CN113234772B - Method for producing glucose by poplar enzymolysis - Google Patents
Method for producing glucose by poplar enzymolysis Download PDFInfo
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- CN113234772B CN113234772B CN202110626444.6A CN202110626444A CN113234772B CN 113234772 B CN113234772 B CN 113234772B CN 202110626444 A CN202110626444 A CN 202110626444A CN 113234772 B CN113234772 B CN 113234772B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C13K1/00—Glucose; Glucose-containing syrups
- C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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Abstract
The invention provides a method for producing glucose by poplar enzymolysis, which firstly utilizes metal chloride AlCl3Or CrCl3The method comprises the steps of pretreating poplar under specific conditions, adding cellulase and surfactant into pretreated residues for enzymolysis, adding the surfactant to assist metal chloride for pretreatment, improving poplar enzymolysis efficiency and glucose yield, reducing enzyme dosage, shortening enzymolysis time, and effectively improving economic benefits, wherein the glucose yield after 24 hours of enzymolysis can reach 86.94% under the condition that the enzyme dosage is 17.5-20 FPU/g of poplar pretreated residues.
Description
Technical Field
The invention belongs to the technical field of biomass pretreatment, and particularly relates to a method for producing glucose by poplar enzymolysis.
Background
With the development of the times, the energy and environmental problems are gradually highlighted, biomass energy is more and more concerned as a new renewable resource, wherein lignocellulose is considered as a good raw material for preparing biofuel due to wide sources, abundant reserves and inedibility, and the method for preparing liquid fuel from lignocellulose biomass is to hydrolyze hemicellulose and cellulose in the raw material into monosaccharides such as xylose and glucose and produce ethanol through fermentation.
The poplar has the advantages of high growth speed, short growth period, strong environmental adaptability, wide planting area and abundant resources, is an important raw material for producing biological energy and biomass chemicals, and has great potential for preparing the biological energy and the chemicals by biotransformation of the poplar. Due to the high lignin resistance of the poplar, in the utilization process of lignocellulose, due to the complex structural characteristics of the lignocellulose, the poplar degradation barrier is strong, and the lignocellulose is difficult to be subjected to effective enzymolysis, so that the pretreatment before the enzymolysis is a key step for improving the hydrolysis efficiency of poplar enzyme.
The pretreatment removes lignin and degrades hemicellulose in a certain mode, so that the chemical components of lignocellulose are changed, the contact area of enzyme and cellulose is increased, and the enzymolysis efficiency is further improved. For example, patent CN201910212819.7 provides a method for improving enzymatic hydrolysis efficiency of poplar pretreated with acetic acid and hydrogen peroxide by adding β -glucosidase, but the method still has long enzymatic hydrolysis time, 72 hours of enzymatic hydrolysis time, large enzyme dosage and high cost, where the enzyme dosage added per gram of dry matter is 20FPU cellulase and the additional 500nkat to 1000nkat β -glucosidase, and thus there is a great need to develop a method capable of reducing economic cost and improving enzymatic hydrolysis efficiency of poplar.
Disclosure of Invention
The invention aims to solve the problems of long enzymolysis time and large cellulase dosage in the enzymolysis process of preparing biological energy by poplar at present, and aims to provide a method for improving the enzymolysis efficiency of poplar by using a surfactant to assist pretreatment of a metal chloride.
The invention aims to provide a method for producing glucose by poplar enzymolysis.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a method for producing glucose by poplar enzymolysis, which comprises the following steps:
s1, poplar pretreatment: taking poplar wood according to a solid-to-liquid ratio of 1: 8-15 are mixed with water, and 0.03-0.15 mol/L of the mixture is addedWater (W)Adding metal chloride according to the amount, carrying out pretreatment reaction at 160-180 ℃, washing a reaction product to be neutral, and separating to obtain poplar pretreatment residues;
s2, enzymolysis: taking poplar pretreatment residues, adding a buffer solution with the pH value of 4.5-5.0, cellulase and a surfactant, and carrying out enzymolysis;
wherein the metal chloride comprises AlCl3Or CrCl3(ii) a Step S2, the mass ratio of the poplar pretreatment residue to the buffer solution to the surfactant is 1 g: 50mL of: 0.1 to 0.4 g.
In the method, the pretreatment of the poplar adopts metal chloride AlCl3And CrCl3Adopting Lewis acid pretreatment to degrade hemicellulose into xylose, removing lignin and further promoting the subsequent enzymolysis process; the buffer solution can maintain the normal pH value required by the enzyme, greatly reduce the change of the pH value and prevent the enzyme activity from being reduced or lost due to the great change of the pH value; the addition of the surfactant can increase the accessible surface area of the cellulose, has small influence on the chemical composition of the lignin, can be adsorbed into the lignin, prevents non-productive adsorption of enzyme, and further improves the yield of glucose.
In some preferred embodiments, in step S1, the solid-to-liquid ratio of poplar to water is 1: 10, see examples 1-12.
In some preferable embodiments, the addition amount of the cellulase is 17.5-20 FPU/gResidue of poplar pretreatmentSee examples 1-12.
In some preferred embodiments, the pretreatment reaction of step S1 is carried out at 200-300 rpm for 10-30 min, see examples 1-12.
Preferably, the water in step S1 is ultrapure water.
Preferably, the poplar in the step S1 is obtained by air-drying and crushing poplar; the poplar and the poplar pretreatment residues are all measured in absolute dry quantity.
Preferably, the buffer solution is an acetic acid-sodium acetate buffer solution.
In some preferred embodiments, the surfactant comprises one or more of whey protein, calcium lignosulfonate, Tween 80, PEG8000, Triton X-100, see examples 1-5.
When the metal chloride is AlCl3When this is the case, the surfactant is most preferably PEG8000, see example 1; when the metal chloride is CrCl3Most preferably, the surfactant is calcium lignosulfonate, see example 6.
In some preferred embodiments, the temperature of the enzymolysis in step S2 is 40-55 ℃, and the rotation speed is 100-250 rpm, as shown in embodiments 1-12.
Most preferably, the temperature of the enzymolysis in step S2 is 50 ℃, and the rotation speed is 150rpm, see example 1.
In some preferred embodiments, the separation in step S1 is a vacuum filtration method.
In some preferred embodiments, the reaction in step S1 is carried out in a reaction kettle.
In addition, the invention also requests to protect the application of the method in the aspect of producing glucose by poplar enzymolysis.
Compared with the prior art, the invention has the beneficial effects that:
the poplar enzymolysis method provided by the invention adopts specific metal chloride to pretreat poplar under specific conditions, then adds cellulase and surfactant to pretreated residues for enzymolysis, improves poplar enzymolysis efficiency and glucose yield by adding the surfactant to assist the pretreatment of the metal chloride, reduces enzyme dosage, shortens enzymolysis time, and reduces enzyme dosage of 17.5-20 FPU/gResidue of poplar pretreatmentAnd the yield of glucose after 24h of enzymolysis can reach 86.94%, and the economic benefit is effectively improved.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.
The poplar wood used in the examples was obtained from a wood factory by air-drying and pulverizing, and the components of the poplar wood were 45.56% cellulose, 14.98% hemicellulose and 22.86% lignin.
Cellulases (the second generation of cellulose research) were purchased from novacin (china) biotechnology limited.
The glucose content in the enzymolysis liquid is analyzed by a high performance liquid chromatography, and the yield is calculated by the following method:
in the formula, Y represents the glucose yield (%) in the enzymolysis liquid; c represents the glucose concentration (g/L) in the enzymolysis solution; v represents the volume of the enzymatic hydrolysate (L); m represents the mass (g) of cellulose in the poplar raw material.
Example 1 method for improving poplar enzymolysis efficiency
S1, poplar pretreatment: 12g (absolute dry weight) of poplar and 0.80g of AlCl3Adding 120mL of ultrapure water, reacting in a reaction kettle at 180 ℃ and 300rpm for 20min, adding deionized water, washing to neutrality, and performing vacuum filtration to obtain poplar pretreatment residues;
s2, enzymolysis: 2g of poplar pretreatment residue (oven-dried amount) was taken, and 100mL of acetic acid-sodium acetate buffer solution having pH of 4.8, 40FPU cellulase (Selengli II), and 0.3g of surfactant PEG8000 were added, and subjected to enzymolysis at 50 ℃ and 150 rpm.
Embodiment 2 method for improving enzymolysis efficiency of poplar
The difference from example 1 is that the surfactant was changed to 0.8g whey protein and 100mL of pH 4.5 buffered acetic acid-sodium acetate solution was added.
Embodiment 3 method for improving enzymatic hydrolysis efficiency of poplar
The difference from example 1 is that the surfactant was changed to 0.2g of calcium lignosulfonate and 100mL of pH 5 acetic acid-sodium acetate buffer solution was added.
Embodiment 4 method for improving enzymolysis efficiency of poplar
The difference from example 1 is that the surfactant was changed to Tween 80 and the pretreatment was carried out for 30min at 200 rpm.
Embodiment 5 method for improving enzymolysis efficiency of poplar
The difference from example 1 is that the surfactant was changed to Triton X-100 and the pretreatment was carried out for 10min at 300 rpm.
Example 6 method for improving poplar enzymolysis efficiency
S1, poplar pretreatment: 12g (absolute dry weight) of poplar and 0.95g of CrCl3Adding 120mL of ultrapure water, reacting in a reaction kettle at 160 ℃ and 300rpm for 20min, adding deionized water, washing to neutrality, and performing vacuum filtration to obtain poplar pretreatment residues;
s2, enzymolysis: 2g of poplar pretreatment residue (oven-dried amount) was taken, 100mL of acetic acid-sodium acetate buffer solution having pH 4.8, 40FPU cellulase (Central second generation) and 0.3g of surfactant calcium lignosulfonate were added, and enzymolysis was carried out at 50 ℃ and 150 rpm.
Example 7 method for improving poplar enzymolysis efficiency
The difference from example 6 is that the surfactant was changed to whey protein in step S2; enzymolysis is carried out at 40 ℃ and the rotating speed of 250 rpm.
Embodiment 8 method for improving poplar enzymolysis efficiency
The difference from example 6 is that the surfactant in step S2 was changed to PEG 8000; enzymolysis is carried out at 55 ℃ and the rotating speed of 100 rpm.
Example 9 method for improving poplar enzymolysis efficiency
The difference from example 6 is that the surfactant is replaced with Tween 80.
Example 10 method for improving poplar enzymolysis efficiency
The difference from example 6 is that the surfactant was changed to Triton X-100.
Example 11 method for improving enzymatic hydrolysis efficiency of poplar
The difference from example 1 is that 0.4g of the surfactant PEG8000 was added at step S2.
Example 12 method for improving poplar enzymolysis efficiency
The difference from example 1 is that 35FPU cellulase (second generation Seiki) was added in step S2.
Comparative example 1
The process of example 6 is followed, except that the metal chloride is replaced by 0.82g of ZnCl2And no surfactant is added in S2.
Comparative example 2
The process as in example 6 is distinguished by the fact that the metal chloride is replaced by 0.57g of MgCl2And no surfactant is added in S2.
Comparative example 3
The same procedure as in example 6, except that the metal chloride was replaced with 0.76g of MnCl2And in S2, no additionAdding a surfactant.
Comparative example 4
The same procedure as in example 6, except that the metal chloride was replaced with 0.97g of FeCl3And no surfactant is added in S2.
Comparative example 5
The same procedure as in example 6, except that the metal chloride was replaced with 0.81g of CuCl2And no surfactant is added in S2.
Comparative example 6
The process of example 6 is followed, except that the metal chloride is replaced by 0.80g of AlCl3And no surfactant is added in S2.
Comparative example 7
The method of example 6 is different in that no surfactant is added in S2.
Comparative example 8
The method of example 1 was different in that no surfactant was added in S2.
Comparative example 9
The process of example 1 is followed, with the difference that 0.40g of AlCl is added3120mL of ultrapure water was added, and no surfactant was added to S2 at 160 ℃ in the reaction vessel.
Comparative example 10
The process is as in example 1, with the difference that 0.56g of AlCl is added3120mL of ultrapure water was added, and no surfactant was added to S2 at 160 ℃ in the reaction vessel.
Comparative example 11
The process of example 1 was repeated except that the reaction temperature in step S1 was 150 ℃ and no surfactant was added in S2.
Comparative example 12
The process of example 1 was repeated except that the reaction temperature in step S1 was 190 ℃ and no surfactant was added in step S2.
Comparative example 13 method for improving enzymolysis efficiency of poplar
The difference from example 1 is that the reaction temperature of step S1 is 170 ℃, and no surfactant is added in S2.
Comparative example 14 method for improving enzymolysis efficiency of poplar
The difference from example 6 is that the reaction temperature of step S1 is 170 ℃, and no surfactant is added in S2.
Comparative example 15 method for improving enzymolysis efficiency of poplar
The difference from example 6 is that the reaction temperature of step S1 is 180 ℃ and no surfactant is added in S2.
Examples of the experiments
In examples 1 to 12 and comparative examples 1 to 15, 1mL of the enzymolysis solution was subjected to inactivation treatment for 10min at 24 hours and 72 hours of enzymolysis, and glucose concentration in the enzymolysis solution was measured by high performance liquid chromatography, and glucose yields at 24 hours and 72 hours were calculated, respectively. The results are shown in table 1:
TABLE 1
As can be seen by comparing examples 1 to 5 with comparative example 8, AlCl with surfactant added3The yield of the pretreated glucose is higher, and the five surfactants have promotion effect on the yield of the glucose, wherein PEG8000 has promotion effect on AlCl3The pre-treatment has the best effect. AlCl with PEG8000 added3The yield of glucose in 24h of pretreatment reaches 86.94%, and the yield of glucose in 72h reaches 88.45%.
As can be seen by comparing example 1 with comparative example 8, AlCl was observed after adding PEG80003The yield of 86.94% after pretreatment in 24h is 81.98% higher than that of 72h without adding surfactant, greatly shortening enzymolysis time and improving enzymolysis yield.
As can be seen by comparing examples 6 to 10 with comparative example 7, CrCl to which a surfactant was added3The yield of the pretreated glucose is higher, and the five surfactants have the promotion effect on the yield of the glucose. Wherein the lignin sulfonic acidCalcium acid para CrCl3The pre-treatment has the best effect. CrCl with calcium lignosulfonate added3The yield of glucose in 24h of pretreatment reaches 69.69%, and the yield of glucose in 72h reaches 85.53%.
Compared with the comparative example 7, the comparison of the example 6 shows that after the calcium lignosulfonate is added, the yield of 69.69% in 24h can reach 65.94% in 72h without adding the surfactant, the highest glucose yield in 72h reaches 85.53%, the enzymolysis time is shortened, and the enzymolysis yield is improved.
As can be seen from the comparison of comparative examples 1 to 7, the effect of different metal chlorides on poplar pretreatment is different at 160 ℃, wherein AlCl is3And CrCl3The effect is relatively good.
As can be seen from the comparison of comparative example 6 with comparative examples 9 to 10, different ion concentrations are applied to AlCl3Pretreatment has an effect in which the glucose yield is reduced by reducing the ion concentration.
Comparison of comparative example 8 and comparative examples 11 to 13 shows that different temperatures are applied to AlCl3Pretreatment has a major impact, with poor glucose yield at too high or too low temperatures, and glucose yield reaching the maximum at 180 ℃.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (9)
1. A method for improving the speed of glucose production by poplar enzymolysis is characterized in that a surfactant is added in the process of glucose production by poplar enzymolysis, and the method comprises the following steps:
s1, poplar pretreatment: taking poplar wood according to a solid-to-liquid ratio of 1: 8-15, mixing with water, adding 0.03-0.05 mol/L of water, adding metal chloride, carrying out pretreatment reaction at 160-180 ℃, washing a reaction product to be neutral, and separating to obtain poplar pretreatment residues;
s2, enzymolysis: taking poplar pretreatment residues, adding a buffer solution with the pH value of 4.5-5.0, cellulase and a surfactant, and carrying out enzymolysis;
wherein the metal chloride is AlCl3Or CrCl3(ii) a Step S2, the mass ratio of the poplar pretreatment residue to the buffer solution to the surfactant is 1 g: 50mL of: 0.1-0.4 g; the addition amount of the cellulase is 17.5-20 FPU/gResidue of poplar pretreatment。
2. The method of claim 1, wherein the pretreatment reaction of step S1 is carried out at 200-300 rpm for 10-30 min.
3. The method of claim 1, wherein the buffer solution is an acetic acid-sodium acetate buffer solution.
4. The method of claim 1, wherein the surfactant comprises any one of whey protein, calcium lignosulfonate, Tween 80, PEG8000, Triton X-100.
5. The method of claim 1, wherein when the metal chloride is AlCl3When the surfactant is PEG 8000; when the metal chloride is CrCl3When the surfactant is calcium lignosulfonate.
6. The method according to claim 1, wherein the temperature of the enzymolysis in step S2 is 40-55 ℃, and the rotation speed is 100-250 rpm.
7. The method of claim 1, wherein the separation in step S1 is a vacuum filtration method.
8. The method as claimed in claim 1, wherein the poplar is obtained by air-drying and pulverizing poplar.
9. The method of claim 1, wherein the reaction of step S1 is performed in a reaction kettle.
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