CN106636227B

CN106636227B - Method for preparing pyruvic acid by converting L-alanine by enzyme method

Info

Publication number: CN106636227B
Application number: CN201610840817.9A
Authority: CN
Inventors: 李斌水; 米造吉; 何连顺; 马静; 杨涛
Original assignee: Jing Jing Pharmaceutical Co ltd
Current assignee: Jing Jing Pharmaceutical Co ltd
Priority date: 2016-09-22
Filing date: 2016-09-22
Publication date: 2020-11-06
Anticipated expiration: 2036-09-22
Also published as: CN106636227A

Abstract

The invention discloses a method for preparing pyruvic acid by converting L-alanine by an enzyme method, which comprises the following steps of 1, adding an acrylamide monomer and a cross-linking agent N, N-methylene bisacrylamide into a wet thallus solution, polymerizing and crosslinking into three-dimensional reticular immobilized enzyme gel under the action of an accelerator tetramethylethylenediamine and a catalyst ammonium persulfate, and further preparing immobilized L-amino acid oxidase; and 2, oxidizing and deaminating the L-alanine serving as a reaction substrate under the action of the immobilized L-amino acid oxidase to obtain L-pyruvic acid. The invention has the advantages of low cost, high purity and mild reaction conditions, and is suitable for the industrial production of pyruvic acid.

Description

Method for preparing pyruvic acid by converting L-alanine by enzyme method

Technical Field

The invention belongs to the technical field of preparation of pyruvic acid, and relates to a method for preparing pyruvic acid by converting L-alanine by an enzyme method.

Background

Pyruvate (pyroglutamic acid) is one of the intermediates involved in the basic metabolism of the whole organism. Pyruvic acid is involved in sugar metabolism, biochemical synthesis and metabolism of colloid, amino acid, protein, etc. and alcohol fermentation of organism, can realize interconversion between sugar, fat and amino acid in vivo through acetyl COA and tricarboxylic acid cycle, and plays an important pivotal role in the metabolic connection of three major nutrients.

Pyruvic acid has important function in the production field, is a main raw material for producing tryptophan, phenylalanine and vitamin B, is also a raw material for synthesizing L-dopa, and has important function in the fields of organic synthesis and biochemical research.

At present, China mainly depends on imported pyruvic acid products, and fermentation methods, chemical synthesis methods and enzyme conversion methods are mainly adopted in the market. The production of L-pyruvic acid is internationally leading in Japan, and a biological fermentation method is mainly adopted, but the conversion rate of preparing pyruvic acid by the fermentation method is often low, because pyruvic acid is in the most active central node in sugar metabolism and is easily converted into other compounds in cells, so the pyruvic acid is difficult to accumulate, and the pyruvic acid is used as a component of a fermentation product mixture and is quite low in concentration, so the separation and extraction of the pyruvic acid from a complex fermentation broth is generally difficult to carry out, and the cost is also expensive. The chemical synthesis method mainly adopts a lactic acid oxidation method, takes lactic acid as a raw material, and produces pyruvic acid by an oxidative dehydrogenation one-step method. However, it is very difficult to directly prepare pyruvic acid from lactic acid, and a proper catalyst must be selected according to different processes. Catalysts that may be selected are iron phosphate, tellurium molybdate, silver, vanadium, and the like. But has the disadvantage of higher cost, about 6 ten thousand yuan per ton.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for preparing pyruvic acid by converting L-alanine by an enzyme method aiming at the defects, and the method has the advantages of low cost, high purity and no pollution.

The invention adopts a method for preparing pyruvic acid by converting L-alanine by an enzyme method, and prepares pyruvic acid by converting L-alanine by immobilized L-amino acid oxidase.

The method for preparing pyruvic acid by converting L-alanine by using the enzyme method comprises the following detailed steps:

step 1, adding an acrylamide monomer and a cross-linking agent N, N-methylene bisacrylamide into a wet thallus solution, and polymerizing and cross-linking to form three-dimensional reticular immobilized enzyme gel under the action of an accelerator tetramethylethylenediamine and a catalyst ammonium persulfate so as to prepare immobilized L-amino acid oxidase;

and 2, oxidizing and deaminating the L-alanine serving as a reaction substrate under the action of the immobilized L-amino acid oxidase to obtain L-pyruvic acid.

The wet cells are E.coli cells containing L-amino acid oxidase.

The wet thallus solution is wet thallus silicone oil-water solution

The concentration of the silicone oil-water solution was 80%.

In the step 1, the method for preparing the wet thallus solution comprises the following steps: uniformly dispersing the wet thallus in a silicone oil-water solution, carrying out low-temperature crushing in an ultrasonic cell crusher, and filtering to remove thallus debris to obtain a wet thallus solution.

In the step 1, the addition amount of the acrylamide monomer is 200-600% of the weight of the wet thallus solution, the addition amount of the cross-linking agent N, N-methylene bisacrylamide is 2.5-5% of the weight of the wet thallus solution, the addition amount of the accelerator tetramethylethylenediamine is 5-8% of the weight of the wet thallus solution, and the addition amount of the catalyst ammonium persulfate is 2-5% of the weight of the wet thallus solution.

In the step 1, the three-dimensional reticular immobilized enzyme gel is uniformly stirred, the gel is washed by water, and a solid is obtained after filtration, namely the immobilized L-amino acid oxidase.

In the step 2, during the oxidative deamination, the reaction temperature, the dissolved oxygen amount and the pH value are controlled, wherein the reaction temperature is controlled to be 25-35 ℃, the pH value is 6.0-7.5, and the dissolved oxygen amount is 410-450 mg/L.

The invention has the following characteristics:

(1) the optimal reaction temperature of the immobilized enzyme catalysis is 30-35 ℃;

(2) the optimum pH value of the immobilized enzyme catalysis is 6.5;

(3) the immobilized enzyme is easily separated from the substrate and the product;

(4) the immobilized enzyme has high use efficiency and low cost.

The invention has the beneficial effects that: the method for preparing pyruvic acid by converting L-alanine by the enzyme method takes L-amino acid as a substrate, and performs biotransformation by using the prepared immobilized enzyme to obtain the product pyruvic acid. The immobilized enzyme is easy to separate the product pyruvic acid and the substrate L-amino acid, the substrate conversion rate is high, the impurities are few, the cost is low, the cost for producing each ton of pyruvic acid is about 1.3 ten thousand yuan, the reaction condition is mild, and the control is easy.

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific examples.

Example 1: uniformly dispersing 80g of wet thallus in 100ml of 80% silicone oil-water solution, crushing at the low temperature of 15 ℃ in 750W Hz ultrasonic waves for 15 minutes in a 3s-2s-3s mode, adding 2 times of acrylamide monomer and 2.5% of cross-linking agent N, N-methylene diacrylamide into the thallus solution, polymerizing and crosslinking into three-dimensional reticular immobilized enzyme gel under the action of an accelerator tetramethyl ethylenediamine (added amount is 5%) and a catalyst ammonium persulfate (added amount is 2%), uniformly stirring, washing the gel with water, and filtering to obtain a solid, namely the immobilized enzyme.

Dissolving 89g of L-alanine in purified water to prepare 8.9% solution, heating to 30 ℃, adding 19g of immobilized enzyme and 774U of enzyme activity into the reaction liquid, controlling the dissolved oxygen content to be 450mg/L, controlling the reaction pH to be 6.0, sampling after reacting for 3h, detecting the concentration (80 g/L) of pyruvic acid in the supernatant and the residue of L-alanine (less than or equal to 0.5g/L) by using a high performance liquid phase, when the residue of L-alanine is less than or equal to 0.5g/L, determining that the reaction is complete, cooling the obtained feed liquid to room temperature, filtering to remove the immobilized enzyme, concentrating the feed liquid to contain 30% of pyruvic acid, adding 10g of activated carbon into the feed liquid, stirring and decolorizing for 30 min, decarburizing and passing through a membrane, and continuously concentrating under reduced pressure to obtain 106g of colorless viscous liquid, namely a crude pyruvic acid product, wherein the conversion rate is greater than or equal to 99.

Example 2: uniformly dispersing 90g of wet thalli in 100ml of 80% silicone oil-water solution, crushing at the low temperature of 15 ℃ in ultrasonic waves of 800W Hz, crushing for 15 minutes in a 3s-2s-3s mode, adding 5 times of acrylamide monomer and 4% of cross-linking agent N, N-methylene bisacrylamide into the thalli solution, polymerizing and crosslinking into three-dimensional reticular immobilized enzyme gel under the action of an accelerator tetramethyl ethylenediamine (the added amount is 6%) and a catalyst ammonium persulfate (the added amount is 4%), stirring uniformly, washing the gel with water, and filtering to obtain a solid, namely the immobilized enzyme.

Dissolving 95g of L-alanine in purified water to prepare 9.5% solution, heating to 32 ℃, adding 22g of immobilized enzyme and 879U of enzyme activity into the reaction liquid, controlling the reaction pH to be 6.5 and the dissolved oxygen to be 410-450mg/L, sampling for 5h of reaction, detecting the concentration (86 g/L) of the pyruvic acid of the supernatant and the residual L-alanine (less than or equal to 0.5g/L) by using a high performance liquid phase, when the residual L-alanine is less than or equal to 0.5g/L, determining that the reaction is complete, cooling the obtained feed liquid to room temperature, filtering to remove the immobilized enzyme, concentrating the feed liquid to contain 30% of pyruvic acid, adding 10g of activated carbon into the feed liquid, stirring and decolorizing for 30 minutes, decarburizing and passing through a membrane, and continuously concentrating under reduced pressure to obtain 115g of colorless viscous liquid, namely a crude pyruvic acid product, wherein the conversion rate is greater than or equal to 99.

Example 3: 100g of wet thalli is uniformly dispersed in 100ml of 80% silicone oil-water solution, the wet thalli is broken at the low temperature of 15 ℃ in ultrasonic waves of 850W Hz, the breaking is carried out for 15 minutes in a 3s-2s-3s mode, 6 times of acrylamide monomer and 5% of cross-linking agent N, N-methylene diacrylamide are added into the thalli solution, the polymerization and the cross-linking are carried out under the action of an accelerator tetramethyl ethylenediamine (the adding amount is 8%) and a catalyst ammonium persulfate (the adding amount is 5%) to form three-dimensional reticular immobilized enzyme gel, then the three-dimensional reticular immobilized enzyme gel is uniformly stirred, the gel is washed by water, and the solid is obtained after the filtration, namely the immobilized enzyme.

Dissolving 100g of L-alanine in purified water to prepare a 10% solution, heating to 35 ℃, adding 25g of immobilized enzyme and 987U of enzyme activity into a reaction liquid, controlling the pH of the reaction to be 7.5 and the dissolved oxygen to be 450mg/L, carrying out reaction for 5h, sampling, detecting the concentration (92 g/L) of pyruvic acid in a supernatant and the residual (less than or equal to 0.5g/L) of L-alanine by using a high performance liquid phase, when the residual of L-alanine is less than or equal to 0.5g/L, determining that the reaction is complete, cooling the obtained feed liquid to room temperature, filtering to remove the immobilized enzyme, concentrating the feed liquid to contain 30% of pyruvic acid, adding 10g of activated carbon into the feed liquid, stirring and decolorizing for 30 min, decarburizing and passing through a membrane, continuously concentrating to obtain 120g of colorless viscous liquid, namely a crude pyruvic acid product, wherein the conversion rate is greater than or equal to.

Claims

1. A method for preparing pyruvic acid by converting L-alanine by an enzyme method is characterized in that:

step 1, uniformly dispersing wet thalli in a silicone oil-water solution, carrying out low-temperature crushing in an ultrasonic cell crusher, and filtering to remove thalli residues to obtain a wet thalli solution; adding an acrylamide monomer and a cross-linking agent N, N-methylene bisacrylamide into the wet thallus solution, and polymerizing and crosslinking into three-dimensional reticular immobilized enzyme gel under the action of an accelerator tetramethylethylenediamine and a catalyst ammonium persulfate to prepare immobilized L-amino acid oxidase;

the addition amount of the acrylamide monomer is 200-600% of the weight of the wet thallus solution, the addition amount of the cross-linking agent N, N-methylene bisacrylamide is 2.5-5% of the weight of the wet thallus solution, the addition amount of the accelerator tetramethyl ethylenediamine is 5-8% of the weight of the wet thallus solution, and the addition amount of the catalyst ammonium persulfate is 2-5% of the weight of the wet thallus solution; the wet thalli is an escherichia coli body for expressing L-amino acid oxidase;

and step 2, taking L-alanine as a reaction substrate, and carrying out oxidative deamination under the action of the immobilized L-amino acid oxidase to obtain L-pyruvic acid.

2. The method for preparing pyruvic acid by converting L-alanine with an enzymatic method according to claim 1, wherein: the wet thallus solution is a wet thallus silicone oil-water solution.

3. The method for preparing pyruvic acid by converting L-alanine with an enzymatic method according to claim 2, wherein: the concentration of the silicone oil-water solution in the wet thallus solution is 80%.

4. The method for preparing pyruvic acid by converting L-alanine with an enzymatic method according to claim 1, wherein: in the step 1, the three-dimensional reticular immobilized enzyme gel is uniformly stirred, the gel is washed by water, and a solid is obtained after filtration, namely the immobilized L-amino acid oxidase.

5. The method for preparing pyruvic acid by converting L-alanine with an enzymatic method according to claim 1, wherein: and 2, during the oxidative deamination, controlling the reaction temperature, the dissolved oxygen and adjusting the pH, wherein the reaction temperature is controlled to be 25-35 ℃, the pH is controlled to be 6.0-7.5, and the dissolved oxygen is 410-450 mg/L.