阅读说明：本技术 一种利用纤维素酶水解处理稻草秸秆的方法 (Method for hydrolyzing rice straw by using cellulase ) 是由 顾斌涛 黄国昌 熊大维 章帅文 于 2021-11-02 设计创作，主要内容包括：本发明公开了一种利用纤维素酶水解处理稻草秸秆的方法,涉及农作物秸秆酶解领域,包括：S1、将稻草秸秆粉碎；S2、将稻草秸秆与离子液体混合,微波加热,并辅助以超声波处理10～30min；S3、将稻草秸秆进行洗涤,干燥至稻草秸秆的湿度为60～70％,然后将稻草秸秆加入密闭容器内,往密闭容器中加入液氨,处理5～20min后释放压力进行爆破；S4、往稻草秸秆中加入pH缓冲溶液,调节pH为4.5～6.5,加入纤维素酶进行酶解。本发明的有益效果是进行了离子液体与氨纤维爆破预处理结合,能够打破木质素与纤维素及半纤维素之间的结合,提高纤维素的可及度及疏松性,促进后续的纤维素酶与底物的接触,提高酶解的效率。(The invention discloses a method for hydrolyzing rice straw by using cellulase, which relates to the field of crop straw enzymolysis and comprises the following steps: s1, crushing straw stalks; s2, mixing the straw stalks with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 10-30 min in an auxiliary manner; s3, washing the straw stalks, drying until the humidity of the straw stalks is 60-70%, adding the straw stalks into a closed container, adding liquid ammonia into the closed container, treating for 5-20 min, and releasing pressure to explode; s4, adding a pH buffer solution into the straw stalks, adjusting the pH to 4.5-6.5, and adding cellulase for enzymolysis. The method has the advantages that the ionic liquid and the ammonia fiber are combined in the blasting pretreatment, so that the combination of lignin, cellulose and hemicellulose can be broken, the accessibility and the looseness of the cellulose are improved, the subsequent contact between the cellulase and a substrate is promoted, and the enzymolysis efficiency is improved.)

1. A method for hydrolyzing rice straw by using cellulase is characterized by comprising the following steps:

s1, crushing straw stalks;

s2, mixing the straw stalks crushed in the step S1 with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 10-30 min in an auxiliary manner;

s3, washing the straw stalks processed in the step S2, drying until the humidity of the straw stalks is 60-70%, adding the straw stalks into a closed container, adding liquid ammonia into the closed container, processing for 5-20 min, and releasing pressure to explode;

s4, adding a pH buffer solution into the straw stalks processed in the step S3, adjusting the pH to 4.5-6.5, and adding cellulase for enzymolysis.

2. The method of claim 1, wherein in step S2, the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid, and the solid-to-liquid ratio of the straw stalks to the ionic liquid is 1: 5-20 g/ml.

3. The method of claim 1, wherein in step S2, the microwave power is 200-300W, the microwave heating temperature is 60-80 ℃, the ultrasonic treatment power is 300-400W, and the frequency is 10-20 KHZ.

4. The method of claim 1, wherein in step S3, the mass ratio of liquid ammonia to straw stalks is 0.5-1.5: 1.

5. The method of claim 1, wherein in step S3, the pressure in the sealed container is 1-4 MPa.

6. The method for hydrolyzing rice straw stalk with cellulase as claimed in claim 1, wherein the step S3 specifically comprises: and (4) washing the straw stalks processed in the step (S2), drying until the humidity of the straw stalks is 60-70%, adding the straw stalks into a closed container, adding liquid ammonia into the closed container, heating the straw stalks to 90-100 ℃, controlling the pressure in the closed container to be 1-4 MPa, and releasing the pressure for blasting after the treatment is carried out for 5-20 min.

7. The method for hydrolyzing rice straw stalks with cellulase according to claim 1, wherein in S4, the addition amount of the cellulase is 5 to 10% of the mass of the rice straw stalks, and the enzyme activity of the cellulase is 1000 to 1500U/g.

8. The method for hydrolyzing rice straw with cellulase according to claim 1, wherein in S4, the enzymolysis temperature is 40-60 ℃ and the enzymolysis time is 20-40 h.

9. The method of claim 1, wherein glutathione, bovine serum albumin, rhamnolipid and cellulase are further added to the S4 for enzymolysis.

10. The method of claim 9, wherein the amount of glutathione added is 0.001-0.0015%, the amount of bovine serum albumin added is 0.002-0.003%, and the amount of rhamnolipid added is 0.001-0.002%.

Technical Field

The invention relates to the technical field of crop enzymolysis, in particular to a method for hydrolyzing rice straw by using cellulase.

Background

The straw stalk is an important renewable biomass resource, and at present, except for the utilization in the aspect of paper industry, most of the straw stalk is abandoned or burnt, thus seriously wasting the resource and polluting the environment.

The main components of the straw stalk comprise cellulose, hemicellulose and lignin, and the cellulose, the hemicellulose and the lignin are mainly present in the form of cell walls. Wherein cellulose is the skeletal material of the fiber, and lignin and hemicellulose are dispersed in and around the fiber in the form of inclusion material. The straw stalk contains about 35% of cellulose, 25% of hemicellulose and 21% of lignin. In addition to cellulose, lignin and hemicellulose, straw stalks contain minor components such as crude proteins, low molecular carbohydrates and inorganic salts.

The research on the hydrolysis of straw stalks to produce alcohol, lactic acid, biodegradable cellulose film and other products is attracting more and more attention, but the structure of plant cell walls is very complex, and lignocellulose forms the plant cell walls to protect the cells. Lignocellulose refers to the general term of cellulose, hemicellulose and lignin, and has a small amount of pectin, gum, algin, agar and other components, and the structure is very complex. Cellulose and hemicellulose are wrapped by lignin layer by layer, the cellulose is linear polysaccharide formed by connecting 1000-10000 beta-D-glucopyranose monomers in a beta-1, 4-glycosidic bond mode, a plurality of molecular layers are arranged in parallel to form a filamentous insoluble microfibril structure, the basic composition unit is cellobiose, and the polymer is the polymer with the most abundant content on the earth. Hemicellulose is mainly composed of xylose, a small amount of arabinose, galactose or mannose. The lignin is a high molecular aromatic compound formed by taking phenylpropane and derivatives thereof as basic composition units, and plays a barrier role in the cellulose hydrolysis process. Thus, the presence of lignin prevents the access of enzymes to cellulose and hemicellulose, and the binding of lignin to hydrolytic enzymes is irreversible, resulting in a reduced rate of hydrolysis. Therefore, to improve the enzymatic efficiency of natural cellulose, a certain pretreatment method must be used to destroy its complex structure before it can be used, so that cellulose can be exposed for hydrolysis by cellulase.

The pretreatment process is to promote the natural polymer structure of the lignocellulose to be decomposed into a structure which is easy to be utilized by microorganisms. The pretreatment method can destroy the package of lignin and hemicellulose on cellulose, remove lignin, degrade hemicellulose, change the crystalline structure of cellulose, improve the accessibility and the looseness of the cellulose, promote the contact of enzyme and a substrate, and improve the efficiency of subsequent enzymolysis and the sugar yield. The conventional pretreatment method mainly adopts acid-base pretreatment and the like, but the acid-base pretreatment method can cause a large amount of waste liquid to be generated, and the waste liquid contains strong acid and strong base which are directly discharged to pollute the environment.

Disclosure of Invention

The invention aims to at least solve one of the technical problems in the prior art and provides a method for treating straw stalks by using cellulase hydrolysis.

The technical solution of the invention is as follows:

a method for hydrolyzing rice straw by using cellulase comprises the following steps:

s1, crushing straw stalks;

s2, mixing the straw stalks crushed in the step S1 with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 10-30 min in an auxiliary manner;

s3, washing the straw stalks processed in the step S2, drying until the humidity of the straw stalks is 60-70%, adding the straw stalks into a closed container, adding liquid ammonia into the closed container, processing for 5-20 min, and releasing pressure to explode;

s4, adding a pH buffer solution into the straw stalks processed in the step S3, adjusting the pH to 4.5-6.5, and adding cellulase for enzymolysis.

Preferably, in the step S2, the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid, and the solid-to-liquid ratio of the straw stalk to the ionic liquid is 1: 5-20 g/ml.

Preferably, in the step S2, the microwave power is 200-300W, the microwave heating temperature is 60-80 ℃, the ultrasonic treatment power is 300-400W, and the frequency is 10-20 KHZ.

Preferably, in the step S3, the mass ratio of the liquid ammonia to the straw stalks is 0.5 to 1.5: 1.

Preferably, in the step S3, the pressure in the closed container is 1 to 4 MPa.

Preferably, the step S3 specifically includes: and (4) washing the straw stalks processed in the step (S2), drying until the humidity of the straw stalks is 60-70%, adding the straw stalks into a closed container, adding liquid ammonia into the closed container, heating the closed container to 90-100 ℃, processing for 5-20 min, and releasing pressure for blasting. .

Preferably, in the S4, the adding amount of the cellulase is 5-10% of the mass of the straw stalks, and the enzyme activity of the cellulase is 1000-1500U/g.

Preferably, in the S4, the enzymolysis temperature is 40-60 ℃, and the enzymolysis time is 20-40 h.

Preferably, glutathione, bovine serum albumin, rhamnolipid and cellulase are further added to the S4 for enzymolysis.

Preferably, the addition amount of the glutathione is 0.001-0.0015% of the mass of the straw stalks, the addition amount of the bovine serum albumin is 0.002-0.003% of the mass of the straw stalks, and the addition amount of the rhamnolipid is 0.001-0.002% of the mass of the straw stalks.

The invention has at least one of the following beneficial effects:

1. the invention combines the ionic liquid and the ammonia fiber blasting pretreatment on the rice straws, firstly, the rice straws and the ionic liquid are mixed and assisted by microwave-ultrasonic treatment, the ionic liquid has good dissolving capacity on cellulose, the microwave treatment comprises a thermal effect and a non-thermal effect, the supermolecular structure of the fiber can be improved, the size of a fiber crystallization area is changed, and lignin and hemicellulose can be partially degraded; the ultrasonic waves can generate a mechanical effect, a cavitation effect and a thermal effect, not only can strengthen mass transfer, increase the contact between the ionic liquid and the surface of the straw stalks, but also can generate structural influence on the straw stalks, so that the straw stalks can be pretreated through the synergistic effect of the ionic liquid and the microwave-ultrasonic treatment, and the combination of lignin, cellulose and hemicellulose is broken; further, the method continuously performs ammonia fiber blasting on the ionic liquid and the straw stalks pretreated by the microwave-ultrasonic waves, and the ammonia fiber blasting can decompose cellulose crystals and depolymerize hemicellulose due to the quenching effect generated by vaporization of liquid ammonia, so that the combination of lignin, the cellulose and the hemicellulose is further broken, the lignin and a small amount of the hemicellulose can be removed, the accessibility and the looseness of the cellulose are improved, the subsequent contact of the cellulase and a substrate can be promoted, and the subsequent enzymolysis efficiency is improved.

2. The cellulase has higher sensitivity, is easy to be influenced by the environment and can be gradually inactivated in the reaction, so the glutathione, the bovine serum albumin, the rhamnolipid and the cellulase are added for enzymolysis together, the glutathione and the rhamnolipid are preferentially adsorbed on the surface of a substrate molecule compared with the cellulase, the enzyme adsorbed on the substrate can be replaced, the enzyme molecule is released into a solution again, the failure caused by the irreversible adsorption of partial enzyme is avoided, the ineffective adsorption of the cellulase can be reduced, the denaturation and inactivation rate of the cellulase is reduced, the bovine serum albumin can improve the activity and the stability of the enzyme, the reaction efficiency is improved, and the hydrolysis efficiency is greatly improved.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1

A method for hydrolyzing rice straw by using cellulase comprises the following steps:

s1, crushing the straw stalks to 50-150 meshes;

s2, mixing the straw stalks crushed in the step S1 with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 10 min; the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid, and the solid-to-liquid ratio of the straw stalk to the ionic liquid is 1:5 g/ml; the microwave power is 300W, the microwave heating temperature is 80 ℃, the ultrasonic treatment power is 400W, and the frequency is 20 KHZ;

s3, washing the rice straw processed in the step S2, drying until the humidity of the rice straw is 60%, then adding the rice straw into a closed container, adding liquid ammonia into the closed container, heating the rice straw to 90 ℃, controlling the pressure in the closed container to be 3MPa, and releasing the pressure to explode after processing for 5 min.

S4, adding a pH buffer solution citric acid-sodium citrate buffer solution into the straw stalks processed in the step S3, adjusting the pH to 4.5, adding cellulase, glutathione, bovine serum albumin and rhamnolipid for enzymolysis, wherein the addition of the cellulase is 5% of the mass of the straw stalks, the enzyme activity of the cellulase is 1500U/g, the addition of the glutathione is 0.001% of the mass of the straw stalks, the addition of the bovine serum albumin is 0.002% of the mass of the straw stalks, and the addition of the rhamnolipid is 0.001% of the mass of the straw stalks; the enzymolysis temperature is 40 ℃, and the enzymolysis time is 20 h.

Example 2

A method for hydrolyzing rice straw by using cellulase comprises the following steps:

s1, crushing the straw stalks to 50-150 meshes;

s2, mixing the straw stalks crushed in the step S1 with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 15 min; the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid, and the solid-to-liquid ratio of the straw stalk to the ionic liquid is 1:10 g/ml; the microwave power is 300W, the microwave heating temperature is 75 ℃, the ultrasonic treatment power is 400W, and the frequency is 20 KHZ;

s3, washing the rice straw processed in the step S2, drying until the humidity of the rice straw is 65%, adding the rice straw into a closed container, adding liquid ammonia into the closed container, heating the rice straw to 92 ℃, controlling the pressure in the closed container to be 1.5MPa, and releasing the pressure to perform blasting after processing for 10 min; the mass ratio of liquid ammonia to straw stalks is 0.8:1, and the pressure in the closed container is 1.5 MPa; the closed container was heated to 90 ℃.

S4, adding a pH buffer solution citric acid-sodium citrate buffer solution into the straw stalks processed in the step S3, adjusting the pH to 5, adding cellulase, glutathione, bovine serum albumin and rhamnolipid for enzymolysis, wherein the adding amount of the cellulase is 6 percent of the mass of the straw stalks, the enzyme activity of the cellulase is 1400U/g, the adding amount of the glutathione is 0.0014 percent of the mass of the straw stalks, the adding amount of the bovine serum albumin is 0.0028 percent of the mass of the straw stalks, and the adding amount of the rhamnolipid is 0.0018 percent of the mass of the straw stalks; the enzymolysis temperature is 55 ℃, and the enzymolysis time is 25 h.

Example 3

A method for hydrolyzing rice straw by using cellulase comprises the following steps:

s1, crushing the straw stalks to 50-150 meshes;

s2, mixing the straw stalks crushed in the step S1 with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 20min in an auxiliary manner; the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid, and the solid-to-liquid ratio of the straw stalk to the ionic liquid is 1:12 g/ml; the microwave power is 250W, the microwave heating temperature is 70 ℃, the ultrasonic treatment power is 350W, and the frequency is 15 KHZ;

s3, washing the rice straw processed in the step S2, drying until the humidity of the rice straw is 65%, then adding the rice straw into a closed container, adding liquid ammonia into the closed container, heating the rice straw to 95 ℃, controlling the pressure in the closed container to be 2.5MPa, and releasing the pressure to explode after processing for 12 min.

S4, adding a pH buffer solution citric acid-sodium citrate buffer solution into the straw stalks processed in the step S3, adjusting the pH to 5.5, adding cellulase, glutathione, bovine serum albumin and rhamnolipid for enzymolysis, wherein the addition of the cellulase is 7%, the enzyme activity of the cellulase is 1300U/g, the addition of the glutathione is 0.0013% of the mass of the straw stalks, the addition of the bovine serum albumin is 0.0025% of the mass of the straw stalks, and the addition of the rhamnolipid is 0.0015% of the mass of the straw stalks; the enzymolysis temperature is 50 ℃, and the enzymolysis time is 30 h.

Example 4

A method for hydrolyzing rice straw by using cellulase comprises the following steps:

s1, crushing the straw stalks to 50-150 meshes;

s2, mixing the straw stalks crushed in the step S1 with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 25 min; the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid, and the solid-to-liquid ratio of the straw stalk to the ionic liquid is 1:15 g/ml; the microwave power is 250W, the microwave heating temperature is 65 ℃, the ultrasonic treatment power is 350W, and the frequency is 15 KHZ;

s3, washing the rice straw processed in the step S2, drying until the humidity of the rice straw is 65%, then adding the rice straw into a closed container, adding liquid ammonia into the closed container, heating the rice straw to 98 ℃, controlling the pressure in the closed container to be 1MPa, and releasing the pressure to perform blasting after processing for 10 min.

S4, adding a pH buffer solution citric acid-sodium citrate buffer solution into the straw stalks processed in the step S3, adjusting the pH to 6, adding cellulase, glutathione, bovine serum albumin and rhamnolipid for enzymolysis, wherein the adding amount of the cellulase is 9% of the mass of the straw stalks, the enzyme activity of the cellulase is 1400U/g, the adding amount of the glutathione is 0.0014% of the mass of the straw stalks, the adding amount of the bovine serum albumin is 0.0028% of the mass of the straw stalks, and the adding amount of the rhamnolipid is 0.0018% of the mass of the straw stalks; the enzymolysis temperature is 55 ℃, and the enzymolysis time is 35 h.

Example 5

A method for hydrolyzing rice straw by using cellulase comprises the following steps:

s1, crushing the straw stalks to 50-150 meshes;

s2, mixing the straw stalks crushed in the step S1 with ionic liquid, heating by microwave, and carrying out ultrasonic treatment for 30 min; the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid, and the solid-to-liquid ratio of the straw stalk to the ionic liquid is 1:20 g/ml; the microwave power is 200W, the microwave heating temperature is 80 ℃, the ultrasonic treatment power is 300W, and the frequency is 15 KHZ;

s3, washing the rice straw processed in the step S2, drying until the humidity of the rice straw is 70%, then adding the rice straw into a closed container, adding liquid ammonia into the closed container, heating the rice straw to 100 ℃, controlling the pressure in the closed container to be 2MPa, and releasing the pressure to explode after processing for 20 min.

S4, adding a pH buffer solution citric acid-sodium citrate buffer solution into the straw stalks processed in the step S3, adjusting the pH to 6.5, adding cellulase, glutathione, bovine serum albumin and rhamnolipid for enzymolysis, wherein the addition of the cellulase is 10% of the mass of the straw stalks, the enzyme activity of the cellulase is 1000U/g, the addition of the glutathione is 0.0015% of the mass of the straw stalks, the addition of the bovine serum albumin is 0.003% of the mass of the straw stalks, and the addition of the rhamnolipid is 0.002% of the mass of the straw stalks; the enzymolysis temperature is 60 ℃, and the enzymolysis time is 40 h.

Comparative example 1

The difference from example 1 is that: in step S2, heating was carried out to 80 ℃ in a conventional manner without microwave and ultrasonic treatment, and the rest was the same as in example 1.

Comparative example 2

The difference from example 1 is that: step S2 is not performed, and the procedure is the same as in example 1.

Comparative example 3

The difference from example 1 is that: step S3 is not performed, and the procedure is the same as in example 1.

Comparative example 4

The difference from example 1 is that: the sequence of steps S2 and S3 is reversed, that is, step S3 is performed first, step S2 is performed again, and the straw stalks processed in step S2 are washed and then step S4 is performed again, which is otherwise the same as in example 1.

Comparative example 5

The difference from example 1 is that: in step S4, glutathione, bovine serum albumin and rhamnolipid were not added, and the procedure was otherwise the same as in example 1.

Comparative example 6

The difference from example 1 is that: steps S2 and S3 were not performed, and glutathione, bovine serum albumin, rhamnolipid were not added in step S4, but the rest were the same as in example 1.

And (4) testing results:

the results of detecting the enzymatic hydrolysis rates in examples 1 to 5 and comparative examples 1 to 6 are shown in table 1:

TABLE 1

As can be seen from table 1, the enzymolysis rate of example 1 is 99.4%, and the enzymolysis rates of examples 2 to 5 are all 100%, which shows that the pretreatment method of the present invention can completely break the combination between lignin, cellulose and hemicellulose, improve the accessibility and the porosity of cellulose, enable cellulase to fully contact with a substrate, and improve the activity and the stability of cellulase by adding glutathione, bovine serum albumin and rhamnolipid, thereby improving the enzymolysis efficiency.

Comparing the example 1 with the comparative examples 1-6, it can be seen that the comparative example 6 has no pretreatment and no glutathione, bovine serum albumin and rhamnolipid are added, the enzymolysis rate is only 8.9%, and the straw stalks need to be pretreated before enzymolysis to obtain higher enzymolysis rate; comparative example 1 no microwave and ultrasonic wave auxiliary treatment was performed, and the enzymolysis rate was 90.2%; comparative example 2 no ionic liquid pretreatment was performed, only ammonia fiber blasting pretreatment was performed, and the enzymolysis rate was 82.4%; comparative example 3, ammonia fiber blasting pretreatment is not carried out, only ionic liquid pretreatment is carried out, and the enzymolysis rate is 85.1%; therefore, whether microwave and ultrasonic wave auxiliary treatment is carried out, whether ionic liquid pretreatment is carried out or not and whether ammonia fiber blasting pretreatment is carried out or not influence the enzymolysis efficiency, and the influence of the ionic liquid pretreatment on the enzymolysis efficiency is the largest. In comparative example 4, the sequence of steps S2 and S3 is simply reversed, the enzymolysis rate is 93.8%, which is slightly lower than that in example 1, thereby illustrating that the ionic liquid pretreatment and the ammonia fiber blasting pretreatment also have an effect on the enzymolysis efficiency, and the mechanism is not clear at all. The comparative example 5 does not add glutathione, bovine serum albumin and rhamnolipid, and the enzymolysis rate is reduced to 84.7%, thereby showing that the glutathione, the bovine serum albumin and the rhamnolipid can influence the activity of cellulase, thereby finally influencing the enzymolysis rate.

The above are merely characteristic embodiments of the present invention, and do not limit the scope of the present invention in any way. All technical solutions formed by equivalent exchanges or equivalent substitutions fall within the protection scope of the present invention.