Preparation method and application of biochar immobilized phytase

Abstract

The invention provides a preparation method and application of biochar immobilized phytase, and relates to the technical field of biological enzyme immobilization. The method comprises the following steps: firing collected pineapple leaves into biochar for later use; adding pectin into water to obtain a pectin aqueous solution; adding the charcoal component into the pectin aqueous solution, uniformly mixing, filtering, washing and drying to obtain pectin charcoal; adding a cross-linking agent into the pectin biochar for cross-linking, then stirring and mixing with a phytase solution, filtering, and freeze-drying to obtain the biochar immobilized phytase. The biochar immobilized phytase can effectively overcome the defects that exogenous phytase is prone to inactivation and loss and difficult to store in soil, the phosphorus dissolving effect of phytase is enhanced, meanwhile, the biological activity and richness of indigenous phosphate-solubilizing bacteria are increased, soil phosphorus circulation is accelerated, soil phosphorus pool mineralization is promoted in the biochemical aspect, and the phosphorus-solubilizing effect is improved. The soil conditioner is a more environment-friendly and efficient soil conditioner and phosphate fertilizer synergist.

Description

TECHNICAL FIELD

[0001] The application provides a preparation method of biochar immobilized phytase and application thereof, and belongs to the technical field of biological enzyme immobilization. BACKGROUND

[0002] Zhanjiang is the main producing area of pineapples in China, and the pineapple leaves belong to agricultural wastes. After pineapple harvesting, more than 5 tons of total stems and leaves can be produced per mu, and the stems and leaves are generally crushed and returned to the field after pineapple harvesting. However, such a treatment method does not bring good benefits to the soil, and therefore, the preparation of biochar from pineapple leaves as raw materials can effectively make up for the defect of low utilization rate of agricultural wastes.

[0003] Phosphorus is one of the essential macro nutrients for plant growth and development, but the utilization rate of phosphorus fertilizer applied to the soil is only 10%-20% due to factors such as leaching loss and soil fixation, thereby causing a large waste of phosphate rock resources and adverse effects of crop yield and quality reduction due to phosphorus deficiency. Phytic acid (inositol hexakisphosphate) is one of the main forms of soil organic phosphorus, accounting for 50%-80% of the content of soil organic phosphorus, and is the main supplier of plant phosphorus. Phytase can hydrolyze phytic acid into biologically available inorganic orthophosphate. However, plants cannot secrete phytase, and the addition of exogenous phytase can promote the mineralization of phytic acid and increase the bioavailability of phytic acid. Natural enzymes have defects such as easy deactivation, loss, instability and difficult preservation in harsh environments.

[0004] Due to the complexity of the soil environment, the application of phytase alone can cause easy deactivation. Therefore, how to use pineapple leaves to enhance the activity of phytase, improve the activity of indigenous phosphorus-solubilizing bacteria, increase the content of available phosphorus in the soil, promote plant growth, and obtain an environmentally friendly and efficient soil conditioner and phosphorus fertilizer synergist with double effects is a technical problem to be solved by the application. SUMMARY

[0005] Based on this, the application provides a preparation method of biochar immobilized phytase and application thereof.

[0006] The application is specifically implemented by using the following technical solutions. A preparation method of biochar immobilized phytase, comprising the following steps: (1) Pineapple leaves collected are burned into biochar for standby use; (2) Pectin is added to water to obtain a pectin aqueous solution; the biochar is added to the pectin aqueous solution, and after uniform mixing, filtration, washing and drying, a pectin biochar is obtained; (3) A crosslinking agent is added to the pectin biochar for crosslinking, and then the phytase solution is stirred and mixed, and after filtration and freeze-drying, biochar immobilized phytase is obtained.

[0007] Preferably, the biochar is prepared by cleaning pineapple stems, drying them in an oven, pulverizing them, and then drying them again to a constant weight; after sieving, the temperature is raised to 300℃~400℃ for pyrolysis treatment, and the biochar is obtained after pulverizing and sieving. More preferably, the heating rate is 20℃·min. -1 The pyrolysis treatment lasts for 4 hours. The pulverized material is then passed through an 18-mesh sieve.

[0008] Preferably, the mass-to-volume ratio of pectin to water in the pectin aqueous solution is 1:5 g / mL.

[0009] Preferably, the mass ratio of pectin to biochar is 1:1.

[0010] Preferably, phytase is dissolved in an acetate buffer solution with a pH of 5.5 to obtain a phytase solution.

[0011] More preferably, the pectin biochar activation treatment involves dispersing the pectin biochar in a crosslinking agent glutaraldehyde solution and performing a shaker crosslinking treatment at room temperature.

[0012] More preferably, the mass ratio of pectin biochar to phytase is 100:30.

[0013] This invention also protects the biochar-immobilized phytase prepared by the above method and its applications, specifically the use of the biochar-immobilized phytase as a soil conditioner. Alternatively, the biochar-immobilized phytase can be used as a phosphate fertilizer synergist.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Pectin, as a natural polysaccharide, has excellent film-forming and biodegradable properties. Existing studies have shown that pectin can increase the activity or abundance of phosphate-solubilizing bacteria in soil, thus accelerating soil phosphorus conversion. Combining pectin with biochar to create a blend can leverage the advantages of both, enhancing phosphorus conversion by phosphate-solubilizing bacteria while simultaneously lowering the pH of the biochar to make it more suitable for phytase activity.

[0015] This invention uses pectin-based biochar as a carrier for plant enzymes, which enhances enzyme stability, facilitates storage and transportation, and promotes soil phosphorus cycling. This study promotes soil phosphorus cycling through both enzyme catalysis and microbial transformation, solving the problem of phytase inactivation upon direct entry into the soil. Simultaneously, the addition of pectin increases the activity of indigenous phosphate-solubilizing bacteria in the soil, thereby increasing the available phosphorus content and promoting crop growth.

[0016] The raw material for this invention, pineapple leaves, is agricultural waste, which is inexpensive. Currently, the method of crushing and returning the leaves to the field has not produced good economic and environmental benefits. Preparing the leaves into biochar can better realize their value.

[0017] The pineapple leaf biochar of this invention contains a high level of phosphorus, making it suitable for use as an exogenous phosphate fertilizer in the soil. The immobilized phytase prepared in this invention exhibits higher temperature and pH tolerance, effectively preventing enzyme activity reduction during production and transportation, and allowing it to adapt to more complex soil environments. The immobilized phytase prepared in this invention can be used as a novel phosphate fertilizer or as a soil conditioner, mineralizing the phosphorus originally fixed in the soil to maximize resource utilization. Attached Figure Description

[0018] Figure 1 The effect of temperature on the activity of phytase immobilized in biochar according to the present invention; Figure 2 The effect of pH on the activity of phytase immobilized in biochar according to the present invention; Figure 3 The effect of different concentrations of glutaraldehyde solution on the enzyme loading efficiency of biochar; Figure 4 The effect of different cross-linking times on enzyme carrying efficiency of biochar; Figure 5 The effect of different mass ratios of enzyme to biochar on enzyme loading efficiency; Figure 6 Scanning electron microscopy, showing the original biochar (a, a1); pectin biochar (b, b1); and immobilized enzyme biochar (c, c1). Figure 7 Changes in available phosphorus in soil of different experimental groups Note: Blank control (ck); Biochar (B); Pectin biochar (BP); Free phytase (Phy); Immobilized phytase (I-Phy) Figure 8 Changes in soil phytase activity in different experimental groups; Figure 9 The abundance coefficient curves of soil bacteria ASV (left: non-rhizosphere soil; right: rhizosphere soil) are shown. Figure 10 Venn diagram showing the distribution of ASV bacteria in soybean non-rhizosphere (left) and rhizosphere (right) soils under different treatments. Figure 11 Distribution of bacteria at the phylum level in soybean non-rhizosphere and rhizosphere soils under different treatments; Figure 12 Distribution of bacteria at the genus level in soybean non-rhizosphere and rhizosphere soils under different treatments. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the preferred embodiments of this invention will be described in further detail below with reference to the examples. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0020] Pineapple stalks were collected from the South Subtropical Crops Research Institute of the Chinese Academy of Tropical Agricultural Sciences, 50 meters east of County Road 668 in Mazhang District, Zhanjiang City, Guangdong Province. After being washed, they were dried in an oven at 100°C until constant weight.

[0021] The dried pineapple straw was crushed and passed through an 18-mesh sieve. 100g of straw powder was placed in a 100ml crucible and pyrolyzed in a muffle furnace at 300℃ (heating rate of 20℃·min). -1 The temperature was raised to 300 °C and kept at that temperature for 4 hours. After cooling to room temperature, 42.90 g of pineapple straw biochar was obtained, with a yield of approximately 43%. The pH of the pineapple straw biochar was measured to be 10.28, indicating that it was slightly alkaline.

[0022] 10g of pectin was weighed and added to 100ml of deionized water, then magnetically stirred until completely dissolved. Next, 10g of biochar was added and magnetically stirred at room temperature for 12 hours to obtain a pectin-biochar composite material. The pH of the pectin-biochar was measured to be 6.78. This indicates that pectin can lower the pH of pineapple stalks, bringing it closer to the optimal pH for phytase (phytase optimal pH = 5.5). This invention uses glutaraldehyde as a cross-linking agent and employs a covalent bonding method to immobilize phytase on pectin-based biochar. The covalent bonding method utilizes the amino and carboxyl groups in the enzyme protein, or the aromatic rings in tyrosine and histidine residues, to form covalent bonds with certain organic groups on the carrier, thus immobilizing the enzyme on the carrier. Pectin, as a polysaccharide, is combined with biochar to prepare a carrier for phytase immobilization, providing more active sites to the biochar, which is beneficial for the covalent bonding between the enzyme and the biochar. Glutaraldehyde concentration, immobilization time, and the biochar-to-enzyme mass ratio are the main factors affecting the efficiency of phytase immobilization on biochar. This invention explores the optimal immobilization conditions for these three aspects.

[0023] Effect of cross-linking agent concentration on enzyme loading: 25 mL solutions of glutaraldehyde with mass concentrations of 2%, 4%, 6%, and 8% were prepared. 0.5 g of pectin biochar was added to each solution, and the mixture was magnetically stirred for 2.5 h. After filtration, the residue was washed three times with deionized water and dried in a 50℃ oven. 15 mg of phytase and 0.5 g of pectin biochar were added to an acetate buffer solution at pH 5.5, and the mixture was shaken on a shaker for 4 hours. The mixture was then filtered, and the enzyme content of the supernatant was measured to calculate the enzyme loading rate. Figure 3 It can be seen that when the glutaraldehyde concentration is 4%, the pectin biochar has the highest loading capacity for phytase, reaching 87%.

[0024] Effect of immobilization time on enzyme loading: 0.5 g of biochar was placed in 25 mL of glutaraldehyde solution and magnetically stirred for 2.5 h, then filtered. The residue was washed three times with deionized water, dried in a 50℃ oven, and 1 mL of enzyme solution (1 mg / mL) was added. 15 mg of phytase and 0.5 g of pectin biochar were added to an acetate buffer solution at pH 5.5. The mixture was shaken on a shaker for 2, 4, 6, and 8 h respectively, then filtered. The enzyme content of the supernatant was measured to calculate the enzyme loading rate. Figure 4 It can be seen that when the immobilization time is 4 hours, the loading capacity of pectin biochar on phytase is the largest, reaching 98%.

[0025] Effect of mass ratio on enzyme loading: 0.5 g of biochar was placed in 25 mL of glutaraldehyde solution and magnetically stirred for 2.5 h, then filtered. Biochar and phytase were then mixed at mass ratios of 100:1, 100:2, 100:3, and 100:4, respectively, shaken for 4 hours, filtered, and the enzyme content of the supernatant was measured to calculate the enzyme loading rate. Figure 5 It can be seen that when the mass ratio of pectin biochar to phytase is 100:3 (3%), the loading capacity of pectin biochar on phytase is the largest, reaching 99%.

[0026] Based on the above results, pectin biochar was activated in 4 wt% glutaraldehyde for 2.5 hours, filtered through filter paper, and dried in an oven at 70°C. 15 mg of solid powdered phytase was dissolved in 15 ml of pH 5.5 acetate buffer to obtain a phytase solution. Then, 1 g of activated pectin biochar was mixed with 15 ml of the phytase solution and shaken for 4 hours. After filtering through filter paper and air-drying at room temperature, the protein content in the residual solution after filtration was determined using the Coomassie brilliant blue method, indicating that the loading efficiency of pectin biochar for phytase was 99.5%.

[0027] Scanning electron microscopy (SEM) was used to examine the original biochar (a, a1), pectin biochar (b, b1), and immobilized enzyme biochar (c, c1) to determine the phytase loading. It was clearly observed that the pineapple straw biochar possessed a rich porous structure and a rough surface. After pectin treatment, the surface became smoother, the pores increased, and some solid pectin remained internally, indicating that the pectin successfully adhered to the biochar and altered its physical structure, increasing its specific surface area. SEM images of the immobilized enzyme on the biochar showed a smoother surface and smaller pores. Phytase attachment was clearly visible on the pectin biochar, with the phytase evenly covering the biochar surface. Phytase activity assays on the carrier materials also confirmed the successful loading of phytase onto the pectin biochar.

[0028] To investigate the application effect of pectin-based biochar immobilized phytase in soil, the immobilized phytase was applied to the soil, changes in the soil environment were observed, and soil microorganisms were measured in the soil samples.

[0029] Initial soil characterization: The experimental soil was taken from the red brick soil of Guangdong Ocean University in Zhanjiang City, Guangdong Province. The pH was 6.5, which is slightly acidic soil, and the water content was 54%. The basic physicochemical properties are shown in Table 1.

[0030] Soil experiment: The soil mass of each treatment group was 50g. Four experimental groups were set up: blank group, biochar group (B), pectin biochar group (BP), free phytase group (Phy), and immobilized phytase group (I-Phy). The collected soil and materials were put into beakers at a ratio of 2:100 and mixed thoroughly for 15min. The soil without added materials was also shaken well. Distilled water was added to adjust the soil moisture content to about 50% of the saturated moisture content. Each experimental group was set up with 4 replicates.

[0031] Adding different materials to soil alters its physicochemical properties. Table 1 shows that the addition of biochar increases soil pH, changing it from slightly acidic to neutral. This is likely because pineapple leaf biochar itself has a high pH, ​​which neutralizes the acidic soil after addition. Simultaneously, soil organic matter and phosphorus levels also increase. This is because biochar itself contains phosphorus and organic matter and has long been used as a soil conditioner. After the addition of materials, available phosphorus and phytase activity in the soil were measured weekly for one month. The results are shown below. Figure 7 and Figure 8 As can be seen, the available phosphorus content in the experimental group with immobilized enzymes increased significantly and continued to increase over a month. In contrast, the available phosphorus content in the free enzyme group increased in the first two weeks but decreased in the following two weeks. This is due to the soil environment, which caused the free phytase to become inactive and unable to continue mineralizing the phosphorus fixed in the soil. Initially, the activity of the free enzyme was greater than that of the immobilized enzyme group, but over time, the activity of the free enzyme group gradually decreased, while the activity of the immobilized enzyme group remained constant, reaching a maximum activity of 11.05 U / g. The addition of immobilized enzymes can significantly increase the available phosphorus content in the soil, and immobilizing phytase before application increases its activity retention time in the soil, continuously mineralizing the phosphorus fixed in the soil and converting it into available phosphorus for plant absorption and utilization.

[0032] Table 1. Basic physicochemical properties of soil from different experimental groups Experimental group pH Moisture content (%) Organic matter (g·kg-1) Total nitrogen (g·kg-1) Available nitrogen (mg·kg-1) Available potassium (mg·kg-1) Total phosphorus (g·kg-1) Available phosphorus (mg·kg-1) Blank 6.5 54 13.2 0.8 62.0 82.4 9.2 7.6 Pectin biochar 7.1 54 15.5 1 83.2 88.2 12.4 9.1 Free phytase 6.5 54 14.2 1.3 77.8 86.3 10.3 9.6 Immobilized enzyme 7.2 54 15.7 1.4 90.3 87.2 13.5 14.8 This study used the Illumina MiSeq sequencing platform to perform high-throughput sequencing of bacterial 16S rRNA genes from 10 soil samples treated with different methods, obtaining a total of 2,579,174 valid sequences. To assess whether the sequencing depth was sufficient, we constructed a dilution curve based on the relationship between the number of ASVs and the sequencing load. Figure 9 The results showed that the curve gradually flattened as the sequencing depth increased, indicating that the current sequencing depth could cover the vast majority of microbial species in the samples, with only a small number of low-abundance taxa remaining uncovered, and the data volume was reasonable and reliable. This result provides sufficient data support for the microbial community diversity analysis in this study.

[0033] Adding different materials to soil affects the structure of soil microbial communities. As shown in Table 2, α-diversity analysis revealed that immobilized phytase treatment (I-Phy) significantly increased species richness (Chao1 index 1379.87), a 30.9% increase compared to biochar treatment (B), confirming the advantage of immobilization technology in maintaining microbial diversity. Biochar primarily optimizes community composition and enhances ecological function by selectively enriching specific functional microbial groups through its porous structure and surface properties; while immobilized phytase, through the continuous release of active phosphate groups, profoundly reshapes the nutrient competition and metabolic networks among microorganisms, inducing a more significant community functional reconstruction. This reconstruction not only changes the dominant bacterial groups but also reconstructs the interaction patterns and functional synergies among microorganisms.

[0034] Table 2. Effects of different treatments on α-diversity of non-rhizosphere and rhizosphere soil bacterial communities in soybean. Experimental group pH Moisture content (%) Organic matter (g·kg-1) Total nitrogen (g·kg-1) Available nitrogen (mg·kg-1) Available potassium (mg·kg-1) Total phosphorus (g·kg-1) Available phosphorus (mg·kg-1) Blank 6.5 54 13.2 0.8 62.0 82.4 9.2 7.6 Pectin biochar 7.1 54 15.5 1 83.2 88.2 12.4 9.1 Free phytase 6.5 54 14.2 1.3 77.8 86.3 10.3 9.6 Immobilized enzyme 7.2 54 15.7 1.4 90.3 87.2 13.5 14.8 Note: The values ​​in the table are mean ± standard error. Different lowercase letters in the same column indicate significant differences (P<0.05). Venn diagram ( Figure 10 Analysis showed that different treatments significantly affected the composition of soybean rhizosphere and non-rhizosphere soil bacterial communities (P<0.05). In non-rhizosphere soil, the immobilized phytase treatment (I-Phy) significantly increased the number of unique ASVs (481, accounting for 42.6%), which was dozens of times higher than other treatments, indicating that immobilized phytase could strongly regulate the microbial community structure; biochar treatment (B) was the second most effective, indicating that biochar also had a certain regulatory effect. In contrast, free phytase (Phy) produced only one unique ASV in the rhizosphere, indicating that the regulatory effect of the unimmobilized enzyme was weaker. The immobilized phytase treatment (I-Phy) still showed the highest number of unique ASVs in the rhizosphere soil (463, accounting for 38.1%), but the proportion decreased, indicating that the rhizosphere microbial community was more stable and its response to intervention was weakened. In addition, the number of shared ASVs between rhizosphere and non-rhizosphere soil increased, suggesting that phytase enhanced the spatial uniformity of rhizosphere effects.

[0035] Distribution of bacteria at the phylum level in soybean non-rhizosphere and rhizosphere soils under different treatments Figure 10 The results showed that Proteobacteria and Actinobacteria were the dominant phyla, with a total relative abundance exceeding 50%. The abundance of Proteobacteria in rhizosphere soil was significantly higher than in non-rhizosphere soil, exhibiting a rhizosphere enrichment effect. Biochar (B) treatment significantly increased the abundance of Acidobacteria in the rhizosphere, while immobilized phytase treatment (I-Phy) significantly increased the abundance of Proteobacteria (including Pseudomonas and Rhizobium) and Actinobacteria in the rhizosphere, and these increases were positively correlated with soil available phosphorus content, indicating their key role in phosphorus promotion. The distribution of bacteria at the genus level in soybean non-rhizosphere and rhizosphere soils under different treatments (…) Figure 11 The results showed significant differences in soil microorganisms at the genus level under different treatments (P<0.05). *Mucilaginibacter*, *Bryobacter*, and *Cyanobacteria* were the core dominant genera, with a total abundance of 32.6%, playing crucial roles in soil structure stability, organic matter degradation, and carbon cycling. *Mucilaginibacter* was significantly enriched in the rhizosphere and influenced by chemotaxis from root exudates. Biochar treatment significantly increased the abundance of *Sphingomonas*, while I-Phy treatment further enriched this genus, and its activity was closely correlated with phytase activity, indicating its prominent function in organophosphate mineralization.

[0036] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing biochar-immobilized phytase, characterized in that, Includes the following steps: (1) Burn the collected pineapple leaves into biochar for later use; (2) Add pectin to water to obtain a pectin aqueous solution; add the biochar to the pectin aqueous solution, mix evenly, filter, wash and dry to obtain pectin biochar; (3) Add a cross-linking agent to the pectin biochar for cross-linking, then stir and mix with phytase solution, filter and freeze dry to obtain biochar immobilized phytase.

2. The method for preparing biochar-immobilized phytase as described in claim 1, characterized in that, The method for preparing biochar is as follows: clean the pineapple stems, dry them in an oven, crush them, and dry them again to constant weight; after sieving, heat the temperature to 300℃~400℃ for pyrolysis treatment, crush them, and then sieve them again to obtain the biochar.

3. The method for preparing biochar-immobilized phytase as described in claim 2, characterized in that, The pectin aqueous solution has a pectin to water mass-volume ratio of 1:5 g / mL.

4. The method for preparing biochar-immobilized phytase as described in claim 1, characterized in that, The mass ratio of pectin to biochar is 1:

1.

5. The method for preparing biochar-immobilized phytase as described in claim 1, characterized in that, Phytase was dissolved in an acetate buffer solution with a pH of 5.5 to obtain a phytase solution.

6. The method for preparing biochar-immobilized phytase as described in claim 5, characterized in that, The pectin biochar activation treatment involves dispersing the pectin biochar in a glutaraldehyde crosslinking agent solution and performing a shaker crosslinking treatment at room temperature.

7. The method for preparing biochar-immobilized phytase as described in claim 6, characterized in that, The mass ratio of pectin biochar to phytase is 100:

30.

8. A biochar-immobilized phytase, characterized in that, It is prepared using any one of the methods described in claims 1 to 7.

9. The application of biochar-immobilized phytase as described in claim 8, characterized in that, The biochar-immobilized phytase was used as a soil conditioner.

10. The application of biochar-immobilized phytase as described in claim 8, characterized in that, The biochar-immobilized phytase was used as a phosphate fertilizer enhancer.

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