Preparation of valuable sialinic acid from cheap sodium lactate by multi-step coupling bio-conversion

A technology of biotransformation and sodium lactate, applied in the direction of fermentation, bacteria, etc., can solve the problems of high production cost, high price, and increased cost

Inactive Publication Date: 2005-02-23
SHANDONG UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, to produce sialic acid by this method, some problems must be paid attention to: (1) ManNAc is expensive and needs to be obtained by isomerization of N-acetylglucosamine (GlcNAc); (2) ManNAc and GlcNAc have similar chemical (3) GlcNAc is an inhibitor of sialic acid aldolase; (4) the Km value of sialic acid aldolase to ManNAc reaction is higher (0.7M); (5) sialic acid aldolase mainly To catalyze the degradation reaction of sialic acid, it is necessary to add 7-10 times excess pyruvic acid to promote the reverse reaction. This way, firstly, the cost is greatly increased, and secondly, the existence of a large amount of pyruvic acid has greatly affected the separation and purification of sialic acid. difficulty
However, the method of forming complexes introduces new impurities that need to be further removed
[0009] As can be seen from the above several synthetic methods, so far, no perfect method has been found to solve a series of problems in the isomerization process and aldolization process, and the production cost is very high, resulting in the price of sialic acid stay high

Method used

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  • Preparation of valuable sialinic acid from cheap sodium lactate by multi-step coupling bio-conversion
  • Preparation of valuable sialinic acid from cheap sodium lactate by multi-step coupling bio-conversion
  • Preparation of valuable sialinic acid from cheap sodium lactate by multi-step coupling bio-conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Cultivation of lactate oxidase-containing strains

[0082] 1. Culture of Pseudomonas sp. ATCC11452

[0083] (1) Lactate oxidase strain: select Pseudomonas sp. ATCC11452;

[0084] (2) Slant culture: Pseudomonas sp. ATCC11452 was inoculated on a solid slant basic medium containing 1.5% mass / volume agar and 1% mass / volume sodium L-lactate at 30°C cultured for 20 hours;

[0085] (3) Primary seed culture: the strains cultivated in step (2) were inoculated into 30 mL liquid basic medium BLM containing L-type sodium lactate with a mass-to-volume ratio of 1% under aseptic conditions, at 30° C. Under the condition of shaking culture on a shaker for 10 hours, first-class seeds were obtained;

[0086] (4) Expanded culture: with an inoculation amount of 5% by volume, the first-class seeds were placed in 500 mL of BLM containing 2% by volume of L-type sodium lactate, and under the condition of 30° C., shaken and cultured on a shaking table for 16 hours to prepare seco...

Embodiment 2

[0104] Example 2: Construction of engineering strain E. coli HB101 (pBV220-ALD)

[0105] Construction of sialyl aldolase strain: Escherichia coli engineering strain, namely E.coli HB101 (pBV220-ALD), was constructed by conventional methods:

[0106]Construction of recombinant engineering strains: Genomic DNA of E.coli K12 strains was prepared by conventional methods. This process can be referred to the method of small-scale preparation of bacterial genomes in the "Guidelines for Molecular Biology" published by Science Press to extract E.coli K12 strains. Genomic DNA of the coli K12 bacterial strain; use synthetic primers to amplify the sialic acid aldolase gene from the genomic DNA of the E.coli K12 bacterial strain; The sialic acid aldolase gene was connected to obtain the recombinant plasmid pBV220-ALD; then the recombinant plasmid pBV220-ALD was transformed into the vector E.coli HB101 to obtain the engineering strain E.coli HB101 (pBV220-ALD);

[0107] Wherein the host is...

Embodiment 3

[0108] Embodiment 3: the cultivation of engineering strain Escherichia coli E.coli HB101 (pBV220-ALD)

[0109] (1) Plate culture: Streak the engineering strain Escherichia coli HB101 (pBV220-ALD) on an ampicillin LB plate containing 100 μg / mL of 1.5% agar in mass volume ratio, and culture at 30° C. for 12 hours;

[0110] (2) First-class seeds: under sterile conditions, use a sterile toothpick to pick a single colony on the plate of step (1), and then inoculate it into 5 mL of liquid medium containing 100 μg / mL ampicillin, for 30 Cultivate with shaking in a shaker for 12 hours;

[0111] (3) Secondary seeds: under aseptic conditions, take the culture solution cultivated in step (2) with a volume ratio of 2% inoculum, inoculate into 240 mL of LB liquid medium containing 100 μg / mL of ampicillin, 30°C shaker culture for 6 hours;

[0112] (4) fermentor culture: under aseptic conditions, get the nutrient solution obtained in step (3) to inoculate the improved M9 liquid medium of 3L...

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Abstract

A method for preparing valuable sialic acid by cheap sodium lactate through multi-step coupling biotransformation is disclosed. It includes culture containing lactic oxidase strain, construction and culture of E.coli HB101(pBV220-ALD), preparation of N-acetylmannosamine, sialic acid converting synthesis by complete cell containing lactic oxidase and sialic aldolase with cheap sodium lactate as precursor, and separation and purification of sialic acid by ion exchange. It achieves low cost, simple operation and extensive converting conditions.

Description

(1) Technical field [0001] The invention relates to a method for preparing sialic acid by catalyzing microorganisms. Specifically, it relates to a method for preparing high-value sialic acid from cheap sodium lactate through multi-step coupled biotransformation of two microbial intact cells containing lactate oxidase and sialyl aldolase respectively. (2) Background technology [0002] Sialic acid (SA), a general term for Neuraminie acid (NA) and its derivatives, is a class of natural carbohydrate compounds widely present in biological systems. Since its identification, a lot of research has been done on its biological and physiological activities. It has been found that sialic acid compounds play an indispensable role in many important biological and physiological processes of life, and they are directly involved in the interaction between cells and cells, cells and microorganisms, cells and toxins, and cells and abzymes. Sialic acid and its derivatives have important appl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/26
Inventor 许平陈净邱建华王鹏马翠卿张奕南郗日沫魏中浩
Owner SHANDONG UNIV
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