CN111424024A - Preparation and use methods of low-temperature cellulase - Google Patents
Preparation and use methods of low-temperature cellulase Download PDFInfo
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- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
The invention relates to a preparation and application method of low-temperature cellulase, which belongs to the technical field of biology, the preparation and application method of the cellulase has clear principle, the enzyme-producing strain used in the invention is hardy brevibacterium, and the strain has the advantages of high growth and reproduction speed, low growth temperature, good enzyme-producing performance and the like; the cellulase has the advantages of simple preparation process, low cost, good acid resistance, stable performance at low temperature and good enzyme activity. The method has no secondary pollution to the environment, can degrade crop straws through cellulase, can obtain required energy substances, has lower temperature required by reaction, has less energy loss, can generate higher economic value, obtains higher economic return at lower cost, has better performance in the aspects of industrial production and utilization of agricultural resource wastes, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a preparation method and a use method of cellulase, and particularly relates to a preparation method and a use method of low-temperature cellulase.
Background
Cellulose is widely existed in nature and is a series of useful chemicals such as glucose and the like, cellulose decomposition is a mode found to be better to utilize cellulose in nature at present, microorganisms utilize cellulose as a nutrient source and need the participation of cellulase, the cellulase is a general name of a multi-component enzyme system and can be divided into three enzyme components, namely endo-1, 4- β -D-glucanase, exo-1, 4- β -D-glucanase and β -glucanase, the cellulose is finally degraded into glucose under the action of the cellulase, and the glucose can be further converted into other useful chemicals such as ethanol, butanol, methane and the like.
At present, the most important application of cellulase is generally considered to be the conversion of cellulose into biofuel ethanol, and with the continuous consumption of natural fossil energy, a series of problems are caused, such as environmental pollution, energy shortage, fuel use cost rise and the like, which compel us to find suitable renewable green energy substitutes to solve the problems, and ethanol is biological energy which is considered to play an important role like petroleum in the future. Ethanol is used as a world-recognized excellent gasoline additive, and is widely used as a mixed component of motor gasoline in some countries such as brazil, the United states and the like, and China is also advancing the process of using the ethanol gasoline. The first generation of fuel ethanol is mainly produced by fermenting corn, cassava or other grains, and has formed a certain scale, but has the problems of high cost, large demand on grains and the like. The second generation fuel ethanol is produced by taking agricultural wastes, crop straws and various plants as raw materials, so that the production cost of ethanol is greatly reduced, and meanwhile, environmental wastes can be better utilized to promote the development of ecological civilization. Cellulases are used in a wide variety of applications, including various industrial processes such as food, textile, laundry, pulp and paper, and agriculture.
The cellulose is an energy substance widely existing in the world, 1.55 × 109t of substances are generated every year according to statistics, mainly comprising agricultural wastes such as straws and the like and industrial wastes such as wood blocks and the like, but the utilization rate of the energy substances is always low and is only limited to simple utilization.
In order to meet the increasing demand of the cellulase, the production potential of the cellulose decomposition bacteria is continuously excavated, and the enzyme yield is improved by optimizing production conditions, constructing engineering bacteria and the like. Cellulases are now widely produced for use in food, feed, energy, paper, textile, environmental and various chemical industries. The method mainly comprises two aspects of physical chemical utilization and biological utilization, wherein the physical chemical methods such as strong acid (alkali), high temperature, ion radiation and the like can degrade cellulose, but the extreme environment is not easy to control and can cause secondary pollution to the environment, and at present, a plurality of strains are applied to the production of cellulase preparations, fungi are mainly used as seed liquid for fermentation production, including mutant strains of the fungi and the like, but the cellulase application environment conditions are relatively severe, the production cost is higher, and the research on the cellulase with strong environmental adaptability is very little, so that a method for preparing the cellulase at low temperature and the application of the cellulase are needed to be designed to solve the problem of energy demand faced at present.
Disclosure of Invention
The invention aims to provide a preparation and use method of low-temperature cellulase aiming at the defects that the way of utilizing cellulose by people is single at the present stage and mainly comprises two aspects of physical and chemical utilization and biological utilization, and the physical and chemical methods such as strong acid (alkali), high temperature, ion radiation and the like can degrade the cellulose, but the extreme environment is not easy to control and can cause secondary pollution to the environment.
The technical scheme of the invention is as follows: the preparation method of the low-temperature cellulase is characterized by comprising the following steps of:
(1) selecting a strain of low-temperature degraded cellulose, namely, a cold-resistant brevibacterium, which is screened in advance, inoculating the strain into a sterile L B culture medium, and shaking the strain for 8 hours at a speed of 220r/min for later use as a subsequent fermentation seed solution;
(2) preparing a fermentation enzyme-producing material: corn straw powder, tryptone, ferrous sulfate and distilled water;
(3) preparation of the required instruments and equipment: a full-temperature shaking flask cabinet, an enzyme-labeling instrument, a centrifuge, a pH meter, a glass rod and a beaker;
(4) selecting low-temperature fermentation hardiness brevibacterium as an experimental object, and performing low-temperature fermentation culture on the strain according to process parameters to prepare cellulase;
(5) the preparation method comprises the following specific steps:
(5-1) adding the fermentation seed liquid obtained in the step (1) into a sterile corn straw fermentation culture medium according to the inoculation amount of 2%;
(5-2) putting the inoculated fermentation enzyme-producing culture medium into a full-temperature shaking cabinet, and carrying out shaking culture at a constant temperature of 180r/min for 24 hours;
(5-3) centrifuging fresh fermentation liquor at 10000r/min for 10min the next day to obtain supernatant which is the low-temperature cellulase enzyme solution of the strain;
(5-4) storing the obtained supernatant at 4 ℃ for later use, and preparing the cellulase.
The technological parameters in the step (4) are that the concentration of the corn straw powder is 3 percent, the concentration of the tryptone is 0.5 percent, the concentration of the ferrous sulfate is 0.5 percent, the PH value of the culture medium is 6, the inoculation amount is 2 percent, the culture temperature is 15 ℃, the corn straw powder is 1.5g, the tryptone is 0.25g, the ferrous sulfate is 0.25g, the seed liquid is 1m L, and the distilled water is 50m L.
The cellulase in the step (5-4) is colorless transparent enzyme liquid, and the enzyme liquid can be prepared at low temperature, has good acid resistance, stable performance at low temperature and good enzyme activity.
The use method of the low-temperature cellulase is characterized by comprising the following steps:
(1) adding 100m L distilled water into every 50g of crop straw powder, stirring and mixing uniformly, transferring into a 1L fermentation bottle, and sterilizing at 121 ℃ for 15 min;
(2) adding the prepared cellulase into sterilized fermentation bottles, wherein the addition amount of the cellulase liquid in each bottle is 20m L, and fully stirring and uniformly mixing;
(3) placing the fermentation bottle into a full-temperature shaking flask cabinet, and carrying out shaking culture at the constant temperature of 15 ℃ and 100r/min for 48 h;
(4) after the fermentation reaction is finished, centrifuging fresh fermentation liquor to obtain supernatant, wherein the supernatant is a product obtained by decomposing crop straws by cellulase;
(5) and (3) measuring the content of glucose in the supernatant, and further separating and purifying the supernatant, wherein the glucose is a main product of straw degradation for subsequent utilization.
The invention has the beneficial effects that: according to the preparation and use method of the low-temperature cellulase provided by the invention, the principle of the preparation and use method of the cellulase is clear, the effect of degrading cellulose by the cellulase is obvious, and the low-temperature cellulase can be used for better degrading crop straws and converting the crop straws into the required material resources; the enzyme-producing strain used in the invention is the hardy brevibacterium, and the strain has the advantages of high growth and reproduction speed, low growth temperature, good enzyme-producing performance and the like; the cellulase has the advantages of simple preparation process, low cost, good acid resistance, stable performance at low temperature and good enzyme activity. The method has no secondary pollution to the environment, can degrade crop straws through cellulase, can obtain required energy substances, has lower temperature required by reaction, has less energy loss, can generate higher economic value, obtains higher economic return at lower cost, has better performance in the aspects of industrial production and utilization of agricultural resource wastes, and has wide application prospect.
Drawings
FIG. 1 is a graph showing cellulase activity assay of crude enzyme in the present invention after each 60min at different temperatures.
FIG. 2 is a graph showing cellulase activity measurements performed after the crude enzyme was subjected to pH3-8 for 60min each.
Detailed Description
The invention will be further described with reference to the accompanying drawings in which:
a method for preparing cellulase under low temperature condition, prepared by fermentation of Brevibacterium fritolerans, comprises the following steps:
selecting low-temperature cellulase-producing strain brevibacterium frigidarium (Brevibacterium frigidarium) which is screened in advance, inoculating the strain into a sterile L B culture medium, and shaking the strain for 8 hours at 220r/min for later use as a subsequent fermentation seed solution;
selecting materials for preparing low-temperature cellulase, namely corn straw powder, tryptone, ferrous sulfate, distilled water, 1 mol/L HCl and 1 mol/L NaOH, and preparing required instruments and equipment, namely a full-temperature shaking flask cabinet, an enzyme-labeling instrument, a centrifuge, a pH meter, a glass rod and a beaker;
selecting low-temperature fermentation hardiness brevibacterium as an experimental object, and performing low-temperature fermentation culture on the strain according to process parameters to prepare cellulase; the components of the culture medium are as follows: the concentration of the corn straw powder is 3%, the concentration of the tryptone is 0.5%, the concentration of the ferrous sulfate is 0.5%, the pH value of a culture medium is 6, the inoculation amount is 2%, the culture temperature is 15 ℃, the raw materials and the addition amount are shown in table 1, and the preparation process of the low-temperature cellulase is as follows:
adding the obtained fermentation seed liquid into a sterile corn straw fermentation culture medium according to the inoculation amount of 2%; placing the inoculated fermentation enzyme-producing culture medium into a full-temperature shaking cabinet, and carrying out shaking culture at the constant temperature of 15 ℃ at 180r/min for 24 hours; centrifuging fresh fermentation liquid at 4 deg.C at 10000r/min for 10min, and centrifuging to obtain supernatant as low temperature cellulase liquid; the obtained supernatant is stored at 4 ℃ for standby, and the cellulase is prepared.
The cellulase prepared by the steps is colorless transparent enzyme liquid, can be prepared at low temperature, and has good acid resistance, stable performance at low temperature and good enzyme activity.
Watch (1)
The use method of the prepared low-temperature cellulase comprises the following steps:
ordinary crop straws are selected, crushed and sieved by 40 meshes, and the obtained straw powder is selected as a degradation raw material to carry out a decomposition experiment.
Adding 100m L distilled water into each 50g of crop straw powder, stirring and mixing uniformly, transferring into a 1L fermentation bottle, and sterilizing at 121 ℃ for 15 min.
Adding the prepared cellulase into sterilized fermentation bottles, wherein the addition amount of the cellulase liquid in each bottle is 20m L, and fully stirring and uniformly mixing.
Placing the fermentation bottle into a full-temperature shaking flask cabinet, and carrying out shaking culture at the constant temperature of 15 ℃ and 100r/min for 48 h.
And after the fermentation reaction is finished, centrifuging fresh fermentation liquor to obtain supernatant, wherein the supernatant is a product obtained by decomposing crop straws by cellulase.
And (3) measuring the content of glucose in the supernatant, and further separating and purifying the supernatant, wherein the glucose is a main product of straw degradation for subsequent utilization.
Example 1
Detecting the cellulase activity of the low-temperature cellulase
In order to explore the yield of the cellulase prepared by the strain under the current condition, bacillus cereus is inoculated into a culture medium under the condition to ferment and produce enzyme according to the condition, three groups of parallel processes are carried out, fresh fermentation liquor is taken to be centrifuged for 10min at 10000r/min at 4 ℃ after the fermentation is finished, the supernatant obtained by centrifugation is low-temperature cellulase enzyme liquid of the strain, the enzyme activity of the cellulase in the enzyme liquid is determined by adopting a 3, 5-dinitrosalicylic acid colorimetric method, the enzyme activity is expressed as 1m L, the cellulase liquid catalyzes a substrate for 1min to generate 1.0 mu g of glucose, and the determination result is shown in a table (2).
Watch (2)
As can be seen from the measurement results in Table (2), when the fermentation medium inoculated with Brevibacterium frigosum is cultured at different fermentation temperatures, the strain can produce cellulase, and the culture is carried out at the temperature of 15 ℃ to obtain higher cellulase activity, which means that the yield of the cellulase is the highest compared with that at other temperatures.
Example 2
Determination of enzymatic Properties of Low temperature cellulases
And (3) measuring the enzyme activity property of the obtained cellulase liquid, and respectively measuring the stability of the cellulase liquid to temperature and pH so as to determine the application condition of the cellulase. The crude enzyme solution obtained by centrifugation was incubated at 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C and 90 deg.C for 60min, the enzyme activity was measured after rapid cooling, and the percentage of residual enzyme activity was calculated with the untreated crude enzyme as 100%, and the results are shown in FIG. 1. The crude enzyme solution obtained by centrifugation was adjusted to different pH (3-8), incubated at 4 ℃ for 60min, and the percentage of residual enzyme activity was calculated using untreated crude enzyme as 100%, the results are shown in FIG. 2.
As shown in fig. 1, the activity of the cellulase is measured after the crude enzyme is placed at different temperatures for 1 hour, so that the stability of the cellulase is reflected, and the relative enzyme activity is preserved to be more than 80% as the temperature stability range; the result shows that the stability of the cellulase is the best under the condition that the temperature is 20 ℃, the displayed cellulose has the highest relative activity, and the relative residual enzyme activity of the cellulase at low temperature within the range of 10-30 ℃ is higher than 80 percent, and the cellulase shows better stability; as shown in FIG. 2, the cellulase activity was measured after the crude enzyme was left at a pH of 3 to 8 for 60min, and it was found that the cellulase stability was the best and the relative cellulose activity was the highest at a pH of 4. The cellulase also shows better stability under the condition of pH 5, and simultaneously, the pH of the culture medium is 4 after the enzyme production culture medium is fully fermented, which is consistent with the experimental result of pH stability, and the cellulase is shown to have good acid resistance, shows better enzyme activity under the acidic condition, and is easy to inactivate under the alkaline condition.
The experiments show that the invention has good enzyme activity in the aspect of preparing low-temperature cellulase, has higher yield at low temperature, and simultaneously the produced cellulase can keep higher enzyme activity at low temperature, has certain tolerance to acid conditions and can cope with different reaction conditions. The cellulase produced by the strain has good development prospect in the aspects of agricultural waste treatment and industrial production.
Claims (4)
1. The preparation method of the low-temperature cellulase is characterized by comprising the following steps of:
(1) selecting a strain of low-temperature degraded cellulose, namely, a cold-resistant brevibacterium, which is screened in advance, inoculating the strain into a sterile L B culture medium, and shaking the strain for 8 hours at a speed of 220r/min for later use as a subsequent fermentation seed solution;
(2) preparing a fermentation enzyme-producing material: corn straw powder, tryptone, ferrous sulfate and distilled water;
(3) preparation of the required instruments and equipment: a full-temperature shaking flask cabinet, an enzyme-labeling instrument, a centrifuge, a pH meter, a glass rod and a beaker;
(4) selecting low-temperature fermentation hardiness brevibacterium as an experimental object, and performing low-temperature fermentation culture on the strain according to process parameters to prepare cellulase;
(5) the preparation method comprises the following specific steps:
(5-1) adding the fermentation seed liquid obtained in the step (1) into a sterile corn straw fermentation culture medium according to the inoculation amount of 2%;
(5-2) putting the inoculated fermentation enzyme-producing culture medium into a full-temperature shaking cabinet, and carrying out shaking culture at a constant temperature of 180r/min for 24 hours;
(5-3) centrifuging fresh fermentation liquor at 10000r/min for 10min the next day to obtain supernatant which is the low-temperature cellulase enzyme solution of the strain;
(5-4) storing the obtained supernatant at 4 ℃ for later use, and preparing the cellulase.
2. The method for preparing low-temperature cellulase of claim 1, wherein the process parameters in step (4) are that the concentration of corn straw powder is 3%, the concentration of tryptone is 0.5%, the concentration of ferrous sulfate is 0.5%, the pH of the culture medium is =6, the inoculation amount is 2%, the culture temperature is 15 ℃, the corn straw powder is 1.5g, the tryptone is 0.25g, the ferrous sulfate is 0.25g, the seed solution is 1m L, and the distilled water is 50m L.
3. The method for preparing and using a low temperature cellulase according to claim 1, wherein the method comprises the following steps: the cellulase in the step (5-4) is colorless transparent enzyme liquid, and the enzyme liquid can be prepared at low temperature, has good acid resistance, stable performance at low temperature and good enzyme activity.
4. The use method of the low-temperature cellulase is characterized by comprising the following steps:
(1) adding 100m L distilled water into every 50g of crop straw powder, fully stirring and uniformly mixing, transferring into a 1L fermentation bottle, and sterilizing at 121 ℃/15 min;
(2) adding the prepared cellulase into sterilized fermentation bottles, wherein the addition amount of the cellulase liquid in each bottle is 20m L, and fully stirring and uniformly mixing;
(3) placing the fermentation bottle into a full-temperature shaking flask cabinet, and carrying out shaking culture at the constant temperature of 15 ℃ and 100r/min for 48 h;
(4) after the fermentation reaction is finished, centrifuging fresh fermentation liquor to obtain supernatant, wherein the supernatant is a product obtained by decomposing crop straws by cellulase;
(5) and (3) measuring the content of glucose in the supernatant, and further separating and purifying the supernatant, wherein the glucose is a main product of straw degradation for subsequent utilization.
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