CN102653752A - Selective inactivation method of cellulase systems - Google Patents
Selective inactivation method of cellulase systems Download PDFInfo
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- CN102653752A CN102653752A CN2012101271069A CN201210127106A CN102653752A CN 102653752 A CN102653752 A CN 102653752A CN 2012101271069 A CN2012101271069 A CN 2012101271069A CN 201210127106 A CN201210127106 A CN 201210127106A CN 102653752 A CN102653752 A CN 102653752A
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Abstract
The invention discloses a selective inactivation method of cellulase systems. The selective inactivation method comprises the steps of adjusting the pH of cellulase liquid to be not less than 8 under a temperature controlled at 5 DEG C to 40 DEG C, and standing the cellulase liquid for 10 to 80 minutes to perform selective inactivation on the cellulase systems, wherein CMCase (carboxymethyl cellulase) and CBH (exo-1,4-beta-D-glucanase) are selectively maintained, and BG ( beta-1,4-D-glucosidase) is selectively inactivated. The selective inactivation method disclosed by the invention is simple and rapid and easy to operate, can realize the selective inactivation on three enzyme systems in the cellulase systems by virtue of a simple pH processing method, serves as a simple and inexpensive directional control technology and method for the vitality of enzyme components, has very important practical values and good practicability, and can generate good economic benefits and social effects.
Description
Technical field
The present invention relates to cellulase, be specifically related to a kind of selectivity method for deactivating of cellulase system.
Background technology
Trichodermareesei (Trichoderma reesei) is one of the most frequently used classical bacterial strain of research and preparation cellulase; Through substrate induce that it can secrete the glycan hydrolase protein component of number of different types with the fermentation control effect, and then form different enzyme systems or prozyme system; Wherein, cellulase system mainly comprises NCE5 (endo-1,4-β-D-glucanase; EC 3.2.1.4 is called for short EG), circumscribed glucomannan enzyme (exo-1,4-β-D-glucanase; EC 3.2.1.91 claims CBH again) and cellose (β-1,4-D-glucosidase; EC 3.2.1.21; Claim beta-glucosidase again, be called for short β-G or BG) three big enzymes are, have had been found that 8 EG (EGI ~ EGVIII), 2 CBH (CBHI and CBHII) and 7 BG (enzyme family of BGI ~ BGVII) at present.
In the enzymic hydrolysis process of lignocellulose raw material glycan class, the synergy through above-mentioned three big enzymes system can catalyse cellulose or semicellulose be that the final hydrolysis of glycan generates monose.Along with functional polysaccharide and oligosaccharides synthetic and the fundamental research in fields such as regulation and control field, food, healthcare products, medicine and feed and fast development of application at cell biological; The research and development of glycan pacemaker enzyme technology for hydrolyzing receives much concern, and the increasing demand of enzyme family with simple function or catalytic activity or enzyme component is increased.The major technique approach one that adopts at present is recombinant expressed through single restriction endonuclease of external source or excision enzyme gene; The 2nd, the natural enzyme preparation is carried out protein split with control and remove the BG enzyme component in cellulose-based; They exist technical sophistication, R&D cycle length, equipment and operational requirement height, early investment and the high deficiency of running cost in large-scale industrial production; Therefore directional control technology and the method for seeking simple cheap enzyme component vigor seem and are even more important to have important theory and practical value.
Summary of the invention
Goal of the invention: to the deficiency that exists in the prior art; The selectivity method for deactivating that the purpose of this invention is to provide a kind of cellulase system; With Trichodermareesei product natural cellulose enzyme and other relevant prozyme systems is material; Method through adopting simple pH value processing is to carry out the selectivity inactivation to handle to its three big enzyme, realizes simple, the quick and selectivity inactivation to cellulase system.
Technical scheme: in order to realize the foregoing invention purpose, the technical scheme that the present invention adopts is following:
A kind of selectivity method for deactivating of cellulase system: 5 ~ 40 ℃ of temperature controls, the pH that regulates cellulase liquid is not less than 8, leaves standstill to handle 10 ~ 80min the plain enzyme of promptly alternative devitalizing fibers system; Wherein, CMCase and CBH selective retention, BG selectivity inactivation.
Described cellulase system is natural cellulose enzyme prozyme system and other the relevant prozyme system that Trichodermareesei produces.
With containing Na
+Alkali lye or damping fluid, or with containing K
+Alkali lye or damping fluid, regulate cellulase liquid pH.
Described pH=8.8 ~ 9.2.
Beneficial effect: the selectivity method for deactivating of cellulase system of the present invention, simple, fast; Operation easily; Only needing just can realize the big enzyme of three in cellulase system system is carried out the processing of selectivity inactivation through the method that adopts simple pH value to handle, is a kind of directional control technology and method of simply cheap enzyme component vigor, has important practical value; Good practicability is arranged, can produce favorable economic benefit and social effect.
Description of drawings
Fig. 1 is the enzyme activity determination figure as a result of cellulase system alkalescence inactivation;
Fig. 2 is influence the as a result figure of treatment temp to cellulase system alkalescence inactivation;
Fig. 3 is influence the as a result figure of treatment time to cellulase system alkalescence inactivation;
Fig. 4 is the dynamic (dynamical) comparative result figure of cellulase hydrolysis before and after handling under the alkaline condition;
Fig. 5 is the sugared composition measuring figure as a result of alkaline purification cellulase directionally hydrolyzing paper pulp.
Embodiment
Below in conjunction with specific embodiment the present invention is done further explanation.
The cellulase system that following examples are to use adopts fermentative prepn; Employed bacterial classification is Trichodermareesei (Trichoderma reesei Rut C-30); Method is: employing Mandels nutritive salt adds 12 g/L paper pulp and steam explosion corn straw mixing substrate is a carbon source; Shake the Trichodermareesei mycelium after adorning liquid 45 mL in the bottle and inserting 5 mL activation at 250 mL glass; Produce enzyme 96h in 30 ℃ and 170 r/min oscillation and fermentation and get fermented liquid, behind centrifugal 10 min of 4000 r/min, get supernatant and promptly obtain cellulase system stoste, freezing is subsequent use after the packing.
The enzyme activity determination method is: adopt two parallel laboratory tests, control parallel error≤5%.Filter paper enzyme activity, NCE5 vigor (CMCase) and exoglucanase vigor (CBH) all adopt the DNS method to measure, and beta-glucosidase (BG) adopts p-NP glucoside method to measure.Above-mentioned various enzyme activity determination all adopts 0.05 mol/L Hydrocerol A-sodium hydrate buffer solution system (pH4.80) and 50 ℃ ± 1.Above-mentioned various enzyme activity values are followed successively by 5.81 FPIU/mL, 38.01 IU/mL, 3.60 IU/mL and 1.13 IU/mL in the cellulase stoste.
Embodiment 1
Under the condition of water bath with thermostatic control and mild stirring; In the Mierocrystalline cellulose original enzyme liquid, slowly drip little amount of N aOH solution and regulate the pH value to stationary value; Enzyme liquid after must handling after sealing and standing is handled adopts the enzyme activity determination method to redeterminate the enzyme activity of three kinds of enzyme components of processing back enzyme liquid again.Adopting two parallel laboratory tests to carry out enzyme is the selectivity inactivation, controls parallel error≤5%.The method of calculation of residual enzyme activity: the various enzyme activity determination value relative percentages with enzyme liquid before and after handling are represented.Cellulase solution spinning condition after the basic treatment: inclining fully behind the cellulase solution 10min after the centrifugal basic treatment of 8000 r/min centrifuged supernatant, cleans centrifugal tube wall with the citrate buffer solution (pH4.80) of 10% (v/v) volume and gets centrifugal sediment.
In the pH value of directly adding 4.0% NaOH or KOH reagent or corresponding buffered liquid (w/w) adjusting cellulase system under 25 ℃ the constant temperature and after leaving standstill processing 60min, measure the enzyme activity of three big enzyme components.The result is as shown in Figure 1, and the enzyme activity of three big enzymes demonstrates two kinds of dissimilar variation characteristics under the basic treatment condition of different pH values.With dividing point, CMCase is two sections stepped with pH8.00, and CBH and BG then are linear attenuation.In the scope of pH5.00 ~ 7.00, the enzyme activity of CMCase and CBH is comparatively stable, can keep more than 90%, and the enzyme activity stability of BG enzyme is lower slightly, continues to improve the pH value and causes the enzyme activity fast linear of CBH and BG to descend.The enzyme activity of CMCase is comparatively stable in being higher than the environment of pH8.00.When the pH value increased to 9.00, the residual enzyme activity of BG was merely 5.6%, and CMCase and CBH still can residual 57.0% and 52.1%, and the relative ratios of the residual enzyme vigor of CMCase, CBH and β-G is respectively 10.3 and 9.4; During to pH10.00, can eliminate the CBH enzyme activity more than BG and 90% basically, but still can keep 48.4% CMCase enzyme activity, promptly can be contained the cellulase preparation of BG enzyme activity hardly.
Embodiment 2
Under the condition of pH9.00, cellulase stoste placed respectively leave standstill synchronously under 5 ~ 40 ℃ ± 1 the water bath with thermostatic control condition and handle 60min, measure the enzyme activity of three big enzyme components.The result is as shown in Figure 2; Under the alkali condition of pH9.00; There is the certain protection effect in the treatment temp that is lower than 10 ℃ to BG enzyme component; Alkaline pH all plays selectivity deactivation preferably to cellulase solution in 15 ~ 30 ℃ TR, but the temperature above 30 ℃ also can cause the remnant enzyme activity of CMCase and CBH to demonstrate downward trend.This helps cellulase and carries out the alkaline purification operation at normal temperatures.Fig. 3 shows different treatment time (10 ~ 80min) result; After handling 30min under the condition of pH9.00 and 25 ℃ ± 1; The residual enzyme activity of three kinds of enzymes just can reach steady state; Wherein the residual enzyme activity of CMCase, CBH and BG is respectively 58.8%, 56.6% and 5.7%, and relative proportion reaches 10.3 and 9.9, can realize the effect of selectivity inactivation.The alkaline purification time, the enzyme activity of CMCase and CBH can show downward trend above behind the 70min, obviously, preferably used 20 ~ 60min, more preferably used 30 ~ 60min.
Embodiment 3
Adopt the enzyme liquid (method with embodiment 1) of two kinds of alkaline conditions after obtaining handling behind 25 ℃ ± 1 processing cellulase solution 30min of pH9.00 ± 0.20 and pH10.0 ± 0.20 respectively; With cellulase stoste is contrast; In the enzymatic hydrolysis system of 10 g/L paper pulp, add three kinds of enzyme liquid of 0.1% (v/v) consumption respectively, carry out enzymatic hydrolysis reaction
Enzymic hydrolysis performance detection: adopt two parallel laboratory tests, control parallel error≤5%.In 100 mL screw socket glass triangle bottles, carry out; Add formulated 10 g/L Whatman filter paper pulp powder (20 order) solution, 25 mL of 0.05mol/L citrate buffer solution (pH 4.80) as the enzymolysis substrate; Add an amount of enzyme liquid and sealing again, in 50 ℃ ± 1 and 100 r/mim oscillatory reactions.1% (v/v) H2SO4 termination enzyme reaction that timing sampling 0.5mL adds 10 μ L gets enzymic hydrolysate, through suitably diluting laggard circumstances in which people get things ready for a trip spectrum analysis.The enzymolysis yield is calculated: multiply by gain factor 0.9 with the material concentration of the product sugar measured in the hydrolysis reaction system again divided by the parent material concentration of paper pulp.
The enzymatic hydrolysis reaction course that compares 3 kinds of enzyme liquid; The result is as shown in Figure 4; Under the reaction conditions of low substrate and less enzyme dosage; Compare with original enzyme liquid, still can preserve CMCase and CBH enzyme activity more than 50% through the zymin after the pH9.00 alkaline purification, the two is close basically to the kinetics and the total reducing sugar yield of paper pulp enzymatic hydrolysis reaction.Because alkaline purification is BG enzyme activity (about residual enzyme activity 5%) in the inhibitory enzyme system optionally, thereby can protect the cellooligosaccharide component that generates in the paper pulp enzymic hydrolysis process effectively.Behind the alkaline purification enzymatic hydrolysis 10g/L paper pulp reaction 24h of 0.1% (v/v) consumption; Various enzymolysis products compositions tend towards stability basically in the reaction system; Corresponding total reducing sugar enzymolysis yield is 8.60%; Wherein the enzymolysis yield of cellooligosaccharide is 6.73%, and the ratio that oligose accounts for total reducing sugar rises to 78.2% by 50.9% of original enzyme liquid, and increase rate reaches 53.0%.This explanation, the selectivity deactivation that the alkalescence reason to the natural cellulose enzyme is can significantly improve the production performance of directionally hydrolyzing preparation of cellulose cellooligosaccharide, can be used for the Production by Enzymes of cellooligosaccharide.Improve enzyme liquid caustic soda degree and can cause the remarkable inactivation of three big enzyme further inactivation, especially CBH to pH10.0, cause the yield of enzymic hydrolysis performance and oligose to descend significantly, but be that the contribution of oligose ratio is less the enzymolysis product quality.
The stratographic analysis of enzymic hydrolysate: determination of glucose adopts HPLC (chromatographic instrument Agilent 1200) according to the NREL method, with Bio-Rad HPX-87H chromatographic column (7.8mm * 300mm) measure.Performance liquid ion-exchange chromatography-pulse ampere method (chromatographic instrument Dionex ICS-3000) is adopted in the assay determination of cellooligosaccharide, with CarboPacTM PA200 anion-exchange column (3mm * 250mm) measure.The standard substance of glucose and cellooligosaccharide are from Magazyme company.
Adopting the high-effect ionic liquid chromatography that enzymolysis product sugar is formed analyzes; The result is as shown in Figure 5; Adopt the plain enzymic hydrolysis paper pulp of alkali devitalizing fibers only to generate cellobiose and a spot of procellose; Can not generate the cellooligosaccharide of other polymerization degree, two kinds of sugars do not account for 90% and 10% of total oligosaccharides.
Claims (6)
1. the selectivity method for deactivating of a cellulase system is characterized in that: 5 ~ 40 ℃ of temperature controls, and the pH that regulates cellulase liquid is not less than 8, leaves standstill to handle 10 ~ 80min the plain enzyme of promptly alternative devitalizing fibers system; Wherein, CMCase and CBH selective retention, BG selectivity inactivation.
2. the selectivity method for deactivating of cellulase system according to claim 1 is characterized in that: described cellulase system is the natural cellulose enzyme that Trichodermareesei produces.
3. the selectivity method for deactivating of cellulase system according to claim 1 is characterized in that: with containing Na
+Alkali lye or damping fluid, or with containing K
+Alkali lye or damping fluid, regulate cellulase liquid pH.
4. the selectivity method for deactivating of cellulase system according to claim 1 is characterized in that: pH=8.8 ~ 9.2.
5. the selectivity method for deactivating of cellulase system according to claim 1, it is characterized in that: described temperature is 15 ~ 30 ℃.
6. the selectivity method for deactivating of cellulase system according to claim 1, it is characterized in that: the described treatment time is 30 ~ 60min.
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Non-Patent Citations (3)
| Title |
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| 于兆海: "里氏木霉纤维素酶的分离纯化与酶学性质研究", 《中国优秀博硕士学位论文全文数据库（硕士） 工程科技I辑》 * |
| 朱年青等: "里氏木霉纤维素酶的分离纯化及酶学性质", 《生物加工过程》 * |
| 江华等: "里氏木霉纤维素酶系的分离及其酶学性质", 《南京林业大学学报（自然科学版）》 * |
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Application publication date: 20120905 |