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Method for producing clostridium perfringens glycerin anhydrase incitant gene and 1,3-propanediol thereof

A technology of Clostridium perfringens and glycerol dehydratase, which is applied in the field of genetic engineering and can solve problems such as inactivation and easy inactivation

Inactive Publication Date: 2008-09-17
南宁中诺生物工程有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] This patent application discloses the gene of glycerol dehydratase. Although the half-life of its enzyme activity is longer than that of the currently disclosed glycerol dehydratase, it will be rapidly inactivated in the presence of the reaction substrate glycerol. It is used to construct the production of 1,3- Propylene glycol engineering bacteria are also easily inactivated during the conversion of glycerol, so the activity of glycerol dehydratase becomes a key factor limiting the conversion rate of glycerol

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0119] 1. Extraction of Clostridium perfringens genomic DNA

[0120] Clostridium perfringens CVCC 2015 (purchased from China Veterinary Microbiological Culture Collection Management Center) was used to inoculate Clostridium perfringens in 20ml blister medium, and cultured overnight at 37°C in an anaerobic incubator. Take 1.5ml culture and centrifuge for 2min, add 567μl TE buffer (TE buffer-100mmol / L Tris-HCl, 1mmol / L EDTA, pH 8.0) and 20μl (100mg / ml) lysozyme to the precipitate, resuspend After mixing, place at 37°C for 2 hours; then add 30 μl of 10% SDS (SDS is sodium dodecyl sulfate) and 3 μl of 20 mg / ml proteinase K, mix well, and incubate at 37°C for 1 hour. Then add 100 μl of 5 mol / L NaCl, mix well, then add 80 μl CTAB / NaCl solution, mix well, and incubate at 65° C. for 10 minutes. The solution was extracted once with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), and then extracted once with an equal volume of chloroform: isoamyl alcohol (24:1). The s...

Embodiment 2

[0137] The 3-phosphate glycerol dehydrogenase and 3-phosphoglycerol lipase genes of Saccharomyces cerevisiae were cloned, and highly expressed in Escherichia coli BL21 (DE3) strain; the engineering bacteria capable of converting glucose to produce glycerol were constructed, and the cloned The glycerol dehydration gene is transformed into the above-mentioned engineering bacteria for co-expression of multiple genes to obtain metabolic engineering bacteria producing 1,3-propanediol.

Embodiment 3

[0139] Using cassava starch as the starting material, after liquefaction by α-amylase, the glucose generated by the action of glucoamylase is used to ferment the obtained glucose according to Example 1 to produce 1,3-propanediol. Experiments show that every 3 kg of starch is processed by α-amylase After liquefaction, glucose is generated by the action of glucoamylase for the fermentation of engineering bacteria. First, deionized water is added to make a 30% solution, and then it is used in the fermentation process of engineering bacteria, and it is fermented at 38°C and pH 7.0. 32-40 hours, while flowing ammonia and citric acid to maintain the stability of pH, can obtain about 500 grams of 1,3-propanediol. The water and impurities in the starch are removed, and the conversion efficiency is the same as that of directly using glucose as a substrate.

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PUM

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Abstract

The invention relates to the clone of a novel glycerol dehydratase activating factor gene and the expression thereof in related host. The activating factor gene, cloned from clostridium perfringens, can activate an inactive glycerol dehydratase under normal biochemical reaction conditions and recover the enzyme activity of the glycerol dehydratase, and can also enable the glycerol dehydratase to be difficult to be inactivated when the glycerol dehydratase is transformed to glycerin in the reaction. On the basis of the invention, related enzyme genes, such as the glycerol dehydratase, 1, 3-propanediol oxidoreductase, can be restructured to colon bacillus so as to get novel metabolic engineering bacteria. The metabolic engineering bacteria transform starch, glycerin or glucose to 1, 3-propanediol through fermentation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a method for producing 1,3-propanediol by a biotransformation method. Background technique [0002] 1,3-propylene glycol is the main raw material for the production of polytrimethylene terephthalate (PPT), and can also be used as a raw material for synthetic plasticizers, detergents, preservatives, and emulsifiers. In particular, the PTT fiber with excellent performance has both the performance of polyethylene terephthalate (PET) and the good resilience and pollution resistance of nylon. It is widely used in carpets, engineering plastics, clothing fabrics and other fields, and has become a hot spot in the development of synthetic fibers in the world. At present, the production method of 1,3-propanediol is mainly a chemical synthesis method, such as using ethylene as a raw material, using silver as a catalyst to oxidize ethylene oxide at high temperature (280°C), and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/10C07K14/195C12N1/21C12P7/18C12R1/19
Inventor 黄日波韦宇拓吴杰群杜丽琴韦旭钦王青艳卢福燊卢运琨
Owner 南宁中诺生物工程有限责任公司
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