CN104278065A - Method for improving yield of enzymatic hydrolysis of plant fiber materials - Google Patents
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Abstract
The invention discloses a method for improving the yield of enzymatic hydrolysis of plant fiber materials. The method comprises the steps of carrying out alkaline pretreatment of the plant fiber materials and preparing monosaccharide by virtue of enzymatic hydrolysis of cellulose and xylanase and is characterized in that when cellulase and xylanase for hydrolyzing the plant fiber materials subjected to alkaline pretreatment are added, 10-50U/g of beta-glucosidase of cellulose is additionally added. Compared with the prior art, the method for improving the yield of enzymatic hydrolysis of the plant fiber materials has the prominent advantages that by excessively adding beta-glucosidase in an enzymatic hydrolysis system of the plant fiber materials subjected to alkaline pretreatment, the concentration of cellobiose in a hydrolysate generated at the initial stage of enzyme hydrolysis is reduced, the inhibition effect of cellobiose generated at the initial stage of enzyme hydrolysis on endoglucanase and exoglucanase is alleviated and thus the enzyme reaction rate and the yield of enzymatic hydrolysis are improved and the production cost for producing biomass sugar from the plant fiber materials subjected to alkaline pretreatment is reduced.
Description
Technical field
The present invention relates to plant fiber material hydrolysis technology field, be specifically related to a kind of method improving plant fiber material enzymic hydrolysis yield.
Background technology
Along with human society is to the growing and overexploitation of fossil resource demand taking petroleum resources as representative, petroleum resources are made inevitably to face exhausted destiny; The Greenhouse effect that the use of a large amount of fossil resource causes simultaneously likely develop into an eco-catastrophe into the mankind.How do the mankind tackle the trend of Future energy shortage, environmental pollution and global warming? the road walking Sustainable development is become a consensus of the international community.Utilizing reproducible vegetable fibre resource to produce bioenergy, bio-based chemical and bio-based materials is the important component part of the strategy of sustainable development.There is abundant agricultural-forestry biomass resource in China, comprises the grain processing waste, city fiber rubbish etc. of agriculture and forestry organic waste material, wood (bamboo) material processing waste, fiber-enriched.Utilize the vegetable fibre resource production biomass sugar (glucose, wood sugar) of these cheapnesss, and as the energy of raw material production necessary for human, chemical and material, the raw material problem that the future biological energy, bio-based chemical and bio-based materials are produced can be solved well.Plant fiber material is produced in the technical system of bioenergy, bio-based chemical and bio-based materials, and the biomass sugar production technology of high enzymic hydrolysis yield, low cost is the prerequisite and the key that determine plant fiber material biorefinery technology heavy industrialization.
The method of plant fiber material production biomass sugar mainly comprises acid-hydrolysis method and enzyme hydrolysis method two kinds, and wherein enzyme hydrolysis method has reaction conditions gentleness, unrestraint material generates, the feature such as low for equipment requirements and have potential prospects for commercial application.Mierocrystalline cellulose in plant fiber material is degraded into glucose under the katalysis of cellulase, and cellulase is a multiply anchor-pile, forms primarily of endoglucanase, exoglucanase and beta-glucosidase.Mierocrystalline cellulose complete hydrolysis completes under the synergy of each component in cellulase system, the cellulolytic mechanism of cellulase synergistic still imperfectly understands, universally recognized viewpoint is endoglucanase first random staple fiber element chain β – 1,4 – glycosidic links, produce new short chain cellulose and cellooligosaccharide; Exoglucanase is hydrolyzed in units of cellobiose successively from the reduction of short chain cellulose and cellooligosaccharide or non-reducing end subsequently; The cellobiose of generation and cellooligosaccharide are thoroughly hydrolyzed into glucose by last beta-glucosidase.Mierocrystalline cellulose is hydrolyzed into simple sugar glucose under the synergy of endoglucanase, exoglucanase and beta-glucosidase in cellulase system, and in cellulase system, a certain component all will affect cellulosic enzymic hydrolysis efficiency containing quantity not sufficient.Trichodermareesei in Trichoderma is the generally acknowledged excellent production bacterial strain of cellulase, but the endoglucanase in trichoderma reesei cellulase system, the ratio of exoglucanase and beta-glucosidase is inharmonious, beta-glucosidase content is relatively low, therefore, during Mierocrystalline cellulose in trichoderma reesei cellulase system degrading plant fibrous material, in hydrolysate, cellobiose content is higher, and at the bioenergy with industrial application value found at present, in bio-based chemical fermentable bacterial strain, most microorganism can not utilize cellobiose, therefore in cellulosic hydrolysates, require that the content of cellobiose is low as far as possible.The problem that in the hydrolysate that beta-glucosidase causes containing quantity not sufficient in Cellulose Enzymolysis of Plant Fiber process cellulase system, cellobiose content is high, to improve in cellulosic hydrolysates fermentability monose ratio for target, carry out large quantifier elimination both at home and abroad, the way addressed this problem mainly adds beta-glucosidase to reduce the content of cellobiose in cellulosic hydrolysates, research shows, in the hydrolyzation system of trichoderma reesei cellulase hydrolyzing plant fiber raw material, the general beta-glucosidase adding 1-5U/g Mierocrystalline cellulose consumption can to realize in hydrolysis sugar liquid cellobiose content lower than 1g/L, usual interpolation can realize above-mentioned effect lower than the beta-glucosidase of 3U/g Mierocrystalline cellulose consumption.In existing bibliographical information, the object of adding beta-glucosidase in cellulase hydrolysis system is the concentration reducing cellobiose in hydrolysis sugar liquid, thus improves the ratio of fermentable sugar in hydrolysate.
Cellulase belongs to the hydrolase of feedback inhibition, become in the process of glucose at cellulase system hydrocellulose, the generation of end product glucose produces restraining effect to the vigor of beta-glucosidase, the accumulation of cellobiose in hydrolytic process is caused after beta-glucoside enzyme activity is suppressed, the accumulation of cellobiose produces restraining effect to endoglucanase and exoglucanase, thus causes cellulase hydrolysis speed of response and be hydrolyzed yield not high.
Applicant finds when studying the enzyme reaction speed in the raw material pretreated process of trichoderma reesei cellulase hydrolyzed alkaline, although the auxiliary cellulosic beta-glucosidase of 3U/g that adds can ensure that in hydrolysate, cellobiose concentration is at below 1g/L in Cellulose Enzymolysis of Plant Fiber system, but in hydrolysis initial stage hydrolyzed solution, cellobiose concentration is higher is the key factor causing cellulase speed of reaction to decline, on this basis, propose excessive auxiliary interpolation beta-glucosidase, to reduce the problem of cellobiose excessive concentration in Cellulose Enzymolysis of Plant Fiber initial stage hydrolyzed solution, alleviate hydrolysis initial fibers disaccharides to endoglucanase, the restraining effect of exoglucanase, thus reach the object improving enzyme reaction speed and hydrolysis yield, simultaneously, along with the raising of cellulose hydrolysis yield, also improve the hydrolysis yield of xylan.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide a kind of method improving plant fiber material enzymic hydrolysis yield, by excessive interpolation beta-glucosidase in alkalescence raw material pretreated enzymic hydrolysis process, reduce the cellobiose concentration in enzymic hydrolysis initial stage hydrolyzed solution, alleviate the restraining effect of enzymic hydrolysis initial fibers disaccharides to endoglucanase and exoglucanase, thus improve enzyme reaction speed and enzymic hydrolysis yield, reduce the production cost of alkaline raw material pretreated production biomass sugar.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is as follows:
A kind of method improving plant fiber material enzymic hydrolysis yield, comprise plant fiber material alkalescence pre-treatment and Mierocrystalline cellulose, xylanase hydrolysis prepare monose step, when adding cellulase and the pretreated plant fiber material of xylanase hydrolysis alkalescence, the cellulosic beta-glucosidase of extra interpolation 10-50U/g.
Described Mierocrystalline cellulose, xylanase hydrolysis prepare monose, substrate w/v concentration 5-20%.
Described Mierocrystalline cellulose, xylanase hydrolysis prepare monose, substrate w/v concentration 15-20%, adopt the method for batch feeding to add substrate.
Described Mierocrystalline cellulose, xylanase hydrolysis prepare monose, and cellulase consumption is not higher than 25FPU/g Mierocrystalline cellulose, and zytase consumption is not higher than 120U/g Mierocrystalline cellulose.
The method of described raising plant fiber material enzymic hydrolysis yield, comprises the steps:
(1) with green liquor, alkaline purification is carried out to air-dry plant fiber material, alkali consumption 2-8%, treatment temp 100-140 DEG C, soaking time 1h, sulphidity 30-40%, the plant fiber material through green liquor pretreatment washes with water, defibrination, filtration;
(2) plant fiber material of green liquor pretreatment is mixed with cellulase and zytase, add water, pH damping fluid, be mixed to substrate w/v concentration 5-20%, control ph is at 4.0-6.0, press the cellulosic amount of 10-50U/g in reaction system and add beta-glucosidase, enzyme digestion reaction 48h under 45-55 DEG C of condition.
In step (2), when enzymolysis substrate w/v concentration is 15-20%, enzymolysis preferably adopts the method for batch feeding.
Described cellulase is with the mixture of one or more enzymes of Trichodermareesei generation.
Beneficial effect: compared with prior art, the method of raising plant fiber material enzymic hydrolysis yield of the present invention, outstanding advantage is by excessive interpolation beta-glucosidase in alkalescence raw material pretreated enzymic hydrolysis system, reduce the cellobiose concentration in enzymic hydrolysis initial stage hydrolyzed solution, alleviate the restraining effect of enzymic hydrolysis initial fibers disaccharides to endoglucanase and exoglucanase, thus improve enzyme reaction speed and enzymic hydrolysis yield, reduce the production cost of alkaline raw material pretreated production biomass sugar.
Accompanying drawing explanation
Fig. 1 adds low beta-glucosidase and adds excessive beta-glucosidase to affect result figure to cellobiose concentration in enzyme reaction process;
Fig. 2 adds low beta-glucosidase and adds excessive beta-glucosidase to affect result figure to average enzyme reaction speed and cellulase hydrolysis yield.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.
In following examples, glucose concn, xylose concentration and cellobiose concentration all adopt high performance liquid chromatography (HPLC) to measure.Chromatographic condition is as follows: chromatographic instrument: Agillent1100 high performance liquid chromatograph; Chromatographic column: Bio-Rad Aminex HPX-87H; Moving phase: 0.005mol/L sulfuric acid, flow velocity: 0.6mL/min; Column temperature: 55 DEG C; Detector: differential refraction detector; Sample size: 10 μ L.External standard method.
Beta-glucosidase vigour-testing method: adopt p-NP-β-D-Glucose glycosides (pNPG) for substrate mensuration.A beta-glucoside enzyme activity unit is defined as under standard reaction condition, the enzyme amount required for per minute hydrolysis generation 1 μm of ol p-NP.
Measuring method is as follows: the enzyme liquid that 0.1mL suitably dilutes is incubated 10min after mixing with the pNPG solution of 0.9mL 5mmol/L, pH 4.80 at 50 DEG C.Add 2mL 1mol/L Na2CO3 solution termination reaction immediately, add the distilled water of 10mL, shake up.Under 400nm, measure absorbancy, replace enzyme liquid to make blank with distilled water.Be calculated as follows beta-glucoside enzyme activity:
Embodiment 1: the pre-treatment of maize straw
Corn stalk powder is broken to 2-5cm, alkali charge 8% (w/w), sulphidity 40%, and solid-to-liquid ratio is 1:6 (w/v), after the 0.5h that dallies, temperature is increased to 140 DEG C at 60 DEG C, insulation 1h.The maize straw of green liquor pretreatment washes residue chemistry medicine with water, defibrination, extracts material, as the substrate of enzymic hydrolysis.Analyze moisture, Mierocrystalline cellulose, the xylan content of substrate, and calculate Mierocrystalline cellulose and the xylan rate of recovery.
Result shows, maize straw through green liquor pretreatment, defibrination, wash, extract after moisture content 76.90%, content of cellulose 55.39% (butt), xylan content 25.43% (butt), green liquor pretreatment material total yield is 63.31%, the Mierocrystalline cellulose rate of recovery is 91.95%, and the xylan rate of recovery is 63.42%.
Embodiment 2: auxiliary add a small amount of and excessive beta-glucosidase to the impact of enzymic hydrolysis yield
Take the green liquor pretreatment maize straw 10.82g (over dry is heavily 2.50g) of embodiment 1 respectively in 12 250mL triangular flasks (enzymolysis concentration of substrate 5% (w/v)), 1mol/L citrate buffer solution 2.5mL is added in each triangular flask, cellulase is added respectively by the cellulosic enzyme dosage of 25FPU/g, zytase is added respectively by the cellulosic enzyme dosage of 120U/g, respectively by 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 37.5, the cellulosic consumption of 50U/g adds beta-glucosidase, in each triangular flask, add appropriate distilled water makes moisture cumulative volume in enzymatic hydrolysis system be 50mL, with glass rod, reaction system is fully mixed rear cover lid, in 150 revs/min, enzymolysis 48h in the constant-temperature table of 50 DEG C.After hydrolysis terminates, respectively by hydrolyzate whizzer centrifugal 10min under 4000 revs/min of conditions, get supernatant liquor mensuration glucose, xylose concentration and cellobiose concentration wherein, and calculate enzymic hydrolysis yield and fermentability glucosyl group ratio.Wherein: cellulase hydrolysis yield (%)=(in hydrolyzed solution in glucose concn g/L × 0.9+ hydrolyzed solution cellobiose concentration g/L × 0.95) × 0.05 ÷ (heavy g × 0.5539 × 100% of substrate over dry; Xylose concentration g/L × 0.88 × 0.05 ÷ (heavy g × 0.2543 of substrate over dry) × 100% in zytase solution yield (%)=hydrolyzed solution; Can glucose concn g/L ÷ in fermented grape glycosyl ratio (%)=hydrolyzed solution (in hydrolyzed solution in glucose concn g/L+ hydrolyzed solution cellobiose concentration g/L) × 100%.In formula: 0.9 is glucose and cellulosic gain factor; 0.95 is cellobiose and cellulosic gain factor; 0.88 is the gain factor of wood sugar and xylan; 0.05 is hydrolysis sugar liquid volume, L; 0.5539 is content of cellulose in raw material, %; 0.2543 is xylan content in raw material, %.The auxiliary influence of beta-glucosidase to enzymic hydrolysis yield of adding is as shown in table 1.
The auxiliary beta-glucosidase that adds of table 1 is on the impact of enzymic hydrolysis
Result shows, trichoderma reesei cellulase hydrolyzed alkaline pre-treatment maize straw, it is higher that hydrolysis terminates cellobiose concentration in rear hydrolyzed solution, reach 4.37g/L, in hydrolyzed solution, fermentability glucosyl group ratio is only 80.41%, illustrate that in trichoderma reesei cellulase system, beta-glucosidase is containing quantity not sufficient, cellobiose concentration in rear hydrolyzed solution is higher, fermentability glucosyl group ratio is low to cause hydrolysis to terminate.The cellulosic beta-glucosidase of 3U/g is added in above-mentioned cellulase hydrolysis system, the hydrolysis cellobiose concentration terminated in rear hydrolyzed solution can be reduced to 0.71g/L from 4.37g/L, fermentability glucosyl group ratio brings up to 96.96% from 80.41%, but cellulose hydrolysis variation of yield is little.Illustrate in trichoderma reesei cellulase hydrolyzation system, a small amount of (1-5U/g Mierocrystalline cellulose) beta-glucosidase of auxiliary interpolation, object is the coordination meeting beta-glucosidase and endoglucanase in cellulase system, exoglucanase ratio, ensure the fermentability glucosyl group ratio in cellulase hydrolysis liquid, and little to the raising effect of cellulase hydrolysis yield.But after excessive auxiliary interpolation beta-glucosidase (addition >10U/g Mierocrystalline cellulose), cellulose hydrolysis yield can be increased substantially, and while raising cellulose hydrolysis yield, also promote the raising of xylanase hydrolysis yield.After adding the cellulosic beta-glucosidase of 10U/g in trichoderma reesei cellulase hydrolyzation system, Mierocrystalline cellulose and xylanase hydrolysis yield improve 6.68 and 2.61 percentage points than hydrolysis yield when adding the cellulosic beta-glucosidase of 3U/g respectively; When after the cellulosic beta-glucosidase of interpolation 20U/g, Mierocrystalline cellulose and xylanase hydrolysis yield reach the highest, be respectively 84.59% and 77.19%, hydrolysis yield respectively during beta-glucosidase more cellulosic than interpolation 3U/g improves 8.52 and 5.81 percentage points, continue the addition improving beta-glucosidase, little to the continuation raising effect of Mierocrystalline cellulose and xylanase hydrolysis yield.
Embodiment 3: excessive auxiliary interpolation beta-glucosidase is on the impact of different concentration of substrate pre-treatment Factor of Enzymolysis Corn Stalk
Take the green liquor pretreatment maize straw 5.41 of embodiment 1 respectively, 10.82, 16.23, 21.65, 27.06, 32.47, (over dry is heavily respectively 1.25 to 43.28g, 2.50, 3.75, 5.00, 6.25, 7.50, 10.00g) in 7 250mL triangular flasks, (correspond to enzymolysis concentration of substrate 2.5%, 5.0%, 7.5%, 10.0%, 12.5%, 15%, 20% (w/v)), 1mol/L citrate buffer solution 2.5mL is added in each triangular flask, cellulase is added respectively by the cellulosic enzyme dosage of 25FPU/g, zytase is added respectively by the cellulosic enzyme dosage of 120U/g, beta-glucosidase is added respectively by the cellulosic consumption of 20U/g, in each triangular flask, add appropriate distilled water makes moisture cumulative volume in enzymatic hydrolysis system be 50mL, with glass rod, reaction system is fully mixed rear cover lid, in 150 revs/min, enzymolysis 48h in the constant-temperature table of 50 DEG C.After hydrolysis terminates, respectively by hydrolyzate whizzer centrifugal 10min under 4000 revs/min of conditions, get supernatant liquor and measure glucose concn wherein and cellobiose concentration, and calculate enzymic hydrolysis yield.Excessive auxiliary interpolation beta-glucosidase on the impact of different concentration of substrate pre-treatment Factor of Enzymolysis Corn Stalk as table 2.
The excessive auxiliary interpolation beta-glucosidase of table 2 is on the impact of different concentration of substrate pre-treatment Factor of Enzymolysis Corn Stalk
Result shows, in the hydrolyzation system of excessive auxiliary interpolation beta-glucosidase, hydrolysis terminates cellobiose concentration in rear hydrolyzed solution and all remains on lower level, and under the condition of concentration of substrate at 2.5%-15%, Mierocrystalline cellulose and xylan all obtain higher hydrolysis yield.It is because along with the increase of concentration of substrate that Mierocrystalline cellulose and xylan hydrolysis yield reduce along with the increase of concentration of substrate, and in hydrolyzed solution, glucose concn increase causes the restraining effect of end product glucose to increase and concentration of substrate increase causes enzymic hydrolysis system mass transfer difficulty to cause.When concentration of substrate is 20%, almost without free water content in hydrolyzation system, enzyme reaction mass transfer is very difficult, and therefore enzymic hydrolysis yield is low, need realize the enzymic hydrolysis of high concentration of substrate by batch feeding technology.
When enzymic hydrolysis concentration of substrate rises to 15% from 2.5%, cellulase hydrolysis yield drops to 79.93% from 92.26%, and xylanase hydrolysis yield drops to 67.53% from 81.49%.But the raising of beta-glucoside enzyme dosage, makes cellobiose remain on lower level always.When enzymic hydrolysis concentration of substrate is 15%, free-water is little, although substrate cannot liquefy for a long time, enzymic hydrolysis still obtains very high hydrolysis yield.In addition, under high concentration of substrate, carry out efficient enzymic hydrolysis, the energy consumption of follow-up distillation can be reduced, reduce the production cost of cellulosic ethanol.
The enzymic hydrolysis of the excessive interpolation cellulosic beta-glucosidase of 20U/g and batch feeding under embodiment 4:20% concentration of substrate
Take the green liquor pretreatment maize straw 43.28g (the heavy 10.00g of over dry) of embodiment 1 respectively, insert reaction system by table 3 adding material mode.Add in 6 50mL triangular flasks respectively by the pre-treatment maize straw addition of table 30h respectively, 1mol/L citrate buffer solution 2.5mL is added in each triangular flask, cellulase is added respectively by the cellulosic enzyme dosage of 25FPU/g, zytase is added respectively by the cellulosic enzyme dosage of 120U/g, beta-glucosidase is added respectively by the cellulosic consumption of 20U/g, in each triangular flask, add appropriate distilled water makes moisture cumulative volume in enzymatic hydrolysis system be 50mL, with glass rod, reaction system is fully mixed rear cover lid, in 150 revs/min, enzymolysis 48h in the constant-temperature table of 50 DEG C, remaining substrate is added by table 3 in enzyme reaction process.After hydrolysis terminates, respectively by hydrolyzate whizzer centrifugal 10min under 4000 revs/min of conditions, get supernatant liquor and measure glucose concn wherein and cellobiose concentration, and calculate enzymic hydrolysis yield.Under 20% concentration of substrate excessive interpolation beta-glucosidase and batch feeding on the impact of pre-treatment Factor of Enzymolysis Corn Stalk as table 4.
Enzymic hydrolysis adding material mode in batches under table 3 20% concentration of substrate
| A | 0h(43.28g) | ? | ? | ? |
| B | 0h(21.64g) | 6h(10.82g) | 12h(10.82g) | / |
| C | 0h(21.64g) | 12h(10.82g) | 24h(10.82g) | / |
| D | 0h(16.23g) | 6h(16.23g) | 12h(10.82g) | / |
| E | 0h(16.23g) | 12h(16.23g) | 24h(10.82g) | / |
| F | 0h(10.82g) | 4h(10.82g) | 8h(10.82g) | 12h(10.82g) |
Result is as shown in table 4, show under 20% (w/v) concentration of substrate, the cellulosic beta-glucosidase of auxiliary interpolation 20U/g in enzyme reaction system, after adopting portion-wise addition method to solve the mass transfer difficult problem of high concentration of substrate, green liquor pretreatment maize straw can obtain higher cellulose hydrolysis yield and xylan hydrolysis yield.Under suitable adding material mode, hydrolysis terminates glucose and xylose concentration in rear hydrolyzed solution and is respectively 94.23g/L and 34.69g/L, Mierocrystalline cellulose and xylan hydrolysis yield are respectively 76.97% and 60.04%, extremely be conducive to follow-up fermenting process containing high density fermentability monose in hydrolysis sugar liquid, the fermentation tank sum product cost for purification of subsequent fermentation can be reduced.
Batch feeding enzymic hydrolysis result under table 4 20% concentration of substrate
Embodiment 5: add beta-glucosidase to the impact of cellobiose concentration and enzyme reaction speed in hydrolytic process
Take the green liquor pretreatment maize straw 21.64g (over dry is heavily respectively 5.0g) of embodiment 1 (enzymolysis concentration of substrate 10% (w/v)) in 2 250mL triangular flasks respectively, 1mol/L citrate buffer solution 2.5mL is added in each triangular flask, cellulase is added by the cellulosic enzyme dosage of 25FPU/g, the cellulosic enzyme dosage of 120U/g adds zytase, beta-glucosidase is added respectively by the cellulosic consumption of 3U/g and 20U/g, in each triangular flask, add appropriate distilled water makes moisture cumulative volume in enzymatic hydrolysis system be 50mL, with glass rod, reaction system is fully mixed rear cover lid, in 150 revs/min, enzymolysis 48h in the constant-temperature table of 50 DEG C.Sample centrifugal 10min under 4000 revs/min of conditions respectively at 20min, 40min, 1h, 2.5h, 6h, 10h, 24h, 36h and 48h, get supernatant liquor and measure glucose concn wherein and cellobiose concentration, and calculate average enzyme reaction speed.Add low beta-glucoside enzyme dosage (3U/g Mierocrystalline cellulose) and Fig. 1 and Fig. 2 is shown in the impact of excessive interpolation beta-glucoside enzyme dosage (20U/g Mierocrystalline cellulose) on cellobiose concentration in enzyme reaction process and average enzyme reaction speed (representing with sugared generating rate).
Result shows, under different beta-glucosidase additions, although after the end of the hydrolysis in hydrolysis sugar liquid cellobiose concentration all lower, in hydrolytic process, cellobiose concentration is different.Be that in the cellulosic hydrolyzation system of 3U/g, cellobiose concentration reaches maximum 5.44g/L at 1.5h at beta-glucosidase addition, keep the slow decline under a higher concentration afterwards; And be in the cellulosic hydrolyzation system of 20U/g at beta-glucosidase addition, cellobiose concentration also reaches maximum at 1.5h, but about being only the half of adding cellobiose concentration in the cellulosic hydrolyzation system of 3U/g, 2.60g/L, remains on the slow decline under a low concentration afterwards.Therefore, in the cellulosic beta-glucosidase hydrolyzation system of interpolation 20U/g, the restraining effect that cellulase is subject to cellobiose is especially hydrolyzed the initial stage lower than the cellulosic beta-glucosidase hydrolyzation system (Fig. 1) of interpolation 3U/g.As can be seen from Figure 2, owing to the addition of in the cellulosic cellulase hydrolysis system of 20U/g excessive, in reaction process especially in hydrolysis reaction initial stage hydrolyzed solution cellobiose concentration lower than the cellobiose concentration of adding in the cellulosic cellulase hydrolysis system of 3U/g, alleviate the restraining effect of cellobiose to endoglucanase and exoglucanase, cause adding the hydrolyzation system cellulase hydrolysis rate of excessive beta-glucosidase and cellulase hydrolysis speed higher than the cellulosic beta-glucosidase hydrolyzation system of interpolation 3U/g.
The enzymic hydrolysis of the cellulosic beta-glucosidase of 3U/g and batch feeding is added under comparative example 1:20% concentration of substrate
Take the green liquor pretreatment maize straw 43.28g (the heavy 10.00g of over dry) of embodiment 1, getting wherein 21.64g adds in 50mL triangular flask, 1mol/L citrate buffer solution 2.5mL is added in triangular flask, enzyme dosage is the cellulosic cellulase of 25FPU/g, enzyme dosage is the cellulosic zytase of 120U/g, the cellulosic beta-glucosidase of enzyme dosage 3U/g, adding appropriate distilled water makes moisture cumulative volume in enzymatic hydrolysis system be 50mL, with glass rod, reaction system is fully mixed rear cover lid, in 150 revs/min, enzymolysis 48h in the constant-temperature table of 50 DEG C, respectively at enzyme reaction 12h, 24h adds pre-treatment maize straw 10.82g.After hydrolysis terminates, respectively by hydrolyzate whizzer centrifugal 10min under 4000 revs/min of conditions, get supernatant liquor and measure glucose concn wherein and cellobiose concentration, and calculate enzymic hydrolysis yield.The enzymic hydrolysis result of the cellulosic beta-glucosidase of 3U/g and batch feeding is added as table 5 under 20% concentration of substrate.
Batch feeding enzymic hydrolysis result under table 5 20% concentration of substrate
Result shows, the green liquor pretreatment corn stalk fiber element enzymic hydrolysis of concentration of substrate 20%, under the beta-glucoside enzyme dosage of auxiliary interpolation 3U/g, hydrolysis terminates that cellobiose concentration in rear hydrolyzed solution is lower, fermentability glucosyl group ratio is higher, but glucose and xylose concentration is lower in hydrolyzed solution, be only 50.41g/L and 21.75g/L, corresponding Mierocrystalline cellulose and xylan hydrolysis yield are respectively 44.43% and 37.64%.And add the enzymic hydrolysis of excessive beta-glucosidase (20U/g Mierocrystalline cellulose) under the same conditions, glucose and xylose is respectively 94.23g/L and 34.69g/L, and corresponding Mierocrystalline cellulose and xylan hydrolysis yield are respectively 76.97% and 60.04% (embodiment 4).
Claims (7)
1. one kind is improved the method for plant fiber material enzymic hydrolysis yield, comprise plant fiber material alkalescence pre-treatment and Mierocrystalline cellulose, xylanase hydrolysis prepare monose step, it is characterized in that: when adding cellulase and the pretreated plant fiber material of xylanase hydrolysis alkalescence, the cellulosic beta-glucosidase of extra interpolation 10-50U/g.
2. the method for raising plant fiber material enzymic hydrolysis yield according to claim 1, is characterized in that: described Mierocrystalline cellulose, xylanase hydrolysis prepare monose, substrate w/v concentration 5-20%.
3. the method for raising plant fiber material enzymic hydrolysis yield according to claim 1 and 2, is characterized in that: described Mierocrystalline cellulose, xylanase hydrolysis prepare monose, substrate w/v concentration 15-20%, adopts the method for batch feeding to add substrate.
4. the method for raising plant fiber material enzymic hydrolysis yield according to claim 1, it is characterized in that: described Mierocrystalline cellulose, xylanase hydrolysis prepare monose, cellulase consumption is not higher than 25FPU/g Mierocrystalline cellulose, and zytase consumption is not higher than 120U/g Mierocrystalline cellulose.
5. the method for raising plant fiber material enzymic hydrolysis yield according to claim 1, is characterized in that, comprise the steps:
(1) with green liquor, alkaline purification is carried out to air-dry plant fiber material, alkali consumption 2-8%, treatment temp 100-140 DEG C, soaking time 1h, sulphidity 30-40%, the plant fiber material through green liquor pretreatment washes with water, defibrination, filtration;
(2) plant fiber material of green liquor pretreatment is mixed with cellulase and zytase, add water, pH damping fluid, be mixed to substrate w/v concentration 5-20%, control ph is at 4.0-6.0, press the cellulosic amount of 10-50U/g in reaction system and add beta-glucosidase, enzyme digestion reaction 48h under 45-55 DEG C of condition.
6. the method for raising plant fiber material enzymic hydrolysis yield according to claim 5, is characterized in that, in step (2), when enzymolysis substrate w/v concentration is 15-20%, enzymolysis adopts the method for batch feeding.
7. the method for the raising plant fiber material enzymic hydrolysis yield according to claim 1,2,4,5 or 6, is characterized in that, described cellulase is with the mixture of one or more enzymes of Trichodermareesei generation.
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| CN110684211A (en) * | 2019-10-14 | 2020-01-14 | 宁夏妙朗生物科技有限公司 | Method for preparing cross-linked dextran gel resistant to hydrolysis by alpha-glucosidase |
| CN114058605A (en) * | 2021-11-10 | 2022-02-18 | 湖北大学 | Method for improving specific activity of cellulase and application thereof |
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