Method for recovering xylanase and xylanase recovery system thereof
By using a two-phase aqueous system composed of betaine-propylene glycol eutectic solvent and dipotassium hydrogen phosphate, the problem of xylanase recovery has been solved, achieving efficient and mild enzyme recovery, reducing costs while maintaining enzyme activity, and making it suitable for the industrial recycling of xylanase.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO UNIV OF SCI & TECH
- Filing Date
- 2026-04-22
- Publication Date
- 2026-05-29
AI Technical Summary
In existing xylanase hydrolysis systems, enzyme recovery is difficult and costly. Traditional methods are complex and result in significant loss of enzyme activity, hindering their industrial application.
A two-phase system consisting of a eutectic solvent (Bet:1,2-Prop) synthesized from betaine and 1,2-propanediol at a molar ratio of 1:3 and inorganic salt K2HPO4 was used to recover xylanase by static phase separation. Optimized conditions included temperature, time, and concentration.
It achieves a high recovery rate of xylanase (over 90%), maintains enzyme activity (over 80%), reduces production costs, simplifies operation procedures, and is suitable for industrial circular applications.
Smart Images
Figure CN122104642A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of enzyme recovery technology, and more specifically, to a method for recovering xylanase using a eutectic solvent / salt aqueous two-phase system and a dedicated xylanase recovery system thereof. Background Technology
[0002] Xylanase is a key enzyme in the hydrolysis of hemicellulose and plays a crucial role in the conversion of lignocellulose resources. With increasing emphasis on environmental protection and resource recycling, the application of xylanase in various industrial fields has garnered widespread attention. Especially in the efficient utilization of agricultural waste, the conversion of biomass energy, and the textile and food processing industries, xylanase has demonstrated enormous application potential. However, traditional hydrolysis systems for xylanase suffer from the drawback of difficult recycling, meaning the enzyme is used only once. The high cost of the enzyme also results in high production costs for the conversion of lignocellulose resources, severely hindering its industrial application.
[0003] Traditional enzyme recovery methods, such as dialysis, ultrafiltration, and electrophoresis, often suffer from problems such as complex operation, high cost, or loss of enzyme activity. Therefore, aqueous two-phase extraction (ATPS) has gradually gained attention as a novel enzyme recovery method. ATPS systems, by adding suitable salts or polymers to a solvent to form two immiscible phases, allow the separation of xylanase from the reaction system without destroying enzyme activity.
[0004] Eutectic solvents (DES), as a novel green solvent, have received widespread attention in recent years for their application in the pretreatment and enzymatic hydrolysis of lignocellulose. DES is a low-melting-point solvent system formed by hydrogen bond acceptors and donors through hydrogen bonding, offering advantages such as low toxicity, recyclability, low cost, and ease of design. Aqueous two-phase extraction systems based on green solvents combine the advantages of green chemistry and efficient separation. Their core advantage lies in using water as the primary medium; by introducing eutectic solvents, a two-phase system can be designed, fundamentally avoiding the use of traditional organic solvents and eliminating safety hazards such as environmental pollution and toxic residues. Simultaneously, this system provides a mild liquid-liquid extraction environment, effectively protecting the structure and function of biomolecules such as enzymes and significantly improving the yield of target products. Furthermore, the aqueous two-phase system exhibits strong tunability; by changing the solvent type, phase-forming salt, or pH value, the partition coefficient between the two phases can be flexibly controlled, achieving highly selective separation of specific components in complex mixtures. It is also easy to scale up and operate continuously, making the separation process more economical and environmentally friendly. Summary of the Invention
[0005] In view of the problems of difficulty in enzyme recovery and high cost in the existing xylanase hydrolysis system, the present invention provides a method and a dedicated recovery system for efficient and gentle recovery of xylanase.
[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for recovering xylanase, characterized by comprising the following steps: adding the enzyme solution containing xylanase to be recovered into a two-phase system composed of a eutectic solvent Bet:1,2-Prop(1:3) synthesized from betaine and 1,2-propanediol in a molar ratio of 1:3 and inorganic salt K2HPO4; mixing thoroughly and allowing the mixture to stand for phase separation; collecting the upper phase rich in the eutectic solvent to obtain the recovered xylanase.
[0007] The present invention also provides a xylanase recovery system, which is composed of a eutectic solvent Bet:1,2-Prop(1:3) and an inorganic salt K2HPO4, the system being used to carry out the above-described method.
[0008] According to the method or system of the present invention, the amounts of Bet:1,2-Prop(1:3) and K2HPO4 in the system are any ratio that allows the system to form a stable aqueous two-phase system; preferably, the final concentration of K2HPO4 in the system is 0.1–0.3 g / mL, and the final volume concentration of Bet:1,2-Prop(1:3) in the system is 30%–50% (v / v). According to the method of the present invention, the temperature for the static phase separation is 15-35°C, preferably 25°C; the static time is 20-60 minutes, preferably 40 minutes.
[0009] In a preferred embodiment of the present invention, the xylanase to be recovered is derived from a commercial enzyme or fermentation broth, and can be present in a simple enzyme solution or in a hydrolysis reaction solution containing substrate hydrolysis products. The method of the present invention allows for the direct addition of K₂HPO₄ and Bet:1,2-Prop (1:3) to the system after the hydrolysis reaction to the desired concentration, without the need for pre-separation of the enzyme solution, making the operation extremely simple.
[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to construct an aqueous two-phase system by combining a betaine-propylene glycol eutectic solvent (Bet:1,2-Prop(1:3)) with dipotassium hydrogen phosphate (K2HPO4) for the recovery of xylanase. This system exhibits excellent extraction selectivity for xylanase, with a recovery rate of over 90%.
[0011] 2. The recovery system provided by this invention has mild conditions (room temperature, no external force required), short extraction time (40 minutes), and can maximize the preservation of xylanase activity. After recovery, the enzyme activity can be retained by more than 80%, which is conducive to the recycling of enzymes and significantly reduces production costs.
[0012] 3. This invention solves the contradiction in existing aqueous two-phase systems where some eutectic solvents (such as choline chloride-based DES) can enhance enzyme activity but cannot effectively recover the enzyme, providing a practical and feasible technical solution for the industrial recycling of xylanase. Attached Figure Description
[0013] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the specific embodiments will be briefly described below: Figure 1 The figure shows the experimental results of the effects of different factors (K2HPO4 concentration, DES concentration, temperature, time, etc.) on the xylanase recovery rate in Example 7 of this invention. Detailed Implementation
[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the scope of protection of the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0015] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific embodiments.
[0016] There are no specific limitations on the xylanase used in this disclosure. Self-made xylanase or commercially available xylanase can be used. The xylanase used in the embodiments of this disclosure is a commercial enzyme, purchased from Shanghai Aladdin (China) Biotechnology Co., Ltd. Other chemical reagents were all commercially available analytical grade. Betaine (Bet), choline chloride (ChCl), 1,2-propanediol (1,2-Prop), 1,3-propanediol (1,3-Prop), 1,4-butanediol (1,4-Bu), and dipotassium hydrogen phosphate (K2HPO4) were all purchased from Sinopharm Chemical Reagent Co., Ltd.
[0017] Example 1: Preparation of a two-phase aqueous phase diagram A certain amount of DES was weighed and added together with the prepared salt solution into a graduated glass bottle. Under constant temperature heating, the mixture was magnetically stirred at 500 r / min for 10 min to ensure thorough mixing of the two phases. Afterward, it was allowed to stand for 40 min to form a stable system. If the system separated into two clear and transparent phases, phase formation was successful. A phase diagram was then drawn, and the phases were determined using turbidity point titration. The phase formation range of the DES system was determined using the phase diagram.
[0018] Example 2: Preparation of xylanase hydrolysis system Betaine (Bet) and 1,2-propanediol (1,2-Prop) were mixed in molar ratios of 1:1, 1:2, 1:3, 1:4, and 1:5, and heated at 80°C with continuous stirring until a colorless, transparent liquid was formed, thus preparing the corresponding eutectic solvents. These eutectic solvents were then prepared into eutectic solvent / acetic acid-sodium acetate aqueous solutions with a volume concentration of 10% in a pH 5.5 acetate-sodium acetate buffer solution. The Bet:1,2-Prop / acetic acid-sodium acetate aqueous solutions at molar ratios of 1:3, 1:4, and 1:5 were stable hydrolysis systems at room temperature. The activity of xylanase in the four systems was determined using the DNS method. The enzyme activity in the Bet:1,2-Prop (1:3) / acetic acid-sodium acetate aqueous solution was 20% higher than that in the conventional buffer solution hydrolysis system without DES. Therefore, this disclosure selects the Bet:1,2-Prop(1:3) / acetic acid-sodium acetate aqueous solution system, which has a better effect on xylanase hydrolysis, as the hydrolysis system.
[0019] Example 3: Preparation of a xylanase recovery system The eutectic solvent Bet:1,2-Prop (1:3) was prepared using the same method as in Example 2. Appropriate amounts of Bet:1,2-Prop (1:3), xylanase, and a certain concentration of K₂HPO₄ solution were mixed and stirred at 500 rpm until fully mixed. The mixture was then allowed to stand for 40 min, and the volumes of the upper and lower phases were recorded. After recovery and separation, the xylanase content in the eutectic solvent-enriched phase and the inorganic salt-enriched phase was determined using the Coomassie Brilliant Blue method. The results showed that the xylanase extraction rate could reach 85%. The above-mentioned eutectic solvent aqueous two-phase system is simple to operate and has a short extraction time; xylanase can be extracted and separated in 40 min, successfully recovering xylanase.
[0020] Comparative Example 1: Effect of different eutectic solvent / salt aqueous two-phase systems on xylanase recovery Choline chloride (ChCl) and 1,2-propanediol (1,2-Prop) were mixed in molar ratios of 1:1, 1:2, 1:3, 1:4, and 1:5, and heated at 80°C with continuous stirring until a colorless and transparent liquid was formed, thus preparing the corresponding eutectic solvents. These eutectic solvents were then prepared into eutectic solvent / acetic acid-sodium acetate aqueous solutions with a volume concentration of 10% in a pH 5.5 acetate-sodium acetate buffer solution. The ChCl:1,2-Prop / acetic acid-sodium acetate aqueous solutions with molar ratios of 1:2, 1:3, 1:4, and 1:5 were stable hydrolysis systems at room temperature. The activity of xylanase in the four systems was determined using the DNS method. The enzyme activity in the ChCl:1,2-Prop (1:4) / acetic acid-sodium acetate aqueous solution was 23% higher than that in the conventional buffer solution hydrolysis system without DES. Therefore, this disclosure selects the ChCl:1,2-Prop(1:4) / acetic acid-sodium acetate aqueous solution system, which has a better effect on xylanase hydrolysis, as the hydrolysis system.
[0021] Then, appropriate amounts of ChCl / 1,2-Prop (1:4), xylanase, and a certain concentration of K₂HPO₄ solution were mixed and stirred at 500 rpm until fully mixed. The mixture was then allowed to stand for 40 min, and the volumes of the upper and lower phases were recorded. After recovery and separation, the xylanase content in the eutectic solvent-enriched phase and the inorganic salt-enriched phase solutions was determined using the Coomassie Brilliant Blue method. The results showed that the xylanase extraction rate was only 61.3%.
[0022] Comparative Example 2: Effect of different eutectic solvent / salt aqueous two-phase systems on xylanase recovery Choline chloride (ChCl) and 1,3-propanediol (1,3-Prop) were mixed in molar ratios of 1:1, 1:2, 1:3, 1:4, and 1:5, and heated at 80°C with continuous stirring until a colorless and transparent liquid was formed, thus preparing the corresponding eutectic solvents. These eutectic solvents were then prepared into eutectic solvent / acetic acid-sodium acetate aqueous solutions with a volume concentration of 10% in a pH 5.5 acetate-sodium acetate buffer solution. The ChCl:1,3-Prop / acetic acid-sodium acetate aqueous solutions with molar ratios of 1:2, 1:3, 1:4, and 1:5 were stable hydrolysis systems at room temperature. The activity of xylanase in the four systems was determined using the DNS method. The enzyme activity in the ChCl:1,3-Prop (1:5) / acetic acid-sodium acetate aqueous solution was 14.3% higher than that in the conventional buffer solution hydrolysis system without DES. Therefore, this disclosure selects the ChCl:1,3-Prop(1:5) / acetic acid-sodium acetate aqueous solution system, which has a better effect on xylanase hydrolysis, as the hydrolysis system.
[0023] Then, appropriate amounts of ChCl:1,3-Prop (1:5), xylanase, and a certain concentration of K2HPO4 solution were mixed. The mixture was stirred at 500 rpm until fully combined, then allowed to stand for 40 min, and the volumes of the upper and lower phases were recorded. After recovery and separation, the xylanase content in the eutectic solvent-enriched phase and the inorganic salt-enriched phase solutions was determined using the Coomassie Brilliant Blue method. The results showed that the xylanase extraction rate was only 36%.
[0024] Preparation of xylanase hydrolysis system in Comparative Example 3 Choline chloride (ChCl) and 1,4-butanediol (1,4-Bu) were mixed in molar ratios of 1:1, 1:2, 1:3, 1:4, and 1:5, and heated at 80°C with continuous stirring until a colorless and transparent liquid was formed, thus preparing the corresponding eutectic solvents. These eutectic solvents were then prepared into eutectic solvent / acetic acid-sodium acetate aqueous solutions with a volume concentration of 10% in a pH 5.5 acetate-sodium acetate buffer solution. The ChCl:1,4-Bu / acetic acid-sodium acetate aqueous solutions with molar ratios of 1:2, 1:3, 1:4, and 1:5 were stable hydrolysis systems at room temperature. The activity of xylanase in the four systems was determined using the DNS method. The enzyme activity in the ChCl:1,4-Bu (1:4) / acetic acid-sodium acetate aqueous solution was 22.5% higher than that in the conventional buffer solution hydrolysis system without the addition of DES. Therefore, this disclosure selects the ChCl:1,4-Bu(1:4) / acetic acid-sodium acetate aqueous solution system, which has a better effect on xylanase hydrolysis, as the hydrolysis system.
[0025] Then, appropriate amounts of ChCl:1,4-Bu (1:4), xylanase, and a certain concentration of K₂HPO₄ solution were mixed and stirred at 500 rpm until fully mixed. The mixture was then allowed to stand for 40 min, and the volumes of the upper and lower phases were recorded. After recovery and separation, the xylanase content in the eutectic solvent-enriched phase and the inorganic salt-enriched phase solutions was determined using the Coomassie Brilliant Blue method. The results showed that the xylanase extraction rate was only 76.2%.
[0026] Although the hydrolysis systems ChCl:1,2-Prop(1:4) / AA-SA, ChCl:1,3-Prop(1:5) / AA-SA, and ChCl:1,4-Bu(1:4) / AA-SA can promote the activity and stability of xylanase, they are not effective as xylanase recovery systems.
[0027] Example 4: Determination of the optimal xylanase recovery system The effects of K₂HPO₄ addition, eutectic solvent addition, xylanase addition, recovery time, and temperature on xylanase recovery efficiency were investigated to screen the optimal aqueous two-phase recovery system. Appropriate amounts of DES solution, inorganic salt solution, and enzyme solution were mixed thoroughly and allowed to stand for 40 minutes. The volumes of the upper and lower phases were recorded separately. The xylanase protein content in the eutectic solvent-enriched phase and the inorganic salt-enriched phase solutions was then determined using the Coomassie Brilliant Blue method. The experimental results are shown in the appendix. Figure 1 The results showed that the extraction rate of xylanase could reach 95% under the optimal hydrolysis conditions. The figure also showed that the optimal aqueous phase recovery extraction system was: K2HPO4 concentration 0.2 g / mL, DES content 40% (v / v), temperature 25℃, and recovery time 40 min. The above recovery system achieved good recovery effect on xylanase.
[0028] Example 5: Recovery of xylanase A Bet:1,2-Prop(1:3) / acetic acid-sodium acetate hydrolysis system was prepared using the same method as in Example 2. Xylanase was added to the system, and the supernatant of acid-pretreated corn cobs was hydrolyzed at 50°C and 150 rpm for 24 h. The amount of xylanase used was 10 U / g, and the mass concentration of the pretreated corn cobs was 2% (w / v). The yield of reducing sugars in the enzyme hydrolysate was 81%.
[0029] A certain amount of eutectic solvent Bet:1,2-Prop (1:3) and K2HPO4 were added to the above enzymatic hydrolysate. After stirring to ensure thorough mixing, the molar concentration of K2HPO4 in the xylanase recovery system reached 0.2 g / mL, and the volume concentration of the eutectic solvent Bet:1,2-Prop (1:3) reached 40% (v / v). The xylanase was recovered by standing at 25℃ for 40 min. The protein content in the final recovered eutectic solvent-enriched phase was 0.42 mg / mL, and the xylanase extraction rate was as high as 92.1%. The activity of the recovered xylanase was measured, and it remained at 87.3% compared to before recovery.
[0030] Please note that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above embodiments only illustrate several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent.
Claims
1. A method for recovering xylanase, characterized in that, Includes the following steps: (1) Prepare a eutectic solvent by mixing hydrogen bond acceptor and hydrogen bond donor and heating; (2) A two-phase aqueous system was constructed by mixing a eutectic solvent, an inorganic salt, and water; (3) Add the enzyme solution containing xylanase to the aqueous two-phase system for extraction and separation.
2. The method as described in claim 1, characterized in that, The preparation method of the eutectic solvent Bet:1,2-Prop(1:3) in the xylan recovery system is as follows: 1,2-propanediol and betaine Bet are mixed in a molar ratio of 1:3 and heated and stirred at 80°C until a clear and transparent liquid is formed, which is stable at room temperature.
3. The method as described in claim 1, characterized in that, Xylanase hydrolyzes the substrate to be hydrolyzed at 50°C for 24 hours.
4. The method as described in claim 1 or 3, characterized in that, The hydrolysis system is a Bet:1,2-Prop(1:3) / acetic acid-sodium acetate hydrolysis system.
5. The method as described in claim 4, characterized in that, The volume concentration of the eutectic solvent in the Bet:1,2-Prop(1:3) / acetic acid-sodium acetate hydrolysis system is 10%.
6. The method as described in claim 1 or 3, characterized in that, The xylanase has an enzyme activity of 10 U / g and the mass concentration of the substrate to be hydrolyzed is 2% w / v.