Production process and application of nano saline-alkali cellulase

Abstract

The invention discloses a production process of nano saline-alkali cellulase, and relates to the technical field of biological fermentation and saline-alkali enzyme culture, and the production process comprises the following steps: culturing halorhodobryum broadcloth, carrying out culture and enzyme activity induction on a strain, pre-cooling, circulating for 2-4 times at 1500 bar, circulating for two minutes each time to obtain crude enzyme, and carrying out graded purification to obtain saline-alkali cellulase; then carrying out molecular domestication with thermal stability, high activity and pH value tolerance to obtain a nano saline-alkali cellulase strain; the obtained nano saline-alkali cellulase strain is subjected to enlarged culture to obtain fermentation liquor, and the fermentation liquor is concentrated and dried to obtain the nano saline-alkali cellulase, the enzyme activity number of the nano saline-alkali cellulase can reach 18000 u / g, the nano saline-alkali cellulase is verified to be remarkable in effect in field tests, large-scale popularization and industrialization promotion can be achieved, and the nano saline-alkali cellulase has good application prospects. The method is not only suitable for saline-alkali soil, but also suitable for desert soil, drought, low temperature and other adversity environments, and has wide stress-resistant potential.

Description

Technical Field

[0001] This invention relates to the fields of bio-fermentation and salt-alkali enzyme culture technology, specifically a production process and application of nano-salt-alkali cellulase. Background Technology

[0002] Traditional methods of soil improvement, such as salt washing and crop rotation, are costly and have limited effectiveness. Therefore, there is a need to research efficient and low-cost technologies for improving saline-alkali land. Nano-salt-alkali cellulase has broad application prospects in four fields: agriculture, industry, medicine, and biomaterials. In agriculture, it can be used to improve the germination rate, growth vitality, and salt tolerance of crops under salt stress, and alleviate cell membrane lipid peroxidation. In industry, it can be used to treat industrial wastewater with a salt content greater than 3.5%, overcoming the inhibition problems of conventional biological methods. In medicine, due to its stable photoelectric conversion properties, it can be used in biosensors and artificial vision devices. In biomaterials, it can be used to synthesize polyhydroxyalkali esters that degrade plastics.

[0003] However, existing nano-salt cellulases are generally liquid formulations, which present inconveniences in terms of transportation, storage, and use. Summary of the Invention

[0004] To address the above problems, the purpose of this invention is to provide a production process for nano-salt cellulase and its application.

[0005] To achieve the above objectives, the present invention employs the following technical solution: A process for producing nano-salt cellulase includes the following steps: S1. Prepare a nutrient medium, aseptically inoculate with Rhodotorula halophila, and then incubate at 28-32℃ for 28-32 hours to obtain the cultured Rhodotorula halophila for later use. S2. Strains culture and enzyme activity induction: Add 3-5% sodium chloride by weight to the broth peptone medium, add the cultured widely distributed halophyte, mix well, and culture with shaking at 30-37℃ for 12-24 hours. Use the halophilic host to induce the halophilic bacteria to secrete, and obtain a mixed bacterial solution. S3. Pre-cool the mixed bacterial solution to 3~5℃, and circulate it at 1500 bar 2~4 times, each cycle for two minutes, to obtain crude enzyme solution. Then, purify it by fractionation to obtain saline cellulase. S4. The saline cellulase obtained in step S3 is subjected to molecular domestication to improve its thermal stability, high activity and pH tolerance, so as to obtain nano saline cellulase strains. The basic domestication culture medium used in molecular domestication culture consists of the following components by weight: 4-6 parts silica, 8-12 parts beef extract, 34-38 parts sodium chloride, and 1000 parts distilled water. S5. The nano-salt cellulase strain obtained in step S4 is cultured in a large-scale manner to obtain a fermentation broth, which is then concentrated and dried to obtain nano-salt cellulase.

[0006] Preferably, the nutrient base is prepared according to the following steps: by weight, 4-6 parts of silicon dioxide, 8-12 parts of beef extract, and 34-38 parts of sodium chloride are added to 1000 parts of distilled water, stirred and mixed, and placed in an Erlenmeyer flask for sterilization at 121°C for 25-30 minutes to obtain a mixture. 0.5% of yeast extract powder by weight is then added to the mixture to obtain the nutrient base.

[0007] Preferably, the fractional purification includes the following steps: The crude enzyme solution was subjected to precipitation with 20% ammonium sulfate, dialysis to remove salt, DEAE-Sepharose FF ion exchange chromatography, Tris elution, and Superdex 200 gel filtration to obtain saline cellulase.

[0008] The purpose of fractional purification is to separate salt-alkali cellulase, salt-alkali catalase, and salt-alkali manganese catalase to obtain salt-alkali cellulase with high purity.

[0009] Preferably, step S4 specifically includes the following steps: S41. Thermal stability culture: Add the hydrochloric acid cellulase obtained in step S3 to the basic acclimatization medium, and gradually increase the temperature at 60℃ for continuous gradient culture, increasing the temperature by 5℃ each time and acclimatizing for 2 hours each time, for a total of 12 acclimatizations, so that the thermal stability of the hydrochloric acid cellulase reaches 110℃, and obtain a hydrochloric acid cellulase with high thermal stability for later use. S42. Salt-tolerant activity culture: The obtained heat-stable salt-alkali cellulase is cultured for another 30 hours. Sodium chloride is added to it until its mass concentration increases to 20%. It is cultured for 28-32 hours. Then, the mass concentration of sodium chloride is increased to 35% in 3 stages, and each culture is carried out for 28-32 hours to obtain highly active salt-alkali cellulase. S43. The obtained highly active salt-alkali cellulase was continuously cultured in a gradient at pH 8.5 for 11-13 hours each time. The pH was adjusted with sodium bicarbonate, increasing by 1 pH value each time, until the tolerance pH reached 10.5, thus obtaining the nano-salt-alkali cellulase strain.

[0010] Preferably, glucose is added during the entire salt tolerance culture of S42 at 70-75 hours of culture; the mass ratio of glucose to basic acclimatization medium is 1-3:100.

[0011] Preferably, the specific steps for expanding the culture of the nano-salt cellulase strain obtained in step S4 are as follows: Under aseptic conditions, the nano-salt cellulase strain was inoculated into the culture medium and cultured at 28-32℃ for 28-32 hours. Then it was transferred to a seed fermenter and cultured at 28-32℃ for 70-75 hours. Finally, it was transferred to a tertiary fermenter and cultured for 28-32 hours to obtain the cultured nano-salt cellulase strain solution. The culture medium for the strain is prepared by mixing silica, amino acids, glucose and water in a mass ratio of 8~12:18~22:4~6:1000; By weight, 100 parts of the cultured nano-salt cellulase inoculum were added to the reactor, along with 8-12 parts of silica, 18-22 parts of amino acids, 4-6 parts of glucose, and 900 parts of sterile water. The mixture was fermented at 28-32°C for 48-52 days, with the pH adjusted to 9-10.5 every 90 hours using sodium bicarbonate to obtain the fermentation broth.

[0012] Preferably, in step S5, a strain protection system is added to the fermentation broth before concentration and drying; the strain protection system consists of glycerol, DTT, and EDTA, wherein the mass ratio of glycerol, DTT, EDTA, and fermentation broth is 18~22: 1.4~1.5: 0.6~0.8: 100; this step can greatly extend the enzyme activity time of nano-salt cellulase, extending the enzyme activity time from 4 months to 24 months.

[0013] Preferably, the concentration and drying process includes the following steps: The fermentation broth is concentrated to a water content of 40-50% and then pumped to an atomizer at the top of a spray drying tower via a high-pressure pump. The atomized broth is dispersed into droplets. Clean, hot, filtered air enters the drying tower in a spiral pattern and comes into contact with the droplets in parallel flow, resulting in dried nano-salt cellulase. The inlet temperature of the spray dryer is 115-120℃, the pressure is 0.3-1MPa, and the velocity of the clean hot air is 4-6 m / s.

[0014] More preferably, the process also includes collecting and cooling the dried nano-salt cellulase. Specifically, the dried nano-salt cellulase descends with the airflow, with most of it being discharged from the bottom of the drying tower, and the remainder being collected by a cyclone separator.

[0015] The present invention also includes the application of nano-salt cellulase obtained by the above-described production process in the improvement of saline-alkali land.

[0016] The present invention has the following advantages over the prior art: This invention involves culturing a widespread halophilic bacterium strain, inducing halophilic bacteria to secrete halophilic enzymes using a halophilic host, isolating and purifying halophilic cellulase, and then subjecting it to molecular domestication to improve its thermal stability, high activity, and pH tolerance, resulting in a nano-sized halophilic cellulase strain. After expansion culture, concentration, and drying, the nano-sized halophilic cellulase is obtained. The nano-halophilic cellulase of this invention is a heat-resistant powder, transforming existing nano-halophilic cellulase formulations from liquid to powder solids, making transportation and storage more convenient and better preserving enzyme activity. The enzyme activity of the nano-halophilic cellulase of this invention can reach 18000 u / g.

[0017] The nano-salt cellulase of this invention has demonstrated significant effects in field trials and can be promoted on a large scale and industrialized. It is not only suitable for saline-alkali land, but also for desert soil, drought, low temperature and other adverse environments, and has broad stress resistance potential. Detailed Implementation

[0018] The purpose of this invention is to provide a production process and application of nano-salt cellulase, which is achieved through the following technical solution: In the buffer protection system, DTT is dithiothreitol and EDTA is ethylenediaminetetraacetic acid.

[0019] The widely distributed haloerythromycete of this invention is an archaea microorganism of the genus Haloerythromycete, with accession number JCM9100, purchased from a microbial culture preservation center.

[0020] The present invention uses a 30KD dialysis membrane or a 30KD dialysis bag for dialysis desalination, with an equivalent pore size of 1 to 5 nm and a permeability of 90%.

[0021] Properties of widely distributed halophyte Halomycetes salivarius is an archaea, not a true bacterium or eukaryote. It is mostly rod-shaped or spherical, Gram-negative, and requires a minimum salt concentration of 1.5 mol / L NaCl for growth, with an optimum of 20-25 wt%. It can grow in near-saturated saline environments. Some species have clustered flagella and are red, purple, or light brown in color. Due to the presence of carotenoids or rhodopsin, its optimal pH is around 8.0, and its optimal temperature is approximately 80℃. Using maltose and maltotriose as substrates, it remains stable in high-salt environments, driving the oxidation of nitrite to nitrate and participating in the nitrogen cycle. It can function continuously in extremely alkaline saline lakes with a pH of 9-10.2, maintaining nitrification processes under high pH conditions, preventing the accumulation and toxicity of nitrite, and scavenging superoxide free radicals produced in plants under stresses such as drought, salinity, and cold damage.

[0022] Salt-eating mechanism of widely distributed haloerythromycetes (1) Ion regulation strategy: Halothrum salicylate employs a "high potassium, low sodium" strategy to cope with osmotic pressure. Its intracellular potassium ion concentration can be up to 100 times that of the external environment, while the sodium ion concentration is much lower than that of the external environment, thereby preventing dehydration and maintaining metabolic activity.

[0023] (2) Cell wall stability depends on sodium ions: The cell wall is composed of glycoproteins rich in acidic amino acids, and its structural integrity depends on high concentrations of sodium ions to maintain ionic bonds. When the sodium ion concentration drops below 1.5 mol / L, the cell wall depolymerizes, leading to cell rupture and autolysis due to water absorption.

[0024] (3) Special membrane structure - purple membrane: Halothrum salicylate contains a purple membrane, and the bacterial rhodopsin within it can act as a light-driven proton pump, establishing a hydrogen ion gradient under light for ATP synthesis, thus providing an additional energy source.

[0025] (4) Salt-tolerant enzyme system: Its enzyme protein is rich in acidic amino acids and requires a high-salt environment to shield negative charges in order to maintain structural stability; once removed from high-salt conditions, the enzyme is easily inactivated.

[0026] (5) Other unique adaptation mechanisms Synthetic osmotic regulators, such as glycerol, sucrose, and trehalose, are small organic molecules that help balance osmotic pressure.

[0027] Gene-level regulation: The ACS gene family and mcrA gene are involved in salt tolerance regulation, enhancing the ability to respond to high salt stress.

[0028] Their nutritional requirements are complex: most require growth factors such as amino acids and vitamins, and they are difficult to survive in basal culture media.

[0029] Halothium salicum accumulates large amounts of potassium ions in its cells to counteract the external hypertonic environment and relies on high concentrations of sodium ions to maintain cell structural stability. Its unique purple membrane structure and salt-tolerant enzyme system enable it to survive at salinity levels of 15-30% or even saturation.

[0030] If the purchased Rhodotorula halophila is in solid form, a suspension needs to be prepared using 1-3% wt% sterile physiological saline; liquid samples can be centrifuged directly (2500-4000 r / min, 15 min) to remove impurities and retain the supernatant.

[0031] The mechanism of action of the nano-salt-alkali cellulase of the present invention in improving saline-alkali land is as follows: I. In-depth analysis of nanoscale penetration effect The core advantage of nano-salt-alkali cellulase lies in its nanoscale ultra-micro size. This scale allows it to penetrate into the microscopic fissures of salt crystals, which are inaccessible to ordinary enzymes. In experiments on the Shule saline-alkali land, cryo-electron microscopy revealed that the nanozyme could penetrate into the pores between soil particles at a depth of 50–200 nm, while traditional enzymes (particle size > 200 nm) could only act on the surface. A 2024 study published in *Nano Letters* showed that this modification increased the enzyme's diffusion coefficient in saline-alkali matrices to 5.8 times that of ordinary enzymes. This means that the nanozyme can act like a "molecular scout," rapidly penetrating deep into the soil to achieve three-dimensional salinization.

[0032] II. Molecular mechanisms of targeted salt ion dissociation The active center of nano-salt cellulase has a dual salt-solubilizing function: ① Electrostatic capture: The densely packed carboxyl groups on the enzyme surface act like "magnets," strongly adsorbing sodium ions through coordination bonds. X-ray absorption fine structure spectroscopy shows that the binding energy between a single carboxyl group and a sodium ion is as high as -28.6 kJ / mol, which is equivalent to three times the strength of an ordinary ionic bond.

[0033] ② Hydrolysis and Activation: The zinc ions at the active center activate water molecules, breaking them down into hydroxyl and hydrogen ions. These active groups act like "molecular scissors," decomposing NaCl crystals into soluble ions. Kinetic studies show that the reaction rate of this process is 17 times that of traditional halophilic enzymes. In an experiment in Dongying, Shandong, the sodium ion concentration in the soil surface decreased by 42% 48 hours after the application of nano-halophilic enzymes, demonstrating its highly efficient and immediate salt-reducing ability.

[0034] The entire process of salt biodegradation by nano-salt cellulase The role of nano-salt cellulase is not a simple biological reaction, but a multi-stage, multi-system synergistic engineering process.

[0035] Phase 1 (0-48 hours): When using nano-salt cellulase in soil with a salinity of 5%, the application rate is 500g per acre, and the salinity drops to 2.1% within 48 hours.

[0036] The second stage (48-96 hours): Applying 500g of nano-salt cellulase per acre easily reduces the salt concentration from 2.1% to 0.5%. Activated salt-tolerant bacteria (such as Halomonas) begin to proliferate, and their secreted extracellular polysaccharides form a biofilm, permanently solidifying free salt ions like "ecological cement." A 2025 study published in *The ISME Journal* confirmed that this process can reduce salt return by 83%.

[0037] IV. Breakthrough Improvement in Environmental Adaptability Compared with traditional halophilic enzymes, nano-salt cellulase has achieved three major breakthroughs in terms of stability in extreme environments: ① Temperature tolerance: It can maintain more than 90% activity in environments ranging from -50℃ frozen soil to 110℃ high temperature. Data from the Taklamakan Desert experimental station in 2025 showed that even when the surface temperature reached 65℃, the enzyme activity was still maintained at 97%.

[0038] ② Depth of action: With the help of the penetrating power of nano-sized particles, its effective depth of action can reach 1.5 meters underground, which is 5 times that of ordinary enzymes. This is of revolutionary significance for the treatment of deep salinization.

[0039] ③ Long-lasting efficacy: Through sustained-release microcapsule technology, the effective period of a single application can last up to 180 days. Comparative trials conducted by the Xinjiang Production and Construction Corps showed that the effect of nano-salt cellulase is more significant when used twice consecutively.

[0040] The present invention will be further described below with reference to specific embodiments. Example 1

[0041] A process for producing nano-salt cellulase includes the following steps: S1. Prepare a nutrient medium, aseptically inoculate with Rhodotorula halophila, and then incubate at 28°C for 32 hours to obtain the cultured Rhodotorula halophila for later use. The nutrient base is prepared according to the following steps: by weight, 4 parts of silicon dioxide, 8 parts of beef extract, and 34 parts of sodium chloride are added to 1000 parts of distilled water, stirred and mixed, and placed in an Erlenmeyer flask for sterilization at 121°C for 25 minutes to obtain a mixture. 0.5% of yeast extract powder by weight is added to the mixture to obtain the nutrient base. S2. Strains culture and enzyme activity induction: Add 3% sodium chloride by weight to broth peptone medium, add the cultured widely distributed halophilic bacteria, mix well, and culture with shaking at 30°C for 24 hours. Use the halophilic bacteria host to induce halophilic bacteria secretion to obtain a mixed bacterial solution. S3. Pre-cool the mixed bacterial solution to 3°C, and cycle it twice at 1500 bar for two minutes each time to obtain crude enzyme solution. Then, purify it by fractionation to obtain saline cellulase. The fractional purification includes the following steps: The crude enzyme solution was subjected to precipitation with 20% ammonium sulfate, dialysis to remove salt, DEAE-Sepharose FF ion exchange chromatography, Tris elution, and Superdex 200 gel filtration to obtain saline cellulase. S4. The saline cellulase obtained in step S3 is subjected to molecular domestication to improve its thermal stability, high activity, and pH tolerance, resulting in a nano-salt-alkali cellulase strain. The basic acclimatization culture medium used in molecular acclimatization culture consists of the following components by weight: 4 parts silica, 8 parts beef extract, 34 parts sodium chloride and 1000 parts distilled water. Specifically, the following steps are included: S41. Thermal stability culture: Add the hydrochloric acid cellulase obtained in step S3 to the basic acclimatization medium, and gradually increase the temperature at 60℃ for continuous gradient culture, increasing the temperature by 5℃ each time and acclimatizing for 2 hours each time, for a total of 12 acclimatizations, so that the thermal stability of the hydrochloric acid cellulase reaches 110℃, and obtain a hydrochloric acid cellulase with high thermal stability for later use. S42. Salt-tolerant activity culture: The obtained high-thermal-stability salt-alkali cellulase was cultured for another 30 hours. Sodium chloride was added to it until its mass concentration increased to 20%, and it was cultured for 28 hours. Then, the mass concentration of sodium chloride was increased to 35% in 3 batches, and each batch was cultured for 28 hours to obtain highly active salt-alkali cellulase. S43. The obtained highly active salt-alkali cellulase was continuously cultured in a gradient at pH 8.5 for 11 hours each time. The pH was adjusted with sodium bicarbonate, increasing by 1 pH value each time, until the tolerance pH reached 10.5, thus obtaining the nano-salt-alkali cellulase strain. S5. The nano-salt cellulase strain obtained in step S4 is expanded and cultured to obtain fermentation broth, which is then concentrated and dried to obtain nano-salt cellulase. The specific steps for expanding the culture of the nano-salt cellulase strain obtained in step S4 are as follows: Under aseptic conditions, the nano-salt cellulase strain was inoculated into the culture medium and cultured at 28°C for 32 hours. Then it was transferred to a seed fermenter and cultured at 28°C for 75 hours. Finally, it was transferred to a tertiary fermenter and cultured for 28 hours to obtain the cultured nano-salt cellulase strain solution. The culture medium for the strain is prepared by mixing silica, amino acids, glucose and water in a mass ratio of 8:18:4:1000. By weight, 100 parts of the cultured nano-salt cellulase inoculum were added to the reaction vessel, along with 8 parts of silica, 18 parts of amino acids, 4 parts of glucose, and 900 parts of sterile water. The mixture was fermented at 28°C for 52 days, with the pH adjusted to 9 every 90 hours using sodium bicarbonate to obtain the fermentation broth. The concentration and drying process includes the following steps: The fermentation broth is concentrated to a water content of 40% and then pumped to an atomizer at the top of a spray drying tower via a high-pressure pump. The atomized broth is dispersed into droplets. Filtered and heated clean hot air enters the drying tower in a spiral pattern and comes into contact with the droplets in parallel flow, resulting in dried nano-salt cellulase. The dried nano-salt cellulase descends with the airflow, with most of it being discharged from the bottom of the drying tower, and the remainder being collected by a cyclone separator. The inlet temperature of the spray dryer is 115℃, the pressure is 0.3MPa, and the velocity of the clean hot air is 4 m / s. Example 2

[0042] A process for producing nano-salt cellulase includes the following steps: S1. Prepare a nutrient medium, aseptically inoculate the widely distributed haloerythromycete, and then incubate at 32℃ for 28 hours to obtain the cultured widely distributed haloerythromycete for later use. The nutrient base is prepared according to the following steps: by weight, 6 parts of silicon dioxide, 12 parts of beef extract, and 38 parts of sodium chloride are added to 1000 parts of distilled water, stirred and mixed, and placed in an Erlenmeyer flask for sterilization at 121°C for 30 minutes to obtain a mixture. 0.5% of yeast extract powder by weight is added to the mixture to obtain the nutrient base. S2. Strains culture and enzyme activity induction: Add 5% sodium chloride by weight to broth peptone medium, add the cultured widely distributed halophilic bacteria, mix well, and culture with shaking at 37°C for 12 hours. Use the halophilic bacteria host to induce halophilic bacteria secretion to obtain a mixed bacterial solution. S3. Pre-cool the mixed bacterial solution to 3°C, and circulate it 4 times at 1500 bar for 2 minutes each time to obtain crude enzyme solution. Then, purify it by fractionation to obtain saline cellulase. The fractional purification includes the following steps: The crude enzyme solution was subjected to precipitation with 20% ammonium sulfate, dialysis to remove salt, DEAE-Sepharose FF ion exchange chromatography, Tris elution, and Superdex 200 gel filtration to obtain saline cellulase. S4. The saline cellulase obtained in step S3 is subjected to molecular domestication to improve its thermal stability, high activity, and pH tolerance, resulting in a nano-salt-alkali cellulase strain. The basic acclimatization culture medium used in molecular acclimatization culture consists of the following components by weight: 6 parts silica, 12 parts beef extract, 38 parts sodium chloride and 1000 parts distilled water. Specifically, the following steps are included: S41. Thermal stability culture: Add the hydrochloric acid cellulase obtained in step S3 to the basic acclimatization medium, and gradually increase the temperature at 60℃ for continuous gradient culture, increasing the temperature by 5℃ each time and acclimatizing for 2 hours each time, for a total of 12 acclimatizations, so that the thermal stability of the hydrochloric acid cellulase reaches 110℃, and obtain a hydrochloric acid cellulase with high thermal stability for later use. S42. Salt-tolerant activity culture: The obtained heat-stable salt-alkali cellulase was cultured for another 30 hours. Sodium chloride was added to it until its mass concentration increased to 20%, and it was cultured for 32 hours. Then, the mass concentration of sodium chloride was increased to 35% in 3 batches, and each batch was cultured for 32 hours to obtain highly active salt-alkali cellulase. S43. The obtained highly active salt-alkali cellulase was continuously cultured in a gradient at pH 8.5 for 13 hours each time. The pH was adjusted with sodium bicarbonate, increasing by 1 pH value each time, until the tolerance pH reached 10.5, thus obtaining the nano-salt-alkali cellulase strain. S5. The nano-salt cellulase strain obtained in step S4 is expanded and cultured to obtain fermentation broth, which is then concentrated and dried to obtain nano-salt cellulase. The specific steps for expanding the culture of the nano-salt cellulase strain obtained in step S4 are as follows: Under sterile conditions, the nano-salt cellulase strain was inoculated into the culture medium and cultured at 32°C for 28 hours. Then it was transferred to a seed fermenter and cultured at 32°C for 70 hours. Finally, it was transferred to a tertiary fermenter and cultured for 32 hours to obtain the cultured nano-salt cellulase strain solution. The culture medium for the strain is prepared by mixing silica, amino acids, glucose and water in a mass ratio of 12:22:6:1000. By weight, 100 parts of the cultured nano-salt cellulase inoculum were added to the reaction vessel, along with 12 parts of silica, 22 parts of amino acids, 6 parts of glucose, and 900 parts of sterile water. The mixture was fermented at 32°C for 48 days, with the pH adjusted to 10.5 every 90 hours using sodium bicarbonate to obtain the fermentation broth. The concentration and drying process includes the following steps: The fermentation broth is concentrated to a water content of 50% and then pumped to an atomizer at the top of a spray drying tower via a high-pressure pump. The atomized broth is dispersed into droplets. Filtered and heated clean hot air enters the drying tower in a spiral pattern and comes into contact with the droplets in parallel flow, resulting in dried nano-salt cellulase. The dried nano-salt cellulase descends with the airflow, with most of it being discharged from the bottom of the drying tower, and the remainder being collected by a cyclone separator. The inlet temperature of the spray dryer is 120℃, the pressure is 1MPa, and the velocity of the clean hot air is 6 m / s. Example 3

[0043] A process for producing nano-salt cellulase includes the following steps: S1. Prepare a nutrient medium, aseptically inoculate with Rhodotorula salina, and then incubate at 29°C for 31 hours to obtain the cultured Rhodotorula salina for later use. The nutrient base is prepared according to the following steps: by weight, 4.5 parts of silicon dioxide, 9 parts of beef extract, and 35 parts of sodium chloride are added to 1000 parts of distilled water, stirred and mixed, and placed in an Erlenmeyer flask for sterilization at 121°C for 26 minutes to obtain a mixture. 0.5% of yeast extract powder by weight is added to the mixture to obtain the nutrient base. S2. Strains culture and enzyme activity induction: Add 5% sodium chloride by weight to broth peptone medium, add the cultured widely distributed halophilic bacteria, mix well, and culture with shaking at 32°C for 20 hours. Use the halophilic bacteria host to induce halophilic bacteria secretion to obtain a mixed bacterial solution. S3. Pre-cool the mixed bacterial solution to 4°C, circulate it three times at 1500 bar for two minutes each time to obtain crude enzyme solution, and then purify it by fractionation to obtain saline cellulase. The fractional purification includes the following steps: The crude enzyme solution was subjected to precipitation with 20% ammonium sulfate, dialysis to remove salt, DEAE-Sepharose FF ion exchange chromatography, Tris elution, and Superdex 200 gel filtration to obtain saline cellulase. S4. The hydrochloric acid cellulase obtained in step S3 is subjected to molecular domestication to improve its thermal stability, high activity and pH tolerance, and nano-hydrochloric acid cellulase strain is obtained. The basic domestication culture medium used in the molecular domestication culture consists of the following components by weight: 4.5 parts silica, 9 parts beef extract, 35 parts sodium chloride and 1000 parts distilled water. Specifically, the following steps are included: S41. Thermal stability culture: Add the hydrochloric acid cellulase obtained in step S3 to the basic acclimatization medium, and gradually increase the temperature at 60℃ for continuous gradient culture, increasing the temperature by 5℃ each time and acclimatizing for 2 hours each time, for a total of 12 acclimatizations, so that the thermal stability of the hydrochloric acid cellulase reaches 110℃, and obtain a hydrochloric acid cellulase with high thermal stability for later use. S42. Salt-tolerant activity culture: The obtained heat-stable salt-alkali cellulase was cultured for another 30 hours. Sodium chloride was added to it until its mass concentration increased to 20%. It was cultured for 29 hours. Then, the mass concentration of sodium chloride was increased to 35% in 3 batches, and each batch was cultured for 29 hours to obtain highly active salt-alkali cellulase. Glucose was added after 70 hours of culture; the mass ratio of glucose to basal acclimatization medium was 1:100.

[0044] S43. The obtained highly active salt-alkali cellulase was continuously cultured in a gradient at pH 8.5 for 12 hours each time. The pH was adjusted with sodium bicarbonate, increasing by 1 pH value each time, until the tolerance pH reached 10.5, thus obtaining the nano-salt-alkali cellulase strain. S5. The nano-salt cellulase strain obtained in step S4 is expanded and cultured to obtain fermentation broth, which is then concentrated and dried to obtain nano-salt cellulase. The specific steps for expanding the culture of the nano-salt cellulase strain obtained in step S4 are as follows: Under aseptic conditions, the nano-salt cellulase strain was inoculated into the culture medium and cultured at 29°C for 30 hours. Then it was transferred to a seed fermenter and cultured at 29°C for 72 hours. Finally, it was transferred to a tertiary fermenter and cultured for 30 hours to obtain the cultured nano-salt cellulase strain solution. The culture medium for the strain was prepared by mixing silica, amino acids, glucose and water in a mass ratio of 9:19:4.5:1000. By weight, 100 parts of the cultured nano-salt cellulase inoculum were added to the reactor, along with 9 parts of silica, 19 parts of amino acids, 4.5 parts of glucose, and 900 parts of sterile water. The mixture was fermented at 28-32°C for 49 days, with the pH adjusted to 9.5 every 90 hours using sodium bicarbonate to obtain the fermentation broth. The concentration and drying process includes the following steps: The fermentation broth is concentrated to a water content of 45% and then pumped through a high-pressure pump to an atomizer at the top of a spray drying tower, where it is dispersed into mist droplets. Filtered and heated clean hot air enters the drying tower in a spiral pattern and comes into contact with the mist droplets in parallel flow, resulting in dried nano-salt cellulase. The dried nano-salt cellulase descends with the airflow, with most of it being discharged from the bottom of the drying tower, and the remainder being collected by a cyclone separator. The inlet temperature of the spray dryer is 116℃, the pressure is 0.5MPa, and the velocity of the clean hot air is 4.5 m / s. Example 4

[0045] A process for producing nano-salt cellulase includes the following steps: S1. Prepare a nutrient medium, aseptically inoculate the widely distributed haloerythromycete, and then incubate at 30℃ for 29 hours to obtain the cultured widely distributed haloerythromycete for later use. The nutrient base is prepared according to the following steps: by weight, 5.5 parts of silica, 9 parts of beef extract, and 35 parts of sodium chloride are added to 1000 parts of distilled water, stirred and mixed, and placed in an Erlenmeyer flask for sterilization at 121°C for 28 minutes to obtain a mixture. 0.5% of yeast extract powder by weight is added to the mixture to obtain the nutrient base. S2. Strains culture and enzyme activity induction: Add 3.5% sodium chloride by weight to the broth peptone medium, add the cultured widely distributed halophilic bacteria, mix well, and culture with shaking at 32°C for 15 hours. Use the halophilic bacteria host to induce halophilic bacteria secretion to obtain a mixed bacterial solution. S3. Pre-cool the mixed bacterial solution to 4°C, and circulate it three times at 1500 bar for two minutes each time to obtain crude enzyme solution. Then, purify it by fractionation to obtain saline cellulase. The fractional purification includes the following steps: The crude enzyme solution was subjected to precipitation with 20% ammonium sulfate, dialysis to remove salt, DEAE-Sepharose FF ion exchange chromatography, Tris elution, and Superdex 200 gel filtration to obtain saline cellulase. S4. The saline cellulase obtained in step S3 is subjected to molecular domestication to improve its thermal stability, high activity, and pH tolerance, resulting in a nano-salt-alkali cellulase strain. The basic domestication culture medium used during molecular domestication culture consists of the following components by weight: 5.5 parts silica, 11 parts beef extract, 37 parts sodium chloride, and 1000 parts distilled water; specifically including the following steps: S41. Thermal stability culture: Add the hydrochloric acid cellulase obtained in step S3 to the basic acclimatization medium, and gradually increase the temperature at 60℃ for continuous gradient culture, increasing the temperature by 5℃ each time and acclimatizing for 2 hours each time, for a total of 12 acclimatizations, so that the thermal stability of the hydrochloric acid cellulase reaches 110℃, and obtain a hydrochloric acid cellulase with high thermal stability for later use. S42. Salt-tolerant activity culture: The obtained high-thermal-stability salt-alkali cellulase was cultured for another 30 hours. Sodium chloride was added to it until its mass concentration increased to 20%, and it was cultured for another 30 hours. Then, the mass concentration of sodium chloride was increased to 35% in three batches, and each batch was cultured for 30 hours to obtain highly active salt-alkali cellulase. Glucose was added after 75 hours of incubation; the mass ratio of glucose to the basal acclimatization medium was 3:100. S43. The obtained highly active salt-alkali cellulase was continuously cultured in a gradient at pH 8.5 for 11.5 hours each time. The pH was adjusted with sodium bicarbonate, increasing by 1 pH value each time, until the tolerance pH reached 10.5, thus obtaining the nano-salt-alkali cellulase strain. S5. The nano-salt cellulase strain obtained in step S4 is expanded and cultured to obtain fermentation broth, which is then concentrated and dried to obtain nano-salt cellulase. The specific steps for expanding the culture of the nano-salt cellulase strain obtained in step S4 are as follows: Under sterile conditions, the nano-salt cellulase strain was inoculated into the culture medium and cultured at 30°C for 30 hours. Then it was transferred to a seed fermenter and cultured at 30°C for 74 hours. Finally, it was transferred to a tertiary fermenter and cultured for 29 hours to obtain the cultured nano-salt cellulase strain solution. The culture medium for the strain is prepared by mixing silica, amino acids, glucose and water in a mass ratio of 11:19:5.5:1000. By weight, 100 parts of the cultured nano-salt cellulase inoculum were added to the reactor, along with 9 parts of silica, 19 parts of amino acids, 5.5 parts of glucose and 900 parts of sterile water. The mixture was fermented at 28-32°C for 48-52 days, with the pH adjusted to 10 every 90 hours using sodium bicarbonate to obtain the fermentation broth. The concentration and drying process includes the following steps: The fermentation broth was concentrated to a water content of 42% and then pumped to an atomizer at the top of a spray drying tower via a high-pressure pump. The atomized broth was dispersed into droplets. Filtered and heated clean hot air entered the drying tower in a spiral pattern and came into contact with the droplets in parallel flow, resulting in dried nano-salt cellulase. The dried nano-salt cellulase descended with the airflow, with most of it being discharged from the bottom of the drying tower, and the remainder being collected by a cyclone separator. The inlet temperature of the spray dryer was 118℃, the pressure was 0.6MPa, and the velocity of the clean hot air was 5.5 m / s. Example 5

[0046] A process for producing nano-salt cellulase includes the following steps: S1. Prepare a nutrient medium, aseptically inoculate the widespread haloerythromycete, and then incubate at 29°C for 29 hours to obtain the cultured widespread haloerythromycete for later use. The nutrient base is prepared according to the following steps: by weight, 4.5 parts of silicon dioxide, 9 parts of beef extract, and 36 parts of sodium chloride are added to 1000 parts of distilled water, stirred and mixed, and placed in an Erlenmeyer flask for sterilization at 121°C for 28 minutes to obtain a mixture. 0.5% of yeast extract powder by weight is added to the mixture to obtain the nutrient base. S2. Strains culture and enzyme activity induction: Add 3% sodium chloride by weight to broth peptone medium, add the cultured widely distributed halophilic bacteria, mix well, and culture with shaking at 32°C for 16 hours. Use the halophilic bacteria host to induce halophilic bacteria secretion to obtain a mixed bacterial solution. S3. Pre-cool the mixed bacterial solution to 4°C, and circulate it three times at 1500 bar for two minutes each time to obtain crude enzyme solution. Then, purify it by fractionation to obtain saline cellulase. The fractional purification includes the following steps: The crude enzyme solution was subjected to precipitation with 20% ammonium sulfate, dialysis to remove salt, DEAE-Sepharose FF ion exchange chromatography, Tris elution, and Superdex 200 gel filtration to obtain saline cellulase. S4. The hydrochloric acid cellulase obtained in step S3 is subjected to molecular domestication to improve its thermal stability, high activity, and pH tolerance, resulting in a nano-hydrochloric acid cellulase strain. The basic domestication culture medium used during molecular domestication culture consists of the following components by weight: 5 parts silica, 10 parts beef extract, 36 parts sodium chloride, and 1000 parts distilled water; specifically, the following steps are included: S41. Thermal stability culture: Add the hydrochloric acid cellulase obtained in step S3 to the basic acclimatization medium, and gradually increase the temperature at 60℃ for continuous gradient culture, increasing the temperature by 5℃ each time and acclimatizing for 2 hours each time, for a total of 12 acclimatizations, so that the thermal stability of the hydrochloric acid cellulase reaches 110℃, and obtain a hydrochloric acid cellulase with high thermal stability for later use. S42. Salt-tolerant activity culture: The obtained high-thermal-stability salt-alkali cellulase was cultured for another 30 hours. Sodium chloride was added to it until its mass concentration increased to 20%, and it was cultured for another 30 hours. Then, the mass concentration of sodium chloride was increased to 35% in three batches, and each batch was cultured for 30 hours to obtain highly active salt-alkali cellulase. Glucose was added after 72 hours of incubation; the mass ratio of glucose to basal acclimatization medium was 1.5:100. S43. The obtained highly active salt-alkali cellulase was continuously cultured in a gradient at pH 8.5 for 12.5 hours each time. The pH was adjusted with sodium bicarbonate, increasing by 1 pH value each time, until the tolerance pH reached 10.5, thus obtaining the nano-salt-alkali cellulase strain. S5. The nano-salt cellulase strain obtained in step S4 is expanded and cultured to obtain fermentation broth, which is then concentrated and dried to obtain nano-salt cellulase. The specific steps for expanding the culture of the nano-salt cellulase strain obtained in step S4 are as follows: Under aseptic conditions, the nano-salt cellulase strain was inoculated into the culture medium and cultured at 30°C for 30 hours. Then it was transferred to a seed fermenter and cultured at 30°C for 72 hours. Finally, it was transferred to a tertiary fermenter and cultured for 30 hours to obtain the cultured nano-salt cellulase strain solution. The culture medium for the strain is prepared by mixing silica, amino acids, glucose and water in a mass ratio of 10:20:5:1000. By weight, 100 parts of the cultured nano-salt cellulase inoculum were added to the reaction vessel, along with 10 parts of silica, 20 parts of amino acids, 5 parts of glucose, and 900 parts of sterile water. The mixture was fermented at 28°C for 50 days, with the pH adjusted to 9.5 every 90 hours using sodium bicarbonate to obtain the fermentation broth. The concentration and drying process includes the following steps: The fermentation broth is concentrated to a water content of 40% and then pumped through a high-pressure pump to an atomizer at the top of a spray drying tower, where it is dispersed into mist droplets. Filtered and heated clean hot air enters the drying tower in a spiral pattern and comes into contact with the mist droplets in parallel flow, resulting in dried nano-salt cellulase. The dried nano-salt cellulase descends with the airflow, with most of it being discharged from the bottom of the drying tower, and the remainder being collected by a cyclone separator. The inlet temperature of the spray dryer is 118℃, the pressure is 0.5MPa, and the velocity of the clean hot air is 5 m / s.

[0047] The activity of the widely distributed halophyte Rhodophyte after S1 culture in Examples 1-5 of this invention was tested and found to be approximately 20 billion / g.

[0048] To prolong the enzyme activity time of nano-salt cellulase, the following operation can be performed in step 5 of Examples 1-5: a strain protection system is added to the fermentation broth before concentration and drying; the strain protection system consists of glycerol, DTT and EDTA, wherein the mass ratio of glycerol, DTT, EDTA and fermentation broth is 18-22:1.4-1.5:0.6-0.8:100; preferably, the mass ratio of glycerol, DTT, EDTA and fermentation broth is 20:1.45:0.73:100; The nano-salt-alkali cellulase obtained in this invention was used to improve saline-alkali land. The initial application rate was 400-500 g / mu, and the application rate after half a month was 300-400 g / mu. The salinity degradation content of the soil was observed, and the crop emergence rate was tracked.

[0049] The nano-salt-alkali cellulase obtained in this invention was used to improve the saline-alkali land in Shule. The original soil data for the experimental field were: salt content 1.28%, pH 7.69. On June 23rd, 400g of nano-salt-alkali cellulase was applied per mu (approximately 0.067 hectares); on July 7th, 300g / mu of nano-salt-alkali cellulase was applied. After improvement, the emergence rate of this year's crop (millet) in the experimental field reached 100%, the soil salt content decreased to 0.40%, and the pH value was 7.35. This saline-alkali land, classified as moderately saline-alkali soil, had been abandoned for many years. After improvement, millet was planted, and the yield was 333.3 kg / mu.

Claims

1. A production process for nano-salt cellulase, characterized in that: Includes the following steps: S1. Prepare a nutrient medium, aseptically inoculate with Rhodotorula halophila, and then incubate at 28-32℃ for 28-32 hours to obtain the cultured Rhodotorula halophila for later use. S2. Strains culture and enzyme activity induction: Add 3-5% sodium chloride by weight to the broth peptone medium, add the cultured widely distributed halophyte, mix well, and culture with shaking at 30-37℃ for 12-24 hours. Use the halophilic host to induce the halophilic bacteria to secrete, and obtain a mixed bacterial solution. S3. Pre-cool the mixed bacterial solution to 3~5℃, and circulate it at 1500 bar 2~4 times, each cycle for two minutes, to obtain crude enzyme solution. Then, purify it by fractionation to obtain saline cellulase. S4. The saline cellulase obtained in step S3 is subjected to molecular domestication culture to improve its thermal stability, high activity and pH tolerance, so as to obtain nano saline cellulase strain. The basic domestication culture medium used in molecular domestication culture consists of the following components by weight: 4-6 parts silica, 8-12 parts beef extract, 34-38 parts sodium chloride, and 1000 parts distilled water. S5. The nano-salt cellulase strain obtained in step S4 is cultured in a large-scale manner to obtain a fermentation broth, which is then concentrated and dried to obtain nano-salt cellulase.

2. The production process of nano-salt cellulase according to claim 1, characterized in that: The nutrient base is prepared according to the following steps: by weight, 4-6 parts of silicon dioxide, 8-12 parts of beef extract, and 34-38 parts of sodium chloride are added to 1000 parts of distilled water, stirred and mixed, and placed in an Erlenmeyer flask for sterilization at 121°C for 25-30 minutes to obtain a mixture. 0.5% of yeast extract powder by weight is then added to the mixture to obtain the nutrient base.

3. The production process of nano-salt cellulase according to claim 1, characterized in that: The fractional purification includes the following steps: The crude enzyme solution was subjected to precipitation with 20% ammonium sulfate, dialysis to remove salt, DEAE-Sepharose FF ion exchange chromatography, Tris elution, and Superdex 200 gel filtration to obtain saline cellulase.

4. The production process of nano-salt cellulase according to claim 1, characterized in that: Step S4 specifically includes the following steps: S41. Thermal stability culture: Add the hydrochloric acid cellulase obtained in step S3 to the basic acclimatization medium, and gradually increase the temperature at 60℃ for continuous gradient culture, increasing the temperature by 5℃ each time and acclimatizing for 2 hours each time, for a total of 12 acclimatizations, so that the thermal stability of the hydrochloric acid cellulase reaches 110℃, and obtain a hydrochloric acid cellulase with high thermal stability for later use. S42. Salt-tolerant activity culture: The obtained heat-stable salt-alkali cellulase is cultured for another 30 hours. Sodium chloride is added to it until its mass concentration increases to 20%. It is cultured for 28-32 hours. Then, the mass concentration of sodium chloride is increased to 35% in 3 stages, and each culture is carried out for 28-32 hours to obtain highly active salt-alkali cellulase. S43. The obtained highly active salt-alkali cellulase was continuously cultured in a gradient at pH 8.5 for 11-13 hours each time. The pH was adjusted with sodium bicarbonate, increasing by 1 pH value each time, until the tolerance pH reached 10.5, thus obtaining the nano-salt-alkali cellulase strain.

5. The production process of nano-salt cellulase according to claim 1, characterized in that: During the entire salt tolerance culture of S42, glucose was added after 70-75 hours of culture; the mass ratio of glucose to basic acclimatization medium was 1-3:

100.

6. The production process of nano-salt cellulase according to claim 1, characterized in that: The specific steps for expanding the culture of the nano-salt cellulase strain obtained in step S4 are as follows: Under aseptic conditions, the nano-salt cellulase strain was inoculated into the culture medium and cultured at 28-32℃ for 28-32 hours. Then it was transferred to a seed fermenter and cultured at 28-32℃ for 70-75 hours. Finally, it was transferred to a tertiary fermenter and cultured for 28-32 hours to obtain the cultured nano-salt cellulase strain solution. The culture medium for the strain is prepared by mixing silica, amino acids, glucose and water in a mass ratio of 8~12:18~22:4~6:1000; By weight, 100 parts of the cultured nano-salt cellulase inoculum were added to the reactor, along with 8-12 parts of silica, 18-22 parts of amino acids, 4-6 parts of glucose, and 900 parts of sterile water. The mixture was fermented at 28-32°C for 48-52 days, with the pH adjusted to 9-10.5 every 90 hours using sodium bicarbonate to obtain the fermentation broth.

7. The production process of nano-salt cellulase according to claim 1, characterized in that: Before concentration and drying, the fermentation broth in step S5 is added to a microbial protection system; the microbial protection system consists of glycerol, DTT and EDTA, wherein the mass ratio of glycerol, DTT, EDTA and fermentation broth is 18~22:1.4~1.5:0.6~0.8:

100.

8. The production process of nano-salt cellulase according to claim 1, characterized in that: The concentration and drying process includes the following steps: The fermentation broth is concentrated to a water content of 40-50% and then pumped to an atomizer at the top of a spray drying tower via a high-pressure pump. The atomized broth is dispersed into droplets. Clean, hot, filtered air enters the drying tower in a spiral pattern and comes into contact with the droplets in parallel flow, resulting in dried nano-salt cellulase. The inlet temperature of the spray dryer is 115-120℃, the pressure is 0.3-1MPa, and the velocity of the clean hot air is 4-6 m / s.

9. The production process of nano-salt cellulase according to claim 8, characterized in that: It also includes the collection and cooling of the dried nano-salt cellulase. Specifically, the dried nano-salt cellulase descends with the airflow, with most of it being discharged from the bottom of the drying tower, and the remainder being collected by a cyclone separator.

10. The application of the nano-salt cellulase obtained by the production process described in claim 1, characterized in that: Used for the improvement of saline-alkali land.