Method for producing D-1, 2, 4-butantriol by simultaneous saccharification and fermentation of corncobs

A simultaneous saccharification and fermentation and corn cob technology, applied in the biological field, can solve the problems of many steps and unfavorable for large-scale fermentation, and achieve the effects of simple operation, large commercial production space, and low production cost

Pending Publication Date: 2021-05-14
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many steps, which is not conducive to mass fermentation

Method used

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  • Method for producing D-1, 2, 4-butantriol by simultaneous saccharification and fermentation of corncobs
  • Method for producing D-1, 2, 4-butantriol by simultaneous saccharification and fermentation of corncobs
  • Method for producing D-1, 2, 4-butantriol by simultaneous saccharification and fermentation of corncobs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Containing the construction of YjhG gene strain

[0032] (1) Use primers at the 5' end and 3' end of the YjhG gene to introduce restriction sites (NcoI and HindIII), perform double restriction digestion on the YjhG gene and the pCWJ plasmid, and then connect the YjhG gene to the pCWJ vector;

[0033] (2) Transfer the above recombinant vector into the competent cells of Escherichia coli Trans 1T1 (Quanshijin Biotechnology Co., Ltd.), spread on LB plates with 50mg / L chloramphenicol resistance, and culture overnight at 37°C;

[0034] (3) Pick a single colony grown on the plate, transfer it to LB medium containing 50mg / L chloramphenicol resistance, then extract the plasmid, and then perform digestion verification with restriction enzymes Spe I and Kpn I , finally obtained the recombinant plasmid pCWJ-YjhG.

[0035] Referring to the above method, proceed to prepare the recombinant plasmids pTRC99a-xylB-mdlC and pTRC99a-yjhG-adhP.

Embodiment 2

[0036] Example 2 Recombinant Escherichia coli uses xylose as a substrate to ferment and produce 1,2,4-butanetriol

[0037] Streak and activate the genetically engineered bacteria containing the recombinant plasmid pTRC99a-xylB-mdlC and the recombinant plasmid PCWJ-yjhG-adhP on the LB plate (streptavidin resistance and ampicillin resistance), pick a single colony, insert 5 mL LB culture culture medium at 37°C, 200 rpm for 7-10 h.

[0038] Take a group of 500ml Erlenmeyer flasks, fill them with 50ml fermentation medium containing 10g / L xylose respectively, inoculate the seed solution prepared by recombinant E. When culturing to OD600=2, add IPTG to a final concentration of 1 mmol / L. After adding IPTG, culture at 33°C for 66 hours until the end of fermentation. Samples are taken every 12 hours to measure the production of D-1,2,4-butanetriol , the result is as figure 1 . As can be seen from the figure, the production rate of the bacterial strain significantly decreased after 6...

Embodiment 3

[0040] Example 3 Synchronous saccharification and fermentation of recombinant Escherichia coli using corn cob as a substrate to produce 1,2,4-butanetriol

[0041] Corncobs were crushed through a 40-mesh sieve, and 1 mol / L sodium hydroxide solution and corncobs were mixed at a solid-to-liquid ratio of 10%. The mixed suspension hydrolyzate was subjected to high-temperature treatment, and the corncob residue was obtained by filtration, and the residue was washed with pure water until neutral.

[0042] Prepare LB medium with 5g / L yeast powder, 10g / L peptone, and 5g / L sodium chloride. Divide into 500mL Erlenmeyer flasks, with a liquid volume of 50mL, add 2g of pretreated corncob residue to LB medium to make a fermentation medium, and sterilize it for use.

[0043] Streak and activate the genetically engineered bacteria containing the recombinant plasmid pTRC99a-xylB-mdlC and the recombinant plasmid PCWJ-yjhG-adhP on the LB plate (streptavidin resistance and ampicillin resistance),...

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Abstract

The invention provides a method for producing D-1, 2, 4-butantriol by synchronous saccharification and fermentation of corncobs, which comprises the following steps: constructing, cloning and expressing genes of 2-keto acid decarboxylase, D-xylose dehydrogenase, xylonic acid dehydratase and alcohol dehydrogenase, transferring the constructed genes into cells of host bacteria to obtain recombinant escherichia coli, and carrying out high-temperature saccharification and fermentation on the recombinant escherichia coli to obtain D-1, 2, 4-butantriol. And culturing the recombinant escherichia coli, inoculating the recombinant escherichia coli into a culture medium containing the pretreated corncob, and fermenting to produce the D-1, 2, 4-butantriol. According to the method, the corncobs are used as raw materials, the butantriol is produced through simultaneous saccharification and fermentation, and the yield of the D-1, 2, 4-butantriol is 6.44 g / L. The 1, 2, 4-butantriol is produced by simultaneous saccharification and fermentation of the corncobs, the operation is simple and convenient, the yield is high, and the method is suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing D-1,2,4-butanetriol by synchronous saccharification and fermentation of corncobs. Background technique [0002] D-1,2,4-Butanetriol is a colorless, odorless, transparent, viscous four-carbon polyol; it has high solubility in water and alcohols and is hygroscopic. In the military, D-1,2,4-butanetriol can be used to synthesize butanetriol trinitrate (BTTN) as a rocket propellant. In medicine, D-1,2,4-butanetriol can be As a sustained-release agent, an intermediate for the preparation of antiviral compounds, D-1,2,4-butanetriol can also be used as a cross-linking agent for polymer materials to increase the strength and hardness of materials. [0003] At present, the production of D-1,2,4-butanetriol mainly focuses on chemical synthesis, that is, using NaBH 4 Reduced malic acid and its derivatives. However, the reaction of chemical synthesis of D-1,2...

Claims

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Application Information

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IPC IPC(8): C12P7/18C12N15/70C12N15/60C12N15/53
CPCC12P7/18C12N15/70C12N15/52C12N9/88C12N9/0006C12Y401/01072C12Y101/01175C12Y402/01082C12Y101/01001
Inventor 陈可泉李孟阳王昕
Owner NANJING UNIV OF TECH
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