CN102191299A - Method for increasing lignocellulose saccharification yield through multi-step enzymolysis - Google Patents
Method for increasing lignocellulose saccharification yield through multi-step enzymolysis Download PDFInfo
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- CN102191299A CN102191299A CN2010101217455A CN201010121745A CN102191299A CN 102191299 A CN102191299 A CN 102191299A CN 2010101217455 A CN2010101217455 A CN 2010101217455A CN 201010121745 A CN201010121745 A CN 201010121745A CN 102191299 A CN102191299 A CN 102191299A
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Abstract
The invention relates to a method for increasing the lignocellulose saccharification yield through multi-step enzymolysis. The method comprises the following main steps: 1) pretreating wood fiber raw material; 2) adding one or more of bio-degrading enzymes in the pretreated wood fiber stepwise or simultaneously, removing the extract component or lignin in the raw material to change the fiber structure of the raw material; 3) adding one or more of hemicellulose degrading enzymes in the lignocellulose which is removed extract or lignin through enzymolysis stepwise or simultaneously, extracting pentose in the raw material; and 4) filtering fiber residue obtained in the step 3) through the enzymolysis, and adding cellulase and/or cellobiase to hydrolyze continuously and obtain glucose hydrolysate. By adopting the method, each component can be recycled and the total reducing sugar yield can be increased.
Description
Technical field
The invention belongs to the cellulase hydrolysis field, relate to a kind of particularly by the method for multistep enzymolysis with raising lignocellulose saccharification yield.
Background technology
Lignocellulose raw material is the very abundant renewable resources of occurring in nature, if lignocellulose can well be utilized, will reduce the cost of industrial chemicals and automobile fuel to a great extent.Yet up to now, people are but very insufficient to the utilization of lignocellulose raw material.This is because not only contain Mierocrystalline cellulose and hemicellulose in the lignocellulose raw material, also contain a certain amount of xylogen and a spot of pectin substance, starch, resin, terpene compound etc., these compositions are closely linked, environment has very strong resistibility to external world, hinder Glycosylase near fiber, limited the hydrolysis and saccharification process of cellulose components.On the other hand, along with the carrying out of enzyme digestion reaction, concentration of reduced sugar increases gradually, and the restraining effect of enzymolysis product strengthens, thereby influences the yield of reducing sugar.
In order to improve the utilization ratio of lignocellulose raw material, launched a series of researchs, and obtained certain progress about wood fibre pre-treatment and coupled enzymatic.The method that has related to acid, alkali, the various processing fibrous materials of oxidation as recent disclosed CN101117777A, CN101255479A, CN101230546A, CN101182551A etc., patent CN101501205A, CN101285106A, CN101381754A have also mentioned and added zytase, cellulase and cellobiase in pretreated fibrous materials, by between synergy improved the yield of reducing sugar.But along with the increase of concentration of reduced sugar, the restraining effect of product is also obvious all the more, and the synergistic product of these enzymes not only has glucose, also has certain pentose, and pentose is difficult to influence utilization ratio of raw materials with the glucose utilization of fermenting together.
Summary of the invention
The object of the present invention is to provide a kind of method of multistep enzymolysis raising lignocellulose saccharification yield, to overcome the deficiency of pre existing treat enzyme Mashing process.
For achieving the above object, multistep enzymolysis provided by the invention improves the method for lignocellulose saccharification yield, it is the preprocessing process that promotes raw material earlier with biological enzyme, the multistep enzymolysis that hemicellulose and Mierocrystalline cellulose substep enzymolysis is extracted pentose and glucose respectively improves the method for reducing sugar yield then, and its concrete steps are as follows:
(1) adopt that diluted acid, alkali, steam explosion, hot water, ammonia are quick-fried, in carbonic acid gas explosion or the change machine slurry method one or more carry out pre-treatment to lignocellulose raw material;
(2) through pretreated lignocellulose substep or add one or more of biodegradable enzymes such as polygalacturonase, lipase, amylase, laccase synchronously, add-on is 100～600U/g, at the substrate mass percent concentration is 2%～12%, enzymolysis pH value 3～7,30～55 ℃ of temperature, reaction times 12～48h divides a step or the pretreated lignocellulose of multistep enzymolysis, to remove extractum composition or the xylogen in the raw material;
(3) enzymolysis is removed the lignocellulose substep behind extractum or the xylogen or add one or more of hemicellulose degrading enzymes such as zytase, arabinofuranosidase/xylosidase, glucuroide, mannosidase, ethyl ester enzyme, glycuronidase synchronously, add-on is 100～600U/g, at the substrate mass percent concentration is 2%～12%, enzymolysis pH value 3～7,40～55 ℃ of temperature, reaction times 24～72h divides half fiber in a step or the multistep hydrolysis material, extracts various pentoses respectively.
(4) fiber residue behind the filtration enzymolysis adds cellulase, cellobiase 200～750U/g, is 2%～12% at the substrate mass percent concentration, enzymolysis pH value 3～7,40～55 ℃ of temperature continue hydrolysis under reaction times 40～72h condition, obtain glucose hydrolysis liquid.
Wherein, the substep enzymolysis can be carried out with any combination for any two steps or choosing any two or more enzymes wherein wherein according to the difference of lignocellulose raw material in step (2)～(4).
Relative prior art, the present invention has following advantage:
1) lignocellulose raw material pre-treatment under relatively mild condition earlier, remove remaining extractum composition and xylogen in the raw material by adding biodegradable enzymes such as polygalacturonase, lipase, amylase, laccase then, can improve cellulosic enzymolysis yield like this, also reduce the growing amount of fermentation inhibitor.
2) with more than half fibers and cellulosic substep enzymolysis, can reach the extraction respectively and the utilization (being used for producing Xylitol, fermentation butyl alcohol etc. as wood sugar) of five-carbon sugar and hexose, solve two kinds of sugar problem of preparing ethanol by fermentation difficulty altogether, the too high restraining effect to enzyme digestion reaction of production concentration in the time of also can reducing synchronous enzymolysis improves utilization ratio of raw materials.
Embodiment
The invention will be further described below in conjunction with example, but the scope of protection of present invention is not limited to the scope that embodiment explains.
Embodiment 1
(treatment condition are: be crushed to the following straw of 0.5cm through the pretreated maize straw of steam explosion, at vapor pressure 1.9MPa, moment explosion behind the pressurize 7min), be divided into two step enzymolysis, the first step enzymatic hydrolysis condition is: concentration of substrate 10%, add the pool, Shandong and give birth to the ZSL type zytase 200ug/g that bio tech ltd is produced, citric acid-sodium citrate buffer is regulated pH value 4.8, place 48h in 50 ℃ of shaking baths, enzymolysis is finished after-filtration and is separated the one-step hydrolysis liquid glucose of winning, and residual residue is carried out the second step enzymolysis.The second step enzymatic hydrolysis condition is: concentration of substrate 10%, the cellulase 300FPU/g, the cellobiase 20IU/g that add Novi's letter, use citric acid-sodium citrate buffer to regulate pH value 4.8, continue, filter to such an extent that second go on foot the enzymolysis liquid glucose at 50 ℃ of shaking bath internal reaction 48h.Two step enzymolysis total reducing sugars yield sums are 28.95% (to substrate).
The same pretreated maize straw of learning from else's experience, at concentration of substrate 10%, add the pool, Shandong synchronously and give birth to ZSL type zytase 200ug/g, cellulase 300FPU/g, the cellobiase 20IU/g of Novi's letter that bio tech ltd is produced, use citric acid-sodium citrate buffer to regulate pH value 4.8, at 50 ℃ of shaking bath internal reaction 96h, filter one the step enzymolysis liquid glucose, the reducing sugar yield is 22.39 (to substrates).Then the total reducing sugars yield of the relative step enzymolysis of two step enzymolysis improves 29.3%.
Embodiment 2
Through 0.5% sulfuric acid at 120 ℃ of manioc wastes of handling 1h, enzymolysis in two steps, the first step enzymatic hydrolysis condition is: concentration of substrate 4%, add the polygalacturonase 100 μ g/g that Dalian Yingsite Biotechnology Co., Ltd. produces, zytase 720U/g, citric acid-sodium citrate buffer is regulated and is placed 48h in 4.8,50 ℃ of shaking baths of pH value, enzymolysis is finished after-filtration and is separated the one-step hydrolysis liquid glucose of winning, and residual residue is carried out the second step enzymolysis.The second step enzymatic hydrolysis condition is: concentration of substrate 4%, the cellulase 350FPU/g, the cellobiase 15IU/g that add Novi's letter, use citric acid-sodium citrate buffer to regulate pH value 4.8, continue, filter to such an extent that second go on foot the enzymolysis liquid glucose at 50 ℃ of shaking bath internal reaction 48h.Two step enzymolysis total reducing sugars yield sums are 29.12% (to substrate).And poly-according to the above same step, do not add polygalacturonase when being the first step enzymolysis, other condition is with above identical, and then two step enzymolysis total reducing sugars yield sums are 26.68%.The enzymolysis yield of the two step enzymolysis yield of adding polygalacturonase when not adding is high by 9.1%.
The same pretreated manioc waste of learning from else's experience, at concentration of substrate 4%, add zytase 720U/g, cellulase 350FPU/g, the cellobiase 15IU/g of Novi's letter that Dalian Yingsite Biotechnology Co., Ltd. produces synchronously, use citric acid-sodium citrate buffer to regulate pH value 4.8, at 50 ℃ of shaking bath internal reaction 96h, filter one the step enzymolysis liquid glucose, the reducing sugar yield is 22.29% (to substrate).Then two step enzyme yield improve 19.7% than a total reducing sugars yield that goes on foot enzymolysis under the same terms.The raw grain yield of always going back that two step enzymolysis yield of interpolation polygalacturonase are compared a step enzymolysis has improved 30.6% especially.
Embodiment 3
Through 0.4% sulfuric acid at 150 ℃ of maize straws of handling 30min, divide two or three step enzymolysis, the first step enzymatic hydrolysis condition is: concentration of substrate 8%, add the laccase 25U/g that Dalian Yingsite Biotechnology Co., Ltd. produces, and be to react 8h under 4.8,65 ℃ of conditions in the pH value.Fiber residue behind the first step enzymolysis adds zytase 650U/g and carries out the second step enzymolysis.Enzymatic hydrolysis condition is 4.8,50 ℃ of pH values, reaction times 40h.Second enzymolysis is finished the after-filtration separation and is obtained the hydrolysis of hemicellulose liquid glucose, and residual residue is carried out the 3rd step enzymolysis.The 3rd step enzymatic hydrolysis condition is: concentration of substrate 8%, the cellulase 370FPU/g, the cellobiase 20IU/g that add Novi's letter, use citric acid-sodium citrate buffer to regulate pH value 4.8, continuation is at 50 ℃ of shaking bath internal reaction 48h, filter the cellulase hydrolysis liquid glucose.Hemicellulose and cellulosic reducing sugar yield sum are 29.46% (to substrate).And according to above same quadrat method, do not add laccase carry out two the step enzymolysis the total reducing sugars yield be 26.84%.The three step enzymolysis yield of adding laccase improve 9.8% than the yield of only using zytase and cellulase to carry out two step enzymolysis.
The same pretreated maize straw of learning from else's experience, at concentration of substrate 8%, add zytase 650U/g, cellulase 370FPU/g, the cellobiase 20IU/g of Novi's letter that Dalian Yingsite Biotechnology Co., Ltd. produces synchronously, use citric acid-sodium citrate buffer to regulate pH value 4.8, at 50 ℃ of shaking bath internal reaction 96h, filter one the step enzymolysis liquid glucose, the reducing sugar yield is 23.82% (to substrate).Then two step enzyme yield improve 12.3% than a total reducing sugars yield that goes on foot enzymolysis under the same terms.And the three step enzymolysis yield of adding laccase are compared the raw grain yield of always going back of a step enzymolysis that does not add laccase and have been improved 23.7%.
Embodiment 4
The maize straw after handling through 200 ℃ of high-temperature-hot-waters, be divided into two step enzymolysis, the first step enzymatic hydrolysis condition is: concentration of substrate 2%, add the zytase 500U/g that Dalian Yingsite Biotechnology Co., Ltd. produces, it is 4.8 that citric acid-sodium citrate buffer is regulated the pH value, place 48h in 50 ℃ of shaking baths, enzymolysis is finished after-filtration and is separated the one-step hydrolysis liquid glucose of winning, and residual residue is carried out the second step enzymolysis.The second step enzymatic hydrolysis condition is: concentration of substrate 2%, the cellulase 380FPU/g, the cellobiase 15IU/g that add Novi's letter, use citric acid-sodium citrate buffer to regulate pH value 4.8, continue, filter to such an extent that second go on foot the enzymolysis liquid glucose at 50 ℃ of shaking bath internal reaction 48h.Two step enzymolysis total reducing sugars yield sums are 23.56% (to substrate).
The same pretreated maize straw of learning from else's experience, at concentration of substrate 4%, add zytase 500U/g, cellulase 380FPU/g, the cellobiase 15IU/g of Novi's letter that Dalian Yingsite Biotechnology Co., Ltd. produces synchronously, use citric acid-sodium citrate buffer to regulate pH value 4.8, at 50 ℃ of shaking bath internal reaction 96h, filter one the step enzymolysis liquid glucose, the reducing sugar yield is 20.47% (to substrate).Then the total reducing sugars yield of the relative step enzymolysis of two step enzymolysis improves 15.1%.
Claims (8)
1. a multistep enzymolysis improves the method for lignocellulose saccharification yield, and key step is as follows:
1) pre-treatment of lignocellulose raw material;
2) substep or add one or more biodegradable enzymes synchronously in pretreated lignocellulose removes extractum composition or xylogen in the raw material, changes the fibrous texture of raw material;
3) enzymolysis is removed the lignocellulose substep behind extractum or the xylogen or add one or more hemicellulose degrading enzymes synchronously, extract the pentose in the raw material;
4) fiber residue behind filtration step 3 enzymolysis adds cellulase and/or cellobiase and continues hydrolysis, obtains glucose hydrolysis liquid.
2. method according to claim 1, wherein, described lignocellulose is maize straw, wheat straw, straw, manioc waste or timber; By weight percentage, fibrous material contains hemicellulose 20%～35%, Mierocrystalline cellulose 35%～45% and xylogen 15%～25%, and all the other are impurity.
3. method according to claim 1, wherein, the lignocellulose raw material pretreatment process in the step 1 comprises the one or more combination in acid treatment, alkaline purification, hot-water cure, steam explosion, ammonia explosion, carbonic acid gas explosion, the chemical mechanical pulping.
4. method according to claim 1, wherein, the biodegradable enzyme in the step 2 is polygalacturonase, lipase, amylase, laccase.
5. according to claim 1 or 4 described methods, wherein, the consumption of biodegradable enzyme is 100～600U/g, and the substrate mass percent concentration is 2%～12%, enzymolysis pH=3～7,30～55 ℃ of temperature, 12～48 hours reaction times.
6. method according to claim 1, wherein, the hemicellulose degrading enzyme in the step 3 is zytase, arabinofuranosidase/xylosidase, glucuroide, mannosidase, ethyl ester enzyme, glycuronidase.
7. according to claim 1 or 6 described methods, wherein, the consumption of hemicellulose degrading enzyme is 100～600U/g, and the substrate mass percent concentration is 2%～12%, enzymolysis pH=3～7,40～55 ℃ of temperature, 24～72 hours reaction times.
8. method according to claim 1, wherein, the cellulase in the step 4, the consumption of cellobiase are 200～750U/g, the substrate mass percent concentration is 2%～12%, enzymolysis pH=3～7,40～55 ℃ of temperature, 40～72 hours reaction times.
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| CN102517355A (en) * | 2011-11-17 | 2012-06-27 | 华南农业大学 | Method for jointly degrading lignocellulose |
| CN102599352A (en) * | 2012-04-09 | 2012-07-25 | 苏州昆蓝生物科技有限公司 | Treating agent for non-fermentative straw feed and application method thereof |
| CN103045679A (en) * | 2011-10-11 | 2013-04-17 | 济南圣泉集团股份有限公司 | Comprehensive utilization method of lignocellulose biomass |
| CN103045693A (en) * | 2011-10-11 | 2013-04-17 | 济南圣泉集团股份有限公司 | Comprehensive utilization method of lignocellulose biomass |
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| CN105886178A (en) * | 2016-06-21 | 2016-08-24 | 淮阴师范学院 | Method for increasing sugar yield of malt by means of laccase pretreatment |
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| CN114574531A (en) * | 2020-12-01 | 2022-06-03 | 国投生物科技投资有限公司 | Method for preparing monosaccharide from lignocellulose raw material |
| CN114317638B (en) * | 2022-01-18 | 2023-09-22 | 中南林业科技大学 | Method for saccharifying lignocellulose by multi-enzyme synergistic surfactant cyclic enzymolysis |
| CN114317638A (en) * | 2022-01-18 | 2022-04-12 | 中南林业科技大学 | Method for saccharifying lignocellulose through cyclic enzymolysis of multiple enzymes and surfactant |
| CN114437927A (en) * | 2022-02-25 | 2022-05-06 | 重庆大学 | Bionic continuous hydrolysis reaction system and method for carrying out enzymolysis |
| CN116219785A (en) * | 2023-02-27 | 2023-06-06 | 安徽金坤达包装集团有限公司 | Method for degrading lignin in situ by manganese oxide coupling laccase |
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