**3. Investigation of the interaction between cellulase and cellulose**

## **3.1. Materials and methods**

Materials: Microcrystalline cellulose, dimethyl sulfoxide (DMSO), and *N*-methylmorpholine -*N*-oxide (NMMO) were purchased from Sigma-Aldrich (St. Louis, MO). Polyvinylamine (PVAM) was purchased from BASF (Florham Park, NJ). The microcantilevers (200 μm × 25 μm × 2 μm, 0.1 N/m) were home-made.

Preparation of the cellulose-coated microcantilever (Zhao et al., 2010): The cellulose II model surface was prepared on the front side of a microcantilever that was made of SiO2. The surface of the microcantilever was first treated with UV ozone for 20 min. It was then immersed in 0.22% PVAM for 60 min followed by rinsing with water. Both the front side and the back side of the microcantilever were coated with PVAM. A suspension of 0.5 mg of microcrystalline cellulose powder in 25 mL of 50% NMMO was heated while stirring until a transparent brown solution was obtained. While still warm, DMSO was added to afford a cellulose solution with a final concentration of 1%. This solution was first evenly applied onto the surface of the front side of the PVAM-coated microcantilever and then allowed to sit for about 1 h. Afterwards a drop of water was added to form the cellulose film. The resulting cellulose-coated microcantilever was soaked in water for additional 4 h, during which the water was replaced every 30 min. Finally the cellulose-coated microcantilever was incubated in an oven at 80°C for 1 h to complete the surface coating.

132 Cellulose – Medical, Pharmaceutical and Electronic Applications

In cellulose, the interaction energy arises from noncovalent interactions (hydrogen bonding and van der Waals interactions) between tightly packed cellulose chains. To release cellulose chains, surface-adsorbed cellulase must break up noncovalent interactions between cellulose chains through decrystallization, which results in a change in overall interaction energy in the cellulose. If cellulose is deposited onto a microcantilever, such dynamic change in interaction energy will be transduced from the cellulose coating into the microcantilever, and will result in a measurable bending of the microcantilever. Thus, the unique link between the enzymatic decrystallization and the resulting energy alteration in cellulose measured by the microcantilever bending has provided a novel strategy to experimentally

Such adsorbate-induced bending often occurs in the presence of a specific interaction (e.g., decrystallization) between an adsorbate (e.g., cellulase) and a substrate (e.g., cellulose) that can alter the internal energy of the substrate. Since a mere adsorption such as the initial binding of cellulase on cellulose has a very minimum impact on the internal energy of cellulose, it will not induce a measurable bending even though the surface-bound cellulase changes the overall gravity (mass) on the microcantilever. Therefore the microcantilever bending can be attributed primarily to enzymatic actions (e.g., enzymatic decrystallization and hydrolytic cleavage) after the initial binding. Because the extent of bending is linearly proportional to the effective concentration of adsorbed species on the microcantilever (Berger et al., 1997; Desikan et al., 2006; Velanki & Ji, 2006), the real-time measurement of the microcantilever bending will reveal the kinetics for enzymatic actions including enzymatic decrystallization by cellulase. To our knowledge, this is the first use of a nanomechanical sensor to study mechanistic enzymology and heterogeneous enzymatic catalysis that

**3. Investigation of the interaction between cellulase and cellulose** 

Materials: Microcrystalline cellulose, dimethyl sulfoxide (DMSO), and *N*-methylmorpholine -*N*-oxide (NMMO) were purchased from Sigma-Aldrich (St. Louis, MO). Polyvinylamine (PVAM) was purchased from BASF (Florham Park, NJ). The microcantilevers (200 μm × 25

Preparation of the cellulose-coated microcantilever (Zhao et al., 2010): The cellulose II model surface was prepared on the front side of a microcantilever that was made of SiO2. The surface of the microcantilever was first treated with UV ozone for 20 min. It was then immersed in 0.22% PVAM for 60 min followed by rinsing with water. Both the front side and the back side of the microcantilever were coated with PVAM. A suspension of 0.5 mg of microcrystalline cellulose powder in 25 mL of 50% NMMO was heated while stirring until a transparent brown solution was obtained. While still warm, DMSO was added to afford a cellulose solution with a final concentration of 1%. This solution was first evenly applied onto the surface of the front side of the PVAM-coated microcantilever and then allowed to

**2.3. Microcantilever in cellulose study** 

examine this unusual enzymatic activity.

involves a solid substrate (e.g., cellulose).

μm × 2 μm, 0.1 N/m) were home-made.

**3.1. Materials and methods** 

AFM imaging (Zhao et al., 2010): To characterize the coverage, morphology, roughness, and thickness of surface coatings, AFM imaging was performed using a Nanoscope IIIa multimode scanning probe microscope from Digital Instruments, Inc. The samples were scanned in contact mode in air using silicon nitride cantilevers (MLCT) manufactured by Veeco (Camarillo, CA), with a nominal spring constant of 0.05 N/m. Images were obtained from at least three different surface areas of the sample with a typical size of 5 μm × 5 μm. The images of both height and deflection modes were captured and the surface morphology was analyzed using the image-processing software.

Microcantilever measurement: All the experiments were performed using a modified commercial Nanoscope III scanning probe microscope (Digital Instruments/Veeco, Santa Barbara, CA). The cellulose-coated microcantilever was mounted with the coating facing down in a liquid cell of the AFM with a volume of 50 μL. The solutions were introduced through injection. The cellulose-coated microcantilever was usually allowed to equilibrate in the assay buffer (25 mM sodium acetate, pH 5.5) for at least 2 h prior to any addition. The enzyme solution was prepared in the same buffer 30 min prior to use. Each assay was run against a reference to allow the subtraction of the background signal and to control for the bulk solvent effect. A desktop PC, running programs written in LabView (National Instruments, Austin, TX), was used to record the microcantilever deflection signal from the AFM via a data acquisition board with a maximum data acquisition rate of 300 kHz. The deflection measurement was monitored using a 5 mW laser diode with a wavelength of 680 nm, and a split position sensitive detector. During a measurement, 100,000 data points were taken every 30 seconds at a rate of 100 kHz. The bending of the microcantilever was obtained by simply averaging the data points.

## **3.2. Preparation of a cellulose-coated microcantilever**

The surface of a microcantilever was coated with a model film of cellulose II having a thickness between 10 to 20 nm. It is generally agreed that the natural substrate is often too complicated to be useful for detailed characterizations of cellulases (Kontturi et al., 2006). Various cellulose model surfaces have therefore been developed in the past decade (Kontturi et al., 2006) and utilized extensively in a variety of studies including the investigation of the interaction between cellulose and cellulase. Among all the model surfaces, cellulose II has been used most often and there is much technical information available about it. In addition, the surface of cellulose II is easy to prepare and characterize. All of these make the cellulose II film an ideal model surface for the current study.

For attaching cellulose to the surface of a microcantilever, an anchoring layer of PVAM was first prepared (Figure 10A). The cellulose dissolved in a hot mixture of NMMO and DMSO was then deposited onto the top of the anchoring layer (Falt et al., 2003; Zhao et al., 2010). The cellulose coating on the microcantilever was characterized with AFM imaging and the

results of the modification of the surface topography after each coating step were shown in the Figures 10B to D. A typical PVAM-coated surface is shown as a layer of small oval particles in Figure 10C. The smoothness and thickness of the coating can be adjusted by changing the coating time. The cellulose II model film on the microcantilever exhibits a cement-like characteristic with a cover of thick, short fibers (Figure 10D).

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 135

which leads to the change in interaction energy. When the cellulose is deposited onto one side of the microcantilever (Figure 10A), the change in the interaction energy in the cellulose coating can exert a differential mechanical stress between the opposite surfaces of the microcantilever, leading to the continuous bending of the microcantilever (Figure 11A). Meanwhile, the change in interaction energy also causes the change in surface morphology

of the cellulose coating as indicated by the image shown in Figure 11D.

**Figure 11.** The morphological changes of the cellulose coating monitored by means of the

increasing NaCl concentration. (B) AFM image of the surface of the cellulose coating on the

**3.4. Detection of the enzymatic decrystallization by cellulase on cellulose** 

2.59 nm with a Z range of 25 nm.

microcantilever technique (Zhao et al., 2010). (A) The bending of the microcantilever increased with the

microcantilever. (C) AFM image of the cellulose surface (as shown in (B)) after being treated with 0.1 M NaCl. (D) The cellulose surface (as shown in (C)) after being treated with 1.0 M NaCl. Mean roughness:

Overall, this study validates that the microcantilever technique is highly sensitive and specific in detecting real-time changes in interaction energy in the surface layer of the cellulose coating on the microcantilever. So, it is feasible of using the microcantilever technique to monitor the dynamic change in interaction energy in the surface layer of the cellulose caused by enzymatic decrystallization. The microcantilever bending also correlates well with the change in molecular structure of the surface region of the cellulose film.

The cellulose coating on a microcantilever was treated with the cellulase CBH I (cellobiohydrolase I, kd = 1 μM, mw = 66 kD), an exoglucanase from *Trichoderma reesei* in 25

**Figure 10.** Surface coating on the microcantilever (Zhao et al., 2010). (A) The coating scheme. (B) to (D) AFM images of surface coatings (5 μm × 5 μm).

## **3.3. Examination of morphological and structural changes of cellulose surface**

The microcantilever coated with cellulose II was treated with a successive exposure of different water/salt solutions. The result of bending of the microcantilever is presented in Figure 11 (Zhao et al., 2010). In the experiment, the cellulose-coated microcantilever was allowed to equilibrate in water for 2 h to achieve a stable baseline. The bending of the microcantilever was measured based on the deflection of the microcantilever at its apex. The measurement was initiated with the injection of water and a constant bending was observed during the first 45 min. The level of bending remained virtually constant even after the injection of a solution of 0.1 M NaCl. Upon the subsequent treatment of higher concentrations of NaCl (0.5 and 1 M), a continuous rise in bending was detected at a pace of roughly 1 nm/min and a cumulative bending of more than 100 nm was observed. To compensate for bulk effects of the buffer and the salt, we measured the differential bending, termed simply ''bending'' hereafter, which is defined as the difference in deflection of the microcantilever with the cellulose coating and without the cellulose coating.

The observed bending of the microcantilever can be attributed to the change in interaction energy within the outlayer of the cellulose surface. It has been shown previously that a cellulose model film undergoes a change in internal charge density when exposed to an electrolyte solution. And the magnitude of the change in charge density depends on the concentration of the electrolyte (Ahola et al., 2008; Freudenberg et al., 2007; Tammelin et al., 2006). Such change likely alters the intermolecular repulsion among cellulose molecules, which leads to the change in interaction energy. When the cellulose is deposited onto one side of the microcantilever (Figure 10A), the change in the interaction energy in the cellulose coating can exert a differential mechanical stress between the opposite surfaces of the microcantilever, leading to the continuous bending of the microcantilever (Figure 11A). Meanwhile, the change in interaction energy also causes the change in surface morphology of the cellulose coating as indicated by the image shown in Figure 11D.

134 Cellulose – Medical, Pharmaceutical and Electronic Applications

AFM images of surface coatings (5 μm × 5 μm).

results of the modification of the surface topography after each coating step were shown in the Figures 10B to D. A typical PVAM-coated surface is shown as a layer of small oval particles in Figure 10C. The smoothness and thickness of the coating can be adjusted by changing the coating time. The cellulose II model film on the microcantilever exhibits a

**Figure 10.** Surface coating on the microcantilever (Zhao et al., 2010). (A) The coating scheme. (B) to (D)

**3.3. Examination of morphological and structural changes of cellulose surface** 

microcantilever with the cellulose coating and without the cellulose coating.

The microcantilever coated with cellulose II was treated with a successive exposure of different water/salt solutions. The result of bending of the microcantilever is presented in Figure 11 (Zhao et al., 2010). In the experiment, the cellulose-coated microcantilever was allowed to equilibrate in water for 2 h to achieve a stable baseline. The bending of the microcantilever was measured based on the deflection of the microcantilever at its apex. The measurement was initiated with the injection of water and a constant bending was observed during the first 45 min. The level of bending remained virtually constant even after the injection of a solution of 0.1 M NaCl. Upon the subsequent treatment of higher concentrations of NaCl (0.5 and 1 M), a continuous rise in bending was detected at a pace of roughly 1 nm/min and a cumulative bending of more than 100 nm was observed. To compensate for bulk effects of the buffer and the salt, we measured the differential bending, termed simply ''bending'' hereafter, which is defined as the difference in deflection of the

The observed bending of the microcantilever can be attributed to the change in interaction energy within the outlayer of the cellulose surface. It has been shown previously that a cellulose model film undergoes a change in internal charge density when exposed to an electrolyte solution. And the magnitude of the change in charge density depends on the concentration of the electrolyte (Ahola et al., 2008; Freudenberg et al., 2007; Tammelin et al., 2006). Such change likely alters the intermolecular repulsion among cellulose molecules,

cement-like characteristic with a cover of thick, short fibers (Figure 10D).

**Figure 11.** The morphological changes of the cellulose coating monitored by means of the microcantilever technique (Zhao et al., 2010). (A) The bending of the microcantilever increased with the increasing NaCl concentration. (B) AFM image of the surface of the cellulose coating on the microcantilever. (C) AFM image of the cellulose surface (as shown in (B)) after being treated with 0.1 M NaCl. (D) The cellulose surface (as shown in (C)) after being treated with 1.0 M NaCl. Mean roughness: 2.59 nm with a Z range of 25 nm.

Overall, this study validates that the microcantilever technique is highly sensitive and specific in detecting real-time changes in interaction energy in the surface layer of the cellulose coating on the microcantilever. So, it is feasible of using the microcantilever technique to monitor the dynamic change in interaction energy in the surface layer of the cellulose caused by enzymatic decrystallization. The microcantilever bending also correlates well with the change in molecular structure of the surface region of the cellulose film.

#### **3.4. Detection of the enzymatic decrystallization by cellulase on cellulose**

The cellulose coating on a microcantilever was treated with the cellulase CBH I (cellobiohydrolase I, kd = 1 μM, mw = 66 kD), an exoglucanase from *Trichoderma reesei* in 25 mM sodium acetate buffer, pH 5.5 at 25°C. Immediately after the addition of 0.15 μM of CBH I, a bell-shaped bending curve was obtained (Figure 12A), which implies that the cellulase is capable of inducing bending of the cellulose-coated microcantilever.

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 137

this technique can be used to probe the dynamic process of the enzymatic decrystallization of cellulose by cellulase. The bending of the microcantilever is likely a result of the change in interaction energy within the cellulose caused by the interaction between cellulase and cellulose (e.g., enzymatic decrystallization), not by the adsorption of cellulase onto cellulose. The innovative microcantilever sensor approach will be used to determine the kinetics of the

This work was supported in part by a grant from the National Science Foundation (NSF) CBET-0843921, Drexel University Career Development award, and subcontract XCO-4- 33099-01 from the National Renewable Energy Laboratory funded by the U.S. DOE Office of

Ahola, S., Salmi, J., Johansson, L. S., Laine, J. & Österberg, M. (2008). Model films from native cellulose nanofibrils. Preparation, swelling, and surface interactions.

Ahola, S., Turon, X., Österberg, M., Laine, J. & Rojas, O. J. (2008). Enzymatic hydrolysis of native cellulose nanofibrils and other cellulose model films: Effect of surface structure.

Berger, R., Delamarche, E., Lang, H. P., Gerber, C., Gimzewski, J. K., Meyer, E. & Guntherodt, H.-J. (1997). Surface stress in the self-assembly of alkanethiols on gold.

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Brown, R. M. J. & Saxena, I. M. (2007). *Cellulose: Molecular and structural biology*, Springer,

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enzymatic decrystallization by cellulase.

*Penn State University, Mont Alto, USA* 

781, ISSN 1470-8728 (Electronic)

ISSN 0925-4005

ISBN 978-1-4020-5332-0, New York, NY

*Drexel University, Department of Chemistry, Philadelphia, USA* 

**Author details** 

Linghao Zhong

Jun Xi and Wenjian Du

**Acknowledgement** 

the Biomass Program.

**5. References** 

**Figure 12.** (A) The progress curves of cellulase actions on cellulose measured by the microcantilever sensor. (B) The progress curves of CBM and cellulase actions on cellulose measured by the microcantilever sensor.

Next, the cellulose coating was treated with 0.9 μM of the carbohydrate binding module (CBM, kd = 0.6 μM, mw = 17 kD) from *Clostridium cellulovorans*, the domain that anchors cellulase to cellulose at 25°C. Figure 12B clearly shows that exposing the cellulose to the CBM does not generate any measurable bending in microcantilever over the course of 120 min. Over the same time frame of the previous experiment, CBH I that contains both CD and CBM domains did induce bending, as shown in Figure 12A. Notably, more weight was probably adsorbed onto the surface of the cellulose coating in the presence of the CBM than in the presence of CBH I due to the difference in protein concentration used in each experiment. This result confirmed that the change in mass (gravity) due to the initial binding of the CBM on the surface of the cellulose does not generate any bending in microcantilever, which was fully expected for a mere protein binding (physisorption) in liquid media. Thus, the cellulase-induced bending shown in Figure 12A was not caused by the initial binding between the cellulase (via the CBM of the cellulase) and the cellulose coating. This bending can therefore be attributed to the result of the cellulase actions that occurred after its initial binding on cellulose. Such actions may include enzymatic decrystallization and/or subsequent hydrolytic cleavage (Figure 6). After the addition of the CBM, the subsequent addition of CBH I (cellulase) did not generate any bending until approximately 6 h later (Figure 12B). This result verified that the CBM was indeed bound to the surface of the cellulose and the cellulose-bound CBM prevented the subsequent binding of CBH I to the cellulose. We believe that the bending in microcantilever beginning after 500 min was due to a slow displacement of the cellulose-bound CBM by CBH I.

#### **4. Conclusion**

These studies have demonstrated that a nanomechanical sensor in microcantilever is capable of detecting the interaction between cellulase and cellulose in real time. More specifically, this technique can be used to probe the dynamic process of the enzymatic decrystallization of cellulose by cellulase. The bending of the microcantilever is likely a result of the change in interaction energy within the cellulose caused by the interaction between cellulase and cellulose (e.g., enzymatic decrystallization), not by the adsorption of cellulase onto cellulose. The innovative microcantilever sensor approach will be used to determine the kinetics of the enzymatic decrystallization by cellulase.

## **Author details**

136 Cellulose – Medical, Pharmaceutical and Electronic Applications

microcantilever sensor.

**4. Conclusion** 

mM sodium acetate buffer, pH 5.5 at 25°C. Immediately after the addition of 0.15 μM of CBH I, a bell-shaped bending curve was obtained (Figure 12A), which implies that the

**Figure 12.** (A) The progress curves of cellulase actions on cellulose measured by the microcantilever

Next, the cellulose coating was treated with 0.9 μM of the carbohydrate binding module (CBM, kd = 0.6 μM, mw = 17 kD) from *Clostridium cellulovorans*, the domain that anchors cellulase to cellulose at 25°C. Figure 12B clearly shows that exposing the cellulose to the CBM does not generate any measurable bending in microcantilever over the course of 120 min. Over the same time frame of the previous experiment, CBH I that contains both CD and CBM domains did induce bending, as shown in Figure 12A. Notably, more weight was probably adsorbed onto the surface of the cellulose coating in the presence of the CBM than in the presence of CBH I due to the difference in protein concentration used in each experiment. This result confirmed that the change in mass (gravity) due to the initial binding of the CBM on the surface of the cellulose does not generate any bending in microcantilever, which was fully expected for a mere protein binding (physisorption) in liquid media. Thus, the cellulase-induced bending shown in Figure 12A was not caused by the initial binding between the cellulase (via the CBM of the cellulase) and the cellulose coating. This bending can therefore be attributed to the result of the cellulase actions that occurred after its initial binding on cellulose. Such actions may include enzymatic decrystallization and/or subsequent hydrolytic cleavage (Figure 6). After the addition of the CBM, the subsequent addition of CBH I (cellulase) did not generate any bending until approximately 6 h later (Figure 12B). This result verified that the CBM was indeed bound to the surface of the cellulose and the cellulose-bound CBM prevented the subsequent binding of CBH I to the cellulose. We believe that the bending in microcantilever beginning after 500

sensor. (B) The progress curves of CBM and cellulase actions on cellulose measured by the

min was due to a slow displacement of the cellulose-bound CBM by CBH I.

These studies have demonstrated that a nanomechanical sensor in microcantilever is capable of detecting the interaction between cellulase and cellulose in real time. More specifically,

cellulase is capable of inducing bending of the cellulose-coated microcantilever.

Jun Xi and Wenjian Du *Drexel University, Department of Chemistry, Philadelphia, USA* 

Linghao Zhong *Penn State University, Mont Alto, USA* 

## **Acknowledgement**

This work was supported in part by a grant from the National Science Foundation (NSF) CBET-0843921, Drexel University Career Development award, and subcontract XCO-4- 33099-01 from the National Renewable Energy Laboratory funded by the U.S. DOE Office of the Biomass Program.

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**Chapter 8** 

© 2013 Dias Marques-Marinho and Duarte Vianna-Soares et al., licensee InTech. This is an open access

© 2013 Dias Marques-Marinho and Duarte Vianna-Soares et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction

chapter distributed under the terms of the Creative Commons Attribution License

The practice of compounding requires not only the drug(s) (active pharmaceutical ingredient, API), but also, the excipient(s) (pharmacological inert component) in order to

in any medium, provided the original work is properly cited.

**Cellulose and Its Derivatives Use in** 

Additional information is available at the end of the chapter

Scotland, Slovenia, Spain, Sweden, Switzerland) [3,4,8-10].

http://dx.doi.org/10.5772/56637

drug therapy benefits [11].

**1. Introduction** 

**the Pharmaceutical Compounding Practice** 

Flávia Dias Marques-Marinho and Cristina Duarte Vianna-Soares

For centuries, the pharmaceutical profession has provided services of fundamental value to society, such as the procurement, storage, compounding and dispensing of drugs. In recent decades, the focus of the pharmacist's role has shifted from compounding medicines to ensuring their safe and effective use by providing information and advice [1,2]. Although compounding activity has decreased over time, it is incontestable that this service is essential in certain patient specific situations, where industrially produced medicine is not available or is inappropriate for a particular reason [3,4]. Thus, compounded medicines are mainly important for paediatric and geriatric patients, and patients with special needs such as those with dermatological diseases [3,5,6,7,8]. In many countries, nowadays, the activity of compounding is a complementary practice to the production of medicines in alternative amounts and diversified dosage forms (liquid, semi-solid, solid) in community pharmacies (United States of America, The Netherlands), as well as in hospital pharmacies (Canada, France, Belgium, Croatia, Denmark, England, Finland, Germany, Ireland, Italy, Norway,

Interestingly, compounded medicines were estimated to make up 10-15% of all dispensed drugs in the Netherlands in the early 90's, 5.5% in 1994 and 6.6% in 1995. By the year 2000 this estimate was 3.7-5.5% [3]. On the other hand, the compounding field appears to have been a considerable and growing business since the 1990's in the United States. These products represented around 1% of all prescriptions dispensed yearly and according to this estimate, 30 million medications would have been compounded in 2003 [4]. This shows that the population has recognized that compounding pharmacies can provide individualized


**Chapter 8** 
