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A kind of method that biotransformation fumaric acid produces l-malic acid

A biotransformation and malic acid technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as those that have not yet been put into industrial production, and achieve convenient subsequent extraction, lower production costs, and high enzyme activity. Effect

Active Publication Date: 2017-08-01
CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the experiment of producing L-malic acid by fermentation of these non-sugar raw materials is still in the laboratory stage and has not yet been put into industrial production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Microbial identification

[0039] 1. Genomic DNA extraction

[0040] (1) Use an inoculation loop to pick a ring of slant bacteria, suspend in 1ml 0.85% NaCl solution, centrifuge at 10000r / min for 2min, and discard the supernatant;

[0041] (2) Resuspend the pellet in 550ul 1×TE, and bathe in boiling water for 10min;

[0042] (3) Add 3ul proteinase K (20ug / ml), incubate at 37°C for 30min;

[0043] (4) Add 30ul 10% SDS, incubate at 37°C for 30min;

[0044] (5) Add 100ul 5M NaCl solution and mix well, then add 80ul CTAB / NaCl, mix well, and bathe in water at 65°C for 10min;

[0045] (6) Add an equal volume of chloroform / isoamyl alcohol (24:1), shake gently to mix, and centrifuge at 10000r / min for 8min;

[0046] (7) Take the supernatant, add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), shake gently; centrifuge at 13000r / min for 8min;

[0047] (8) Repeat step 6;

[0048] (9) Take the supernatant, add 0.6-0.8 times the volume of isopropanol, mix gently...

Embodiment 2

[0056] Slant medium (g / L): malt extract powder 90.0, agar 15.0, pH 6.0.

[0057] Seed medium (g / L): glucose 40.0, yeast powder 3.0, ammonium sulfate 4.0, potassium dihydrogen phosphate 2.0, magnesium sulfate heptahydrate 0.2, adjust pH 6.0.

[0058] Fermentation medium (g / L): glucose 70.0, yeast powder 6.0, ammonium sulfate 6.0, potassium dihydrogen phosphate 2.0, magnesium sulfate heptahydrate 0.2, adjust pH 6.0.

[0059] Take a loop of Corynespora portuguese CICC 33054 slant strain and inoculate it into a Erlenmeyer flask containing 30 mL of seed medium, and culture it at 29°C for 18 hours on a shaking table at 200 rpm to obtain an inoculum. Take 3mL inoculum solution, inoculate it into a Erlenmeyer flask containing 300mL fermentation medium, and culture it at 200rpm for 28h to obtain the fermentation broth of C. acid solution (sodium hydroxide to adjust the pH value to 6.0), then add 0.2mL of toluene, and biotransform at 30°C for 31h to generate L-malic acid. Add calcium ...

Embodiment example 3

[0061] Slant medium (g / L): malt extract powder 150.0, agar 15.0, pH 7.0.

[0062] Seed medium (g / L): glucose 70.0, yeast powder 8.0, ammonium sulfate 8.0, potassium dihydrogen phosphate 5.0, magnesium sulfate heptahydrate 0.6, adjust the pH value to 7.0.

[0063] Fermentation medium (g / L): glucose 120.0, yeast powder 15.0, ammonium sulfate 12.0, potassium dihydrogen phosphate 5.0, magnesium sulfate heptahydrate 0.6, adjust the pH to 7.0.

[0064] Inoculate one loop of Corynespora portuguese CICC 33054 slant strain into a 1L Erlenmeyer flask containing 300mL of seed medium, shake at 200rpm, and incubate at 33°C for 24h to obtain the inoculum, and combine to obtain 600mL of fermentation broth. Take 600mL inoculum solution, inoculate it into a 50L fermenter with 30L fermentation medium, culture at 33°C, ventilation rate 1v / v.min, 500rpm for 24h to obtain the fermentation broth of C. Add 60L of 139g / L fumaric acid solution (sodium hydroxide to adjust the pH to 6.0) to the cells, ...

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Abstract

The invention relates to a method for bioconverting fumaric acid to produce L-malic acid. Using the self-selected Corynespora lusitaniae CICC 33054 (Clavispora lusitaniae CICC 33054), the yeast cells were obtained by fermentation and centrifugation. Add sodium fumarate solution to yeast cells for biotransformation, replace precipitation with calcium fumarate, filter, sulfuric acid hydrolysis, filter, concentrate, crystallize and dry to obtain L-malic acid product. Corynespora portuguese CICC 33054 is easy to cultivate and has high transformation efficiency, and its use in the production of L-malic acid can improve production efficiency and reduce production costs.

Description

technical field [0001] The invention relates to a method for producing L-malic acid by using fumaric acid, in particular to a method for producing L-malic acid by biotransformation of fumaric acid by Corynespora portuguese yeast CICC 33054. Background technique [0002] L-malic acid, also known as hydroxysuccinic acid or hydroxysuccinic acid, has the molecular formula C 4 h 6 o 5 , with a molecular weight of 134.09. At room temperature, L-malic acid is white crystal or crystalline powder, odorless, with refreshing sour taste, specific gravity 1.601, melting point 98~99℃. At 20°C, 34% L-malic acid aqueous solution has no optical activity, and it can appear levorotatory after diluting with water, and dextrorotatory when its concentration exceeds 34%. [0003] Since the molecule of L-malic acid contains -OH group and -COOH group, it has high solubility in polar solvents and relatively active chemical properties. It is easily soluble in ethanol at room temperature, and its ...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12P7/46C12P7/42C12R1/645
CPCC12P7/42C12P7/46C12N1/145C12R2001/645
Inventor 程池黄玲裴疆森刘勇张京涛徐友强王哲
Owner CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD
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