Biocatalysis method for preparing pyruvic acid from L-alanine

A biocatalysis and alanine technology, which is applied in the biological field, can solve the problems such as the lack of L-amino acid oxidase pyruvate, the difficulty of separation and extraction of pyruvate, and the low concentration of pyruvate, and achieves low cost, high chemical purity, and high separation efficiency. simple craftsmanship

Active Publication Date: 2012-07-18
CHONGQING UNIV OF POSTS & TELECOMM +1
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Problems solved by technology

The disadvantages of producing pyruvate by fermentation are: as the final product of the glycolysis pathway, pyruvate is at the key metabolic fulcrum in the metabolic pathway, and it is easily metabolized into other products in cells, which is difficult to accumulate; the concentration of pyruvate in the fermentation broth is relatively high. Low, and there are organic heteroacids such as polypyruvate and α-ketoglutaric acid, and the separation and extraction of pyruvic acid is difficult
So far, there is no report about the use of L-amino acid oxidase for the preparation of pyruvate

Method used

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  • Biocatalysis method for preparing pyruvic acid from L-alanine

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Embodiment 1

[0019] Example 1: Cell Culture

[0020] (1) Incline culture: Alcaligenes faecalis Alcaligens faecali 1. The 1799 strain was inoculated on sterilized solid medium and cultured statically at 30°C for 48h. Solid medium: peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, agar 15 g / L, pH 7.4-7.6.

[0021] (2) Primary culture: inoculate the cells cultured on the slant into a test tube containing 5ml of sterile medium, and culture on a shaker at 30°C for 24 hours. Medium: peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, pH 7.0.

[0022] (3) Expansion culture: inoculate the primary cultured cells in 50 mL sterile expansion medium containing inducer (L-alanine) at 10% inoculum, and culture on a shaker at 30°C for 24 hours. Medium: peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, L-alanine 10 g / L, pH 7.0. The culture solution was centrifuged for 15min (8000rpm) to collect the wet cells.

Embodiment 2

[0023] Example 2: Cell Permeabilization Treatment

[0024] Add 50mL of 30% acetone aqueous solution to 50g of wet bacteria, treat at room temperature for 5-20min, centrifuge for 15min (8000rpm), and wash twice with normal saline to obtain 48g of permeable cells.

Embodiment 3

[0025] Embodiment 3: preparation of pyruvic acid

[0026] Suspend 20g of permeabilized wet cells in 1L of distilled water, add 17.8g (0.2mol) L-alanine, react at 30°C for 6h, monitor the reaction by HPLC until the complete conversion of L-alanine. After the reaction is completed, centrifuge for 15min (8000rpm) to remove the cells. The concentration of pyruvate in the supernatant was determined by HPLC. The concentration of pyruvic acid in the reaction solution was 16.7g / L, and the yield was 95%.

[0027] The HPLC conditions are:

[0028] Column: C18;

[0029] Mobile phase: a solution mixed with 10mmol / L sodium acetate and methanol at a volume ratio of 80:20, pH3.5;

[0030] Detection wavelength: 214nm;

[0031] Temperature: 25°C.

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Abstract

The invention provides a biocatalysis method for preparing pyruvic acid from L-alanine. The method comprises the steps of: carrying out permeability treatment on Alcaligens faecalisl.1799 cells; catalyzing oxidative deamination of L-alanine by utilizing L-amino acid oxidase in the cells in the presence of air to form pyruvic acid, ammonia and hydrogen peroxide; catalyzing the hydrogen peroxide formed in the reaction by utilizing catalase in the cells to realize pyruvic acid accumulation without producing by-products. The method provided by the invention has the advantages of low cost, high product yield and purity and environment friendliness, and is suitable for industrial production of pyruvic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a novel process for preparing ketoacids by microorganism catalysis. Specifically, the present invention relates to a biocatalytic method for preparing pyruvate by using Alcaligenes faecalis cells to catalyze the oxidative deamination of L-alanine. Background technique [0002] Pyruvate is one of the most important α-oxocarboxylic acids. It not only plays a very important role in bioenergy metabolism, but also is an important pharmaceutical and chemical product. And scientific research fields have a wide range of uses. In view of the huge market demand of pyruvate, the research on its production technology has become a hot spot in chemical industry and biotechnology. [0003] The preparation methods of pyruvic acid mainly include chemical synthesis and biotechnology. [0004] Chemical synthesis mainly includes tartaric acid method and lactic acid oxidation method. The tartaric acid ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/40C12R1/05
Inventor 夏仕文何从林徐红梅方国兰
Owner CHONGQING UNIV OF POSTS & TELECOMM
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