CN113388653A - Method for improving yield of moso bamboo holocellulase hydrolysis glucose - Google Patents
Method for improving yield of moso bamboo holocellulase hydrolysis glucose Download PDFInfo
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Abstract
The application discloses a method for improving yield of moso bamboo holocellulose enzymatic hydrolysis glucose, wherein before moso bamboo holocellulose is subjected to enzymolysis, microwave-assisted eutectic solvent is adopted to pretreat moso bamboo. The microwave-assisted metal ion eutectic solvent pretreatment used in the method can effectively improve the yield of glucose hydrolyzed by enzyme, and has obvious effects on breaking high aggregation state and high crystallinity of cellulose; meanwhile, the method has the advantages of simple flow, mild reaction conditions, environmental friendliness, easiness in operation and the like, and the used method has the characteristics of short time and high efficiency and has practicability for producing the fermentable sugar by pretreating bamboo wood raw materials.
Description
Technical Field
The application belongs to the technical field of efficient conversion and utilization of biomass resources, and relates to a method for improving yield of moso bamboo holocellulase hydrolysis glucose.
Background
Nowadays, the demand of people for fuel resources is increasing day by day, and the traditional non-renewable fossil fuel resources are gradually exhausted after the exploitation of people for many years, and novel green and environment-friendly biomass fuel is gradually concerned by people. China has abundant agriculture and forestry biomass resources, and the cheap, easily-obtained and renewable resources are utilized to produce and convert energy substances required by human beings, so that the problem of shortage of future energy raw materials can be solved to a certain extent. The wood fiber mainly comprises cellulose, hemicellulose and lignin, wherein the cellulose and the hemicellulose can be converted into various monosaccharides through enzymolysis, and various platform compounds are generated through further fermentation and utilization. The lignocellulose is complex in structure and difficult to be directly utilized, so that the effective degradation and separation of the complex structure become key steps in the efficient utilization of the lignocellulose. One is gradually turning the line of sight to efficient pretreatment methods and understanding the intrinsic mechanisms that they efficiently break down the lignocellulosic biomass degradation barrier.
The dissociation of lignocellulose by various pretreatment methods is to increase the hydrolysis rate of cellulose by destroying the chemical components and physical structure of the cell wall of lignocellulose to increase the accessibility of enzymes to cellulose. Typical thermochemical pretreatments include hydrothermal, dilute acid and organic solvent pretreatments, which mainly result in depolymerization of the polymeric matrix surrounding the long cellulose chains, such as delignification and hemicellulose degradation. However, the conventional pretreatment method generates a large amount of waste liquid and is difficult to recover, and causes great pollution to the environment, so that a new environment-friendly method and reagent for pretreatment of lignocellulose are urgently needed to be found.
In recent years, eutectic solvents (DES) having properties similar to those of ionic liquids have attracted more and more attention due to their advantages of low cost, simple synthesis process, good biodegradability in biorefinery research, and the like. DES is made by combining a Hydrogen Bond Donor (HBD) (such as an amine, amide, alcohol, or carboxylic acid) and a quaternary ammonium salt as a Hydrogen Bond Acceptor (HBA). They have ionic properties, but the melting point of such organic substances is significantly lower than that of the individual components. In the existing literature reports, choline chloride is used as HBA, and metal ions are used as DES of HBD, so that the composite has strong capacity of dissolving lignin and cracking lignin-carbohydrate complexes. Recent studies have shown that DES containing metal ions can be used for heterogeneous esterification and degradation modification of cellulose, for example, in DES composed of ChCl-zinc chloride (1: 2), the substitution degree of modified cellulose is 0.64-2.74 [ ABBOTT A P, BELL T J, HANDA S, et al.O-acetic acid of cellulose and monosachhorides using a zinc based ionic liquid [ J ]. Green Chemistry, 2005, 7 (10): 705. DOI: 10.1039/b511691k ]. Meanwhile, the metal-based DES has the capability of catalyzing and degrading activated lignin [ Lisan, eutectic ionic liquid and mechanism research of activating lignin by plasma [ D ]. Nanjing university of forestry, 2014 ]. By utilizing the characteristic, the metal ion DES is successfully applied to the pretreatment of lignocellulose. Although DES pretreatment has proven to be expected to enhance the deconstruction and hydrolysis efficiency of biomass, the reaction process requires considerable time and higher temperatures and is not satisfactory for use.
Disclosure of Invention
Aiming at the defects in the prior art, the method for improving the yield of the glucose hydrolyzed by the phyllostachys edulis heald cellulase can effectively shorten the reaction time while improving the yield of the glucose hydrolyzed by the enzyme.
In order to solve the technical problem, the technical scheme adopted by the application is as follows:
a method for improving the yield of moso bamboo holocellulose enzyme hydrolysis glucose is characterized in that before moso bamboo holocellulose is subjected to enzymolysis, a microwave-assisted eutectic solvent is adopted to pretreat moso bamboo.
The hydrogen bond donor of the eutectic solvent is zinc acetate, the hydrogen bond acceptor is choline chloride, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1: 1-2.
The microwave radiation frequency is 600W, and the treatment temperature is 80-120 ℃.
The particle size of the raw material of the moso bamboo is less than 80 meshes.
The method for improving the yield of the moso bamboo holocellulase hydrolysis glucose comprises the following steps:
(1) preparing a eutectic solvent;
(2) taking a eutectic solvent to mix with the moso bamboo raw material in a solid-liquid manner, and carrying out microwave radiation pretreatment;
(3) carrying out solid-liquid separation on the pretreated mixture to obtain solid bamboo cellulose residues;
(4) and preparing glucose by enzyme hydrolysis.
In the step (1), the hydrogen bond donor of the eutectic solvent is zinc acetate, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor and the hydrogen bond acceptor are respectively mixed according to the molar ratio of 1: 1-2, and are stirred at the temperature of 60-100 ℃ after being mixed to react for 2 hours, so that the eutectic solvent is prepared.
In the step (2), the mass ratio of the moso bamboo to the eutectic solvent pretreatment liquid is 1: 10.
In the step (2), the microwave radiation frequency is 600W, the treatment temperature is 80-120 ℃, and the treatment time is 3 minutes.
In the step (3), the solid-liquid separation is that the moso bamboo powder is cooled to room temperature, washed alternately by acetone and water, and then centrifuged.
In the step (4), the pretreated moso bamboo raw material is mixed with cellulase CTec.2, pH buffer solution is added into the mixture according to the solid-liquid mass-volume ratio of 1: 10g/mL, and 20FPU/g cellulase CTec.2 is added into the mixture; the temperature is kept at 48 ℃ for 72 hours in a rotary shaker at 150 rpm.
Has the advantages that: compared with the prior art, the microwave-assisted metal ion eutectic solvent pretreatment used in the method can effectively improve the yield of glucose hydrolyzed by enzyme, and has obvious effects on breaking high aggregation state and high crystallinity of cellulose; meanwhile, the method has the advantages of simple flow, mild reaction conditions, environmental friendliness, easiness in operation and the like, and the used method has the characteristics of short time (reaction time of 3min) and high efficiency, and has practicability for producing fermentable sugar by pretreating bamboo wood raw materials.
Drawings
FIG. 1 is a graph showing the results of cellulase enzymolysis in example 1;
FIG. 2 is a graph showing the results of cellulase enzymolysis in example 2;
FIG. 3 is a diagram showing the results of cellulase enzymolysis of moso bamboo holocellulose pretreated by different eutectic solvents.
Detailed Description
In order that the present application may be more readily understood, the following detailed description of the present application is given by way of illustration only, and not by way of limitation, in conjunction with the accompanying examples.
The grain size of the phyllostachys edulis cellulose powder used in the following examples was 20 to 80 mesh, and the major components thereof were 74.0% in terms of cellulose, 14.5% in terms of hemicellulose (mainly xylan), and about 5% in terms of lignin content in the holocellulose, by component analysis.
The enzymolysis efficiency in the following examples is calculated by using glucose yield, and the glucose content in the hydrolysate is determined by using ion chromatography, and the specific disclosure is as follows:
the glucose yield (%) -, glucose content (g) in the hydrolysate/cellulose content (g) in the original enzymolysis material.
Example 1
A method for improving the yield of moso bamboo holocellulase hydrolysis glucose comprises the following steps:
(1) preparation of eutectic solvent (DES): zinc Acetate (ZA) is taken as a hydrogen bond donor, choline chloride (ChCl) is taken as a hydrogen bond acceptor, the zinc acetate and the choline chloride (ZA) are uniformly mixed according to the molar ratio of 1: 1 of the donor to the acceptor, and the mixture is heated for 2 hours at 80 ℃ by magnetic stirring until a uniform transparent liquid appears.
(2) Pretreatment: 6g of Phyllostachys pubescens holocellulose powder and 60g of DES (ZAC-1) were added to a Teflon container of a microwave reactor, and stirred and mixed uniformly using a glass rod, and 3 parts of the mixture were heated to 80 ℃ (ZAC-1-80 ℃), 100 ℃ (ZAC-1-100 ℃) and 120 ℃ (ZAC-1-120 ℃) respectively at a microwave irradiation power of 600W, and then subjected to constant temperature treatment for 3 minutes. After pretreatment the mixture was cooled to room temperature, washed alternately with acetone and water, centrifuged and the powder residue dried in an oven at 60 ℃ to constant weight.
(3) Enzymolysis: 1g of pretreated Phyllostachys Pubescens powder was taken, and 1g of non-pretreated Phyllostachys Pubescens powder (HC) was used as a control, 20FPU/g of cellulase CTec.2 and 10mL of a buffer (50mM sodium acetate, pH 4.8) were added thereto, and the mixture was reacted for 72 hours in an air shaking incubator maintained at 48 ℃ for 150 rpm. After the reaction, the filtrate was filtered with suction until the filtrate became neutral, and a sample was taken for component analysis, the results of which are shown in FIG. 1.
Example 2
A method for improving the yield of moso bamboo holocellulase hydrolysis glucose comprises the following steps:
(1) preparation of DES: zinc Acetate (ZA) is taken as a hydrogen bond donor, choline chloride (ChCl) is taken as a hydrogen bond acceptor, the zinc acetate and the choline chloride (ZA) are uniformly mixed according to the molar ratio of 1: 2 of the donor to the acceptor, and the mixture is heated for 2 hours at 80 ℃ by magnetic stirring until a uniform transparent liquid appears.
(2) Pretreatment: 6g of Phyllostachys pubescens holocellulose powder and 60g of DES (ZAC-2) were added to a Teflon container of a microwave reactor, and stirred and mixed uniformly using a glass rod, and 3 parts of the mixture were heated to 80 ℃ (ZAC-1-80 ℃), 100 ℃ (ZAC-1-100 ℃) and 120 ℃ (ZAC-1-120 ℃) respectively at a microwave irradiation power of 600W, and then subjected to constant temperature treatment for 3 minutes. After pretreatment the mixture was cooled to room temperature, washed alternately with acetone and water, centrifuged and the powder residue dried in an oven at 60 ℃ to constant weight.
(3) Enzymolysis: 1g of pretreated Phyllostachys Pubescens powder was taken, and 1g of non-pretreated Phyllostachys Pubescens powder (HC) was used as a control, 20FPU/g of cellulase CTec.2 and 10mL of buffer (50mM sodium acetate, pH 4.8) were added thereto, and the mixture was reacted for 72 hours in an air shaking incubator maintained at 48 ℃. After the reaction, the filtrate was filtered with suction until the filtrate became neutral, and a sample was taken for component analysis, the results of which are shown in FIG. 2.
Example 3
As shown in figure 1, figure 2 and figure 3, the cellulase enzymolysis result of the moso bamboo holocellulose pretreated by the eutectic solvent shows that the glucose yield of the untreated raw material (HC) is only 50.30 percent after 72 hours of enzyme hydrolysis reaction, and the glucose yields after microwave-assisted pretreatment of ZAC-1-80 ℃, ZAC-1-100 ℃, ZAC-1-120 ℃, ZAC-2-80 ℃, ZAC-2-100 ℃ and ZAC-2-120 ℃ are respectively improved to 65.11 percent, 67.33 percent, 63.80 percent, 64.22 percent, 66.83 percent and 65.98 percent. The results showed that of the six microwave-assisted DES, ZAC-1-100 ℃ gave the highest glucose yield of 67.33%, followed by ZAC-2-100 ℃ (66.83%), ZAC-2-120 ℃ (65.98%), ZAC-1-80 ℃ (65.11%), ZAC-2-80 ℃ (64.22%) and ZAC-1-120 ℃ (63.80%), respectively.
Therefore, the method can obviously promote enzymatic hydrolysis and improve the yield of glucose, and has the advantages of simple pretreatment process condition, short operation time, high efficiency and practical operation value.
Example 4
The process is the same as in examples 1 and 2, except that:
1. the microwave-assisted treatment is changed into oil bath heating, the reaction temperature and the reaction time are the same as those of the example and the example 2, and the yield of the glucose hydrolyzed by the enzyme of the obtained sample for 72 hours is lower and is similar to that of the glucose (HC) hydrolyzed by the enzyme of the original moso bamboo holocellulose.
2. The pretreatment was carried out while changing the microwave treatment power to 200W and 400W, and the other conditions were the same as in examples 1 and 2, and the yields of glucose by the enzymatic hydrolysis of the obtained samples for 72 hours were as shown in Table 1.
TABLE 172 hours yield of enzymatically hydrolyzed glucose
It should be noted that the embodiments of the present application are not limited by the above-mentioned examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present application should be regarded as equivalent replacements and are included in the protection scope of the present application.
Claims (10)
1. A method for improving the yield of moso bamboo holocellulose enzymatic hydrolysis glucose is characterized in that before moso bamboo holocellulose enzymatic hydrolysis, a microwave-assisted eutectic solvent is adopted to pretreat moso bamboo.
2. The method for improving the yield of the moso bamboo holocellulase-hydrolyzed glucose according to claim 1, wherein a hydrogen bond donor of the eutectic solvent is zinc acetate, a hydrogen bond acceptor is choline chloride, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1: 1-2.
3. The method for improving the yield of the moso bamboo holocellulase-hydrolyzed glucose according to claim 1, wherein the microwave radiation frequency is 600W, and the treatment temperature is 80-120 ℃.
4. The method for improving the yield of the moso bamboo holocellulase-hydrolyzed glucose according to claim 1, wherein the particle size of the moso bamboo raw material is less than 80 meshes.
5. The method for improving the yield of the moso bamboo holocellulase-hydrolyzed glucose according to claim 1, characterized by comprising the following steps:
(1) preparing a eutectic solvent;
(2) taking a eutectic solvent to mix with the moso bamboo raw material in a solid-liquid manner, and carrying out microwave radiation pretreatment;
(3) carrying out solid-liquid separation on the pretreated mixture to obtain solid bamboo cellulose residues;
(4) and preparing glucose by enzyme hydrolysis.
6. The method for improving the yield of the moso bamboo holocellulase hydrolysis glucose according to claim 5, characterized in that in the step (1), the hydrogen bond donor of the eutectic solvent is zinc acetate, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor and the hydrogen bond acceptor are respectively mixed in a molar ratio of 1: 1-2, and are stirred at 60-100 ℃ after being mixed, and react for 2h to prepare the eutectic solvent.
7. The method for improving the yield of the moso bamboo holocellulase-hydrolyzed glucose in the claim 5, wherein in the step (2), the mass ratio of the moso bamboo to the eutectic solvent pretreatment solution is 1: 10.
8. The method for improving the yield of the moso bamboo holocellulase-hydrolyzed glucose according to claim 5, wherein in the step (2), the microwave radiation frequency is 600W, the treatment temperature is 80-120 ℃, and the treatment time is 3 minutes.
9. The method for improving the yield of the moso bamboo holocellulase-hydrolyzed glucose according to claim 5, wherein in the step (3), the solid-liquid separation is that the moso bamboo powder is cooled to room temperature, washed by acetone and water alternately and then centrifuged.
10. The method for improving the yield of the moso bamboo holocellulase hydrolysis glucose according to claim 5, characterized in that in the step (4), the pretreated moso bamboo raw material is mixed with the cellulase CTec.2, pH buffer solution is added at a solid-liquid mass-volume ratio of 1: 10g/mL, 20FPU/g cellulase CTec.2 is added; the temperature is kept at 48 ℃ for 72 hours in a rotary shaker at 150 rpm.
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