Method for preparing xylitol by utilizing whole-cell catalysis

A technology for catalytic preparation of xylitol, applied in biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve problems such as increasing production costs, and achieve cost-saving and high application value effects

Active Publication Date: 2018-12-07
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Preparation or purification of enzymes and expensive

Method used

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  • Method for preparing xylitol by utilizing whole-cell catalysis
  • Method for preparing xylitol by utilizing whole-cell catalysis
  • Method for preparing xylitol by utilizing whole-cell catalysis

Examples

Experimental program
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Effect test

Embodiment 1

[0020] The construction of embodiment 1 recombinant escherichia coli

[0021] 1. Find the xylose reductase sequence (GenBank: AB002105.2) of Candida tropicalis IFO 0618 and the glucose dehydrogenase sequence (GenBank: BC4715) of Bacillus cereus ATCC 14579 in the NCBI database, and optimize the codon corresponding to Escherichia coli , the nucleotide sequence of the optimized xylose reductase gene XR is shown in SEQ ID NO:1, and the nucleotide sequence of the optimized glucose dehydrogenase gene GDH is shown in SEQ ID NO:2. It should be pointed out that there are many homologous genes in the above-mentioned xylose reductase gene XR and glucose dehydrogenase gene GDH, including orthologous genes and paralogous genes. Homologous genes are capable of catalyzing the same reaction and share similarity in nucleotide sequence (and therefore similarity in protein sequence). Specifically, a homologous gene refers to a gene having more than 90% similarity in sequence with the above-ment...

Embodiment 2

[0053] Example 2 whole cell catalysis

[0054] The whole cell catalytic reaction was added according to the following system:

[0055] Table 5 whole cell catalytic system

[0056]

[0057] The product is heated to inactivate the enzyme, and the product components are detected in the liquid phase after being filtered through a water-based filter membrane. The liquid phase conditions: ①The chromatographic column is Aminex HPX-87C, the mobile phase is ultrapure water, the flow rate is 0.6mL / min, and the column temperature is 80°C . Detected with a refractometer RID. The results showed that xylose and glucose had been completely converted within 24h. ② The chromatographic column is Aminex HPX-87H, and the mobile phase is 5mM H 2 SO 4 , the flow rate is 0.4mL / min, the column temperature is 45°C, and the content of xylitol is detected with a refractometer RID. The yield of xylitol in the product is about 0.97 (g / g), and the concentration can reach 145.81g / L, and the yield o...

Embodiment 3

[0058] Example 3 Utilization of Whole Cell Catalysis in Straw Hydrolyzate

[0059] Crushed corn stalks (derived from Huai'an, Jiangsu) and then dried, and processed in a reactor: 10g dry weight corn stalks were added with 60vt.% ethanol aqueous solution, and 4wt.% NaOH aqueous solution as a catalyst, and the reaction was controlled at 110°C for 90min . After the reaction, the solid was washed twice with 25 mL of pure water, dried at 105°C until the mass was constant, and pulverized with a pulverizer. Add 1kg of pretreated corn stalks to a 20L hydrolysis kettle, add deionized water to 10L, adjust the pH to 4.8 with sulfuric acid, and then add cellulase (from Henan Nanyang Tianguan Group, whose filter paper enzyme activity 245FPU / mL), 50°C, 150rpm, constant temperature hydrolysis for 24h, the glucose content of the obtained straw hydrolyzate is: 51.57g / L, the xylose content is 32.04g / L, and the pH is about 4.6; adjust the pH to 7 with NaOH Catalyzes reactions in whole cells. ...

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Abstract

The invention discloses a method for preparing xylitol by utilizing whole-cell catalysis. The method comprises the following steps: (1) culturing recombinant Escherichia coli containing xylose reductase genes XR and glucose dehydrogenase genes GDH in an LB culture medium until the OD is 0.6-0.8, adding IPTG (Isopropyl-beta-d-Thiogalactoside) to induce expressions of the xylose reductase and glucose dehydrogenase, and centrifuging to obtain whole cells; and (2) enabling an aqueous solution containing xylose and glucose, the whole cells obtained in the step (1) and CaCO3 to form a biological catalysis system, and catalyzing the xylose to carry out a reduction reaction so as to produce the xylitol. The method disclosed by the invention can be effectively applied to comprehensive utilization of straws, the reaction system is applied to treating crop straw hydrolyte, and the substrate of the hydrolyte can be completely consumed within 20 hours. According to the method disclosed by the invention, extraction of enzymes in cells and addition of exogenous cofactors can be saved, and the process and cost for producing the xylitol by biological catalysis can be greatly simplified.

Description

technical field [0001] The invention relates to a preparation method of xylitol, in particular to a method for preparing xylitol by catalyzing whole cells. Background technique [0002] Xylitol is widely used in chemical, food, pharmaceutical and other industries. For example, in the field of pharmacy, xylitol surfactants are used as excipients of medicaments; It can also be used for parenteral nutrition, diabetes treatment, liver disease treatment, etc. Xylitol is a five-carbon sugar alcohol widely found in fruits and vegetables, but its content is extremely low, and it is uneconomical to extract directly from it. In 2012, the market size of xylitol reached about 540 million US dollars, based on 24,000 yuan / ton, the market demand was 150,000 tons. my country's annual output is less than 30,000 tons, and there is still a high demand for xylitol. [0003] Chemical catalytic hydrogenation is currently the main method for industrial production of xylitol, but the process re...

Claims

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Application Information

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IPC IPC(8): C12P19/02C12N1/21C12R1/19
CPCC12N9/0006C12P19/02C12Y101/01307C12Y101/9901
Inventor 应汉杰柳东常子月
Owner NANJING UNIV OF TECH
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