阅读说明：本技术 一种聚合物固定化木聚糖酶及其制备方法和应用 (Polymer immobilized xylanase, preparation method and application thereof ) 是由 王俊 胡艳 石灿阳 黄博荣 游帅 盛晟 吴福安 于 2020-12-10 设计创作，主要内容包括：一种聚合物固定化木聚糖酶及其制备方法和应用,将水溶性引发剂加入木聚糖酶缓冲液中,低温条件下使引发剂的羧基与木聚糖酶表面的氨基酸残基共价反应获得木聚糖酶大分子引发剂,温敏性聚合物的单体在催化剂的作用下在木聚糖酶大分子引发剂主链上聚合,获得聚合物固定化木聚糖酶。将聚合物固定化酶与纤维素酶协同水解桑皮、玉米芯、甘蔗渣、麸皮等农业废弃物获得低聚木糖。本发明选用具有良好生物相容性的聚合物,确保固定化酶维持较高的活力,同时温敏性聚合物增加了固定化酶的重复利用率。本发明可以解决农业废弃物资源利用的问题,有望产生良好的经济效应。(A polymer immobilized xylanase and a preparation method and application thereof are disclosed, wherein a water-soluble initiator is added into a xylanase buffer solution, carboxyl of the initiator and amino acid residues on the surface of the xylanase are subjected to covalent reaction at low temperature to obtain a xylanase macro-initiator, and monomers of a temperature-sensitive polymer are polymerized on the main chain of the xylanase macro-initiator under the action of a catalyst to obtain the polymer immobilized xylanase. And (3) carrying out synergistic hydrolysis on the polymer immobilized enzyme and cellulase on agricultural wastes such as mulberry bark, corncob, bagasse, bran and the like to obtain the xylo-oligosaccharide. The invention selects the polymer with good biocompatibility, ensures that the immobilized enzyme maintains higher activity, and simultaneously increases the repeated utilization rate of the immobilized enzyme by the temperature-sensitive polymer. The invention can solve the problem of resource utilization of agricultural wastes and is expected to produce good economic effect.)

1. A preparation method of polymer immobilized xylanase is characterized by comprising the following specific steps: dissolving an initiator in a xylanase buffer solution with the pH value of 8.0-9.0, wherein the initiator is N-2 bromo-2-methylpropionyl-beta-alanine N' -oxysuccinimide ester, the molar ratio of the initiator to amino groups on xylanase is (3-10): 1, and stirring and reacting at the temperature of 0-8 ℃ to obtain a xylanase macro-initiator; simultaneously blowing the water solution containing the xylanase macroinitiator and the polymer monomer with a dissolved catalyst 1 and a catalyst 2 by using nitrogen to deoxidize in an ice bath, and transferring the solution containing the xylanase macroinitiator and the polymer monomer into the water solution of the dissolved catalyst, wherein the xylanase macroinitiator comprises the following components in percentage by weight: polymer monomer (b): catalyst 1: the molar ratio of the catalyst 2 is 1 (50-500) to 1.3:1.9, and the polymer immobilized xylanase solution is obtained by polymerizing for 5-30 hours at the temperature of 0-8 ℃, wherein the catalyst 1 is as follows: CuBr, CuCl or CuBr₂The catalyst 2 is: me₆TREN, HMTETA, TPMA or bpy, dialyzing to remove unreacted monomers, and drying the solution under reduced pressure to obtain immobilized xylanase solid powder.

2. The method for producing a polymer-immobilized xylanase according to claim 1, characterized in that: the mol ratio of the initiator to the amino groups on the xylanase protein in the step (1) is 4: 1, the concentration of the buffer solution is 0.1mol/L, the pH value is 8.5, and the reaction temperature is 4 ℃.

3. The method for producing a polymer-immobilized xylanase according to claim 1, characterized in that: the polymer monomers in the step (1) are as follows: n-isopropylacrylamide (NIPAAm), dimethylaminoethyl acrylate (DMAEA) or 3-Dimethylaminopropylamine (DMAPA).

4. A polymer-immobilized xylanase prepared according to the process of any one of claims 1-3.

5. The use of claim 5, wherein: immersing agricultural wastes in 0.1-15 wt.% alkaline solution, treating at 60-90 ℃ for 12-36 h, adjusting the pH to be neutral, and drying; mixing the pretreated agricultural waste with a buffer solution according to a solid-to-liquid ratio of 1 (2-15), wherein the unit g/mL and the pH value of the buffer solution are 3.0-9.0, adding immobilized xylanase or a composition of the immobilized xylanase and cellulase, and performing synergistic hydrolysis to obtain xylooligosaccharide, wherein the ratio of the enzyme activity units of the immobilized xylanase and the cellulase is as follows: 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9: 1; and (3) centrifuging the hydrolyzed solution, taking supernatant, raising the temperature to 50-60 ℃ to make the immobilized enzyme hydrophobically separated out, and collecting the immobilized enzyme.

6. Use according to claim 6, characterized in that: the alkaline solution is sodium hydroxide, calcium hydroxide or ammonia water.

7. Use according to claim 6, characterized in that: the agricultural waste is mulberry bark, mulberry twigs, corncobs, corn stalks, bagasse or bran.

8. Use according to claim 6, characterized in that: the pH value of the buffer solution is 5.0, and the agricultural wastes and the buffer solution are mixed according to the solid-to-liquid ratio of 1:15, wherein the unit is g/mL.

9. Use according to claim 6, characterized in that: the immobilized enzyme was collected at 55 ℃.

Technical Field

The invention belongs to the field of immobilized enzymes, and particularly relates to a polymer immobilized xylanase, and a preparation method and application thereof.

Background

Xylo-oligosaccharide, also called xylo-oligosaccharide, is a degradation product of xylan, is a heterogeneous polysaccharide formed by connecting 2-7 xylose molecules through beta-1, 4-glycosidic bonds, and is an important member in a functional oligosaccharide family. Xylo-oligosaccharide, especially xylo-disaccharide and xylo-trisaccharide, has the effect of proliferating bifidobacterium which is 20 times of that of other functional saccharides, and can effectively stimulate intestinal peristalsis and promote calcium absorption (Green Chemistry,2019,21: 3213-3231). Meanwhile, xylo-oligosaccharide has toxic action on human leukemia cells, is beneficial to type II diabetes, and can be used as an antioxidant (Bioresource Technology,2018,249: 395-. Therefore, the xylo-oligosaccharide is considered as a novel prebiotic and has wide application prospect in the fields of food, feed and the like.

At present, xylo-oligosaccharide is mainly prepared by degrading xylan, and the common methods are as follows: hydrothermal extraction, acid extraction and enzymolysis extraction. Because acid-resistant and corrosion-resistant equipment is needed in the acid extraction process, and carcinogenic toxic substances may be contained in the generated by-products, the investment cost for extracting xylooligosaccharide by the acid method is high, the technical requirement is high, and the method is not suitable for large-scale industrial production. And the hydrothermal method needs high-temperature resistant equipment for extraction, and the produced xylo-oligosaccharide has dark color, which increases difficulty for subsequent treatment, so the application in industrial production is less. The enzymolysis technology of xylanase is widely accepted in the production of xylooligosaccharide. Xylanases are a type of enzyme system that degrades xylan by hydrolyzing xylose molecules beta-1, 4-glycosidic bonds to hydrolyze xylan into xylooligosaccharides, including xylooligosaccharides such as xylobiose and xylotriose, and small amounts of arabinose (Carbohydrate Polymers,2019,207: 34-43). However, the free xylanase has some problems in practical application, such as poor stability, low recycling rate and the like, and the use cost is high. Therefore, in the application research of xylanase, the research of immobilized enzyme is an emerging technology in the field of biotechnology. Immobilization of enzymes is a technique in which free enzymes are physically or chemically blocked in a solid material or confined to a certain area for active, specific catalysis, and can be recovered for long-term use. Compared with free enzyme, the immobilized enzyme not only can maintain high-efficiency catalytic property, but also has high storage stability and can be repeatedly used. The series of advantages lead the immobilized enzyme to be developed rapidly in the fields of biological engineering, chemistry, medicine and the like and meet the strategic requirements of sustainable development.

At present, how to improve the stability of xylanase, improve the recovery rate of enzyme activity and find a better immobilization method and a carrier is the trend and direction of xylanase immobilization research. Invention CN 106434620A immobilization xylanase on 3-aminopropyl triethoxy silane modified Fe₃O₄@SiO₂The temperature tolerance range of the xylanase is improved on the surface of the nano-particle carrier. The invention CN 107012137A prepares the sodium alginate-chitosan immobilized xylanase, and effectively improves the temperature stability and the pH stability of the xylanase. It has also been reported that xylanase grafted onto magnetic chitosan has increased thermal stability and pH stability compared to the free enzyme and can be reused three times (Applied Biochemistry and Biotechnology,2(188): 395) 409). In the existing xylanase immobilization technology, a lot of reports of immobilized xylanase with a carrier exist, but no report of preparation of immobilized xylanase without a carrier exists.

Polymer immobilized enzymes are an active research area that has developed rapidly in the last decade. The attachment of polymers allows the preparation of proteins for specific applications and confers properties which cannot be provided by themselves (Biomaterials,2016,107: 115-. The influence of covalently linked polymer chains on enzymes ranges from increased solubility, enhanced biocompatibility and stability to modulated enzymatic activity. Polymer immobilized lipases have been reported to be prepared using N-isopropylacrylamide, and only 10% of the activity of the lipase is lost after 6 days of stabilization in organic solvents (Physical Chemistry Chemical Physics,2012,27(14): 9594-. It has also been reported that acrylamide is selected to prepare polymer immobilized horseradish peroxidase, which has improved 90% stability at 55 deg.C (Acta biomaterials, 2011,5(7): 2131-. Therefore, polymers with good biocompatibility have become the most important representative of the carrier-free immobilized enzyme technology, and have become the focus of research. The research selects polymers with temperature sensitivity to prepare polymer immobilized xylanase, and the polymer immobilized xylanase is applied to degrading agricultural wastes so as to improve the resource utilization rate.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a polymer immobilized xylanase, a preparation method and application thereof, and aims to solve the problems of poor stability, easy inactivation, difficult diffusion, low reusability and the like of free xylanase in practical application.

The technical scheme is as follows: a preparation method of polymer immobilized xylanase comprises the following specific steps: dissolving an initiator in a xylanase buffer solution with the pH value of 8.0-9.0, wherein the initiator is N-2 bromo-2-methylpropionyl-beta-alanine N' -oxysuccinimide ester, the molar ratio of the initiator to amino groups on xylanase is (3-10): 1, and stirring and reacting at the temperature of 0-8 ℃ to obtain a xylanase macro-initiator; simultaneously blowing the water solution containing the xylanase macroinitiator and the polymer monomer with a dissolved catalyst 1 and a catalyst 2 by using nitrogen to deoxidize in an ice bath, and transferring the solution containing the xylanase macroinitiator and the polymer monomer into the water solution of the dissolved catalyst, wherein the xylanase macroinitiator comprises the following components in percentage by weight: polymer monomer (b): catalyst 1: catalyst 2 molar ratio of 11.3:1.9, and polymerizing for 5-30 hours at 0-8 ℃ to obtain a polymer immobilized xylanase solution, wherein the catalyst 1 is as follows: CuBr, CuCl or CuBr₂The catalyst 2 is: me₆TREN, HMTETA, TPMA or bpy, dialyzing to remove unreacted monomers, and drying the solution under reduced pressure to obtain immobilized xylanase solid powder.

The mol ratio of the initiator to the amino groups on the xylanase protein in the step (1) is 4: 1, the concentration of the buffer solution is 0.1mol/L, the pH value is 8.5, and the reaction temperature is 4 ℃.

The polymer monomer in the step (1) is: n-isopropylacrylamide (NIPAAm), dimethylaminoethyl acrylate (DMAEA) or 3-Dimethylaminopropylamine (DMAPA).

The polymer immobilized xylanase prepared by the method.

The specific application steps are as follows: immersing agricultural wastes in 0.1-15 wt.% alkaline solution, treating at 60-90 ℃ for 12-36 h, adjusting the pH to be neutral, and drying; mixing the pretreated agricultural waste with a buffer solution according to a solid-to-liquid ratio of 1 (2-15), wherein the unit g/mL and the pH value of the buffer solution are 3.0-9.0, adding immobilized xylanase or a composition of the immobilized xylanase and cellulase, and performing synergistic hydrolysis to obtain xylooligosaccharide, wherein the ratio of the enzyme activity units of the immobilized xylanase and the cellulase is as follows: 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9: 1; and (3) centrifuging the hydrolyzed solution, taking supernatant, raising the temperature to 50-60 ℃ to make the immobilized enzyme hydrophobically separated out, and collecting the immobilized enzyme.

The alkaline solution is sodium hydroxide, calcium hydroxide or ammonia water.

The agricultural waste is mulberry bark, mulberry twigs, corncobs, corn stalks, bagasse or bran.

The pH value of the buffer solution is 5.0, and the agricultural wastes and the buffer solution are mixed according to the solid-to-liquid ratio of 1:15, and the unit is g/mL.

The immobilized enzyme was collected at 55 ℃.

Has the advantages that: according to the invention, xylanase is covalently connected through an initiator to obtain a xylanase macro-initiator, and then a polymer monomer grows from a macro-initiator main chain to obtain the polymer immobilized xylanase. The immobilized xylanase prepared by the method has high immobilization efficiency, and greatly increases the thermal stability and the repeated use times. Moreover, the xylooligosaccharide can be used in cooperation with cellulase, so that the yield of xylooligosaccharide in agricultural wastes is greatly increased.

Drawings

FIG. 1 is a schematic representation of the xylanase immobilized enzyme of example 1;

FIG. 2 is a temperature stability curve for xylanase free and immobilized enzymes;

FIG. 3 is a graph plotting pH stability of xylanase free enzyme and immobilized enzyme;

FIG. 4 is a graph of the recycling performance of polymer immobilized xylanases.

Detailed Description

The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

The calculation method of the yield of the polymer immobilized xylanase in the embodiment comprises the following steps:

wherein: m represents the mass (g) of the polymer immobilized enzyme;

m_XYL-mass of xylanase (g);

m_initiator-mass of initiator (g).

Example 1

This example illustrates the preparation of a polymer immobilized xylanase.

20mg of xylanase was weighed out and dissolved in 5mL of citric acid-disodium hydrogen phosphate buffer (0.1mol/L, pH8.0), 100mg of initiator (N-2 bromo-2-methylpropionyl-beta-alanine N' -oxysuccinimide ester) was added) And stirring and reacting for 5 hours at 4 ℃ to prepare the xylanase macro-initiator. Mixing bottle A containing macroinitiator and NIPAAm with bottle A containing CuBr and Me₆TREN and a B-flask of deionized water were simultaneously purged with nitrogen under an ice bath to deoxygenate. Transferring the mixture in the bottle A to the bottle B, wherein the molar ratio of each component is macroinitiator: NIPAAm: and (3) CuBr: me₆TREN ═ 1:100:1.3: 1.9. Polymerizing for 12h at 4 ℃ to obtain an immobilized enzyme solution. Dialyzing with 10mmol buffer solution for 2 days to remove unreacted monomer, drying the solution under reduced pressure to obtain immobilized xylanase solid powder with polymer immobilized enzyme yield of 90%, and the schematic diagram of the polymer immobilized xylanase is shown in FIG. 1.

Example 2

This example illustrates the preparation of a polymer immobilized xylanase.

Weighing 20mg of xylanase, dissolving the xylanase in 5mL of citric acid-disodium hydrogen phosphate buffer solution (0.1mol/L, pH8.5), adding 100mg of initiator (N-2 bromo-2-methylpropionyl-beta-alanine N' -oxysuccinimide ester), and stirring and reacting at 4 ℃ for 3 hours to obtain the xylanase macroinitiator. Mixing bottle A containing macroinitiator and NIPAAm with bottle A containing CuBr and Me₆TREN and a B-flask of deionized water were simultaneously purged with nitrogen under an ice bath to deoxygenate. Transferring the mixture in the bottle A to the bottle B, wherein the molar ratio of each component is macroinitiator: NIPAAm: and (3) CuBr: bpy is 1:200:1.3: 1.9. Polymerizing for 15h at 4 ℃ to obtain an immobilized enzyme solution. Dialyzing with 10mmol buffer solution for 2 days to remove unreacted monomer, and drying the solution under reduced pressure to obtain immobilized xylanase solid powder with a polymer immobilized enzyme yield of 93%.

Example 3

This example illustrates the determination of enzymatic activity, thermal stability and recycling properties of polymer immobilized xylanases.

1. The enzyme activity determination method of the polymer immobilized xylanase comprises the following steps: xylanase activity was determined using DNS. 50. mu.L of the enzyme solution obtained by appropriately diluting the immobilized polymer enzyme prepared in example 1 was added to 450. mu.L of beech xylan as a substrate, and reacted at 50 ℃ for 10 min. Adding 750 μ L of terminator DNS, boiling in boiling water for 5min, and measuring absorbance at 540nm wavelength.

2. The thermostability of the polymer immobilized xylanase was investigated: the free xylanase and the polymer immobilized xylanase prepared in example 1 are taken, treated for 2, 5, 10, 20, 30 and 60min at different temperatures (80 and 90 ℃), quickly placed on ice, and the residual enzyme activity is measured under the conditions of 50 ℃ and pH 4.5, so as to measure the thermal stability of the xylanase before and after polymer immobilization. Temperature stability curves of the polymer immobilized xylanase and free enzyme were made, as shown in FIG. 2. The thermal stability of the polymer immobilized xylanase at 90 ℃ is improved by 20 percent.

3. The pH stability of the polymer immobilized xylanase was studied: free xylanase and the polymer immobilized xylanase prepared in example 1 were incubated at 37 ℃ for 1h in buffers of different pH values and the remaining enzyme activity was measured at pH 4.5 and 50 ℃. A pH stability curve of the polymer immobilized xylanase and free enzyme was prepared, as shown in FIG. 3. The xylanase immobilized by the polymer improves the pH tolerance range (pH2.0-8.0) of the xylanase.

4. The recycling performance of the polymer immobilized xylanase was studied: taking xylanase free enzyme and the polymer immobilized xylanase prepared in the example 1, measuring enzyme activity, adding the xylanase free enzyme and the polymer immobilized xylanase prepared in the example 1 into a substrate beech xylan solution, reacting for 10min, adding acetone, obtaining immobilized enzyme and free enzyme after centrifugal separation, measuring the enzyme activity for the 2 nd time, repeating the operation, measuring the enzyme activity for 8 times, and taking the enzyme activity value measured for the 1 st time as 100 percent to obtain the reusability of the xylanase as shown in a figure 4. After repeated use for 5 times, the activity of the polymer immobilized xylanase can still be kept above 60%.

Example 4

This example illustrates the production of xylo-oligosaccharides from mulberry bark treated with a polymer-immobilized xylanase in combination with cellulase.

Immersing mulberry bark in 15 wt.% sodium hydroxide solution, and treating at 90 deg.C for 10 h; then the pH value is adjusted to be neutral, and the mixture is dried at 70 ℃. 2g of the pretreated mulberry bark was mixed with 30mL of a buffer solution having pH 5.0, and the polymer-immobilized xylanase prepared in example 1 and cellulase, xylanase: cellulase (U) ═ 50:50, 0: 100. After 20h, the reducing sugar content in the mulberry bark hydrolysate treated by the cellulase alone is 4.83mg/mL, and the reducing sugar content in the mulberry bark hydrolysate treated by the cellulase synergistically is 9.13mg/mL, which is improved by 1.9 times.

Example 5

This example illustrates the production of xylo-oligosaccharides by co-treatment of corn cobs with polymer-immobilized xylanase and cellulase.

Immersing mulberry bark in 10 wt.% sodium hydroxide solution, and treating at 80 deg.C for 12 h; then the pH value is adjusted to be neutral, and the mixture is dried at 70 ℃. 2g of the pretreated mulberry bark was mixed with 30mL of a buffer solution having pH 4.5, and the polymer-immobilized xylanase prepared in example 1 and cellulase, xylanase: cellulase (U) ═ 60:40, 0: 100. After 20h, the reducing sugar content in the corn cob hydrolysate treated by the cellulase independently is 6.32mg/mL, and the reducing sugar content in the corn cob hydrolysate treated by the cellulase synergistically is 15.74mg/mL, which is improved by 2.5 times.