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Clostridium perfringen glycerol dehydrase gene, and its 1,3-propylene glycol producing method

A technology of Clostridium perfringens and glycerol dehydratase, applied in the field of genetic engineering

Inactive Publication Date: 2010-05-12
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

[0020] Some of the above-mentioned documents are previous studies of the present inventors, disclosing the selection of Klebsiella, culturing and isolating the gene encoding glycerol dehydratase, cloning it into an expression vector, and then introducing it into Escherichia coli to produce 1,3- Propylene glycol, but there are still some problems in the research introduced in these literatures, such as only the research on the enzymatic properties of Klebsiella dehydratase and the expression in E. Promising Glycerol Dehydratase Research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] 1. Extraction of Clostridium perfringens genomic DNA

[0141] 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 of the culture and centrifuge for 2min, add 567μl of TE buffer and 20μl (100mg / ml) lysozyme to the precipitate, resuspend and mix well and place it at 37°C for 2 hours; then add 30μl of 10% SDS and 3μl of 20mg / 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 supernatant was extracted with 0.6 times The volume of isopropanol was u...

Embodiment 2

[0159] Using glycerol as starting material, carry out fermentation to produce 1,3-propanediol, add glycerol concentration to the culture medium in Example 1 to be 1%, ferment and cultivate under the condition of 35°C, 200rpm, ventilation rate of 0.9vvm, 8 hours Then start to add glycerin, and keep the pH stable at 6-7 by adding ammonia and citric acid. After 32-40 hours of continuous fermentation, use HPLC to analyze the content of glycerol and 1,3-propanediol in the fermentation broth, and stop the fermentation when the amount of glycerol in the fermentation broth no longer decreases and the amount of 1,3-propanediol no longer increases . According to HPLC detection, the engineering bacteria consume about 1.5kg of glycerol after 32-40 hours of fermentation, and the output of 1,3-propanediol can reach 30-35 grams per liter of fermentation broth, and a total of about 600 grams of 1,3-propanediol is obtained. The conversion efficiency About 40%.

Embodiment 3

[0161] Starch is used as the starting material, and after being liquefied 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. Ferment for 32-40 hours under the conditions of 32-38°C and pH 6-7, and about 500 grams of 1,3-propanediol can be obtained. 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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Abstract

The invention relates to new glycerin dehydrase gene cloning and its expression in relative host. The enzyme gene is cloned form clostridium perfringens which can transform the glycerin into 3-hydroxyl propionic aldehyde at normal biological chemical reacting condition. Based on the invention, it can recombine the 1,3-propylene glycol redox enzyme relative gene into Escherichia coli to gain a newmetabolism engineering fungus which can be transformed into 1, 3-propylene glycol by fermenting tapioca hydrolyzate, glucose, or glycerol.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a method for producing 3-hydroxypropionaldehyde and 1,3-propanediol by using 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 a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N9/02C12N15/52C12N1/21C12P7/18
Inventor 黄日波韦宇拓吴杰群齐向辉孟晓蕾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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