid deaminase expression strain; m: DNA Marker; lane 1: PvLAAD strain.

FIG. 3: SDS-PAGE verifies that the D/L-lactate dehydrogenase and the formate dehydrogenase co-express the engineering strain; (a) in (1), M: DNAmarker; lane 1: CbFDH-PaDLDH; (b) in (1), M: DNA Marker; lane 1: CbFDH-BalLDH.

FIG. 4: the (R) -HMTBA is produced by step-by-step cascade catalysis.

FIG. 5, times of material feeding and synchronous cascade catalytic production of (R) -HMTBA.

FIG. 6: fed-batch synchronous cascade catalytic production of (R) -HMTBA.

FIG. 7: the (S) -HMTBA is produced by step-by-step cascade catalysis.

FIG. 8 is a series of simultaneous batch-wise catalytic cascades for producing (S) -HMTBA.

FIG. 9: fed-batch synchronous cascade catalytic production of (S) -HMTBA.

Detailed Description

The experiments described below all employ conventional experimental methods and the materials for carrying out the experiments are commercially available.

Sample pretreatment: the transformation solution was centrifuged at 12000rpm for 10min to collect the supernatant, and a standard solution was prepared using (R/S) -HMTBA as a standard. Filtering the supernatant and the standard solution after appropriate dilution with 0.22 μm microporous membrane, and detecting with high performance liquid chromatography.

Determination of the content of Ketone methionine and (R/S) -HMTBA: high performance liquid chromatography, mobile phase composition: 5mmol/L dilute sulfuric acid, the flow rate is 0.6 mL/min; a chromatographic column: aminex HPX-87H Ion Exclusion Column, 300X 7.8 mm; a detector: and an ultraviolet detector with the wavelength of 210 nm.

Chiral identification of (R/S) -HMTBA: high performance liquid chromatography, mobile phase composition: 0.1% triethylamine solution (pH adjusted to 3.0 with phosphoric acid)/pure methanol volume ratio 4: 6, flow rate: 0.2 mL/min; a chromatographic column: CHIRALPAK IG-3,0.46 cmI.D.. times.25 cm L.times.3 μm; a detector: an ultraviolet detector with the wavelength of 205 nm; ee ═ C_R-C_S)/(C_R+C_S) (wherein CR and CS are peak areas for R-isomer and S-isomer, respectively).

The method for measuring the activity of the amino acid deaminase comprises the following steps: 0.5g of wet cells were weighed into a 250mL conical flask, and 30mL of a preheated L-methionine solution was added thereto, followed by reaction in a shaker at 30 ℃ and 200rpm for 15 min. After the reaction is finished, taking a proper amount of reaction liquid for rapid centrifugal dilution and carrying out liquid phase detection. The enzyme activity unit is defined as the amount of enzyme required for the conversion to 1. mu. mol ketomethionine in 1 min.

The method for measuring the activity of the R/S-stereoselective dehydrogenase comprises the following steps: the reaction system (200. mu.L) was determined by UV spectrophotometry: Tris-HCl 100mM pH 7.0, L-methionine 20mM, NADH 0.25mM totaling 180. mu.L, added with D/L-lactate dehydrogenase 20. mu.L after appropriate dilution, reacted for 10min and sampled to detect the concentration of (R/S) -HMTBA produced. The enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol (R/S) -HMTBA per minute.

The method for measuring the activity of the formate dehydrogenase comprises the following steps:

the reaction system (200. mu.L) was determined by UV spectrophotometry: the reaction premix contained a sodium formate solution of pH 7.5, 0.167mmol/L and 1.67mmol/L NAD⁺Add 20. mu.L of diluted sample to be tested, start the reaction and time, record absorbance values every 30 seconds at 340nm, enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol NADH per minute.