CN114058605A

CN114058605A - Method for improving specific activity of cellulase and application thereof

Info

Publication number: CN114058605A
Application number: CN202111327122.8A
Authority: CN
Inventors: 江正兵; 冉秋萍; 宋慧婷; 李华南; 刘家书; 周樱
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2022-02-18
Anticipated expiration: 2041-11-10
Also published as: CN114058605B

Abstract

The invention relates to a method for improving specific activity of cellulase and application thereof, in particular to cellulase with improved specific activity obtained after the cellulase is incubated by phenolic acid, and the cellulase with improved specific activity can be used for improving the enzymatic hydrolysis saccharification efficiency of biomass. Efficient biorefineries of lignocellulosic biomass are limited due to the negative effects of expensive cellulases and lignin degradation products on cellulase hydrolysis. The lignin derived phenolic acid compounds, such as vanillic acid, syringic acid, ferulic acid, isovanillin and the like can effectively improve the specific activity of the cellulase, effectively improve the saccharification efficiency of different biomasses under the condition of not increasing the dosage of the enzyme, and effectively reduce the use cost of the enzyme for enzymolysis and saccharification of the biomasses.

Description

Method for improving specific activity of cellulase and application thereof

Technical Field

The invention relates to the field of cellulase activity modification and biomass saccharification and fermentation, in particular to a method for improving specific activity of cellulase and application thereof.

Background

Resource, energy and environmental issues are becoming more serious. Therefore, there is an urgent need to develop clean renewable energy sources to replace fossil resources. Lignocellulosic biomass is the most abundant renewable resource on earth and can be converted to high-value chemicals or biofuels by sugar platforms. Commodities produced by utilizing renewable resources can replace products based on petroleum supply, thereby being beneficial to improving energy safety and reducing the influence on the environment. Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. The production of organisms from lignocellulosic biomass involves three steps, namely pretreatment, enzymatic hydrolysis and subsequent fermentation. Enzymatic hydrolysis is considered to be the most economical and environmentally friendly way to degrade lignocellulose to produce fermentable sugars. The structural complexity of lignocellulose and the presence of lignin polymers are obstacles to the enzymatic digestion of biomass. The physical and chemical barriers of lignocellulose need to be removed by pretreatment to make the cellulose susceptible to enzymatic hydrolysis.

Pretreatment changes the chemical or physical properties of the biomass, improves the accessibility of cellulase to cellulose, and breaks biomass resistance. Pretreatment typically degrades hemicellulose to produce pentose, hexose, sugar acids, fatty acids, furan aldehydes, and the like, while lignin is typically degraded into a variety of soluble phenolic compounds (syringaldehyde, vanillin, ferulic acid, vanillic acid, gallic acid, and the like) and oligomeric lignins (ellagic acid, tannic acid, and the like). Lignocellulose-derived by-products produced by the pretreatment inhibit downstream enzymatic saccharification and bioconversion. Phenolic substances have a stronger inhibitory effect than soluble sugars, furan derivatives and organic acids because phenolic substances cause precipitation and irreversible inhibition of enzymes and exist as a strong inhibitor in the hydrolysis process, which can hinder the hydrolysis of enzymes and reduce the yield of sugars, and small phenolic compounds in hot water pretreated lignocellulose hinder enzymatic hydrolysis. At a proper concentration, vanillin has a reversible inhibition effect on the activity of cellulase. Cinnamic acid was found to adhere to the substrate and cause inhibition, as cellulase was unable to bind cellulose by this attachment. In contrast to these negative effects, some studies report that lignin-degrading phenolic compounds may also have a promoting effect on biological enzymes. Syringic acid can coat cellulose and change its structure, making it easier to hydrolyze. The low concentration of small phenolic compounds increases the hydrophobicity of the cellulase surface, facilitating the binding of the cellulase to the substrate. The carboxyl group and the methoxy group of the phenolic acid are probably related to the promotion effect in the enzymolysis process, but at present, no clear theory exists for the promotion or inhibition mechanism of the lignin-derived phenolic compounds on the enzymolysis process. The influence of phenolic acid on cellulase is mainly researched by adding phenolic acid in the enzymolysis process, and the antibacterial property of phenolic compounds can inhibit the growth of microorganisms in the subsequent fermentation process, so that the influence on the subsequent fermentation process is in many aspects.

According to the invention, the cellulase is incubated by using the phenolic compound (phenolic acid), so that the specific activity of the cellulase is effectively improved, and the enzymolysis efficiency of different biomasses can be effectively improved by using the cellulase after incubation by using the phenolic acid. The use of phenolic compounds in the enzymolysis process is avoided, so that the inhibition effect of phenolic acid on microorganisms in the subsequent fermentation process is avoided, and the method has important significance for reducing the use cost of cellulase and improving the biomass enzymolysis and biorefinery efficiency.

Disclosure of Invention

Aiming at the defects, the invention provides a method for improving the specific activity of cellulase and application thereof in biomass enzymolysis and saccharification.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for improving specific activity of cellulase specifically comprises the step of incubating cellulase by using phenolic acid to obtain the cellulase with improved specific activity.

Further, the phenolic acid is any one or more of syringic acid, ferulic acid, vanillic acid and isovanillin.

Furthermore, the content of phenolic acid in the incubation is 1.25-40mmol phenolic acid/g enzyme.

Further, the content of phenolic acid during incubation was 5mmol phenolic acid/g enzyme.

The method is applied to improving the enzymolysis saccharification efficiency of the biomass.

Has the advantages that: according to the invention, phenolic acid is adopted to incubate the cellulase, the specific activity of the enzyme after phenolic acid incubation is effectively improved, the incubated cellulase is used for biomass hydrolysis saccharification, the enzymatic hydrolysis saccharification efficiency of different types of biomass is obviously improved under the condition of not increasing the enzyme dosage, and the use cost of the cellulase can be reduced. The phenolic acid and the cellulase are pre-incubated, so that the specific activity of the cellulase can be greatly improved, and the saccharification efficiency of the cellulase on the lignocellulose biomass is improved.

The present invention will be described in detail below with reference to the accompanying drawings and examples.

Drawings

FIG. 1 is a graph showing the effect of phenolic acid on hydrolytic phosphoric acid swelling cellulose (a) and microcrystalline cellulose (b);

FIG. 2 is a graph of the effect of different concentrations of syringic acid and hydrolysis time on cellulase hydrolysis;

FIG. 3 shows the stability of cellulase at 50 ℃;

FIG. 4 shows the improvement of specific activity of cellulase after pre-incubation of four phenolic acids (vanillic acid, syringic acid, ferulic acid, isovanillin) with cellulase at 50 ℃;

FIG. 5 shows specific activities of FP enzyme (a) and CMC enzyme (b) with filter paper as substrate, and pNPG enzyme (c) with pNPG as substrate, respectively, after the syringic acid and isovanillin are respectively incubated with cellulase at the ratio of 1.25, 2.5, 5, 10, 20, 40mmol phenolic acid/g cellulase. Obtaining corresponding specific activity improvement rate by taking cellulase without phenolic acid as a reference;

FIG. 6 is the sugar yield (a) of cellulase after syringic acid incubation for hydrolysis of microcrystalline cellulose at different enzyme loadings (20mg enzyme/g substrate and 10mg enzyme/g substrate). The hydrolysis rate (b) of different cellulases;

FIG. 7 shows the promotion rate of cellulase on the saccharification efficiency of different substrates after phenolic acid incubation.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1.1 Effect of phenolic acid on enzymatic efficiency of cellulase

1.1.1 Effect of phenolic acids on cellulase hydrolysis

The influence of phenolic acid on the enzymolysis efficiency of the cellulase is analyzed by respectively taking phosphoric acid swelling cellulose and microcrystalline cellulose as substrates. Adding 25mM phenolic acid into an enzymolysis reaction system, and judging the enzymolysis efficiency of the cellulase by detecting the release amount of reducing sugar after 24 hours of enzymolysis. The enzyme digestion efficiency of the control group without added phenolic acid was defined as 100%. In an enzymolysis system using phosphoric acid swelling cellulose as a substrate, the enzymolysis efficiency in a reaction system added with vanillic acid, syringic acid, ferulic acid and isovanillin is respectively improved by 44.27%, 50.05%, 42.67% and 39.53% (fig. 1 a). In the enzymolysis system using microcrystalline cellulose as substrate, the enzymolysis efficiency in the reaction system added with vanillic acid, syringic acid, ferulic acid and isovanillin is respectively improved by 42.33%, 40.25%, 51.44% and 44.69% (fig. 1 b). Different types of phenolic acid added into an enzymolysis reaction system have similar promotion effects on enzymolysis efficiency, but the promotion effects of different phenolic acids have no significant difference.

1.1.2 Effect of different concentrations of phenolic acid on cellulase hydrolysis

Considering that different phenolic acids have no significant difference in the promotion effect on the enzymolysis, syringic acid is selected to analyze the influence of phenolic acids with different concentrations and different hydrolysis time on the enzymolysis efficiency. Phosphoric acid swelling cellulose and microcrystalline cellulose are respectively used as substrates, and 0mM, 5mM, 10mM, 15mM and 25mM syringic acid are respectively added into a hydrolysis reaction system. In an enzymolysis system using phosphoric acid swelling cellulose as a substrate, the reaction is carried out for 72 hours, the reaction system without adding syringic acid is used as a control group, the yield of reducing sugar is 5.52mg/ml, the yields of reducing sugar in the reaction systems with adding 5mM, 10mM, 15mM and 25mM syringic acid are respectively 6.72mg/ml, 8.06mg/ml, 8.84mg/ml and 9.14mg/ml, and the enzymolysis efficiency is respectively improved by 32.53%, 45.92%, 60.01% and 65.52%. (FIG. 2a/b), the reducing sugar yield shows an increasing trend along with the increase of the concentration of the syringic acid, but the effect of the high concentration of the syringic acid on the reducing sugar yield is not obvious. In an enzymolysis system using a microcrystalline cellulose substrate, the yield of reducing sugar in the reaction system without adding syringic acid is 3.41mg/ml after reaction for 72 hours, the yields of reducing sugar in the reaction systems with adding 5mM, 10mM, 15mM and 25mM syringic acid are 4.40mg/ml, 5.07mg/ml, 5.67mg/ml and 5.90mg/ml respectively, and the enzymolysis efficiency is improved by 28.90%, 48.59%, 66.26% and 73.03% respectively. (FIG. 2 c/d). The influence of the syringic acid on the enzymolysis efficiency shows the same trend in hydrolysis systems of different substrates, the promotion effect is not obvious within 24 hours in a reaction system of 5mM of the syringic acid, and the promotion effect is only improved by 19.35 percent and 17.01 percent respectively in a system taking phosphoric acid swelling cellulose and microcrystalline cellulose as substrates. In the biomass enzymolysis process, when the concentration of phenolic acid is lower than 5mM, the enzymolysis efficiency is hardly promoted.

1.2 activating Effect of phenolic acid on cellulase specific Activity

1.2.1 Effect of phenolic acids on cellulase enzyme Activity

And the enzyme activity is detected by taking the filter paper as a substrate to represent the total enzyme activity of the cellulase, and the enzyme activity is detected by taking the CMC as a substrate to represent the enzyme activities of the endoglucanase and the exoglucanase. The cellulase used in the present invention has good thermal stability at 50 ℃. The filter paper enzyme activity and the CMC enzyme activity remained stable after 24 hours, 48 hours and 72 hours incubation at 50 ℃ respectively (FIG. 3). Cellulase is not affected by high temperature, which helps to better illustrate the effect of phenolic acid on cellulase activity during incubation. The specific activity of the filter paper enzyme after phenolic acid incubation is improved by 9.26-15.54% (figure 4a), and the specific activity of CMC enzyme is improved by 10.40-24.69% (figure 4 b). The activation effects of the four phenolic acids do not show significant difference, and the promotion effect of the phenolic acids on the specific activity of the CMC enzyme is stronger than the promotion effect on the specific activity of the filter paper enzyme. There was also no significant difference in phenolic acid activation of enzyme activity at different incubation times (12 hours, 24 hours, 48 hours and 72 hours). That is, in the present invention, it can be considered that the enzyme activity of cellulase is stabilized at 12 hours of incubation, so that the incubation time is set to 12 hours in the subsequent experiment.

1.2.2 Effect of the incubation ratio of phenolic acid to enzyme on cellulase specific Activity

The syringic acid and isovanillic acid are used as raw materials, the influence of the phenolic acid and cellulase incubation ratio on the activity is researched, the determination of the pNPG enzyme activity is supplemented, and the pNPG enzyme activity is used for representing the enzyme activity of the beta-D glucosidase after incubation. The incubation ratios of different phenolic acids and enzymes were obtained by setting cellulase concentrations at different concentrations and phenolic acids at fixed concentrations for incubation (table 1). After the incubation, the specific activities of the filter paper enzyme, the CMC enzyme and the pNPG enzyme are detected, and the enzyme which does not contain phenolic acid and is incubated at the same time is used as a control. Different ratios of phenolic acid had different effects on the specific activity of cellulase after incubation (figure 5). At low incubation ratios of phenolic acid to cellulase (1.25mmol phenolic acid/g enzyme), the specific activities of all three substrates were not significantly promoted. The filter paper enzyme specific activities of the enzymes after syringic acid and isovanillin incubation are respectively improved by 14.75% and 13.91% (fig. 5a), the CMC enzyme specific activity is respectively improved by 31.49% and 28.81% (fig. 5b), and the filter paper enzyme specific activity does not influence the pNPG enzyme specific activity within an error range (fig. 5 c). As the ratio of phenolic acid to enzyme increases, the corresponding activation rate also changes. There is an optimum activation incubation ratio for filter paper enzyme specific activity and CMC enzyme specific activity. As shown in fig. 5, syringic acid and isovanillin increased the filter paper enzyme specific activity by 57.06% and 50.08%, respectively, and the CMC enzyme specific activity by 125.82% and 136.79%, respectively, when incubated with 5mmol phenolic acid/g enzyme. When the ratio of phenolic acid to enzyme is increased again, the activation rates for filter paper enzyme specific activity and CMC enzyme specific activity are not increased, but the activation effect of phenolic acid on cellulase still exists. For the specific activity of the pNPG substrate detection, the activation rate of the pNPG specific activity is gradually increased along with the increase of the phenolic acid ratio. Under the proportion of 40mmol phenolic acid/g enzyme, the specific activity of the syringic acid to the pNPG enzyme is improved by 482.48%, and the specific activity of the isovanillin to the pNPG enzyme is improved by 406.02%. The total enzyme activity of the cellulase is reacted by the enzyme activity of the filter paper enzyme, the CMC enzyme reflects the activity of the endoglucanase and the exoglucanase, and the influence on the whole mixed enzyme system is comprehensively considered, so that the optimal use amount of phenolic acid is obtained when the incubation is carried out at the ratio of 5mmol of phenolic acid/g of enzyme in the incubation process, and the specific enzyme activity of the pNPG incubated by the syringic acid and the isovanillin is respectively improved by 103.88 percent and 110.61 percent under the incubation ratio, and the effect of improving the specific activity of the whole cellulase is very obvious. The gradual slowing of cellulase hydrolysis during biorefinery is a fundamental problem in cellulase production. One major cause of deceleration is product inhibition. Under high substrate loading conditions, product inhibition during cellulose hydrolysis is significant. High substrate loading results in the production of high concentrations of products such as glucose, cellobiose, and other cellooligosaccharides. Non-productive adsorption and inactivation of lignin by enzymes during hydrolysis can also adversely affect saccharification efficiency. The phenolic acid has obvious promotion effect on the activity of the cellulase. The phenolic acid is added in the hydrolysis process, which is helpful for improving the activity of enzymes such as beta-D-glucosidase and the like, so that the oligosaccharide inhibitor can be rapidly hydrolyzed in the later hydrolysis stage.

TABLE 1 ratio of phenolic acids used in preincubation with cellulase

1.3 hydrolysis of cellulase after preincubation with phenolic acid

1.3.1 hydrolysis of cellulase after Pre-incubation with phenolic acid at different times

After incubation with phenolic acid, the specific activity of the cellulase is obviously improved, and the activation effects of the four phenolic acids on the enzymatic activity are similar. In order to examine the hydrolysis performance of the cellulase after phenolic acid incubation, syringic acid is used for incubating the cellulase, and a saccharification experiment is carried out at different times by using microcrystalline cellulose as a substrate. Enzyme after syringic acid incubation with Cellulase_SAShowing that the enzyme incubated in buffer was the control and Celluase was used₀And (4) showing. Hydrolysis was carried out for 72 hours and the reducing sugar yield was determined at different reaction times (FIG. 6 a). To determine the effect of a single reaction time on the saccharification yield of cellulose, the rate of enzymatic hydrolysis, i.e., the amount of reducing sugars released by cellulase from microcrystalline cellulose at a particular time point, was calculated (fig. 6 b). High enzyme load (20mg of enzyme/g substrate) and low enzyme load (10mg of enzyme/g substrate) were included in this experiment. The reaction time was 72 hours, the saccharification yield of the enzyme after the syringic acid incubation under high enzyme load (20mg enzyme/g substrate) was 4.54mg/ml, and the control groupThe saccharification yield of (2) was 3.77mg/ml, the saccharification yield of the enzyme after incubation with syringic acid at low enzyme load (10mg enzyme/g subatrate) was 3.30mg/ml, and the saccharification yield of the control group was 2.71mg/ml (FIG. 6 a). The result shows that the saccharification efficiency of the enzyme after the syringic acid incubation is obviously improved compared with that of a control enzyme. Under high enzyme load, the saccharification efficiency of the enzyme incubated with syringic acid in different time is improved by 13.84-20.49%, and the improvement rate is increased along with the increase of time. Under low enzyme load, the saccharification efficiency of the enzyme after syringic acid incubation in different time is improved by 19.42-25.14%, and the enzyme reaches the maximum in 24 hours (figure 6 b). In order to maximize hydrolysis during depolymerization of lignocellulosic biomass, a synergistic effect of at least three cellulases is generally required. Exoglucanases release cellobiose from the reducing and non-reducing ends of the cellulose chain, while endoglucanases randomly hydrolyze glycosidic bonds from within the cellulose chain, reducing cellulose length, and beta-D-glucosidase hydrolyzes cellobiose and cellooligomers to glucose. The specific enzyme activity of the enzyme after phenolic acid incubation is higher than that of a control enzyme, so that the enzyme after phenolic acid incubation has higher saccharification yield under the condition of using the same enzyme amount, the difference caused by insufficient enzyme load is reduced to a great extent, and the hydrolysis cost can be effectively reduced. The concentration of the phenolic acid remaining in the cellulase after phenolic acid incubation in an enzymolysis system is 0.15-0.3 mM, the influence of the phenolic acid concentration on enzymolysis can be ignored, and the concentration is far lower than the minimum bacteriostatic concentration and has no influence on subsequent biological fermentation.

1.3.2 hydrolysis of different substrates by cellulase after incubation with phenolic acid

To investigate the saccharification effect of phenolic acid incubated enzymes on different biomasses, the enzymes were incubated using the optimal ratio of activated phenolic acid to cellulase (5mmol phenolic acid/g enzyme). And (3) carrying out hydrolysis reaction on the incubated enzyme with phosphoric acid swelling cellulose, microcrystalline cellulose and pretreated corn straws, carrying out hydrolysis for 24 hours at 50 ℃ by taking the enzyme hydrolysis yield after the incubation of the buffer solution as a reference, and detecting the yield of reducing sugar in the supernatant after the reaction is finished. The saccharification efficiency of the cellulase after phenolic acid pre-incubation on three different substrates is obviously improved. Wherein, the enzymes after phenolic acid incubation showed the greatest saccharification efficiency promoting effect when hydrolyzing phosphoric acid swollen cellulose, and the saccharification efficiencies of the enzymes after vanillic acid, syringic acid, ferulic acid and isovanillin incubation were respectively improved by 44.97%, 36.07%, 28.96% and 45.13% (fig. 7). This is similar to the boosting effect of adding 25mM phenolic acid during hydrolysis (FIG. 1). This means that the enzymes incubated with phenolic acids achieve the same accelerating effect as high concentrations of phenolic acids. The use of high concentrations of phenolic acids in enzymatic processes is impractical and detrimental to downstream biological fermentation processes. The saccharification efficiency of the incubated enzyme is obviously improved by taking microcrystalline cellulose as a substrate, and the saccharification efficiency of the enzyme incubated by vanillic acid, syringic acid, ferulic acid and isovanillin is respectively improved by 22.33%, 23.00%, 25.46% and 33.92%. When the pretreated corn straws are used as a substrate, the saccharification efficiency of the enzyme incubated by vanillic acid, syringic acid, ferulic acid and isovanillin is respectively improved by 10.81%, 8.22%, 6.94% and 11.53% (fig. 7). The specific activity of the enzyme after phenolic acid incubation is effectively improved, and the saccharification efficiency is effectively improved under the condition of using the same enzyme amount for different types of biomass materials.

The specific activity of the cellulase can be greatly improved by incubating the phenolic acid and the enzyme together, and the saccharification efficiency of the cellulase on the lignocellulose biomass is improved. Previous studies on lignin-derived phenolic compounds have shown that the inhibitory or accelerating effect is generally exerted on a co-system of phenolic acid, enzyme and substrate. The low concentration of phenolic acid contained in the pretreated straws can form a hydrophobic layer on the surface of protein, so that the hydrophobicity of the surface of the cellulase is increased, and more interaction sites are formed with crystalline cellulose through van der Waals force and aromatic ring polarization, thereby promoting the hydrolysis of the cellulase. Cinnamic acid was found to attach to the substrate and cause inhibition as by this attachment cellulose became inaccessible to cellulase whereas syringic acid, an activator, almost completely covered the cellulose and changed its structure, allowing the substrate to be better hydrolysed. Phenolic compounds are in the same system as the enzyme and substrate, whether the phenolic acid alters the hydrolysis conditions during hydrolysis to facilitate enzymatic hydrolysis or the phenolic compound destroys and covers the substrate to allow more complete hydrolysis. The presence of phenolic compounds in the system is an obstacle to subsequent biological fermentation and product separation. According to the invention, the specific activity of the cellulase is obviously improved by using the vanillic acid, the syringic acid, the ferulic acid, the isovanillin and other phenolic acids for incubation, and the specific activity of the enzyme can be improved by improving the specific activity of the enzyme even if the phenolic acid or phenolic acid with extremely low concentration does not exist in a hydrolysis system through a pre-incubation strategy of the cellulase and the phenolic acid. The discovery is favorable for reducing the use cost of the cellulase in biological enzymolysis and realizing the high-efficiency utilization of lignocellulose and derivative compounds thereof. It is also of great significance to the production and preparation of cellulase.

Through the experiment, the following results are found:

1. the four phenolic acids with the same concentration are added into the hydrolysis system, the same promotion effect is generated on the hydrolysis of microcrystalline cellulose and phosphoric acid swelling cellulose substrate, and no significant difference is shown between the promotion effects of the phenolic acids. In an enzymolysis system taking phosphoric acid swelling cellulose as a substrate, vanillic acid, syringic acid, ferulic acid and isovanillin are added, so that the enzymolysis efficiency is respectively improved by 44.27%, 50.05%, 42.67% and 39.53%. In an enzymolysis system taking microcrystalline cellulose as a substrate, vanillic acid, syringic acid, ferulic acid and isovanillin are added, so that the enzymolysis efficiency is improved by 42.33%, 40.25%, 51.44% and 44.69%.

2. The phenolic acid with different concentrations is added into the hydrolysis system, and the promotion rate of the phenolic acid in the buffer solution is gradually increased along with the increase of the concentration of the phenolic acid, so that the phenolic acid with low concentration in the hydrolysis system has no influence on the hydrolysis.

3. The specific activity of the cellulase after phenolic acid incubation at 50 ℃ is obviously improved. And incubating for 12-72 h. The specific activity of the filter paper enzyme of the cellulase after the vanillic acid incubation is improved by 9.26-15.03 percent, and the specific activity of the CMC enzyme is improved by 15.26-24.69 percent; the specific activity of the filter paper enzyme of the enzyme incubated by the syringic acid is improved by 9.78-15.54 percent, and the specific activity of the CMC enzyme is improved by 10.92-18.13 percent; the specific activity of the filter paper enzyme of the enzyme incubated by the ferulic acid is improved by 10.07-14.92%, and the specific activity of the CMC enzyme is improved by 11.77-15.80%; the specific activity of the filter paper enzyme of the enzyme incubated with isovanillin is improved by 9.69-15.26%, and the specific activity of the CMC enzyme is improved by 10.40-16.29%. There was no significant difference in specific activity after incubation with the four phenolic acids. The specific activities of the cells were not significantly different in 12h, 24h, 48h and 72 h.

4. And incubating the cellulase by using phenolic acid with different concentrations to obtain the optimal usage amount of the phenolic acid, wherein the optimal usage amount of the phenolic acid is 5mmol of phenolic acid/g of the cellulase during incubation, and the specific activity of the cellulase can be effectively improved when the usage amount of the phenolic acid is within the range of 1.25-40mmol of phenolic acid/g of the cellulase.

5. The cellulase incubated with the phenolic acid is used for hydrolysis of biomass, and compared with the enzyme not incubated with the phenolic acid, the saccharification efficiency is obviously improved. The saccharification efficiency of the enzyme incubated by vanillic acid, syringic acid, ferulic acid and isovanillin is respectively improved by 44.97%, 36.07%, 28.96% and 45.13% by taking phosphoric acid swelling cellulose as a substrate. The saccharification efficiency of the enzyme incubated by vanillic acid, syringic acid, ferulic acid and isovanillin is respectively improved by 22.33%, 23.00%, 25.46% and 33.92% by taking microcrystalline cellulose as a substrate. When the pretreated corn straws are used as a substrate, the saccharification efficiency of the enzyme incubated by vanillic acid, syringic acid, ferulic acid and isovanillin is respectively improved by 10.81%, 8.22%, 6.94% and 11.53%.

And (4) conclusion:

the phenolic acid can effectively improve the hydrolysis capacity of the cellulase in the hydrolysis system of the cellulase, the promotion effect is better along with the increase of the concentration of the phenolic acid, and the promotion effect is not obvious at low concentration. The specific activity of the cellulase can be obviously improved by pre-incubating the cellulase with phenolic acid, and the optimal usage amount of the phenolic acid is 5mmol of phenolic acid/g of cellulase. The cellulase incubated with the phenolic acid is used for hydrolyzing different biomasses, the saccharification efficiency of the cellulase can be obviously improved, the concentration of the residual phenolic acid in an enzymolysis system is extremely low, and no negative influence is generated on subsequent biotransformation and fermentation. The method for pre-incubating phenolic acid and cellulase is an effective method for improving the specific activity of the cellulase and improving the enzymolysis saccharification efficiency of biomass.

The foregoing is illustrative of the best mode of the invention and details not described herein are within the common general knowledge of a person of ordinary skill in the art. The scope of the present invention is defined by the appended claims, and any equivalent modifications based on the technical teaching of the present invention are also within the scope of the present invention.

Claims

1. The method for improving the specific activity of the cellulase is characterized in that the cellulase with improved specific activity is obtained by incubating the cellulase with phenolic acid.

2. The method for improving the specific activity of the cellulase according to claim 1, wherein the phenolic acid is any one or more of syringic acid, ferulic acid, vanillic acid and isovanillin.

3. The method for improving the specific activity of the cellulase according to claim 1, wherein the content of the phenolic acid during the incubation is 1.25-40mmol of phenolic acid per gram of the enzyme.

4. The method for improving the specific activity of cellulase according to claim 3, wherein the phenolic acid content during the incubation is 5mmol phenolic acid/g enzyme.

5. Use of the method of claim 1 to increase the efficiency of enzymatic saccharification of biomass.