CN110540982B

CN110540982B - Fermentation method for improving activity of Thermobacteroid cellulase

Info

Publication number: CN110540982B
Application number: CN201910942909.1A
Authority: CN
Inventors: 邓禹; 毛银; 陆春波; 卫亚峰; 李国辉; 赵运英; 周胜虎
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2021-11-02
Anticipated expiration: 2039-09-30
Also published as: CN110540982A

Abstract

The invention discloses a fermentation method for improving the activity of a Thermus fusiforme cellulase, belonging 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.

Description

Fermentation method for improving activity of Thermobacteroid cellulase

Technical Field

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

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, lg/L of sodium sulfate, lg/L of monopotassium phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, lg/L of yeast extract, 50g/L of magnesium chloride hexahydrate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, lg/L of cysteine monohydrate, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, and 0.002g/L of vitamin B₁₂0.02g/L, vitamin B_l0.002g/L, 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.

EXAMPLE 1 preparation of cellulases at different fermentation temperatures

Setting different fermentation temperatures of 30, 40, 50, 55 and 60 ℃ respectively, fermenting for 72 hours, and measuring the activity of the cellulase after the fermentation is finished. The results are shown in Table 1, and the relative enzyme activity of the strain can reach more than 90% under the condition of 50-60 ℃.

TABLE 1 Effect of different fermentation temperatures on enzyme Activity

EXAMPLE 2 preparation of cellulases at different fermentation times

Setting different culture times of 24h, 48h, 60h, 72h, 84h and 96h, respectively, culturing at 60 ℃, and determining the activity of the cellulase after the fermentation is finished. The results are shown in Table 2, the enzyme activity is in an increasing trend along with the increase of the fermentation time, the enzyme activity reaches the peak value after fermentation for 60 hours, and the enzyme activity begins to decrease for 96 hours. Therefore, the fermentation is stopped after 60 hours, and the fermentation cost can be greatly saved.

TABLE 2 Effect of different fermentation times on enzyme Activity

Example 3 preparation of cellulases at different initial pH

Controlling other conditions to be unchanged, setting different initial pH values of the culture medium to be 5.5, 6, 6.5, 7, 7.5 and 8 respectively, fermenting at 60 ℃ for 72 hours, and determining the cellulase activity after the fermentation is finished. The results are shown in Table 3, and when the initial pH of the culture medium is 6.5-8, the relative enzyme activity can reach more than 90%.

TABLE 3 Effect of initial pH of different media on enzyme Activity

EXAMPLE 4 fermentative preparation of cellulase

Fermenting for 60h under the optimized conditions, namely at 55 ℃ and under the condition that the pH of the initial culture medium is 7, and measuring that the enzyme activity is 5.12U/mL and is 1.5 times of the unoptimized enzyme activity

EXAMPLE 5 cellulase fermentation under amplification Process

Sucking 200 mu L of glycerol tube storage strain, transferring to 25mL of seed culture medium, culturing at constant temperature of 55 ℃, activating the strain, transferring the activated strain to 50mL of fermentation culture medium according to the inoculum size of 2%, and culturing at constant temperature of 55 ℃ for 24h to obtain first-grade seed liquid.

Fermentation medium: 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, 1.5g/L of sodium sulfate, 0.1g/L of potassium dihydrogen phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, 0.2g/L of calcium chloride dihydrate, 0.2g/L of cysteine hydrochloride monohydrate, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin and vitamin B₁₂0.02g/L, vitamin B_l0.002g/L, and natural pH value. Sterilizing at 115 deg.C by wet heat.

After sterilization, nitrogen was introduced into the fermentor to remove the medium and oxygen from the fermentor. Adjusting the culture pH to 7, controlling the tank temperature to 55 deg.C, inoculating the first-stage seed liquid, stopping aeration, and intermittently stirring to prevent thallus precipitation. After the fermentation is finished, the enzyme activity is measured to be 12.37U/mL, which is 1.14 times of the enzyme activity in the state without controlling the pH value.

EXAMPLE 6 preparation of cellulase by batch fermentation

Since continuous fed-batch fermentation is easy to pollute mixed bacteria, has high degeneration and small application range, the research on batch and fed-batch fermentation methods is carried out.

Batch fermentation: inoculating the preserved strain into a seed culture medium, then inoculating the strain into a fermentation culture medium by 2% of inoculation amount to obtain a first-stage seed solution, inoculating the seed solution into a 5L fermentation tank filled with 2L of fermentation culture medium by 5% of inoculation amount, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, and carrying out intermittent stirring. The enzyme activity is 12.37U/mL after fermentation for 60 h.

Example 7 preparation of cellulase by fed-batch fermentation at different feed times

Inoculating the strain into a seed culture medium, then inoculating the strain into a fermentation culture medium by 2% of inoculation amount to obtain a first-stage seed solution, inoculating the seed solution into a 5L fermentation tank filled with 3L of fermentation culture medium by 5% of inoculation amount, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, and carrying out intermittent stirring. According to the growth of the cells and the consumption of sugar, the cellobiose solution was slowly added to the fermentation system at the time points of 16h, 20h and 24h after the start of the fermentation at a flow rate of 6mL/h and at a concentration of 200 g/L. Compared with the enzyme production condition, the sugar consumption and the growth condition are slow in 0-12 h, and after 16h, the thalli grow rapidly and the sugar consumption enters a rapid consumption stage, so 16h, 20h and 24h are selected as the feeding points. By comparing the enzyme production conditions at different feeding time points, the enzyme activity is the highest when feeding is started from a 16h point, and after fermentation is carried out for 48h, the thalli reach a stable period, the sugar consumption is slowed, and the feeding significance is not large at the moment, so that the feeding is carried out for 48 h. The feeding time is determined to be 16-48 h.

TABLE 5 Effect of different feed time points on enzyme activity

Example 8 preparation of cellulase by fed-batch fermentation with different carbon sources

Inoculating the preserved strain into a seed culture medium, then inoculating the strain into a fermentation culture medium by 2% of inoculation amount to obtain a first-stage seed solution, inoculating the seed solution into a 5L fermentation tank filled with 3L of fermentation culture medium by 5% of inoculation amount, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, and carrying out intermittent stirring. Within the 16 th-48 th time period of fermentation, cellobiose, microcrystalline cellulose and xylan solution are respectively added, the flow rate is 6mL/h, the concentration of the carbon source solution is 200g/L, and the fermentation is carried out for 60 h. Comparing the enzyme production, see table 5, the result shows that cellobiose is the best carbon source for feeding.

TABLE 5 Effect of initial pH of different media on enzyme Activity

Example 9 preparation of cellulase by Fed-batch fermentation

Inoculating the strain into a seed culture medium, then inoculating the strain into a fermentation culture medium by 2% of inoculation amount to obtain a first-stage seed solution, inoculating the seed solution into a 5L fermentation tank filled with 3L of fermentation culture medium by 5% of inoculation amount, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, and carrying out intermittent stirring. Slowly adding a cellobiose solution into the fermentation system within 16-48 h after the fermentation is started, wherein the flow rate is 6mL/h, the concentration of the cellobiose solution is 200g/L, and the fermentation time is 60 h. After the fermentation is finished, the enzyme activity of the fermentation liquor is measured, the enzyme activity is measured to be 16.56U/mL, the condition that no control is carried out in the fermentation process is taken as a reference, and through comparison, the enzyme activity measured by adopting the fermentation process is improved by 52.62 percent relative to the reference.

Example 10 preparation of cellulases from different strains under fed-batch fermentation conditions

The obtained Thermus fusiforme ATCC 27405, BNCC 134251 and BNCC 340971 are put into a tank for fermentation according to the method, the temperature is 55 ℃, the constant pH value is controlled to be 7, cellobiose solution is slowly added for 16-48 hours, the flow rate is 6mL/h, the concentration of the cellobiose solution is 200g/L, the fermentation time is 60 hours, the comparison with the fermentation process without any control is carried out, the enzyme activity condition is shown in Table 6, and the enzyme activity is respectively improved by 52.1%, 50.9% and 53.4% through the process.

TABLE 6 enzyme activity after fermentation optimization of different strains

EXAMPLE 11 preparation of enzyme preparation

Preparation of enzyme preparation: performing solid-liquid separation on the fermentation liquor, removing thalli, concentrating the filtrate to 30-40% of the original volume at normal temperature by using a rotary evaporator, for example, concentrating to 20% of the original volume, then performing ammonium sulfate precipitation, adding 30% saturated ammonium sulfate, standing for 3h, removing the precipitate, adding 60% saturated ammonium sulfate, standing for 6h, removing the supernatant to obtain a cellulase crude product, then performing gel chromatography on the cellulase crude product for purification, balancing by using distilled water, passing the enzyme liquid obtained in the previous step through a column, eluting by using 0.2mol/L phosphoric acid buffer solution, adding 5% lactose and 5% skim milk (by mass) serving as a protective agent, pre-cooling at the temperature of-80 ℃, and performing freeze drying to obtain a cellulase preparation, wherein the enzyme activity is 2100000U/g.

The enzyme preparation is used as a bioactive substance, and the activity exertion function is a key point, so the heat stability experiment and the damp-heat resistance experiment are carried out according to the experimental scheme of feed additive nicotinic acid stability experimental research published by Wangheiying and the like. As seen from the results in tables 7, 8 and 9, the enzyme preparation has excellent heat resistance and moisture resistance, and the enzyme activity is maintained at 90% or more after three months of storage.

TABLE 7 thermostability testing of enzyme preparations

TABLE 8 moisture resistance testing of enzyme formulations

TABLE 9 preservation experiments of enzyme preparations

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for increasing the growth rate of Thermus fuscusClostridium thermocellum) The fermentation method of the cellulase enzyme activity 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 h, and the material supplement is carried out after the fermentation is carried out for 16 h;

the Thermus fusiforme is ATCC 27405 or BNCC 340971;

the feed is a feed cellobiose solution; the flow rate of the fed batch is controlled to be 4-6 mL/h, and the concentration of the cellobiose solution is 200 g/L.

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

3. The method according to claim 2, 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.

4. The method of claim 3, wherein the seed medium comprises: cellobiose, corn steep liquor, tripotassium citrate, citric acid, sodium sulfate, potassium dihydrogen 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 B1.

5. The method of claim 1, wherein the fermentation is conducted in a fermentation medium; the fermentation medium contains cellobiose, microcrystalline cellulose and corn steep liquor.

6. The method of claim 2, wherein the fermentation medium comprises cellobiose, microcrystalline cellulose, and corn steep liquor.

7. 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 5, then the enzyme solution is concentrated, and a protective agent is added.

8. The method of claim 7, wherein the protective agent is lactose or skim milk.