CN113388653B - Method for improving glucose hydrolysis yield of moso bamboo heald cellulase - Google Patents
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Abstract
The application discloses a method for improving glucose hydrolysis yield of moso bamboo heald cellulose, which comprises the step of preprocessing moso bamboo by adopting a microwave-assisted eutectic solvent before enzymolysis of Mao Zhuzeng cellulose. The microwave-assisted metal ion eutectic solvent pretreatment used in the method can not only effectively improve the yield of glucose hydrolyzed by enzyme, but also has remarkable effect on breaking the high aggregation state and high crystallinity of cellulose; meanwhile, the method has the advantages of simple flow, mild reaction conditions, environment 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 fermentable sugar by pretreating bamboo 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 glucose hydrolysis yield of moso bamboo comprehensive cellulase.
Background
Today, the demand for fuel resources is increasing, and traditional non-renewable fossil fuel resources are gradually exhausted after years of exploitation, and new green and environment-friendly biomass fuels are gradually attracting attention. China has rich agriculture and forestry biomass resources, and can utilize the cheap and easily available renewable resources to produce energy substances required by human beings, so that the shortage problem of future energy raw materials can be solved to a certain extent. The lignocellulose 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 has a complex structure and is difficult to directly utilize, so that the effective degradation and separation of the complex structure become key steps in the efficient utilization of lignocellulose. The line of sight is increasingly being turned to efficient pretreatment methods and understand their inherent mechanisms of efficient deconstructing of the lignocellulosic biomass degradation barrier.
The dissociation of lignocellulose by various pretreatment methods increases the accessibility of enzymes to cellulose by destroying the chemical composition and physical structure of the cell wall of lignocellulose to increase the rate of hydrolysis of cellulose. Typical thermochemical pretreatments include hydrothermal, dilute acid and organic solvent pretreatments, which primarily result in depolymerization of the polymeric matrix surrounding the long chains of cellulose, such as delignification and hemicellulose degradation. However, the conventional pretreatment method can generate a large amount of waste liquid, is difficult to recycle and can cause huge pollution to the environment, so that a novel environment-friendly method and reagent for pretreatment of lignocellulose are required to be searched.
In recent years, a eutectic solvent (DES) similar to ionic liquids in nature has been receiving more and more attention due to its advantages of low cost, simple synthesis process, good biodegradability in biorefinery and the like. DES is formed 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 materials is significantly lower than that of the individual components. In the prior literature report, the DES which takes choline chloride as HBA and metal ions as HBD has strong capability of dissolving lignin and cracking lignin-carbohydrate complex. 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 degree of substitution of modified cellulose is 0.64-2.74[ABBOTT A P,BELL T J,HANDA S,et al.O-Acetylation of cellulose and monosaccharides using a zinc based ionic liquid [ J ]. Green Chemistry,2005,7 (10): doi:10.1039/b511691k ]. Meanwhile, the metal-based DES has the capacity of catalyzing and degrading and activating lignin [ Li Shan ], the mechanism research [ D ] of eutectic ionic liquid and plasma activated lignin, university of Nanjing forestry, 2014 ]. With this feature, the metal ion DES is successfully applied to the pretreatment of lignocellulose. While DES pretreatment has proven promising to enhance the efficiency of biomass deconstruction and hydrolysis, the reaction process requires considerable time and higher temperatures and is not satisfactory for use.
Disclosure of Invention
Aiming at the defects existing in the prior art, the application aims to provide a method for improving the glucose hydrolysis yield of the moso bamboo comprehensive cellulase, which can effectively shorten the reaction time while improving the glucose hydrolysis yield of the enzyme.
In order to solve the technical problems, the technical scheme adopted in the application is as follows:
a method for improving glucose hydrolysis yield of moso bamboo heald cellulase comprises pretreating moso bamboo with microwave-assisted eutectic solvent before Mao Zhuzeng cellulose is hydrolyzed.
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 grain size of the moso bamboo raw material is smaller than 80 meshes.
The method for improving the yield of the Mao Zhuzeng cellulase hydrolyzed glucose comprises the following steps:
(1) Preparing a eutectic solvent;
(2) Mixing eutectic solvent with Phyllostachys Pubescens raw material solid-liquid, and performing microwave radiation pretreatment;
(3) Carrying out solid-liquid separation on the pretreatment mixture to obtain solid phyllostachys pubescens cellulose residues;
(4) Glucose is prepared by enzymatic 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 the mixture is stirred at 60-100 ℃ and reacts for 2 hours to prepare the eutectic solvent.
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 carried out by cooling moso bamboo powder to room temperature, washing with acetone and water alternately, and centrifuging.
In the step (4), the pretreated phyllostachys pubescens raw material is mixed with cellulase CTec.2, a pH buffer solution is added according to the solid-liquid mass volume ratio of 1:10 g/mL, and 20FPU/g of cellulase CTec.2 is added; the reaction was continued in a 150 rpm rotary shaker at 48℃for 72h.
The beneficial effects are that: compared with the prior art, the microwave-assisted metal ion eutectic solvent pretreatment used in the application can not only effectively improve the yield of glucose hydrolyzed by enzyme, but also has remarkable effect on breaking the high aggregation state and high crystallinity of cellulose; meanwhile, the method has the advantages of simple flow, mild reaction conditions, environment friendliness, easiness in operation and the like, and the used method has the characteristics of short time (reaction time is 3 min) and high efficiency, and has practicability for producing fermentable sugar by pretreating bamboo raw materials.
Drawings
FIG. 1 is a graph showing the results of enzymolysis with cellulase in example 1;
FIG. 2 is a graph showing the results of enzymolysis with cellulase in example 2;
FIG. 3 is a graph showing the results of cellulase enzymatic hydrolysis of Mao Zhuzeng cellulose pretreated with different eutectic solvents.
Detailed Description
In order that the present application may be more readily understood, the following detailed description of the embodiments will be presented by way of illustration and not limitation.
The Mao Zhuzeng cellulose powder used in the following examples, having a particle size of 20 to 80 mesh, was composed of cellulose 74.0% and hemicellulose 14.5% (mainly xylan) as main components, and had a lignin content of about 5% in the total cellulose, by component analysis.
The enzymolysis efficiency in the following examples is calculated by using the glucose yield, and the glucose content in the hydrolysate is measured by using ion chromatography, and is specifically shown as follows:
glucose yield (%) =glucose content (g) in hydrolysate/cellulose content (g) of original enzymatic hydrolysate.
Example 1
A method for improving the glucose hydrolysis yield of moso bamboo heald cellulase comprises the following steps:
(1) Preparation of the eutectic solvent (DES): zinc Acetate (ZA) was taken as hydrogen bond donor and choline chloride (ChCl) as hydrogen bond acceptor, and the mixture was uniformly mixed in a molar ratio of donor to acceptor of 1:1, and heated with magnetic stirring at 80 ℃ for 2h until a uniform transparent liquid appeared.
(2) Pretreatment: 6g Mao Zhuzeng cellulose powder and 60g DES (ZAC-1) were added to a Teflon container of a microwave reactor, and stirred and mixed uniformly by using a glass rod, and 3 parts of the mixture was heated to 80℃C (ZAC-1-80 ℃) 100℃C (ZAC-1-100 ℃) 120℃C (ZAC-1-120 ℃) respectively under 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 was dried to constant weight in an oven at 60 ℃.
(3) Enzymolysis: taking 1g of pretreated moso bamboo powder, taking 1g of non-pretreated moso bamboo powder (HC) as a reference, adding 20FPU/g of cellulase CTec.2 and 10mL of buffer solution (50 mM sodium acetate, pH=4.8), and carrying out 150-revolution/minute reaction for 72 hours in an air shaking incubator maintained at 48 ℃. After the reaction, the filtrate was filtered to neutrality, and the sample was taken for component analysis, and the result is shown in FIG. 1.
Example 2
A method for improving the glucose hydrolysis yield of moso bamboo heald cellulase comprises the following steps:
(1) Preparation of DES: zinc Acetate (ZA) was taken as hydrogen bond donor and choline chloride (ChCl) as hydrogen bond acceptor, and the mixture was uniformly mixed in a molar ratio of donor to acceptor of 1:2, and heated with magnetic stirring at 80 ℃ for 2h until a uniform transparent liquid appeared.
(2) Pretreatment: 6g Mao Zhuzeng cellulose powder and 60g DES (ZAC-2) were added to a Teflon container of a microwave reactor, and stirred and mixed uniformly by using a glass rod, and 3 parts of the mixture was heated to 80℃C (ZAC-1-80 ℃) 100℃C (ZAC-1-100 ℃) 120℃C (ZAC-1-120 ℃) respectively under 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 was dried to constant weight in an oven at 60 ℃.
(3) Enzymolysis: taking 1g of pretreated moso bamboo powder, taking 1g of non-pretreated moso bamboo powder (HC) as a reference, adding 20FPU/g of cellulase CTec.2 and 10mL of buffer solution (50 mM sodium acetate, pH=4.8), and reacting for 72h in an air shaking incubator maintained at 48 ℃ at 150 rpm. After the reaction, the filtrate was filtered to neutrality, and the sample was taken for component analysis, and the result is shown in FIG. 2.
Example 3
As shown in FIG. 1, FIG. 2 and FIG. 3, the cellulose enzymatic hydrolysis result of Mao Zhuzeng cellulose pretreated with eutectic solvent shows that the glucose yield of raw material (HC) after 72h enzymatic hydrolysis reaction is only 50.30%, while the glucose yields after microwave-assisted pretreatment of ZAC-1-80deg.C, ZAC-1-100deg.C, ZAC-1-120deg.C, ZAC-2-80deg.C, ZAC-2-100deg.C and ZAC-2-120deg.C are respectively increased to 65.11%, 67.33%, 63.80%, 64.22%, 66.83% and 65.98%. The results showed that among the six microwave assisted DESs, the highest glucose yields were 67.33% at ZAC-1-100deg.C, followed by ZAC-2-100deg.C (66.83%), ZAC-2-120deg.C (65.98%), ZAC-1-80deg.C (65.11%), ZAC-2-80deg.C (64.22%), and ZAC-1-120deg.C (63.80%), respectively.
Therefore, the method can obviously promote the enzymatic hydrolysis, improve the glucose yield, has simple pretreatment process conditions, short operation time and high efficiency, and has practical operation value.
Example 4
The process is the same as in example 1 and example 2, except that:
1. the microwave auxiliary 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 obtained sample has lower yield of the enzymatic hydrolysis glucose in 72 hours and is similar to the yield of the original Mao Zhuzeng cellulase hydrolysis glucose (HC).
2. Pretreatment was performed by changing the microwave treatment power to 200W and 400W, and other conditions were the same as in examples 1 and 2, and the obtained samples were subjected to enzymatic hydrolysis for 72 hours to give glucose yields as shown in Table 1.
TABLE 1 glucose yield by 72 hours enzymatic hydrolysis
It should be noted that the embodiments of the present application are not limited by the examples described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present application should be made by equivalent substitution methods, and are included in the scope of the present application.
Claims (5)
1. A method for improving glucose hydrolysis yield of moso bamboo heald cellulase is characterized in that a microwave-assisted eutectic solvent is adopted to pretreat moso bamboo before Mao Zhuzeng cellulose is subjected to enzymolysis; 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, the treatment temperature is 80-120 ℃, and the treatment is carried out for 3 minutes.
2. The method for increasing the yield of Mao Zhuzeng cellulase hydrolysis glucose of claim 1, wherein the moso bamboo feedstock has a particle size of less than 80 mesh.
3. The method for increasing the yield of Mao Zhuzeng cellulase hydrolysis glucose of claim 1, comprising the steps of:
(1) Preparing a eutectic solvent;
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 mol ratio of 1:1-2, and the mixture is stirred at 60-100 ℃ and reacts for 2 hours to prepare the eutectic solvent
(2) Mixing eutectic solvent with Phyllostachys Pubescens raw material solid-liquid, and performing microwave radiation pretreatment;
the mass ratio of the moso bamboo to the eutectic solvent pretreatment liquid is 1:10, the microwave radiation frequency is 600W, the treatment temperature is 80-120 ℃, and the treatment time is 3 minutes;
(3) Carrying out solid-liquid separation on the pretreatment mixture to obtain solid phyllostachys pubescens cellulose residues;
(4) Glucose is prepared by enzymatic hydrolysis.
4. The method for increasing the yield of Mao Zhuzeng cellulase hydrolysis glucose according to claim 3, wherein in the step (3), the solid-liquid separation is performed by cooling moso bamboo powder to room temperature, washing with acetone and water alternately, and centrifuging.
5. The method for increasing the yield of the hydrolysis glucose of the Mao Zhuzeng cellulase according to claim 3, wherein in the step (4), the pretreated phyllostachys pubescens raw material and the cellulase CTec.2 are mixed, a pH buffer solution is added according to the solid-liquid mass volume ratio of 1:10 g/mL, and 20FPU/g of the cellulase CTec.2 is added; the reaction was continued in a 150 rpm rotary shaker at 48℃for 72h.
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