阅读说明：本技术 一种利用pH自平衡催化体系制备木糖醇的方法 (Method for preparing xylitol by utilizing pH self-balancing catalytic system ) 是由 应汉杰 柳东 张迪 王振宇 雷鸣 陈勇 牛欢青 于 2019-11-05 设计创作，主要内容包括：本发明公开了一种利用pH自平衡催化体系制备木糖醇的方法,其特征在于,木糖、葡萄糖、甲酸类化合物、木糖还原酶、葡萄糖脱氢酶、甲酸脱氢酶、电子载体和水形成生物催化体系,催化木糖反应生成木糖醇。与现有技术相比,本发明通过耦合反应过程,实现反应体系中酸性和碱性产物的相互中和,能够稳定反应过程的pH中和,省去了生产过程pH的调控,因此反应过程更简单、稳定、高效,可以在纯水中进行,从而提高了生产效率、降低了生产成本和操作难度；同时,采用由甲酸脱氢酶、葡萄糖脱氢酶和木糖还原酶构成的酶偶合反应系统催化生产木糖醇,24小时内从2M木糖中获得了278.4g/L的木糖醇产量,产率高达11.6g/L/h。(The invention discloses a method for preparing xylitol by utilizing a pH self-balancing catalytic system, which is characterized in that xylose, glucose, formic acid compounds, xylose reductase, glucose dehydrogenase, formate dehydrogenase, an electronic carrier and water form a biological catalytic system to catalyze xylose to react to generate xylitol. Compared with the prior art, the method realizes mutual neutralization of acidic and alkaline products in a reaction system through a coupling reaction process, can stabilize pH neutralization in the reaction process, and omits regulation and control of pH in the production process, so that the reaction process is simpler, more stable and more efficient, and can be carried out in pure water, thereby improving production efficiency and reducing production cost and operation difficulty; meanwhile, an enzyme coupling reaction system consisting of formate dehydrogenase, glucose dehydrogenase and xylose reductase is adopted to catalyze and produce the xylitol, the yield of the xylitol of 278.4g/L is obtained from 2M xylose within 24 hours, and the yield reaches 11.6 g/L/h.)

1. A method for preparing xylitol by utilizing a pH self-balancing catalytic system is characterized in that xylose, glucose, formate compounds, xylose reductase, glucose dehydrogenase, formate dehydrogenase, an electronic carrier and water form a biological catalytic system to catalyze xylose to react to generate xylitol.

2. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein the initial concentration of xylose in the biocatalytic system is 0.1-3M.

3. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein the initial concentration of glucose in the biocatalytic system is 0.1-2M.

4. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein the formic acid compound is formic acid or sodium formate; the initial concentration of the formic acid compound in the biological catalysis system is 0.1-3M.

5. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein the dosage of xylose reductase in the biological catalytic system is 3-6U/mL.

6. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein the dosage of glucose dehydrogenase in the biocatalytic system is 0.5-2.5U/mL.

7. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein the xylose reductase and the glucose dehydrogenase are expressed by recombinant Escherichia coli, and the steps of expression and extraction of the xylose reductase and the glucose dehydrogenase are as follows:

(1) respectively carrying out codon optimization on a xylose reductase gene and a glucose dehydrogenase gene, wherein the nucleotide sequences are respectively shown as SEQ ID NO 1 and SEQ ID NO 2;

(2) subcloning the optimized xylose reductase gene and glucose dehydrogenase gene obtained in the step (1) to plasmid pET-28a respectively to obtain recombinant plasmids;

(3) introducing the recombinant plasmid obtained in the step (2) into E.coli.BL21 to obtain recombinant escherichia coli;

(4) inoculating the recombinant escherichia coli obtained in the step (3) into an LB liquid culture medium containing kanamycin to activate;

(5) transferring the activated recombinant escherichia coli obtained in the step (4) to an LB liquid culture medium containing kanamycin for continuous culture, and when OD600 reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) into the LB liquid culture medium to induce the expression of xylose reductase and glucose dehydrogenase, wherein the concentration of the IPTG in the LB liquid culture medium is 0.8-1.2 mM;

(6) and (5) collecting the thalli obtained in the step (5), carrying out ultrasonic crushing, and then carrying out centrifugation, wherein the supernatant is the crude enzyme solution.

8. The method for preparing xylitol by utilizing a pH self-balancing catalytic system as claimed in claim 1, wherein the dosage of formate dehydrogenase in the biocatalytic system is 0.5-2.5U/mL.

9. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein said electron carrier is NAD⁺、NADP⁺The initial concentration of the electronic carrier in the biological catalysis system is 0.5-10 mM.

10. The method for preparing xylitol by utilizing a pH self-balancing catalytic system according to claim 1, wherein the reaction temperature is 20-40 ℃ and the reaction time is 2-72 h.

Technical Field

The invention relates to a method for preparing xylitol, in particular to a method for preparing xylitol by utilizing a pH self-balancing catalytic system.

Background

Xylitol is an important functional sugar alcohol, widely used in food, pharmaceutical and chemical industries, and listed as one of twelve preferentially developed and utilized platform compounds by the renewable resources office of the U.S. department of energy. In recent years, the global demand for xylitol is steadily increasing, and the efficient production of xylitol draws great attention.

Currently, xylitol is produced primarily by hydrogenating highly purified xylose under stringent conditions. The chemical synthesis method has the defects of high energy consumption, requirements on complex equipment, environmental pollution risks and the like. Compared to chemical processes, enzymatic processes are milder and cleaner, while xylitol can still be selectively obtained. Thus, the enzymatic production of xylitol is an attractive alternative to chemical synthesis.

In the enzymatic process, Xylose Reductase (XR) is the key enzyme for the one-step reduction of xylose to xylitol using the cofactor nad (p) H as reducing capability, whereas nad (p) H has to be regenerated by coupling the reactions catalyzed by dehydrogenases. Previously, a process with a coupled XR and NADPH dependent Glucose Dehydrogenase (GDH) and a process with a coupled XR and NADH dependent Formate Dehydrogenase (FDH) were tried for the enzymatic production of xylitol. FDH-catalyzed oxidation of formate compounds produces hydroxides or bicarbonates resulting in alkaline pH changes in the reaction solution, while GDH-catalyzed oxidation of glucose produces gluconic acid resulting in acidic pH changes. Thus, in both methods, production must be carried out in buffer solution, or complex pH control strategies at relatively low production rates. Therefore, it is necessary to develop a reaction process for preparing xylitol with stable pH so as to simplify the process for preparing xylitol by a biological method and reduce the production cost.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a method for preparing xylitol by utilizing a pH self-balancing catalytic system aiming at the defects of the prior art.

The invention idea is as follows: since the base produced by FDH and the gluconic acid produced by GDH neutralize each other, the process can be simply carried out in pure water without pH adjustment, and the pH neutralization process is highly efficient for the enzymatic production of xylitol, a reaction process consisting of coupled XR-FDH-GDH was developed for the enzymatic production of xylitol.

In order to solve the technical problem, the invention discloses a method for preparing xylitol by utilizing a pH self-balancing catalytic system, wherein xylose, glucose, formic acid compounds, Xylose Reductase (XR), Glucose Dehydrogenase (GDH), Formate Dehydrogenase (FDH), an electronic carrier and water form a biological catalytic system to catalyze xylose to react to generate xylitol.

Glucose is catalyzed by GDH to generate an acidic product gluconic acid, and the process provides electrons to an electron carrier; formic acid or sodium formate generates CO gas under catalysis of FDH₂And the alkaline product hydroxide, which is also capable of providing electrons to the electron carrier. Therefore, products of the glucose dehydrogenase and the formate dehydrogenase can be mutually neutralized in the reaction, so that the pH of a reaction system is stabilized, and electrons provided by the glucose and the formate compounds can be transferred to xylose under the catalytic action of xylose reductase through an electron carrier to further generate the xylitol. FIG. 1 further illustrates the process of pH self-balancing of the reaction system of the present invention.

Wherein the initial concentration of xylose in the biocatalysis system is 0.1-3M.

Wherein the glucose dehydrogenase is derived from Bacillus cereus, Exiguobacterium sibiricum 255-15, Thermoplasma acidophilum DSM 1728 and Bacillus subtilis; the initial concentration of glucose in the biological catalysis system is 0.1-2M; preferably 1M.

Wherein, the formic acid compound is formic acid or sodium formate; the initial concentration of the formic acid compound in the biological catalysis system is 0.1-3M.

Wherein said xylose reductase is derived from Candida tenuis CBS 4435, Rhizopus oryzae, Candida tropicalis IFO 0618 or Thermomyces lanuginosus SSBP; the dosage of the xylose reductase in the biological catalysis system is 3-6U/mL; preferably 5U/mL; wherein, the enzyme activity of the xylose reducing sugar is defined as follows: the amount of enzyme required to consume 1mM NADH per minute at 25 ℃ at pH 7.

Wherein the dosage of the glucose dehydrogenase in the biological catalysis system is 0.5-2.5U/mL; preferably 1.5U/mL; wherein, the enzyme activity of the glucose dehydrogenase is defined as follows: detecting the enzyme activity at 340nm by using an ultraviolet spectrophotometer under the following detection conditions: the reaction was carried out at 25 ℃ in 100mM tris-HCl buffer pH7, and the enzyme activity was expressed as the amount of NADH produced in 1 minute. Glucose dehydrogenase was purchased from Amono and the enzyme was extracted from Bacillus cereus.

The xylose reductase and the glucose dehydrogenase are expressed by recombinant escherichia coli, and the expression and extraction steps of the xylose reductase and the glucose dehydrogenase are as follows:

(1) respectively carrying out codon optimization on the xylose reductase gene and the glucose dehydrogenase gene;

(2) subcloning the optimized xylose reductase gene and glucose dehydrogenase gene obtained in the step (1) to plasmid pET-28a respectively to obtain recombinant plasmids;

(3) introducing the recombinant plasmid obtained in the step (2) into E.coli.BL21 to obtain recombinant escherichia coli;

(4) inoculating the recombinant escherichia coli obtained in the step (3) into an LB liquid culture medium containing kanamycin to activate;

(5) transferring the activated recombinant escherichia coli obtained in the step (4) to an LB liquid culture medium containing kanamycin for continuous culture, and when OD600 reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) into the LB liquid culture medium to induce the expression of xylose reductase and glucose dehydrogenase, wherein the concentration of the IPTG in the LB liquid culture medium is 0.8-1.2 mM;

(6) and (5) collecting the thalli obtained in the step (5), carrying out ultrasonic crushing, and then carrying out centrifugation, wherein the supernatant is the crude enzyme solution.

In step (1), the codon optimization steps are as follows: analysis of rare codons: rare codons were mainly analyzed by the software e.coli Codon Usage Analysis 2.0 to obtain codons used with relative frequency below the threshold (10 ‰) in e. Rare codons were optimized: codons with a frequency of usage below the threshold are optimized according to the frequency of usage of the different codons in e. The xylose reductase gene and formate dehydrogenase gene sequences are optimized according to the codon preference of escherichia coli, and the optimized nucleotide sequences are respectively shown as SEQ ID NO 1 and SEQ ID NO 2.

In the step (2), the optimized xylose reductase gene and formate dehydrogenase gene are subcloned between EcoRI and HindIII enzyme cutting sites of plasmid pET-28a respectively to form recombinant plasmids.

And (5) adding IPTG into the LB liquid culture medium in the step (5), and culturing at 20 ℃ and 200rpm for 12h to perform induced expression of xylose reductase and formate dehydrogenase.

Wherein the formate dehydrogenase is derived from Candida Boidinii, Pichia pastoris GS115, Clostridium ljungdahlii, Desulfovibrio desuifuricans ATCC 27774; the dosage of the formate dehydrogenase in the biological catalysis system is 0.5-2.5U/mL; preferably 1.5U/mL; wherein, the enzyme activity of the formate dehydrogenase is defined as follows: the amount of enzyme required to produce 1mM NADH per minute at 25 ℃ at pH 7.

Wherein the electron carrier is NAD⁺、NADP⁺FAD, FMN and methyl viologen, preferably NAD⁺(ii) a The initial concentration of the electron carrier in the biological catalysis system is 0.5-10 mM.

FIG. 1 further illustrates the process of pH self-balancing the reaction system of the present invention, XR reduces xylose to xylitol, where NADH is consumed, NADH is regenerated in the reaction catalyzed by FDH and GDH, the alkaline product of FDH from sodium formate and the acidic product of GDH from glucose neutralize each other, and thus the process can be simply carried out in pure water.

Wherein the reaction temperature is 20-40 ℃, and the reaction time is 2-72 h.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) according to the invention, through the coupling reaction process, mutual neutralization of acidic and alkaline products in the reaction system is realized, pH neutralization in the reaction process can be stabilized, and regulation and control of pH in the production process are omitted, so that the reaction process is simpler, more stable and more efficient, and can be carried out in pure water, thereby improving the production efficiency, and reducing the production cost and the operation difficulty.

(2) The invention adopts an enzyme coupling reaction system consisting of formate dehydrogenase, glucose dehydrogenase and xylose reductase to catalyze and produce the xylitol, obtains the xylitol yield of 278.4g/L from 2M xylose within 24 hours, and the yield reaches 11.6 g/L/h.

Drawings

FIG. 1 is a schematic diagram of the reaction system of the present invention showing pH self-equilibrium.

FIG. 2 is a map of recombinant plasmid pET-28 a-teXR.

FIG. 3 is a map of recombinant plasmid pET-28 a-FDH.

FIG. 4 is an electrophoresis diagram of xylose reductase and formate dehydrogenase expressed by recombinant E.coli.

FIG. 5 is a graph showing the change of xylitol concentration and pH with time in comparative example 1.

FIG. 6 is a graph showing the change of xylitol concentration and pH with time in comparative example 2.

FIG. 7 is a graph showing the time-course changes of the substrate concentration and the xylitol concentration and pH in example 3.

FIG. 8 is a graph of glucose and xylitol concentrations over time for reactions catalyzed by three different types of cofactors in example 4.

FIG. 9 is a graph showing the effect of different enzyme dosages on glucose and xylitol concentrations in example 5.

FIG. 10 is a graph showing the effect of xylose concentration on glucose and xylitol concentration in example 6.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.