阅读说明：本技术 一种3d打印木聚糖酶催化剂的方法及其应用 (Method for 3D printing of xylanase catalyst and application thereof ) 是由 江伟 裴蕊 于 2019-10-29 设计创作，主要内容包括：本发明公开了一种3D打印木聚糖酶催化剂的方法及其应用,属于生物法催化降解难分解化合物技术领域。本发明确定了3D打印木聚糖酶催化剂的材料、3D打印材料对酶活的影响、3D打印的粒径、3D打印木聚糖酶催化剂的酶活及重复利用率、3D打印木聚糖酶催化剂在降解木质纤维素方面的应用。本发明操作方便,在生物催化降解木糖和制备具有空间结构可控的3D打印催化剂方面具有较好的工业应用前景,对于今后生物催化专用3D催化剂的开发及生物大分子的降解的研究具有较为重要的意义。(The invention discloses a method for 3D printing of a xylanase catalyst and application thereof, and belongs to the technical field of catalytic degradation of difficultly-decomposed compounds by a biological method. The invention determines the material of the 3D printing xylanase catalyst, the influence of the 3D printing material on enzyme activity, the grain size of 3D printing, the enzyme activity and the repeated utilization rate of the 3D printing xylanase catalyst and the application of the 3D printing xylanase catalyst in the aspect of degrading lignocellulose. The method is convenient to operate, has good industrial application prospects in the aspects of degrading xylose by biocatalysis and preparing the 3D printing catalyst with controllable spatial structure, and has important significance in the development of special 3D catalysts for biocatalysis and the research of the degradation of biomacromolecules in the future.)

1. A method for 3D printing of a xylanase catalyst, comprising the steps of:

1) ink preparation: (1) according to the weight ratio of 0.1-0.5: weighing xylanase powder and deionized water at a ratio of 10g/ml to completely dissolve xylanase in the deionized water, centrifuging and taking supernatant to obtain enzyme solution; (2) weighing sodium alginate, dissolving in enzyme solution with concentration of 0.5-2 wt%, and dissolving completely; (3) preparing 1.5-2.5 wt% calcium chloride solution;

2)3D printing: (1) controlling with 3D printer, selecting 0.21-0.41um needle under 0.25-0.4Mpa, extruding and printing uniform sodium alginate-xylanase microspheres with particle size of 1.5-3.5mm, and dripping the microspheres into calcium chloride solution for crosslinking and curing for 5-15 min;

3) and (3) crosslinking and curing post-treatment: (1) fishing out the solidified microspheres, and (2) washing away the residual calcium chloride on the surface by using deionized water to obtain the immobilized sodium alginate-xylanase microspheres.

2. The method of 3D printing a xylanase catalyst according to claim 1, wherein: the step 1) is as follows: ink preparation: (1) weighing 200mg of xylanase powder, dissolving the xylanase powder in 10ml of deionized water, shaking and shaking for 10min to enable the xylanase to be completely dissolved in the deionized water, and centrifuging to obtain a supernatant; (2) weighing 100mg of sodium alginate in an enzyme solution with the concentration of 1 wt%, and carrying out ultrasonic treatment to fully dissolve the sodium alginate; (3) meanwhile, 2 wt% calcium chloride solution is prepared.

3. The method of 3D printing a xylanase catalyst according to claim 1, wherein:

the step 2) is as follows: 3D printing: (1) controlling by using a 3D printer, selecting a needle head of 0.21um and a pressure of 0.4Mpa, extruding and printing the uniform sodium alginate-xylanase microspheres with the particle size of 1.5mm, and dropping the microspheres into a calcium chloride solution for crosslinking and curing for 10 min.

4. The method of 3D printing a xylanase catalyst according to claim 1, wherein:

the step 3) is as follows: and (3) crosslinking and curing post-treatment: (1) fishing out the cured microspheres with mechanical strength; (2) and (3) washing away the residual calcium chloride on the surface by using deionized water to obtain the immobilized sodium alginate-xylanase microspheres, namely the 3D printing xylanase catalyst with the particle size of 1.5 mm.

5. Use of a method of 3D printing a xylanase catalyst according to any of claims 1 to 4 for degrading xylan.

6. A3D-printed xylan catalyst prepared according to the method of 3D-printed xylanase catalyst according to any of the claims 1 to 4.

7. Use of the 3D printing xylanase catalyst of claim 6 for the catalytic degradation of xylan.

Technical Field

The invention relates to a method for 3D printing of a xylanase catalyst and application thereof.

Background

The biocatalyst is widely used in the fields of chiral drug synthesis, high value-added chemical preparation, degradation of difficultly-degraded biological macromolecules and the like, has excellent performance and higher repeated utilization rate, and has important theoretical and economic benefits. In past research, the development of biocatalysis has a great leap every time the research on the biocatalyst has been greatly improved. Lignocellulose, as a huge potential green resource, is the most widely distributed and abundant renewable high-energy polymer on the earth, which is mainly present in straws and wood, is a main byproduct of the agriculture and forestry industry, and generates about 1500 million tons of lignocellulose worldwide every year. However, the utilization rate of agricultural and forestry wastes such as lignocellulose is low at present, and the traditional method can bring huge resource waste, environmental pollution and the like. Because the structure of lignocellulose is complex and is formed by polymerization of tens of thousands of glucose molecules, the natural degradation speed of lignocellulose is slow, and the traditional methods such as incineration and the like have high cost, low utilization rate and environmental pollution. In contrast, the method for degrading lignocellulose by using the biological enzyme method has the advantages of high degradation efficiency, strong specificity, environmental protection, lower cost and the like, so that the method is widely applied and becomes a research hotspot for developing and utilizing lignocellulose. Current research indicates that microorganisms such as fungi are the major organisms for degrading lignocellulose, but relatively few studies have been made on specific functional enzymes. The biological enzyme method for degrading lignocellulose cannot separate from a biological catalyst, namely enzyme, so that the development and preparation of the biological catalyst with good performance are hot spots and difficulties for solving the problems.

Xylanase (D-xylenylanhydrohydrolase EC3.2.1.8) belongs to hydrolase, and is a kind of xylan degrading enzyme. Complete degradation of xylan requires a synergistic completion between the various enzymes in the xylanase system, and xylanase (. beta. -1, 4-D-xylanase) is one of the most critical hydrolases. The xylanase acts on beta-1, 4-xyloside bonds in the main chain of the xylan in an endo mode, hydrolysis products of the xylanase are mainly a small amount of xylose and xylo-oligosaccharide, and the reaction plays an important role in utilizing the xylan. The xylanase has high application value and can be widely applied to the fields of medicine, papermaking, feed, food and the like. Therefore, the development and research of xylanase have important commercial value. The xylanase is mainly derived from fungi, and the optimal reaction conditions of most of the xylanase are neutral, so that the application of the xylanase in the industrial extreme environment in the fields of medicine, paper making, feed, food and the like is limited to a certain extent. Therefore, it is of great significance to obtain xylanase with excellent performance and high repeated utilization rate.

The 3D printing technology is one of the important marks of the third industrial revolution, and has been applied to the technical field of bone printing and the like, so as to bring high technical progress to the existing technology, and the application value of the technology is gradually enhanced. The 3D printing technology is a process of establishing a model by using a computer, copying by using a special new material for rapid forming and combining a traditional manufacturing process. The 3D printing technology utilizes a computer to design a three-dimensional model, can fully stimulate imagination of people, reduces manufacturing complexity and shortens research and development period through model calculation and material simulation, and provides conditions for technological innovation, new product development and the like. The repeated utilization rate and the catalytic efficiency of the biocatalyst are the key for determining the industrial value of the biocatalyst in the using process, the 3D printing technology can realize the space controllability of a printing model, and entities can be printed by the 3D printing technology in a macroscopic view, so that the repeated utilization rate and the recovery rate of microscopic materials can be improved. Therefore, the application of the compound in the field of biocatalysts is of great significance.

CN 201610057210.3A method for immobilizing xylanase by sodium alginate-chitosan. The method comprises the following steps: weighing a certain amount of sodium alginate in a raw material mixing step, fully dissolving the sodium alginate in distilled water to form a colloidal solution, adding a proper amount of xylanase liquid into the colloidal solution, adjusting the pH value to 6.0-7.0, fully stirring and uniformly mixing to obtain a mixed solution 1; respectively weighing a certain amount of chitosan and calcium chloride, dissolving the chitosan and the calcium chloride in distilled water, adjusting the pH value to 3.0-6.0, and fully stirring and uniformly mixing to obtain a mixed solution 2; (2) forming and coating microspheres: dripping the mixed solution 1 obtained in the step (1) into the mixed solution 2 obtained in the step (1) by using a peristaltic pump with a needle head to obtain smooth microspheres with the diameter of 2-3 mm; (3) and (3) low-temperature curing: placing the smooth microspheres obtained in the step (2) in an environment of 3-10 ℃ for low-temperature curing to obtain cured microspheres, wherein the low-temperature curing time is 1-4 hours; (4) and (3) an elution filtration step: washing the cured microspheres prepared in the step (3) with distilled water to remove residual impurities on the surfaces to obtain elution microspheres; (5) and (3) low-temperature drying: placing the eluted microspheres in a constant-temperature drying box at 35-45 ℃ for low-temperature drying to obtain approximately spherical particles, wherein the drying time is 4-8 hours; (6) and (3) particle forming: and (5) screening the immobilized enzyme particles which are similar to the spheres and obtained in the step (5).

The disadvantages of the invention are: (1) the steps of immobilizing the enzyme are more, the required time is long, and constant-temperature drying and the like are required, so that the activity of the enzyme is influenced; (2) the size of the microspheres is not accurately controlled, which is not beneficial to accurately regulating and controlling the reaction.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a method for 3D printing of a xylanase catalyst and application thereof, and solves the problems in the background technology.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for 3D printing of a xylanase catalyst, comprising the steps of:

1): ink preparation: (1) according to the weight ratio of 0.1-0.5: weighing xylanase powder and deionized water at a ratio of 10g/ml to completely dissolve xylanase in the deionized water, centrifuging and taking supernatant to obtain enzyme solution; (2) weighing sodium alginate, dissolving in enzyme solution with concentration of 0.5-2 wt%, and dissolving completely; (3) preparing 1.5-2.5 wt% calcium chloride solution;

2): 3D printing: (1) controlling with 3D printer, selecting 0.21-0.41um needle under 0.25-0.4Mpa, extruding and printing uniform sodium alginate-xylanase microspheres with particle size of 1.5-3.5mm, and dripping the microspheres into calcium chloride solution for crosslinking and curing for 5-15 min;

3): and (3) crosslinking and curing post-treatment: (1) fishing out the solidified microspheres, and (2) washing away the residual calcium chloride on the surface by using deionized water to obtain the immobilized sodium alginate-xylanase microspheres.

Preferably, step 1) is: ink preparation: (1) weighing 200mg of xylanase powder, dissolving the xylanase powder in 10ml of deionized water, shaking and shaking for 10min to enable the xylanase to be completely dissolved in the deionized water, and centrifuging to obtain a supernatant; (2) weighing 100mg of sodium alginate (1 wt%) in an enzyme solution, and performing ultrasonic treatment to fully dissolve the sodium alginate; (3) meanwhile, 2 wt% calcium chloride solution is prepared.

Preferably, step 2) is: 3D printing: (1) controlling by using a 3D printer, selecting a needle head of 0.21um and a pressure of 0.4Mpa, extruding and printing the uniform sodium alginate-xylanase microspheres with the particle size of 1.5mm, and dropping the microspheres into a calcium chloride solution for crosslinking and curing for 10 min.

Preferably, step 3) is: and (3) crosslinking and curing post-treatment: (1) fishing out the cured microspheres with mechanical strength; (2) and (3) washing away the residual calcium chloride on the surface by using deionized water to obtain the immobilized sodium alginate-xylanase microspheres, namely the 3D printing xylanase catalyst with the particle size of 1.5 mm.

The invention also provides application of the 3D printing xylanase catalyst in degrading xylan.

The invention also provides a 3D printing xylan catalyst, which is prepared by the method for preparing the 3D printing xylanase catalyst.

The invention also provides application of the 3D printing xylanase catalyst in catalytic degradation of biomass compounds such as xylan.

The invention determines the material (sodium alginate) of the 3D printing xylanase catalyst, the influence of the 3D printing material on enzyme activity, the grain size (1.5mm, 2.5mm and 3.5mm) of 3D printing, and the enzyme activity and the reutilization ratio of the 3D printing xylanase catalyst.

The 3D xylanase catalyst can catalyze and degrade xylan. The 3D xylanase catalyst is subjected to selection of a 3D printing material, determination of influence of the 3D printing material on enzyme activity, selection of 3D printing particle size, determination of enzyme activity and recycling rate of the 3D xylanase catalyst, and the method and the constructed 3D xylanase catalyst are used for catalytically degrading xylan.

Compared with the background technology, the technical scheme has the following advantages:

(1) the 3D printing xylanase catalyst is a 3D catalyst which is designed by utilizing a 3D printing technology and has controllable space and scale, optimizes the application of the catalyst in the aspect of catalyzing and degrading xylan by inspecting the selection of a 3D printing material, determining the influence of the 3D printing material on enzyme activity, selecting 3D printing particle size, determining the enzyme activity and the repeated utilization rate of the 3D printing xylanase catalyst and the like, can catalyze and degrade xylan, has a simple repeated use method, and has the potential of degrading biomass macromolecules such as xylan and the like;

(2) the method for 3D printing of the xylanase catalyst has the advantages of convenience in operation, high efficiency, simple equipment, reusability, controllable spatial structure and the like, and has good industrial application prospects in the fields of degrading xylan by biocatalysis and preparing 3D printing catalysts with controllable spatial structure.

(3) The method has the advantages of few steps and short time, and the xylanase can keep higher activity; (2) the size of the microspheres can be precisely controlled.

(4) The xylanase catalyst prepared by the invention has better repeated utilization rate and can be used for multiple times.

Drawings

FIG. 13D effect of printing material on enzyme activity, a is effect of time and b is effect of temperature.

FIG. 2 is an analysis chart of enzyme activity and recycling rate of xylanase catalysts printed by 3D printing with different particle sizes.

Detailed Description