CN-122012469-A - Production process and application of nano saline-alkali cellulase

CN122012469ACN 122012469 ACN122012469 ACN 122012469ACN-122012469-A

Abstract

The invention discloses a production process of nano saline-alkali cellulase, which relates to the technical field of biological fermentation and saline-alkali enzyme culture, and comprises the following steps of culturing rhodobacter salsa, culturing bacterial strains, inducing enzyme activity, precooling, circulating for 2-4 times at 1500bar for two minutes each time to obtain crude enzyme liquid, purifying in a grading manner to obtain the saline-alkali cellulase, carrying out molecular domestication with high heat stability, high activity and pH value tolerance to obtain nano saline-alkali cellulase strain, carrying out enlarged culture on the obtained nano saline-alkali cellulase strain to obtain fermentation liquor, concentrating and drying to obtain the nano saline-alkali cellulase.

Inventors

LI XIAOYE
YANG ZHIJUN
HUANG GUANGHUI
QIU TAOYU
XU JIANGUO
DU JINMIN
LI HONGYE
SUN GUIXIANG
BU DONGSHENG
WANG PANPAN

Assignees

河北著微生物科技有限公司

Dates

Publication Date: 20260512
Application Date: 20260213

Claims (10)

1. A production process of nano-saline-alkali cellulase is characterized by comprising the following steps: s1, preparing a nutrient medium, carrying out aseptic inoculation on the rhodobacter salsa, and then culturing for 28-32 hours at 28-32 ℃ to obtain the cultured rhodobacter salsa for later use; S2, strain culture and enzyme activity induction, namely adding sodium chloride with the mass of 3-5% into a broth peptone culture medium, adding the cultured rhodobacter salsa, uniformly mixing, carrying out shake culture for 12-24 hours at the temperature of 30-37 ℃, and inducing the secretion of halophage by utilizing a halophage host to obtain a mixed bacterial solution; s3, pre-cooling the mixed bacterial liquid to 3-5 ℃, circulating for 2-4 times at 1500bar for two minutes each time to obtain crude enzyme liquid, and carrying out fractional purification to obtain saline-alkali cellulase; S4, performing molecular domestication culture on the saline-alkali cellulase obtained in the step S3, wherein the molecular domestication culture is high in thermal stability and activity and resistant to pH value, so as to obtain nano saline-alkali cellulase strains; the basic domestication culture medium adopted in the molecular domestication culture consists of, by weight, 4-6 parts of silicon dioxide, 8-12 parts of beef extract, 34-38 parts of sodium chloride and 1000 parts of distilled water; and S5, performing enlarged culture on the nano saline-alkali cellulase strain obtained in the step S4 to obtain fermentation liquor, concentrating and drying to obtain the nano saline-alkali cellulase.
2. The production process of the nano saline-alkali cellulase is characterized in that the nutrient medium is prepared by adding 4-6 parts by weight of silicon dioxide, 8-12 parts by weight of beef extract and 34-38 parts by weight of sodium chloride into 1000 parts by weight of distilled water, stirring and mixing uniformly, placing a triangular flask and sterilizing at 121 ℃ for 25-30 minutes to obtain a mixed solution, and adding yeast extract with the mass of 0.5% into the mixed solution to obtain the nutrient medium.
3. The process for producing nano-grade saline-alkali cellulose according to claim 1, wherein the step of classifying and purifying comprises the following steps: And (3) carrying out ammonium sulfate precipitation, dialysis desalting, DEAE-Sepharose FF ion exchange chromatography, tris elution and Superdex 200 gel filtration on the crude enzyme solution with the mass concentration of 20% to obtain the saline-alkali cellulase.
4. The production process of the nano-saline-alkali cellulase according to claim 1, wherein the step S4 specifically comprises the following steps: S41, performing heat stability culture, namely adding the saline-alkali cellulase obtained in the step S3 into a basic domestication culture medium, gradually increasing the temperature on the basis of 60 ℃ for continuous gradient culture, wherein the temperature is increased by 5 ℃ each time, and each time of heat resistance domestication is performed for 2 hours, and 12 times of heat resistance domestication are performed continuously, so that the heat resistance value of the saline-alkali cellulase reaches 110 ℃, and the saline-alkali cellulase with high heat stability is obtained for standby; S42, salt-tolerant activity culture, namely continuously culturing the obtained high-heat-stability saline-alkali cellulase for 30 hours, adding sodium chloride into the saline-alkali cellulase until the mass concentration of the saline-alkali cellulase is increased to 20%, culturing for 28-32 hours, and then dividing the saline-alkali cellulase into 3 echelons, and increasing the mass concentration of the sodium chloride to 35%, wherein the saline-alkali cellulase is cultured for 28-32 hours each time to obtain the high-activity saline-alkali cellulase; s43, continuously carrying out gradient culture on the high-activity saline-alkali cellulase on the basis of pH value of 8.5, regulating the pH value by sodium bicarbonate for 11-13 hours each time, and increasing the pH value by 1 time each time to enable the tolerance pH value to reach 10.5, so as to obtain the nano saline-alkali cellulase strain.
5. The production process of the nano saline-alkali cellulase is characterized in that glucose is added in the whole salt-tolerant activity culture of S42 at the time of 70-75 hours of culture, and the mass ratio of the glucose to a basic domestication culture medium is 1-3:100.
6. The production process of the nano saline-alkali cellulase according to claim 1, wherein the specific steps of performing the enlarged culture on the nano saline-alkali cellulase strain obtained in the step S4 are as follows: inoculating nano saline-alkali cellulose bacteria into a bacteria culture medium under the condition of sterility, culturing for 28-32 hours at 28-32 ℃, transferring into a seed fermentation tank, culturing for 70-75 hours at 28-32 ℃, transferring into a three-stage fermentation tank, and culturing for 28-32 hours to obtain a cultured nano saline-alkali cellulose bacteria liquid; the strain culture medium is prepared by mixing silicon dioxide, amino acid, glucose and water according to a mass ratio of 8-12:18-22:4-6:1000; Adding 100 parts by weight of the cultured nano saline-alkali cellulose bacterial strain liquid into a reaction kettle, adding 8-12 parts by weight of silicon dioxide, 18-22 parts by weight of amino acid, 4-6 parts by weight of glucose and 900 parts by weight of sterile water, fermenting for 48-52 days at 28-32 ℃, and adjusting the pH to 9-10.5 by using sodium bicarbonate every 90 hours to obtain fermentation liquor.
7. The production process of the nano saline-alkali cellulase is characterized in that a strain protection system is added into the fermentation broth in the step S5 before concentration and drying, wherein the strain protection system consists of glycerin, DTT and EDTA, and the mass ratio of the glycerin to the DTT to the EDTA to the fermentation broth is 18-22:1.4-1.5:0.6-0.8:100.
8. The process for producing nano-grade saline-alkali cellulose according to claim 1, wherein the concentrating and drying steps comprise: concentrating the fermentation liquor until the water content is 40-50%, pumping the fermentation liquor to an atomizer at the top of a spray drying tower through a high-pressure pump, dispersing the fermentation liquor into mist-shaped liquid drops, enabling filtered and heated clean hot air to enter the drying tower in a spiral mode, enabling the filtered and heated clean hot air to contact the mist-shaped liquid drops in parallel flow, and obtaining the dried nano saline-alkali cellulose, wherein the inlet temperature of spray drying is 115-120 ℃, the pressure is 0.3-1 MPa, and the speed of the clean hot air is 4-6 m/s.
9. The process for preparing nano-salt-alkali cellulase according to claim 8, wherein the process further comprises collecting and cooling the dried nano-salt-alkali cellulase, wherein the dried nano-salt-alkali cellulase descends along with the airflow, most of the nano-salt-alkali cellulase is discharged from the bottom of the drying tower, and the rest of the nano-salt-alkali cellulase is complemented by a cyclone separator.
10. The application of the nano-saline-alkali cellulase obtained by the production process of claim 1 is characterized in that the nano-saline-alkali cellulase is used for improving saline-alkali soil.

Description

Production process and application of nano saline-alkali cellulase Technical Field The invention relates to the technical field of biological fermentation and saline-alkali enzyme culture, in particular to a production process and application of nano saline-alkali cellulase. Background The traditional improvement means such as salt washing and rotation have high cost and limited improvement effect, so that the efficient and low-cost saline-alkali soil improvement technology needs to be researched. The nano saline-alkali cellulose has wide application prospects in four fields of agriculture, industry, medicine and biological materials, is used for improving germination rate and growth activity salt tolerance of crops under salt stress and relieving cell membrane lipid peroxidation in agriculture, is used for treating industrial wastewater with salt content of more than 3.5% in industry and overcoming the problem that a conventional biological method is inhibited, can be used for a biosensor, an artificial vision device and the like due to stable photoelectric conversion performance in medicine, and can be used for synthesizing polyhydroxyalkanoate of degradable plastics in biological materials. However, the existing nano saline-alkali cellulose is generally a liquid preparation, and the liquid preparation has the problem of inconvenient transportation, storage and use. Disclosure of Invention In order to solve the problems, the invention aims to provide a production process and application of nano-saline-alkali cellulose. The invention aims to achieve the aim, and the aim is achieved by the following technical scheme: A production process of nano-saline-alkali cellulase comprises the following steps: s1, preparing a nutrient medium, carrying out aseptic inoculation on the rhodobacter salsa, and then culturing for 28-32 hours at 28-32 ℃ to obtain the cultured rhodobacter salsa for later use; S2, strain culture and enzyme activity induction, namely adding sodium chloride with the mass of 3-5% into a broth peptone culture medium, adding the cultured rhodobacter salsa, uniformly mixing, carrying out shake culture for 12-24 hours at the temperature of 30-37 ℃, and inducing the secretion of halophage by utilizing a halophage host to obtain a mixed bacterial solution; s3, pre-cooling the mixed bacterial liquid to 3-5 ℃, circulating for 2-4 times at 1500bar for two minutes each time to obtain crude enzyme liquid, and carrying out fractional purification to obtain saline-alkali cellulase; s4, carrying out molecular domestication on the alkaline cellulase obtained in the step S3, wherein the molecular domestication is high in thermal stability, high in activity and tolerant in pH value, so as to obtain nano alkaline cellulase strains; the basic domestication culture medium adopted in the molecular domestication culture consists of, by weight, 4-6 parts of silicon dioxide, 8-12 parts of beef extract, 34-38 parts of sodium chloride and 1000 parts of distilled water; and S5, performing enlarged culture on the nano saline-alkali cellulase strain obtained in the step S4 to obtain fermentation liquor, concentrating and drying to obtain the nano saline-alkali cellulase. The nutrient medium is prepared by adding 4-6 parts by weight of silicon dioxide, 8-12 parts by weight of beef extract and 34-38 parts by weight of sodium chloride into 1000 parts by weight of distilled water, stirring and mixing uniformly, placing a triangular flask for sterilization at 121 ℃ for 25-30 minutes to obtain a mixed solution, and adding yeast extract powder with the mass of 0.5% into the mixed solution to obtain the nutrient medium. Preferably, the fractional purification comprises the steps of: And (3) carrying out ammonium sulfate precipitation, dialysis desalting, DEAE-Sepharose FF ion exchange chromatography, tris elution and Superdex 200 gel filtration on the crude enzyme solution with the mass concentration of 20% to obtain the saline-alkali cellulase. The purpose of the grading purification is to separate the saline-alkali cellulase, the saline-alkali catalase and the saline-alkali manganese catalase to obtain the high-purity saline-alkali cellulase. Preferably, the step S4 specifically includes the following steps: S41, performing heat stability culture, namely adding the saline-alkali cellulase obtained in the step S3 into a basic domestication culture medium, gradually increasing the temperature on the basis of 60 ℃ for continuous gradient culture, wherein the temperature is increased by 5 ℃ each time, and each time of heat resistance domestication is performed for 2 hours, and 12 times of heat resistance domestication are performed continuously, so that the heat resistance value of the saline-alkali cellulase reaches 110 ℃, and the saline-alkali cellulase with high heat stability is obtained for standby; S42, salt-tolerant activity culture, namely continuously culturing the obtained high-heat-stability saline-alkali cellulase for 30 ho