A kind of method that takes sucrose as raw material biotransformation to produce mannitol

A technology of mannitol and mannitol dehydrogenase, which is applied in the biological field, can solve the problems of unfavorable industrial production, long fermentation cycle, and high production cost, and achieve the effects of shortening the fermentation cycle, low production cost, and simple ingredients

Active Publication Date: 2016-06-08
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In 2010, Jin Hongxing and others used sucrose as raw material to ferment Leuconostoc to produce mannitol, but the mannitol output was only 7.31g / L
Korakli, Niklasvon Weymarn and others isolated or screened lactic acid bacteria that can produce high mannitol. The carbon source used is mostly fructose-glucose mixture or molasses-fructose syrup mixture. The yield is high, but they all contain fructose and have a long fermentation period. , leading to higher production costs, which is not conducive to industrial production
In 2009, Wang Fang and others cloned and expressed mannitol dehydrogenase in Escherichia coli, using fructose as a substrate. Fructose is used for both energy metabolism and mannitol conversion. The conversion rate is 26.67%, which is relatively low. Low

Method used

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  • A kind of method that takes sucrose as raw material biotransformation to produce mannitol
  • A kind of method that takes sucrose as raw material biotransformation to produce mannitol
  • A kind of method that takes sucrose as raw material biotransformation to produce mannitol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Recombinant Escherichia coli induced expression of sucrose hydrolase and mannitol dehydrogenase

[0029] Pick a single colony of recombinant Escherichia coli pET28a-sacA-mdh / BL21 from the LB plate and inoculate it in LB liquid medium, culture it at 37°C overnight, then inoculate it in 100mL LB medium according to the inoculation amount of 1%-2%, at 37°C After culturing, when the OD600 reaches 0.5-0.8, add the inducer IPTG (0.05-0.2mmol / L), and the induction time is 6-10h.

Embodiment 2

[0031]Preparation of crude enzyme solution: take the induced bacterial solution and centrifuge at 6000r / min at 4°C for 10min, discard the supernatant. The precipitate was washed twice with sodium acetate buffer (pH 5.3), and the washed precipitate was resuspended in lysis buffer and ultrasonically disrupted on ice at 55 Hz for 3 s with an interval of 4 s; a total of 30 min of sonication. The sonicated bacterial solution was centrifuged at 6000r / min, 4°C for 10min. The supernatant was taken as the crude enzyme solution, and the enzyme activities of sucrose hydrolase and mannitol dehydrogenase were determined.

[0032] Sucrose hydrolase enzyme activity assay method: with sucrose as substrate, the total volume of enzyme reaction is 3.0mL, which contains 2.7mL, pH value 5.0, 0.2mol / L disodium hydrogen phosphate-citric acid buffer, 270μL 5% sucrose, 30 μL crude enzyme solution. Incubate in a constant temperature water bath at 42°C for 30 minutes, then measure the amount of reduci...

Embodiment 3

[0034] Example 3 Recombinant Escherichia coli uses sucrose as a substrate to produce mannitol by batch fermentation

[0035] Pick a single colony of recombinant Escherichia coli pET28a-sacA-mdh / BL21 from the LB plate and inoculate it in LB liquid medium, cultivate overnight at 37°C, then inoculate 2% inoculum in 100mL LB medium, culture at 37°C, OD 600 When it reaches 0.8, add inducer IPTG0.2mmol / L and substrate sucrose with different concentrations of 30-120g / L, culture temperature is 23°C, pH=6, rotation speed is 200r / min, and fermentation time is 10h. The bacterial liquid after induced expression was centrifuged at 6000r / min, 4°C for 10min, and the supernatant was taken, and the production of mannitol and the residual amount of sugar were determined by high performance liquid chromatography (HPLC).

[0036] High-performance liquid chromatography analysis conditions: the chromatographic column is TSK-GELAmide-80, the column temperature is maintained at 80°C, the mobile phase...

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Abstract

The invention discloses a recombinant bacterial strain pET28a-sacA-mdh / BL21 expressing sucrose hydrolase and mannitol dehydrogenase and a method for producing mannitol by fermenting the bacterial strain. The method includes: (1) optimizing the fermentation conditions of the strain in a sucrose-containing fermentation medium; (2) carrying out batch fermentation with different concentrations of sucrose, and investigating the production level of mannitol produced by the strain and the conversion of sucrose (3) Using different methods to produce mannitol by fed-batch fermentation can improve production efficiency and mannitol yield. The method not only does not generate by-product sorbitol, but also has low production cost, short cycle, wide source of raw materials and simple process, which provides a new method for the industrial preparation of mannitol.

Description

technical field [0001] The invention relates to a recombinant bacterial strain pET28a-sacA-mdh / BL21 expressing sucrose hydrolase and mannitol dehydrogenase and a method for producing mannitol by fermenting the bacterial strain, belonging to the field of biotechnology. Background technique [0002] Mannitol (Mannitol) scientific name hexyl alcohol, wood honey alcohol, is a hexavalent sugar alcohol, isomers of sorbitol. Molecular formula is C 6 h 14 o 6 , the molecular weight is 182.17. Mannitol is a colorless, white needle-like or columnar crystalline powder, odorless, with a cool and sweet taste. Melting point 166°C, density 1.489g / cm 3 (20°C), easily soluble in water, soluble in pyridine, aniline and hot ethanol, almost insoluble in most common organic solvents. Unlike other polyols, mannitol is the only crystalline non-hygroscopic. [0003] Mannitol is widely used in food, medicine, light industry and chemical industry and other fields. The production methods of ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/18C12R1/19
Inventor 李玉路福平陈艳王正祥田康明
Owner TIANJIN UNIV OF SCI & TECH
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