Trichoderma reesei during Hydrolysis of Mi - American

Chapter 8

Absorption of Endoglucanase I and Cellobiohydrolase I of Trichoderma reesei during Hydrolysis of Microcrystalline Cellulose 1

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H. Ding , E . Vlasenko , C. Shoemaker , and S. Shoemaker Downloaded by NANYANG TECHNOLOGICAL UNIV on June 21, 2016 | http://pubs.acs.org Publication Date: October 17, 2000 | doi: 10.1021/bk-2001-0769.ch008

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Department of Food Science and Technology andCaliforniaInstitute of Food and Agricultural Research, University of California, Davis, CA 95616

Endoglucanase I and cellobiohydrolase I (EG I and CBH I) were purified from a commercial Trichoderma cellulase preparation. Adsorption of EG I and CBH I, alone and in combination, was investigated using microcrystalline cellulose. Capillary zone electrophoresis was developed as a sensitive (picomolar range) and reliable method for quantifying individual adsorbed enzymes. Adsorption kinetics as well as adsorption isotherms were evaluated for EG I and CBH I. The adsorption data followed the Langmuir adsorption equation. Adsorption parameters, A and K , were calculated for EG I and CBH I. C B H I showed higher binding affinity to Avicel, compared to E G I. The hydrolysis kinetic parameters, V and K , were determined for EG I and CBH I, and found to correlate with adsorption parameters. max

max

p

m

Cellulose is the most abundant natural polymer on earth. The disposal of cellulosecontaining waste materials, such as agricultural residues, waste wood, and municipal solid wastes, poses a serious problem. Considerable efforts have been directed toward developing bioprocesses to convert cellulosic wastes to ethanol and other chemicals (1-6). The key and rate-limiting step in cellulose-to-ethanol processes is enzymatic depolymerization of cellulose (2,7). Despite the tremendous efforts of researchers in this area, the mechanism of cellulose hydrolysis is not entirely understood. The adsorption of cellulase is very important because adsorption is the prerequisite step in the enzymatic hydrolysis of cellulose. A good understanding of the adsorption of cellulases onto cellulose will provide further insights into the overall reaction mechanism and the observed synergism of cellulase components (8-11). Also, adsorption of cellulases may be utilized as a method of enzyme recycling (12-14).

© 2001 American Chemical Society Himmel et al.; Glycosyl Hydrolases for Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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132 Previously reported studies examining adsorption of cellulase mostly have used the cellulase system of Trichoderma (8,9,12,14,15-21). Adsorption studies based on a mixture of cellulase components do not give information on the adsorption of any particular component. Some studies using purified cellulase components have been reported (10,11, 22,23,24). However, these studies relied on total protein (10,11) and cellulase activity measurements (10,24) and did not allow specific measurement of cellulase components in their reconstituted mixtures. Thus, it was not possible to compare the adsorption of cellulases as single components and in reconstituted mixtures. Recently, fast protein liquid chromatography (FPLC) was suggested for quantitative determination of cellulase components in mixtures (25,26). There are only a few reports in the literature using FPLC to quantify the adsorption of cellulases during hydrolysis of lignocellulose (16) and microcrystalline cellulose (22). More recently, capillary zone electrophoresis (CZE) was reported to be a powerful analytical method to fractionate and quantify biomolecules such as proteins, DNA and polysaccharides (27-31). CZE is electrophoresis in free homogeneous solution. It separates substances based on their physico-chemical properties. The sensitivity of CZE with fluorescent detection is greater than most HPLC methods (30,31). In this paper, purified E G I and CBH I from Trichoderma reesei cellulases were used for adsorption studies. The enzymes were studied alone and in reconstituted mixtures. Capillary zone electrophoresis was used to determine the amount of enzyme remaining in solution (free enzyme) so as to calculate the amount of enzyme adsorbed onto microcrystalline cellulose (bound enzyme). Adsorption parameters were obtained using the Langmuir adsorption equation. Hydrolysis kinetic parameters were also determined for EG I and CBH I based on the Michaelis-Menten equation, and the two sets of parameters were compared with each other.

Materials and Methods Substrate. Avicel® PH-101 was purchasedfromFMC Corporation. Enzyme Purification. EG I and CBH I were purified from Spezyme® CP cellulase (Genencor International, Inc.). The purification procedures are given in Figure 1. Isoelectric focusing (PhastGel IEF 4-6.5, Pharmacia) with Coomassie blue staining gave a single band for CBH I, and a predominant single band and a slight contaminating band for EG I. The isoelectric points of the purified EG I and CBH I, based on PI standards, were found to be 4.58 and 4.18, respectively. SDS gel electrophoresis (PhastGel Homogeneous 7.5, Pharmacia) with silver staining gave single bands for both E G I and CBH I. The molecular weight of the purified EG I and CBH I, based on molecular weight standards, were found to be 56,000 and 61,000 Da, respectively. These values were close to what has been previously reported (32). The purified cellulase components showed single peaks on FPLC Mono Q column (Pharmacia) at pH 6.5, as well as using capillary zone electrophoresis at pH 3.0 on a μ 8 Π . ^ capillary (J & W Scientific). Purified EG I and CBH I were concentrated by ultrafiltration, and the buffer was changed to 50 mM sodium acetate buffer, pH 5.0.

Himmel et al.; Glycosyl Hydrolases for Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

133 Protein contents were measured by the Lowry method (33). Enzyme activities were measured using carboxymethyl cellulose, Avicel, and H P0 -swollen cellulose as substrates (Table 1). 3

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Table 1. Activities of E G I and CBH I purified from Trichoderma reesei cellulase Specific Activity (U/mg) towards Downloaded by NANYANG TECHNOLOGICAL UNIV on June 21, 2016 | http://pubs.acs.org Publication Date: October 17, 2000 | doi: 10.1021/bk-2001-0769.ch008

Enzyme

EG I CBH I

Avicel

CM-cellulose 74.6 0.07

0.12 0.11

H P0 -swollen cellulose 24.3 0.21 3

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Specific activities were measured at 50 °C, pH 5.0 using the substrates, CM-cellulose, Avicel and H P0 -swollen cellulose. 3

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Spezyme® CP

DEAE Bio-Gel A, pH5.0 (NaCl Gradient)

DEAE Bio-Gel A, pH6.0 (NaCl Giadient) FPLC Mono Q column, pH 6.5 (NaCl Gradient) FPLC Mono Q column, pH 6.5 (NaCl Gradient)

Figure 1. Flow chart of the purification of EG I and CBH I from a commercial cellulase preparation.

Quantitative Determination of E G I and C B H I. EG I and CBH I in solution were quantitatively determined by capillary zone electrophoresis using a 50 μπι* 25 cm (iSIL-FC capillary in a BioFocus®-3000 system (BioRad). Cartridge temperature was set at 20°C. Sodium acetate buffer (50 mM, pH 4.0) was used as the running buffer. Running voltage was set at 25 kV. Sample injection was performed at 15 psi*sec for


134 all samples. Ultraviolet (UV) absorbance of the eluent was followed at 200 nm. The separation of EG I from CBH I is shown in the electropherogram (Figure 2). Calibration curves based on peak areas were used to determine enzyme concentration in the injected sample.


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I 6.00

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Figure 2. Electropherogram of sample containing EG I and CBH I. Sample was applied on a 50 μτη* 25 cm pSIL-FC capillary, using 50 mM, pH 4.0 sodium acetate buffer as the running buffer.

Adsorption Kinetics. Avicel PH-101 was used as the cellulose adsorbent. Avicel (25 mg) was suspended in 50 mM sodium acetate buffer, pH 5.0, and preincubated at 25 °C for 30 minutes. After preincubation, a specified amount of E G I and/ or CBH I was added to bring the total reaction volume to 5 mL. The enzyme-to-substrate ratio was set at 0.27 μπιοΐ/g for both E G I and CBH I (based on the calculated molecular weight for E G I and CBH I of 56,000 g/mol and 61,000 g/mol, respectively). Experiments were performed with E G I and CBH I, alone and in equimolar mixtures. The reaction mixture was contained in a 10 mL glass bottle with a screw cap, and stirred on a magnetic stirring plate. Samples were taken throughout a 60-minute incubation and centrifuged for 2 minutes at 10,000 rpm. The amount of free enzyme in the supernatant was determined by capillary zone electrophoresis. Adsorbed enzyme was calculated by subtracting the free enzyme remaining after time (t ) from the initial enzyme (to). x


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Adsorption Isotherms. Avicel PH-101 was used in the adsorption isotherm studies. Avicel concentration was set at 5 mg/mL (as described in adsorption kinetic studies), and the enzyme-to-substrate ratio was varied from 0.27 μπιοΐ/g to 1.34 μπιοΐ/g (0.27, 0.40, 0.50, 0.60, 0.80, 1.34 μπιοΐ/g). The incubation time was set at 30 minutes in order to attain adsorption equilibrium. Experiments were performed with EG I and CBH I alone and in equal molar mixtures. Hydrolysis Kinetics. Avicel PH-101 was used as the substrate for hydrolysis kinetics studies. Avicel was incubated at 25 °C in 50 mM sodium acetate buffer, pH 5.0, for 30 minutes, then a specified amount of EG I or CBH I was added to start the reaction. Substrate concentration varied in a range of 0.1 to 5.0 mgf mL. Enzyme concentration was 10 ug/ mL for EG I, and 50 ug/ mL for CBH I. Total reaction volume was 20 mL. The reaction mixtures were contained in 25-mL flasks, and stirred on a magnetic stirring plate. Samples were taken over a 30-minute reaction time, reducing sugars produced were measured by the BCA method (34), and the initial rates were calculated.

Results and Discussion Analytical Method. Capillary zone electrophoresis was found to be a reliable method to quantify the amount of enzymes. At the conditions established E G I was separated from CBH I completely (Figure 2), so that it was possible to quantify EG I and CBH I in mixtures. Purified E G I and CBH I were used as the standards to construct the calibration curves based on peak areas. Using the calibration curves, the enzyme concentration in the samples, ranging of 10 to 200 μg/mL, was determined. It was found that the standard deviation calculated from three injections was less than 6%. Capillary zone electrophoresis is defined as electrophoresis in free homogeneous solution. In CZE no interactions between the substances and a gel or a micellar pseudo-stationary phase are present. Thus, CZE must be considered as the simplest electrophoretic technique since the substances are simply separated on the basis of the applied electric field and as a function of the physico-chemical properties of the substance, such as their charge-to-radius ratio and their relationship with the composition of the solution, i.e., the ionic strength and the pH (27). CZE offers some advantages when compared with chromatographic techniques: the sample required is very small (injection volume less than 1 yûL), and the separation is highly efficient, rapid and quantitative (27). Capillary zone electrophoresis gives a more direct separation and analysis when compared to chromatography techniques because it does not involve the chromatographic process of adsorbing proteins on the column followed by elution with a sodium chloride gradient or pH gradient. Using UV detection, the sensitivity of capillary zone electrophoresis is similar to that of FPLC and other chromatographic methods (picomolar range). The sensitivity of capillary zone electrophoresis can be further increased using fluorescence detection.


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Adsorption Kinetics. Adsorption of E G I and CBH I on Avicel was determined by capillary zone electrophoresis during the 60-minute incubation at 25 °C. The enzyme to substrate ratio was set at 0.27 μπιοΐ/g for both EG I and CBH I. EG I and CBH I were used alone and in reconstituted mixtures. When these enzymes were used in mixtures, a fixed 1:1 molar ratio was used in order to compare the results obtained using the enzymes alone and in reconstituted mixtures. We have not compared these conditions with conditions present in Trichoderma cellulase systems where CBH I and EG I are about 60% and 15% of total protein by weight, respectively. In earlier adsorption studies, the required time to reach equilibrium varied from 2 minutes (35) to 2 hours (36). The required time depends on the characteristics of the substrate, the origin of the enzyme and the enzyme to substrate ratio. In this study, 30 minutes was found to be adequate to reach adsorption equilibrium in all cases. Figure 3 shows the adsorption process for E G I and CBH I, alone and in equimolar mixture. When used alone, about 92% of the initially added CBH I was adsorbed on Avicel at equilibrium, while about 75% of the total EG I was adsorbed. CBH I demonstrated a higher binding capacity on Avicel, compared with E G I. Comparing the adsorption of CBH I when used alone and when used together with equimolar EGI, the amount of adsorption was similar. This pattern also was observed for E G I, alone and in combination with equimolar CBH I (Figure 3). There are various statements in the literature describing the interactions between enzyme components during adsorption on crystalline cellulose. Tomme et al. (10) reported synergistic adsorption of CBH I and CBH II on Avicel. They suggested that a "loose complex" between CBH I and CBH II occurs in solution prior to adsorption, thus more adsorption occurs when both enzymes are present. In contrast, Ryu et al. (8) and Kyriacou et al.(23) found competition between different cellulase components for adsorption onto cellulose. In the present study, the results in Figure 3 did not support the "loose complex" between E G I and CBH I, neither did it show competition between EG I and CBH I during adsorption. Instead, the results in Figure 3 suggest that there are some specific binding sites on Avicel for EG I and CBH I, respectively. At a low enzyme to substrate ratio, both E G I and CBH I mostly bound to their specific binding sites, so that no matter whether they were used alone or in an equimolar mixture, the amount of adsorption was similar. However, it should be noted that this phenomenon was found using a low enzyme to substrate ratio. Adsorption Isotherms. In adsorption isotherm studies, EG I and CBH I were again used alone as well as in equimolar mixtures. The enzyme to substrate ratio was varied from 0.27 μπιοί enzyme / g Avicel to 1.34 μπιοί enzyme / g Avicel. The reaction mixtures were incubated at 25 °C for 30 minutes to ensure equilibrium conditions for enzyme adsorption under all conditions of this study. Table 2 shows the conversion of the substrate during the 30-minute incubation at 25 °C. When E G I and CBH I were used alone, the conversion was less than 2% at the highest enzyme to substrate ratio. When E G I and CBH I were used together in the equimolar mixture, 4.6% substrate was converted with the highest enzyme to substrate ratio. These data reflect conditions where there was no significant degradation or


137 structural change on the substrate, conditions that are very important to interpreting adsorption data.

— · — CBHI alone —•—EC! alone —*— CBHI in mixture —*— E d in mixture


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Trichoderma reesei during Hydrolysis of Mi - American

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