CN104894087A - Customized compounding method of cellulase preparation for efficiently hydrolyzing biomass raw materials in agriculture and forestry - Google Patents
Customized compounding method of cellulase preparation for efficiently hydrolyzing biomass raw materials in agriculture and forestry Download PDFInfo
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- CN104894087A CN104894087A CN201510266804.0A CN201510266804A CN104894087A CN 104894087 A CN104894087 A CN 104894087A CN 201510266804 A CN201510266804 A CN 201510266804A CN 104894087 A CN104894087 A CN 104894087A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a customized compounding method of a cellulase preparation for efficiently hydrolyzing biomass raw materials in agriculture and forestry. The method comprises the following steps: pretreating with a glycerol organic solvent to break the complex composition and dense structure of the biomass raw materials in agriculture and forestry so that the biomass raw materials can be easily decomposed by enzymes; carrying out Trichoderma reesei liquid fermentation to obtain a cellulase; and finally, mixing the cellulase with an auxiliary enzyme and additives to obtain the compound cellulase preparation which is used for carrying out enzymolysis on the biomass matrixes pretreated by the glycerol organic solvent. Compared with the commercial cellulase, the cellulase used by the method has the advantages of economical sources, low required enzyme amount for enzymolysis and high enzymolysis ratio for matrixes.
Description
Technical field
The invention belongs to the utilization of agricultural-forestry biomass raw material resources and biological technical field, relate to the method that cellulose sugar prepared by a kind of economical and efficient hydrolysis agricultural-forestry biomass raw material.
Technical background
Although biorefinery has become study hotspot in recent years, the real Biomass Economy epoch have not yet arrived, and utilize lignocellulose to produce bioenergy and biobased products and are still faced with many bottleneck problems, and wherein to produce sugared problem particularly outstanding for Matrix lysis.The biomass such as agricultural wastes (maize straw, paddy rice and wheat etc.), fast-growing crop (switchgrass and Fast growth poplar etc.) and forest (dragon spruce and pine tree) have pre-treatment selectivity, same kind raw material is caused on the Nomenclature Composition and Structure of Complexes, to there is the significance difference opposite sex after different pretreatments, and different genera biomass material is larger through the pretreated otherness of different methods, this just considerably increases the difficulty of follow-up enzymolysis.But, Current commercial cellulase is made by fermentation low-kappa number maize straw after producing primarily of aerobic cellulose degradation fungi (Trichoderma reesei and Humicola insolens), they are extremely similar in enzymic activity with composition, can not meet the needs of different sources lignocellulosic material in biorefinery, and production cost is higher.
Although Jie Neng section and Xin Liang company of Novi almost announce the cellulase cost of cellulosic ethanol production to be dropped to about 0.2 dollar from former 5.4 dollars every barrel in recent years simultaneously, but many reports think that enzyme cost is still higher, must continue to reduce costs to 0.03 ~ 0.05 dollar every barrel.Due to, (1) the matrix otherness of different genera and different methods preprocessing lignocellulose is comparatively large, and the identical commercial fibres similar with the composition element in source zymin can not meet substrate specificity demand at present; (2) the single enzyme class in different sources cellulase and between each component cooperative ability there are differences; (3) key enzyme component/activated protein content in total protein of lignocellulose hydrolysis is on the low side, and Rate activity is not high, and these reasons cause lignocellulose enzymolysis and produce sugared cost costly.
In cellulase, add some auxiliary enzymes and additive can partly solve above-mentioned proposed problem, its reason is, some auxiliary enzymes can improve the synergy between cellulase, better a certain specific lignocellulosic material of hydrolysis; Some additives can improve the stability of the accessibility of raw material, the ineffective adsorption reducing xylogen and raising enzyme, reach the effect improving cellulase utilization ratio.
Summary of the invention
A kind of economical and efficient hydrolysis agricultural-forestry biomass raw material is the object of the present invention is to provide to prepare the method for cellulose sugar.By adding auxiliary enzymes and additive from cellulase-producing, carry out enzymolysis to the pretreated wheat straw of glycerine organic solvent, enzyme carrying capacity used is low, obtains high wheat straw enzymatic hydrolyzation, for the efficient and rational utilization of agricultural-forestry biomass raw material is laid a good foundation.
The composite method for customizing of a kind of effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin that the present invention relates to, comprises the following steps:
1, agricultural-forestry biomass raw material pulverizing: wheat straw, after naturally drying, is cut into the small segment of length 1 ~ 5cm, 60 ~ 80 DEG C of dry for standby.
2, raw materials glycerine organic solvent pre-treatment: material concentration is 5% ~ 15%, initial aqueous glycerin solution concentration is 30 ~ 100%, and boiling temperature 130 ~ 240 DEG C maintains 1 ~ 4h.Then wash twice with the aqueous glycerin solution of 50%, wash twice from the beginning, the matrix of acquisition is dried and is recorded water content, tests for enzymolysis.
3, the preparation of cellulase: a certain amount of spore inoculating of picking 200 ~ 240rpm, 28 ~ 32 DEG C of cultivation 24 ~ 30h in seed culture medium from Trichodermareesei test tube slant.Seed liquor is seeded in fermention medium according to the ratio of 8% ~ 12%, 220 ~ 240rpm, 30 DEG C of fermentations 7 ~ 9 days, the centrifugal rear refrigerated storage of gained fermentation liquor, as the cellulase of experiment, and records its filter paper enzyme activity, CMC enzyme is lived, and beta-glucosidase is lived and xylanase activity.
Seed culture medium: potato 20g, glucose 20g, deionized water 1000mL.
Fermention medium is: Microcrystalline Cellulose 5 ~ 20g, groundnut meal 1 ~ 5g, cupric chloride 0.05 ~ 0.3g, trehalose 0.001 ~ 0.5g, L-AA 0.01 ~ 0.5g, PEG10,0000.5 ~ 5g, 480mL citrate buffer solution, 500mL liquid microelement, 20mL trace elements liquid.
Described citrate buffer solution (0.1M): Na
2hPO
42H
2o 17.80g/L, citric acid 21.01g/L.
Described micro-salts solution: (NH
4)
2sO
42.8g/L, KH
2pO4 4.0g/L, MgSO
47H
2o 0.6g/L, CaCl
22H
2o 0.8g/L.
Described trace elements: FeSO47H
2o 250mg/L, MnSO4H2O 85mg/L, ZnSO
47H
2o 70mg/L, CoCl
22H
2o 100mg/L, it is 2 that the vitriol oil is transferred to pH, filtration sterilization.
The various enzyme of obtained cellulose enzyme liquid is lived and is respectively: FPA enzyme 0.6 ~ 0.8U/mL, CMC enzyme alive 10 ~ 25U/mL alive, beta-glucosidase 0.6 ~ 1.0U/mL, xylanase activity 40 ~ 60U/mL alive.
4, enzymolysis produces sugar: in 2 ~ 15% substrate concns, add cellulase 2 ~ 30FPU/g matrix, beta-glucosidase 5 ~ 10U/g matrix, zytase 100 ~ 120U/g matrix, N,O-Diacetylmuramidase 50 ~ 100U/g matrix, cationic polyacrylamide 1 ~ 10mg/g matrix, tea saponin 1 ~ 10mg/g matrix, at 40 ~ 50 DEG C, enzymolysis 48 ~ 96h under 100 ~ 160rpm condition after mixing.
5, the mensuration of enzymatic hydrolyzation: respectively at pre-treatment wheat straw enzymolysis when 6h, 12h, 24h and 48h, gets after Partial digestion liquid is diluted to suitable multiple and measures concentration of reduced sugar wherein by DNS method, and extrapolate enzymatic hydrolyzation thus.
The invention has the advantages that:
1) the glycerine organic solvent pretreatment process that matrix treatments is used, eliminate most of xylogen, Mierocrystalline cellulose retention rate is high, is conducive to efficient enzymolysis.
2) cellulase production technique is simple, and cost is low.
3) enzyme carrying capacity used during enzymolysis is little, and enzymolysis efficiency is high.
Embodiment
Be below several specific examples of the present invention, further describe the present invention, but the present invention be not limited only to this.
Embodiment 1
1, the preparation of glycerine organic solvent pre-treatment wheat straw: the wheat straw taking 10g oven dry, in the Erlenmeyer flask of 500mL, adds 147g industry glycerol and 53g deionized water (glycerol concentration 70%).On heating jacket, 200rpm stirs, and adds prolong when being heated to 220 DEG C, maintains 3h.After time of arrival, remove heating jacket and add 150mL boiling water after temperature be down to about 120 DEG C, 150rpm stir 10min, 500mL boil 50% aqueous glycerin solution suction filtration twice, then 500mL boiling water suction filtration twice.
2, the preparation of cellulase: a certain amount of spore inoculating of picking 220rpm, 30 DEG C of cultivation 24h in seed culture medium from the test tube slant of Trichodermareesei Rut C-30 mutant strain.Seed liquor is seeded in fermention medium according to the ratio of 10%, 220rpm, 30 DEG C fermentation 7 days, the centrifugal rear refrigerated storage of gained fermentation liquor as experiment cellulase.
Seed culture medium: potato 20g, glucose 20g, deionized water 1000mL.
Fermention medium is: Microcrystalline Cellulose 15g, groundnut meal 1g, cupric chloride 0.276g, trehalose 0.025g, PEG10000 2.5g, L-AA 0.025g, 480mL citrate buffer solution, 500mL liquid microelement, 20mL trace elements liquid.
The performance of obtained cellulase product is, the various enzymes of cellulose enzyme liquid are lived and are respectively: FPA enzyme 0.61U/mL, CMC enzyme alive 18.72U/mL, BG enzyme alive 0.62U/mL, xylanase activity 51.82U/mL alive.
3, enzymolysis: take be equivalent to 0.75g dry weight glycerine organic solvent pre-treatment wheat straw wet basis (substrate concn is 5%) in 50mL triangular flask, add by following compositions: cellulase 2.5FPU/g matrix, Novozyme 188 7.5U/g matrix, Htec 115U/g matrix, N,O-Diacetylmuramidase 75U/g matrix, cationic polyacrylamide 2mg/g matrix, tea saponin 8mg/g matrix, add the buffered soln of pH 4.8 to 15g, laying temperature 50 DEG C and rotating speed 120rpm shaking bath carry out enzymolysis.
4, measuring method: sample after glycerine organic solvent pre-treatment wheat straw enzymolysis 6h, 12h, 24h, 48h respectively, centrifuging and taking supernatant is diluted to suitable multiple, measure the sugar degree in supernatant liquor by DNS method, and be converted into the enzymatic hydrolyzation of glycerine organic solvent pre-treatment wheat straw.
Embodiment 2
Cellulase is identical with embodiment 1 with the preparation of glycerine organic solvent pre-treatment wheat straw, is 5FPU/g matrix unlike the cellulase carrying capacity of adding during enzymolysis.According to the pretreated wheat straw of method enzymolysis 5% glycerine organic solvent of embodiment 1.
Embodiment 3
Cellulase is identical with embodiment 1 with the preparation of glycerine organic solvent pre-treatment wheat straw, is commercial fibres element enzyme (Novi letter Cellic CTec2) of 5FPU/g matrix unlike what add during enzymolysis.According to the pretreated wheat straw of method enzymolysis 5% glycerine organic solvent of embodiment 1.
In embodiment 1 ~ 3, the enzymatic hydrolyzation of glycerine organic solvent pre-treatment wheat straw is as shown in table 1.
The enzymolysis of customization cellulase and commercial fibres element enzyme optimized by table 1
As can be seen from embodiment 2 and embodiment 3, the compound cellulose zymin of identical filter paper enzyme activity comparatively commercial fibres element enzyme more can effective enzymolysis lignocellulose.With the pretreated wheat straw of compound cellulose enzymic hydrolysis glycerine organic solvent of 5FPU/g matrix, 6h enzymatic hydrolyzation reaches 66.3%, obtains higher enzymatic hydrolyzation in the short period of time; The hydrolysis result of its 12h, more than the plain enzyme of 48h commercial fibres, reaches 91.4%; During 48h, intramatrical Mierocrystalline cellulose and hemicellulose almost discharge completely, and enzymatic hydrolyzation reaches 97.3%.Commercial fibres element enzyme (Cellic CTec2) of 5FPU/g matrix is better than from the effect of the compound cellulose enzymic hydrolysis glycerine organic solvent pre-treatment wheat straw of embodiment 1 and embodiment 3,2.5FPU/g matrix.Therefore, the method adopting a kind of economical and efficient hydrolysis agricultural-forestry biomass raw material provided by the invention to prepare cellulose sugar can obtain high enzymatic hydrolyzation when low enzyme carrying capacity, is conducive to the reasonable efficiency utilization of agricultural-forestry biomass raw material.
It is to be understood that: although above embodiment to be contrasted detailed description to the present invention; but these illustrate, just to simple declaration of the present invention, instead of limitation of the present invention; any innovation and creation do not exceeded in connotation of the present invention, all fall within the scope of protection of the present invention.
Claims (10)
1. a composite method for customizing for effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin, is characterized in that method steps is as follows:
1) agricultural-forestry biomass raw material pulverizing: agricultural-forestry biomass, after naturally drying, is cut into the small segment of length 1 ~ 5cm, 60 ~ 80 DEG C of dry for standby.
2) raw materials glycerine organic solvent pre-treatment: raw material and aqueous glycerin solution are mixed by a certain percentage, washed twice with aqueous glycerin solution after heat treated for some time, then with washing twice from the beginning.
3) cellulase fermentations: more additional product enzyme promotor in the fermentation medium, Li's Trichoderma strains is accessed after sterilizing, at 28 ~ 32 DEG C and 200 ~ 240rpm condition bottom fermentation 7 ~ 9 days, after fermentation, fermentation liquor 6000 ~ 8000rpm is centrifugal, and supernatant liquor is as enzymolysis cellulase used.
4) enzymolysis produces sugar: in 2 ~ 15% substrate concns, add cellulase 2 ~ 30FPU/g matrix, to add after a certain amount of auxiliary enzymes and additive enzymolysis 48 ~ 96h under 40 ~ 50 DEG C and 100 ~ 160rpm condition.
2. the composite method for customizing of a kind of effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin according to claim 1, its step 1) described in agricultural-forestry biomass raw material refer to wheat straw, straw, maize straw, corn cob, cotton stalk, bagasse, hardwood, cork fibrous matter waste, and draft class plant and their any mixing.
3. the composite method for customizing of a kind of effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin according to claim 1, its step 2) the pretreated material concentration of glycerine organic solvent is 5% ~ 15%, initial aqueous glycerin solution concentration is 30 ~ 100%, and boiling temperature 130 ~ 240 DEG C maintains 1 ~ 4h.
4. the composite method for customizing of a kind of effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin according to claim 1, its step 2) pre-treatment of glycerine organic solvent relates to autocatalysis and extra catalyst, and wherein extra catalyst comprises acid, alkali and metal-salt.
5. the composite method for customizing of a kind of effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin according to claim 1, its step 3) added promoter is cupric ion 0.3 ~ 2.5m mol/L in fermention medium, trehalose 0.001 ~ 0.5g/L, L-AA 0.01 ~ 0.5g/L, PEG series of surfactants 0.5 ~ 5g/L.
6. the composite method for customizing of a kind of effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin according to claim 1, its step 4) in auxiliary enzymes used be beta-glucosidase, zytase and N,O-Diacetylmuramidase.
7. auxiliary enzymes according to claim 6, its addition is respectively: beta-glucosidase 0.5 ~ 20U/g matrix, zytase 100 ~ 300U/g matrix, N,O-Diacetylmuramidase 30 ~ 150U/g matrix.
8. the composite method for customizing of a kind of effectively hydrolyzing agricultural-forestry biomass raw cellulose zymin according to claim 1, its step 4) additive is cationic polyacrylamide and tea saponin.
9. additive according to claim 7, its addition is respectively: cationic polyacrylamide 1 ~ 10mg/g matrix, tea saponin 1 ~ 10mg/g matrix.
10. a kind of enzymolysis according to claim 1 produces liquid glucose, and its characteristic is: glucose concn 10 ~ 150g/L, xylose concentration 2 ~ 50g/L, enzymatic hydrolyzation 60 ~ 99%.
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| CN110157755A (en) * | 2019-05-31 | 2019-08-23 | 江南大学 | A method of strengthening agricultural-forestry biomass raw material thick mash enzymatic hydrolysis and produces sugar |
| CN110358722A (en) * | 2019-08-16 | 2019-10-22 | 中南林业科技大学 | Preparation method of litsea cubeba mesophyll cell protoplast |
| CN113402312A (en) * | 2021-06-23 | 2021-09-17 | 兰州大学 | Nano composite catalyst, device and method for treating agricultural wastes |
| CN114941254A (en) * | 2022-06-16 | 2022-08-26 | 淮阴工学院 | Pretreatment method and application of lignocellulose biomass and method for improving enzymolysis efficiency of lignocellulose biomass |
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Cited By (7)
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