CN109837319B - Method for improving poplar enzyme hydrolysis efficiency - Google Patents
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- CN109837319B CN109837319B CN201910212819.7A CN201910212819A CN109837319B CN 109837319 B CN109837319 B CN 109837319B CN 201910212819 A CN201910212819 A CN 201910212819A CN 109837319 B CN109837319 B CN 109837319B
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 27
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 24
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 21
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 28
- 229940088598 enzyme Drugs 0.000 claims abstract description 26
- 108010059892 Cellulase Proteins 0.000 claims abstract description 25
- FVTRDWMTAVVDCU-UHFFFAOYSA-N acetic acid;hydrogen peroxide Chemical compound OO.CC(O)=O FVTRDWMTAVVDCU-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229940106157 cellulase Drugs 0.000 claims abstract description 25
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 24
- 239000002994 raw material Substances 0.000 claims abstract description 24
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 20
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 69
- 239000000243 solution Substances 0.000 claims description 16
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 230000003301 hydrolyzing effect Effects 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000002023 wood Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 230000003197 catalytic effect Effects 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 17
- 239000008103 glucose Substances 0.000 abstract description 17
- 230000008569 process Effects 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000036983 biotransformation Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 229920001503 Glucan Polymers 0.000 abstract 1
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 22
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 11
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 11
- 239000000758 substrate Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- 229920005610 lignin Polymers 0.000 description 6
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 5
- 238000002203 pretreatment Methods 0.000 description 5
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- 230000004888 barrier function Effects 0.000 description 2
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000002608 ionic liquid Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
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- 238000001179 sorption measurement Methods 0.000 description 2
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- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
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Abstract
The invention provides a method for improving the enzymatic hydrolysis efficiency of poplar, which takes poplar pretreated by acetic acid hydrogen peroxide as a raw material and solves the problems of large usage amount of cellulase and low enzymatic hydrolysis efficiency in the existing hydrolysis process. The method additionally adds beta-glucosidase in an enzyme hydrolysis reaction system on the basis of the existing cellulase, so that the glucose yield of the poplar can reach 99.4 percent to the maximum, and glucan in the poplar is almost completely hydrolyzed. The method has the advantages of high efficiency, low cost and simple operation, almost realizes the complete enzymatic hydrolysis of the poplar, and lays a foundation for preparing biological energy and chemicals by the biotransformation of the poplar.
Description
Technical Field
The invention relates to a biomass pretreatment and enzyme hydrolysis technology, in particular to a method for improving the enzyme hydrolysis efficiency of poplar pretreated by acetic acid hydrogen peroxide by adding beta-glucosidase.
Background
The poplar is a fast-growing and high-yield tree species, has a large-area planting in China, and has high growth speed, rich resources and high cellulose content, so that the biological energy and chemicals prepared by biotransformation of the poplar have great potential. The pretreatment is an important step for improving the enzymatic hydrolysis efficiency of the poplar, and the pretreatment of the poplar is mainly divided into methods such as acid, alkali, ionic liquid, steam explosion and the like.
After treatment with 6.5% acetic acid at 172 ℃ for 27 minutes by Huang et al, 2018, J Wood Chem technol.38(5): 371-.
Kim et al, 2017, Biotechnol Biofuels.10:101, using an ionic liquid [ C2C1Im][OAc]After poplar was treated at 160 ℃ for 3 hours, the glucose yield was less than 35%.
Tian D et al, 2017, Biotechnol biofuels.10:157, pretreated poplar in two steps, first soaked overnight at room temperature with 0.7% sulfuric acid, then steam exploded for 10 minutes at 190 ℃, with a glucose yield of only 70% for poplar with cellulase additions up to 32mg/g dry matter.
The Bengxin article (the influence of green liquor pretreatment on poplar lignin and enzymatic hydrolysis, Nanjing university of forestry, 2014) research shows that after poplar is pretreated by green liquor, the dosage of cellulase is 40FPU/g, and the glucose is only 89.9%.
As can be seen, the above methods all have problems of incomplete hydrolysis of the starting material or large enzyme dosage.
Compared with other pretreatment methods, the pretreatment of the acetic acid hydrogen peroxide has obvious effect of removing the lignin of the raw material, can reduce the space barrier effect of the lignin on the enzyme and the ineffective adsorption of the lignin on the enzyme, and has certain application prospect in the aspect of improving the enzymatic hydrolysis efficiency.
Wi et al found that the lignin content of the raw materials was less than 6% and the glucose conversion of the raw materials was 85.0% by simultaneous fermentation after pretreatment of straw, pine and oak with acetic acid hydrogen peroxide in 2015, Biotechnol biofuels.8: 228.
Luo et al, 2018, Biomass Conv Bioref (https:// doi. org/10.1007/s 13399-018. about. 0364-0), pretreated poplar with acetic acid hydrogen peroxide, gave a glucose yield of less than 80.0% at a cellulase dose of 20mg/g cellulose.
The method also shows that the problems of large cellulase usage amount and low enzymatic hydrolysis efficiency still exist after the poplar is treated by the acetic acid and the hydrogen peroxide, and the difference with the expected poplar enzymatic hydrolysis effect is certain, but the reason is not clear at present.
Disclosure of Invention
The invention aims to solve the defects of large cellulase usage and low enzymatic hydrolysis efficiency in the existing poplar hydrolysis process, and provides a method for improving the enzymatic hydrolysis efficiency of poplar pretreated by acetic acid hydrogen peroxide.
The invention conception of the invention is as follows:
based on the prior art, the problems that after poplar is pretreated by acetic acid dioxygen water, which is regarded as good application prospect, the cellulase usage amount is large and the enzymatic hydrolysis efficiency is low cannot be known, and the inventor conducts intensive research on the problems, and finally finds that after poplar is pretreated by acetic acid dioxygen water, a certain amount of cellobiose is accumulated in the enzymatic hydrolysis process, so that the hydrolysis capacity of cellulase is inhibited, and the problems are caused. Therefore, the inventor considers that the beta-glucosidase is additionally added in the enzymatic hydrolysis process, and the strong hydrolysis capability of the beta-glucosidase on cellobiose is utilized, so that the cellobiose can be effectively degraded, the inhibition effect of the cellobiose is relieved, and the enzymatic hydrolysis efficiency is further improved.
In order to achieve the purpose, the technical solution provided by the invention is as follows:
the method for improving the hydrolyzing efficiency of the poplar enzyme is characterized by comprising the following steps:
step 1) pretreatment
Pretreating the raw materials by using acetic acid and hydrogen peroxide;
step 2) enzymatic hydrolysis
Performing enzymatic hydrolysis reaction on the pretreated raw material by adopting cellulase; the enzyme hydrolysis reaction system is additionally added with beta-glucosidase.
Further, in order to make the pretreatment effect better, the specific steps of step 1) are as follows:
1.1) mixing acetic acid with the purity of 99% and hydrogen peroxide with the purity of 30% to prepare an acetic acid hydrogen peroxide solution with the volume concentration of 80-100%;
1.2) mixing the raw materials with the acetic acid hydrogen peroxide solution according to the material-liquid ratio of 1:10, and pretreating for 2 hours at the temperature of 60-80 ℃;
1.3) cooling after pretreatment, carrying out solid-liquid separation, collecting filter residues, and washing the filter residues to be neutral.
Further, in the 1.1), a catalytic amount of sulfuric acid is also added; the molar concentration of the sulfuric acid is 100 mmol/L.
Further, the specific steps of step 2) are as follows:
2.1) placing the raw materials pretreated in the step 1) into a reaction bottle, and adding a sodium citrate buffer solution with the pH value equal to 5.0 into the reaction bottle to ensure that the mass concentration of the raw materials is 2%;
2.2) adding cellulase and beta-glucosidase into the solution obtained in the step 2.1) to carry out enzymatic hydrolysis reaction; shaking uniformly, placing in a constant temperature shaking table, and hydrolyzing at 50 deg.C for 72 hr;
wherein the enzyme dosage added per gram of dry matter is 20FPU cellulase and 500 nkat-1000 nkat beta-glucosidase.
Furthermore, the cellulase is a second-generation enzyme (Cellic CTec2), and the cellulase is convenient to purchase in the market and has good product performance.
Further, in order to reduce the loss of xylan components in the acetic acid hydrogen peroxide pretreatment process, the raw material used in the step 1) is poplar filter residue pretreated by acetic acid; the acetic acid pretreatment refers to adding acetic acid with the purity of 5% into poplar, and treating at 170 ℃ for 30min, so that xylan in the poplar can be degraded to prepare xylo-oligosaccharide, and the component utilization efficiency of raw materials is improved.
Further, post-treatment in step 3)
After completion of the hydrolysis, the solution obtained in step 2) was heated in a boiling water bath for about 10 minutes to inactivate the enzyme, cooled to room temperature, and centrifuged to obtain a supernatant.
The invention has the advantages that:
1. the method has simple process and environmental protection, and firstly, the raw material is pretreated by utilizing the advantage of acetic acid hydrogen peroxide pretreatment, so that the space barrier effect of lignin on enzyme and ineffective adsorption on the enzyme are reduced; and secondly, the beta-glucosidase is additionally added into an enzymatic hydrolysis reaction system, so that the hydrolysis capacity of the beta-glucosidase is stable, the inhibition of cellobiose in the enzymatic hydrolysis process can be reduced, the dry matter per gram can be almost completely hydrolyzed under the action of 20FPU cellulase, the poplar enzyme hydrolysis efficiency is greatly improved, the use amount of the cellulase is reduced, the hydrolysis material cost is reduced, and the application prospect is good.
2. The raw material pretreated by the acetic acid hydrogen peroxide is the poplar filter residue pretreated by the acetic acid, namely, the acetic acid pretreatment is carried out before the acetic acid hydrogen peroxide is pretreated to prepare the xylo-oligosaccharide, and the method can improve the utilization efficiency of the xylan component in the raw material.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples:
example one
A method for improving the hydrolyzing efficiency of poplar enzymes comprises the following steps:
step 1) pretreatment
1.1) measuring 60mL of 99% acetic acid and 60mL of 30% hydrogen peroxide, mixing, adding a catalytic amount of 100mmol/L sulfuric acid, and uniformly mixing to prepare an acetic acid hydrogen peroxide pretreatment solution;
1.2) weighing 10 grams of absolute dry poplar residues after acetic acid pretreatment, placing the absolute dry poplar residues in a polytetrafluoroethylene lining steel cylinder, adding an acetic acid hydrogen peroxide solution with the volume concentration of 100% according to the material-liquid ratio of 1:10, and pretreating for 2 hours at the temperature of 60-80 ℃; the acetic acid pretreatment refers to adding acetic acid with the purity of 5% into poplar wood, and treating for 30min at 170 ℃;
1.3) cooling after the pretreatment is finished, performing solid-liquid separation by using a centrifugal machine, collecting filter residues, and washing the filter residues to be neutral for biological enzyme hydrolysis;
step 2) enzymatic hydrolysis
2.1) weighing 1 g of pretreated raw material into a conical flask, and adding a sodium citrate buffer solution with the pH value equal to 5.0 into the conical flask to ensure that the mass concentration of the raw material is 2%;
2.2) adding 20FPU cellulase and 500nkat beta-glucosidase into the solution obtained in the step 2.1), shaking uniformly, placing in a constant temperature shaking table, and hydrolyzing for 72 hours at 50 ℃; the cellulase can be a second generation enzyme (Cellic CTec 2);
step 3) post-treatment
After the hydrolysis is finished, boiling the solution obtained in the step 2) for 10 minutes by adopting a boiling water bath to inactivate the enzyme; cooling to room temperature, and centrifuging to obtain supernatant;
detecting monosaccharide in the supernatant by adopting high performance liquid chromatography, and calculating the hydrolysis rate of the pretreated raw material monosaccharide according to the following formula:
the cellulose and xylan contents in the formula substrate are determined according to the American national renewable energy laboratory method;
if only 20FPU cellulase is added during hydrolysis, the calculated glucose yield of the poplar without any pretreatment is 11.4 percent, and the xylose yield is 8.8 percent; after pretreatment with acetic acid, the calculated glucose yield was 20.0% and xylose yield was 27.7%; after pretreatment with acetic acid and acetic acid hydrogen peroxide, the calculated glucose yield was 79.2% and xylose yield was 98.9%. After 500nkat of beta-glucosidase is additionally added to each gram of substrate, the glucose yield of the poplar pretreated by acetic acid and acetic acid hydrogen peroxide is improved to 91.9 percent, and the xylose yield is 99.0 percent.
Example two
The pretreatment method and the reaction conditions are basically the same as those in the first embodiment, and the difference is that the volume concentration of the acetic acid hydrogen peroxide solution in the acetic acid hydrogen peroxide pretreatment is 80%; if only 20FPU cellulase is added during hydrolysis, the calculated glucose yield is 67.2 percent and the xylose yield is 72.4 percent. After 500nkat of beta-glucosidase is additionally added to each gram of substrate, the glucose yield of enzyme hydrolysis is improved to 91.1 percent, and the xylose yield is improved to 94.9 percent.
EXAMPLE III
The pretreatment method and reaction conditions were substantially the same as in example one except that in the enzymatic hydrolysis, 1000nkat of β -glucosidase was added per gram of substrate, and the final glucose yield was 97.5% and xylose yield was 99.7%.
Example four
The pretreatment method and reaction conditions were substantially the same as in example two except that in the enzymatic hydrolysis, 1000nkat of β -glucosidase was added per gram of substrate, and the final enzymatic hydrolysis yield of glucose was 99.4% and xylose was 100.0%.
EXAMPLE five
The reaction conditions are basically the same as the first embodiment, and the difference is that the raw materials are not pretreated by acetic acid before being pretreated by using the acetic acid hydrogen peroxide, but 10 g of completely dried poplar is directly weighed and placed in a polytetrafluoroethylene lining steel cylinder, and the pretreatment is carried out by using the acetic acid hydrogen peroxide solution; the final glucose yield was 85.3% and xylose yield was 82.4%. After 500nkat of beta-glucosidase is additionally added to each gram of substrate, the glucose yield is improved to 87.9 percent, and the xylose yield is 94.4 percent.
EXAMPLE six
The pretreatment method and reaction conditions were substantially the same as those in example five except that in the enzymatic hydrolysis, 1000nkat of β -glucosidase was added per gram of substrate, and the final enzymatic hydrolysis yield of glucose was 98.8% and xylose was 99.1%.
Therefore, the acetic acid pretreatment is carried out before the acetic acid hydrogen peroxide pretreatment on the poplar, the volume concentration of the acetic acid hydrogen peroxide solution in the acetic acid hydrogen peroxide pretreatment is 80%, and when 20FPU of cellulase and 1000nkat of beta-glucosidase are added to each gram of substrate, the hydrolysis efficiency is higher.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications or substitutions can be easily made by those skilled in the art within the technical scope of the present disclosure.
Claims (3)
1. A method for improving the hydrolyzing efficiency of poplar enzymes is characterized by comprising the following steps:
step 1) pretreatment
Pretreating the raw materials by using acetic acid and hydrogen peroxide;
step 2) enzymatic hydrolysis
Performing enzymatic hydrolysis reaction on the pretreated raw material by adopting cellulase; beta-glucosidase is also added into the enzyme hydrolysis reaction system;
the specific steps of the step 1) are as follows:
1.1) mixing acetic acid with the purity of 99% and hydrogen peroxide with the purity of 30% to prepare an acetic acid hydrogen peroxide solution with the volume concentration of 80-100%;
1.2) mixing the raw materials with the acetic acid hydrogen peroxide solution according to the material-liquid ratio of 1:10, and pretreating for 2 hours at the temperature of 60-80 ℃;
the raw material is poplar filter residue pretreated by acetic acid;
the acetic acid pretreatment refers to adding acetic acid with the purity of 5% into poplar wood, and treating for 30min at 170 ℃;
1.3) cooling after pretreatment, carrying out solid-liquid separation, collecting filter residues, and washing the filter residues to be neutral;
the specific steps of the step 2) are as follows:
2.1) placing the raw materials pretreated in the step 1) into a reaction bottle, and adding a sodium citrate buffer solution with the pH value equal to 5.0 into the reaction bottle to ensure that the mass concentration of the raw materials is 2%;
2.2) adding cellulase and beta-glucosidase into the solution obtained in the step 2.1), shaking uniformly, placing in a constant temperature shaking table, and hydrolyzing for 72 hours at 50 ℃;
wherein, the enzyme dosage added per gram of dry matter is 20FPU cellulase and 500 nkat-1000 nkat beta-glucosidase;
the cellulase is a second enzyme of Selence.
2. The method for improving the hydrolyzing efficiency of poplar wood enzyme according to claim 1, wherein the method comprises the following steps: in the 1.1), a catalytic amount of sulfuric acid is also added; the molar concentration of the sulfuric acid is 100 mmol/L.
3. The method for improving the efficiency of poplar enzymatic hydrolysis according to claim 1, further comprising:
step 3) post-treatment
After hydrolysis, boiling the solution obtained in step 2) in a boiling water bath to inactivate the enzyme, cooling to room temperature, and centrifuging to obtain a supernatant.
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| CN111534556B (en) * | 2020-04-30 | 2023-04-25 | 南京林业大学 | Method for preparing high-concentration monosaccharide solution by using poplar enzyme method |
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| CN107475467B (en) * | 2017-09-14 | 2020-12-22 | 齐鲁工业大学 | Efficient extraction method of poplar xylose |
| CN107988441A (en) * | 2017-12-25 | 2018-05-04 | 广州市楹晟生物科技有限公司 | A kind of production method of liquid glucose |
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