Cardamine violifolia processing by-product-based charcoal immobilized cellulase as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomass resource utilization, and discloses cardamine violifolia processing by-product-based charcoal immobilized cellulase as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing ferric chloride with the cardamine violifolia processing byproduct, and performing pyrolysis carbonization treatment to obtain a pyrolysis product; the pyrolysis product is subjected to acid pickling, water washing and drying treatment in sequence, and charcoal is obtained; and carrying out immobilization treatment on cellulase by taking the biochar as a carrier to obtain the cardamine violifolia processing byproduct-based biochar immobilized cellulase. The method provided by the invention not only realizes resource utilization of cardamine violifolia processing byproducts, but also provides a new path for preparation of immobilized cellulase with high loading capacity and high stability in the field of enzymatic preparation of glucose.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomass resource utilization, and more specifically relates to a biochar-immobilized cellulase based on a byproduct of Cardamine viola processing, as well as a preparation method and application thereof. Background Art

[0002] Selenium is an essential trace element for humans, animals, and plants. Selenium deficiency may lead to a variety of diseases, including heart disease, immune disorders, cognitive decline, and hypothyroidism. Cardamine violae is a super-selenium-accumulating plant unique to my country. It has extremely strong selenium accumulation and tolerance capabilities. Its intrinsic selenium-rich proteins and polysaccharides have high nutritional value and biological activities, such as antioxidant, anti-inflammatory, and anti-tumor properties. Currently, extracting selenium-rich proteins and polysaccharides from Cardamine violae is an important way to increase its value-added utilization. However, the above-mentioned extraction process produces a large amount of by-products, and there is currently a lack of reasonable ways to utilize them.

[0003] On the one hand, enzymatic production of glucose from biomass is an important resource for its utilization, offering a viable solution to current resource shortages and environmental challenges. However, the free cellulase used in traditional enzymatic hydrolysis processes is difficult to separate and recover within the reaction system, requiring significant cellulase consumption for each reaction, increasing production costs. Furthermore, free cellulase exhibits poor stability, particularly at high temperatures or other extreme conditions, where its activity and structure are easily destroyed, limiting its industrial application.

[0004] Enzyme immobilization technology uses physical or chemical means to fix enzyme molecules on solid supports, which can improve enzyme stability and enable enzyme recovery and reuse. Some studies have reported on the preparation of immobilized cellulases, in which the design of the immobilization support is crucial. However, the preparation process of the cellulase immobilization supports reported in these studies is generally complex and costly, and the cellulase loading capacity of the immobilized enzyme is generally low, and the stability needs to be improved. Therefore, the optimized design of immobilized cellulases urgently needs further research. Summary of the Invention

[0005] The present invention addresses the shortcomings of existing technologies and proposes a biochar-immobilized cellulase derived from a Cardamine viola processing byproduct, as well as a preparation method and application thereof. This method not only realizes the resource utilization of Cardamine viola processing byproducts but also provides a new approach for the preparation of high-load, highly stable immobilized cellulase in the enzymatic production of glucose.

[0006] In order to achieve the above object, the present invention provides a first aspect of a method for preparing biochar-immobilized cellulase based on a byproduct of Cardamine viola processing, the preparation method comprising the following steps:

[0007] S1: mixing ferric chloride with a by-product of Cardamine viola processing and performing pyrolysis and carbonization treatment to obtain a pyrolysis product;

[0008] S2: sequentially performing acid washing, water washing, and drying on the pyrolysis product to obtain biochar;

[0009] S3: performing an immobilization treatment on cellulase using the biochar as a carrier to obtain the Cardamine viola processing byproduct-based biochar-immobilized cellulase.

[0010] In the present invention, the Cardamine viola processing by-products refer to by-products obtained after extracting selenium-rich proteins and selenium-rich polysaccharides from Cardamine viola. The extraction method can be any method known in the art.

[0011] According to the present invention, preferably, the mass ratio of the ferric chloride to the by-products of Cardamine viola processing is (1-2):5.

[0012] According to the present invention, preferably, the conditions of the pyrolysis carbonization treatment are: heating from 25-30°C to 600-800°C at a heating rate of 5-15°C / min under a nitrogen atmosphere, and then keeping the temperature for 30-60 minutes.

[0013] According to the present invention, preferably, the equipment for pyrolysis carbonization treatment is a tubular furnace.

[0014] In the present invention, the pyrolysis product is solid.

[0015] According to the present invention, preferably, the detergent used in the pickling is a hydrochloric acid aqueous solution with a concentration of 0.5-1 mol / L, and the dosage ratio of the hydrochloric acid aqueous solution to the Cardamine viola processing by-product is (29-31): 1 mL / g.

[0016] According to the present invention, preferably, the water washing is to filter and wash the acid-washed product with deionized water until the pH value of the filtrate obtained by filtration is neutral.

[0017] According to the present invention, preferably, the drying process is carried out at a temperature of 45-55° C. and for a time of 22-30 hours.

[0018] According to the present invention, preferably, the temperature of the immobilization treatment is 45-55° C. and the time is 9-12 hours.

[0019] According to the present invention, preferably, the mass ratio of the biochar to the cellulase is (5-15):8.

[0020] The second aspect of the present invention provides Cardamine viola processing by-product-based biochar-immobilized cellulase prepared by the method for preparing Cardamine viola processing by-product-based biochar-immobilized cellulase.

[0021] The third aspect of the present invention provides the use of the Cardamine viola processing by-product-based biochar-immobilized cellulase in the enzymatic hydrolysis of biomass to produce glucose.

[0022] The technical solution of the present invention has the following beneficial effects: the present invention converts Cardamine violae processing byproducts into biochar with specific physical and chemical properties, uses the biochar as a carrier to immobilize cellulase, and then applies it to the enzymatic production of glucose. This method not only achieves resource utilization of Cardamine violae processing byproducts but also provides a new approach for the preparation of high-load, highly stable immobilized cellulase in the enzymatic production of glucose. Specifically:

[0023] 1. The preparation process of the biochar carrier in the biochar-immobilized cellulase of the present invention is relatively simple and has a high loading capacity for cellulase. In addition, the obtained biochar-immobilized cellulase has strong stability. The principle is as follows: a porous carbon material rich in iron can be obtained by pyrolyzing a mixture of ferric chloride and Cardamine violaceum processing by-products, and further appropriate acid washing can remove most of the iron-containing substances such as Fe2O3 and Fe3O4 in the carbon material, thereby obtaining a mesoporous biochar with a larger specific surface area and a small amount of doping with iron elements such as Fe3C. The biochar of the present invention achieves efficient loading of cellulase and improved stability through physical adsorption of rich mesopores and chemical adsorption of iron elements such as Fe3C.

[0024] 2. The present invention not only realizes the resource utilization of Cardamine violacea processing by-products, but also provides a new path for the preparation of high-load and high-stability immobilized cellulase in the field of enzymatic preparation of glucose.

[0025] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0027] Figure 1 The pH stability comparison of free cellulase and biochar-immobilized cellulase prepared in Example 1 of the present invention is shown.

[0028] Figure 2 The temperature stability comparison of free cellulase and biochar-immobilized cellulase prepared in Example 1 of the present invention is shown.

[0029] Figure 3The graph shows a comparison of the storage stability of free cellulase and the biochar-immobilized cellulase prepared in Example 1 of the present invention.

[0030] Figure 4 The results show the recycling performance of the biochar-immobilized cellulase prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Instead, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0032] In the following embodiments and comparative examples:

[0033] The Cardamine viola processing byproducts refer to byproducts obtained after extracting selenium-rich proteins and selenium-rich polysaccharides from Cardamine viola. The extraction method can be any method known in the art.

[0034] The ferric chloride and hydrochloric acid were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0035] The cellulase was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0036] Example 1

[0037] This embodiment provides a method for preparing biochar-immobilized cellulase based on a byproduct of Cardamine viola processing, comprising the following steps:

[0038] S1: mixing ferric chloride and a by-product of Cardamine violacea processing in a mass ratio of 2:5, and then adding the mixture into a tube furnace for pyrolysis and carbonization treatment to obtain a pyrolysis product;

[0039] The pyrolysis carbonization treatment conditions are as follows: heating from 25°C to 800°C at a heating rate of 10°C / min under a nitrogen atmosphere, and then keeping the temperature for 60 minutes;

[0040] S2: washing the pyrolysis product with a 1 mol / L hydrochloric acid solution (the ratio of the hydrochloric acid solution to the Cardamine violaceum processing byproduct is 30:1 mL / g), filtering and washing the acid-washed product with deionized water until the pH value of the filtrate reaches neutral; drying the washed product at 50° C. for 24 hours to obtain biochar;

[0041] S3: Immobilizing the cellulase using the biochar as a carrier at 50° C. for 12 hours to obtain the Cardamine viola processing byproduct-based biochar-immobilized cellulase, wherein the mass ratio of the biochar to the cellulase is 5:4.

[0042] Comparative Example 1

[0043] The only difference between this comparative example and Example 1 is that the mass ratio of the ferric chloride to the by-product of Cardamine viola processing is 0:1.

[0044] The method for preparing biochar-immobilized cellulase based on a byproduct of Cardamine viola processing in this comparative example comprises the following steps:

[0045] S1: adding the byproducts of Cardamine viola processing into a tube furnace for pyrolysis and carbonization treatment to obtain a pyrolysis product;

[0046] The conditions of the pyrolysis carbonization treatment are the same as those in Example 1;

[0047] S2: Same as Example 1;

[0048] S3: Same as Example 1.

[0049] Comparative Example 2

[0050] This comparative example provides a method for preparing biochar-immobilized cellulase based on a byproduct of Cardamine viola processing, comprising the following steps:

[0051] S1: Same as Example 1;

[0052] S2: Using the pyrolysis product obtained in step S1 as a carrier, immobilizing cellulase at 50° C. for 12 hours to obtain the Cardamine viola processing byproduct-based biochar-immobilized cellulase, wherein the mass ratio of the biochar to the cellulase is 5:4.

[0053] Test Example 1

[0054] This test example tests the pore characteristics of the biochar in the biochar-immobilized cellulase prepared in Example 1 and Comparative Examples 1-2 and the loading amount of the cellulase on the biochar. The results are shown in Table 1.

[0055] in:

[0056] The test method for pore characteristics is:

[0057] The pore characteristics of the carriers of cellulase in Example 1 and Comparative Examples 1-2 were analyzed using a JW-BK122 specific surface area and pore size analyzer from Beijing Jingwei Gaobo Science and Technology Co., Ltd. under nitrogen atmosphere at 77K. Before the analysis, the samples were first degassed at 200 °C for 3 hours, and then adsorption and desorption experiments were carried out. The main experimental data processing methods are as follows: The specific surface area of the sample was calculated by the Brunauer-Emmet-Teller (BET) formula (0.01 < P / P0 < 0.1), and the total pore volume (V Figure 1-4 , ,

[0066] , ,

[0065] ) was determined by the single-point adsorption amount at P / P0 = 0.988. The density functional theory (DFT) calculation method was used to determine the pore size distribution, and the average value of all pores was the average pore size.

[0058] The test method for the loading amount of cellulase on biochar is as follows:

[0059] The immobilization amount of cellulase was determined by the Bradford method.​​​​​​​​​​​​​​​​​​​​​​Free cellulase and the biochar-immobilized cellulase prepared in Example 1 were placed in 0.05 mol / L acetate buffer at pH 5. After mixing in a ratio of cellulase solution to buffer of 1:5, 10 mL of 1% (w / w) sodium carboxymethyl cellulose (CMC) solution was added. The mixed solution was placed in a constant temperature water bath shaker and shaken at 150 rpm and 50°C for 30 minutes. After the reaction was completed, 2 mL of the supernatant and 2 mL of DNS reagent were placed in a test tube, and the mixture was immediately cooled to room temperature (25°C) in an ice bath after boiling in a water bath for 10 minutes. The volume was finally adjusted to 25 mL, shaken, and allowed to stand. The ultraviolet absorbance was measured at a wavelength of 540 nm, and the reducing sugar content was calculated. Finally, the activity of the free cellulase and the biochar-immobilized cellulase prepared in Example 1 was obtained.

[0067] from Figure 1-3 It can be seen that compared with free cellulase, the pH stability, temperature stability and storage stability of the biochar-immobilized cellulase prepared in Example 1 are significantly improved. Figure 4 As shown, the biochar-immobilized cellulase prepared in Example 1 also has good recycling performance. After recycling 10 times, its relative activity is 51% of the initial activity.

[0068] The present invention produces biochar-immobilized cellulase with high loading capacity and high stability. The biochar-immobilized cellulase can be applied to the enzymatic hydrolysis of biomass to produce glucose, resolving the issues of poor stability and difficulty in recycling free cellulase. Furthermore, the present invention provides a new technical approach for the high-value, comprehensive utilization of Cardamine viola processing byproducts.

[0069] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing biochar-immobilized cellulase based on a byproduct of Cardamine viola processing, characterized in that: The preparation method comprises the following steps: S1: mixing ferric chloride with a by-product of Cardamine viola processing and performing pyrolysis and carbonization treatment to obtain a pyrolysis product; S2: sequentially performing acid washing, water washing, and drying on the pyrolysis product to obtain biochar; S3: performing an immobilization treatment on cellulase using the biochar as a carrier to obtain the Cardamine viola processing byproduct-based biochar-immobilized cellulase.

2. The method for preparing biochar-immobilized cellulase based on Cardamine violacea processing byproducts according to claim 1, wherein: The mass ratio of the ferric chloride to the by-products of Cardamine violacea processing is (1-2):

5.

3. The method for preparing biochar-immobilized cellulase based on Cardamine violacea processing byproducts according to claim 1, wherein: The conditions of the pyrolysis carbonization treatment are: heating from 25-30°C to 600-800°C at a heating rate of 5-15°C / min under a nitrogen atmosphere, and then keeping the temperature for 30-60 minutes; The equipment for the pyrolysis carbonization treatment is a tubular furnace.

4. The method for preparing biochar-immobilized cellulase based on Cardamine violacea processing byproducts according to claim 1, wherein: The detergent used for the pickling is a hydrochloric acid aqueous solution with a concentration of 0.5-1 mol / L, and the dosage ratio of the hydrochloric acid aqueous solution to the Cardamine violacea processing by-product is (29-31): 1 mL / g.

5. The method for preparing biochar-immobilized cellulase based on Cardamine violacea processing byproducts according to claim 1, wherein: The water washing is to filter and wash the acid-washed product with deionized water until the pH value of the filtrate obtained by filtration is neutral.

6. The method for preparing biochar-immobilized cellulase based on Cardamine violacea processing byproducts according to claim 1, wherein: The drying temperature is 45-55° C. and the drying time is 22-30 hours.

7. The method for preparing biochar-immobilized cellulase based on Cardamine violacea processing byproducts according to claim 1, wherein: The temperature of the immobilization treatment is 45-55° C., and the time is 9-12 hours.

8. The method for preparing biochar-immobilized cellulase based on Cardamine violacea processing byproducts according to claim 1, wherein: The mass ratio of the biochar to the cellulase is (5-15):

8.

9. Cardamine viola processing by-product-based biochar-immobilized cellulase prepared by the method for preparing Cardamine viola processing by-product-based biochar-immobilized cellulase according to any one of claims 1 to 8.

10. Use of the biochar-immobilized cellulase based on the by-product of Cardamine viola processing as claimed in claim 9 in the preparation of glucose by enzymatic hydrolysis of biomass.

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