阅读说明：本技术 一种提高梭热杆菌纤维素酶产量发酵方法 (Fermentation method for increasing yield of Thermobacteroides cellulase ) 是由 邓禹 毛银 陆春波 卫亚峰 李国辉 赵运英 周胜虎 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种提高梭热杆菌纤维素酶产量发酵方法,属于发酵过程领域。本发明对梭热杆菌进行发酵工艺的优化,使纤维素酶的酶活由10.85U/mL提高到16.56U/mL,纤维素酶活比未优化前提高了52％。并根据菌体生长情况,进行补料添加碳源,大大提高了其纤维素酶活,并节省了成本,适合工业化生产。本发明制作的酶制剂具有极高的耐热性和耐湿性,且在保藏三个月之后,酶活力依然保持在90％以上,适合工业化生产。(The invention discloses a fermentation method for increasing yield of a clostridium cellulolytic enzyme, and belongs to the field of fermentation processes. The invention optimizes the fermentation process of the clostridium thermocellum, so that the enzyme activity of the cellulase is improved to 16.56U/mL from 10.85U/mL, and the cellulase activity is improved by 52 percent compared with that before the optimization. And according to the growth condition of the thalli, a carbon source is supplemented and added, so that the cellulase activity of the thalli is greatly improved, the cost is saved, and the method is suitable for industrial production. The enzyme preparation prepared by the invention has extremely high heat resistance and moisture resistance, and the enzyme activity is still kept above 90% after the enzyme preparation is stored for three months, so that the enzyme preparation is suitable for industrial production.)

1. A fermentation method for increasing the yield of cellulase of clostridium thermobacter is characterized in that the fermentation temperature is controlled to be 50-60 ℃, the initial pH value is 6.5-8, the fermentation is carried out for 48-72 hours, and the material supplement is carried out after the fermentation is carried out for 16 hours.

2. The method of claim 1, wherein the feeding is with a supplemental carbon source; the carbon source includes, but is not limited to, cellobiose.

3. The method according to claim 1 or 2, wherein the feeding is performed at a controlled flow rate of 4-6 mL/h and the cellobiose solution concentration is 180-250 g/L.

4. The method according to claim 1 or 3, wherein the seed solution is inoculated into the fermentation medium in an inoculum size of 1-2%.

5. The method according to claim 4, wherein the seed solution is obtained by inoculating the strain into a seed culture medium according to an inoculation amount of 1-2%, and performing activated culture at 50-60 ℃ for 2-3 generations.

6. The method of claim 5, wherein the seed medium comprises: cellobiose, corn steep liquor, tripotassium citrate, citric acid, sodium sulfate, monopotassium phosphate, sodium bicarbonate, ammonium chloride, urea, yeast extract, magnesium chloride, ferrous chloride, calcium chloride, cysteine hydrochloride, pyridoxamine dihydrochloride, p-aminobenzoic acid, D-biotin, vitamin B12 and vitamin Bl.

7. The method of claim 1 or 4, wherein the fermentation medium comprises cellobiose, microcrystalline cellulose, corn steep liquor.

8. A method for preparing a cellulase preparation, which is characterized in that the cellulase is prepared by the fermentation method of any one of claims 1 to 7, enzyme solution is concentrated, and a protective agent is added.

9. The method according to claim 8, characterized in that the protective agent lactose, skim milk.

10. A cellulase preparation produced by the method of claim 8 or 9.

Technical Field

The invention relates to a fermentation method for improving yield of a clostridium cellulolytic enzyme, and belongs to the field of fermentation engineering.

Background

The lignocellulose raw material has wide sources and is a renewable resource with abundant reserves. Cellulase is a general name of a group of enzymes capable of degrading cellulose into glucose, lignocellulose can be hydrolyzed by cellulase to generate sugar, the sugar can be directly used for fermentation of microorganisms, the cellulose can be converted into valuable primary metabolites, and the cellulase is often applied to industries such as food and feed industries, paper making industries, medicine aspects, biological energy sources and the like, and has good application prospect.

The cellulase-producing bacteria include bacteria, fungi, actinomycetes, etc., and among these microorganisms, a microorganism called Thermobifida (fusobacterium) is known for its high cellulolytic activity and cellulose degradation rate is comparable to Trichoderma reesei. The strain has the characteristics of wide available substrates, strong environmental adaptability, difficult pollution and the like, can generate a complex enzyme system for degrading cellulose, comprises endoglucanase, exoglucanase, hemicellulase and the like, and is a strain with the greatest potential of positive economic influence.

At present, the application of cellulase is often limited by high production cost, the high production cost of the cellulase is mainly focused on two aspects, firstly, the enzyme activity of a strain for producing the cellulase is not high and far meets the production requirement, so that the screening of the strain with high cellulase activity is a key point. Secondly, the currently frequently used strain is trichoderma reesei, the enzyme production capacity is equivalent to that of the clostridium thermocellum, but due to factors such as characteristics, culture conditions and the like, the cellulase fermentation time is long, the efficiency is low, the cost is overhigh, the cellulase is produced by adopting liquid fermentation at present and is suitable for large-scale production, but the enzyme activity of the fermentation liquid is low, so that the price of the cellulase product produced by adopting the liquid method is high, and the wide application of the cellulase in industry is limited.

Disclosure of Invention

the invention provides a fermentation method for improving the yield of cellulase of clostridium thermocellum, which comprises the steps of controlling the fermentation temperature to be 50-60 ℃, controlling the initial pH value to be 6.5-8, fermenting for 48-72 hours, and starting to perform continuous feeding after fermenting for 16 hours.

In one embodiment of the invention, the feeding is a supplemental carbon source.

In one embodiment of the invention, the feed is supplemented with cellobiose.

In one embodiment of the present invention, the feeding is performed at a flow rate of 4-6 mL/h, and the concentration of the cellobiose solution is 180-250 g/L.

In one embodiment of the invention, the fermentation is inoculated with a seed solution in an inoculum size of 1-2%.

In one embodiment of the invention, the seed solution is obtained by inoculating 1-2% of strains into a seed culture medium and performing activated culture at 50-60 ℃ for 2-3 generations.

In one embodiment of the present invention, the seed solution is cultured in a seed medium comprising: 10g/L of cellobiose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, 1.5g/L of sodium sulfate, 0.1g/L of monopotassium phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, 0g/L of yeast extract, 50g/L of magnesium chloride hexahydrate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, 0.02g/L of cysteine monohydrochloride, 0.004g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, 0.02g/L of vitamin B120, and 0.002g/L of vitamin Bl.

In one embodiment of the invention, the fermentation medium used for the fermentation contains: 10g/L of cellobiose, 10g/L of microcrystalline cellulose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, 1.25g/L of sodium sulfate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, 5g/L of urea, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, 0.02g/L of vitamin B120, and 0.002g/L of vitamin Bl.

The second purpose of the invention is to provide a method for preparing the cellulase preparation, wherein the method is used for preparing the cellulase by the fermentation method, then the enzyme solution is concentrated, and a protective agent is added.

In one embodiment of the invention, the protective agent is lactose, skim milk.

In one embodiment of the invention, the cellulase preparation is prepared by mixing cellulase with lactose with a final concentration of 3-5% and skim milk with a final concentration of 3-5%, and freeze-drying the mixture.

The invention has the beneficial effects that: the invention optimizes the fermentation process of the clostridium thermocellum, optimizes the fermentation culture conditions, greatly improves the cellulase activity of the clostridium thermocellum from the original 10.85U/mL to 16.56U/mL, supplements the clostridium thermocellum timely according to the growth condition of thalli, greatly improves the cellulase activity of the clostridium thermocellum, saves the cost, and is suitable for industrial production. The enzyme preparation prepared by the invention has extremely high heat resistance and moisture resistance, and the enzyme activity is still kept above 90% after the enzyme preparation is stored for three months, so that the enzyme preparation is suitable for industrial production.

Drawings

FIG. 1A plot of Thermobactase growth;

Detailed Description

1. Seed culture medium: 10g/L of cellobiose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, 1.25g/L of sodium sulfate, 0.1g/L of monopotassium phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, 0g/L of yeast extract, 50g/L of magnesium chloride hexahydrate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, 0.02g/L of cysteine monohydrochloride, 0.004g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, 0.02g/L of vitamin B120, 0.002g/L of vitamin Bl, and natural pH value.

2. Strain activation: inoculating the strain into a seed culture medium according to the inoculation amount of 1%, activating and culturing at 60 ℃, activating for 2-3 generations according to the same manner, and continuously culturing in the seed culture medium to obtain the bacterial liquid of the fusobacterium.

3. Fermentation medium: under the basic condition of a seed culture medium, 10g/L of microcrystalline cellulose is added.

4. Inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2%, fermenting and culturing for 72h at 60 ℃, and determining the enzyme activity after the fermentation is finished. The enzyme activity was measured to be 3.41U/mL.

5. Enzyme activity assay (filter paper enzyme activity): 50mg of filter paper is taken, 1ml of citric acid buffer solution with the pH value of 60.1 mol/L is added, 0.5ml of proper diluted enzyme solution is added, the reaction is carried out for 1h at the temperature of 50 ℃, and 3ml of DNS reagent is added for measuring sugar. The enzyme activity is calculated after reducing sugar in the fermentation liquor is deducted, the enzyme activity adopts international units, and the enzyme activity is defined as that the enzyme amount for generating 1 mu mol glucose by hydrolysis in each minute is 1 activity unit.