Measurement of Cellulase Activity with Piezoelectric Resonators

Chapter 30

Measurement of Cellulase Activity with Piezoelectric Resonators

Downloaded by UNIV OF BATH on July 2, 2016 | http://pubs.acs.org Publication Date: April 16, 2007 | doi: 10.1021/bk-2007-0954.ch030

Orlando J. Rojas, Changwoo Jeong, Xavier Turon, and DimitrisS.Argyropoulos Department of Forest Biomaterials Science and Engineering, North Carolina State University, Raleigh, NC 27695-8005

The dynamics of cellulase binding and the activity on thin films of cellulose by using a piezoelectric sensing device (Quartz Crystal Microbalance with Dissipation monitoring, Q C M - D ) were examined. Upon exposure of the cellulose film to enzyme mixtures, a reduction in the sensor's frequency due to molecular binding is observed. Thereafter the frequency increases due to the loss of effective mass caused by the degradation of the film (enzymatic attack). In this study, it is demonstrated that the rate at which degradation of the film occurs can be monitored and quantified in situ and in real time as a function of enzyme concentration, temperature and pH of the incubating solution. Also, mass transport effects can be investigated by changing the flow conditions within the Q C M — D reaction cell. The use of piezoelectric sensing to characterize and monitor the mechanisms and kinetics of enzyme activity on cellulosic substrates adds another dimension to our knowledge and to the methods available for such investigations.

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© 2007 American Chemical Society Argyropoulos; Materials, Chemicals, and Energy from Forest Biomass ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


479 Over the last two decades there has been remarkable progress in understanding the mechanisms by which enzymes interact with natural substrates such as lignocellulosic materials. Research has been carried out to manipulate and exploit enzymatic hydrolysis with the aim of producing fermentable sugars mainly driven by concerns over the escalating cost and reduced availability of fossil fuels. Such interest in cellulose conversion to energy and chemicals is not surprising since cellulose is the most abundant carbohydrate in the biosphere. Use of enzymes is also relevant to other industrial activities including wood pulping (/) and delignification (2); cellulose pulp bleaching (3,4,5); textile printing (6) and cotton pre-treatment (7); treatment of waste liquid effluents and detergency (8). Most of the current work dealing with enzymes is focused on their adsorption and formation of enzyme-substrate complexes and on the development of enzyme binding domains and specificity (9). Active areas of research also include the three-dimensional structure of cellulolytic enzymes and the mechanisms and kinetics of cellulose binding and hydrolysis (10). Lignocellulose degradation is a multienzymatic process due to the complex nature of lignified plant materials. Such degradation is an important step for the carbon cycle in the biosphere involving both hydrolytic and oxidative enzymes. Lignin removal is the key step for natural biodégradation of lignocellulosic materials, and is also required for converting wood into pulp for paper manufacture. In this area cellulases are the most important enzymes due to their ability to cleave cellulose polymers into smaller units or simple sugars via hydrolysis reactions. Different oxidoreductases such as laccases and peroxidases which are involved in lignin degradation and other hydrolytic enzymes such as xylanases are widely used in industrial processes, especially in pulp and paper industries.

Cellulases At present, cellulases are mainly used in food, beer and wine, animal feed, textile and laundry, pulp and paper, as well as in agricultural industries. The demand for these enzymes is growing rapidly, and this has driven extensive efforts to understand their mechanisms of activity. Some microorganisms including fungi and bacteria produce various types of cellulase components. In several industrial applications genetic material is transferred to a host organism for commercial production. Because the original organism evolved to metabolize a mixed substrate containing cellulose, hemicellulose, lignin and extractives, the lower cost commercial enzymes are often a mixture of different specific isozymes.

Argyropoulos; Materials, Chemicals, and Energy from Forest Biomass ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


480 Commercial cellulase mixes usually contain one or more exoglucanases such as cellobiohydrolase (CBH) which will proceed from either the reducing end or non reducing end of the cellulose chain and produce a shortened chain and cellobiose. The cellulase mixture may also contain several endoglucanases (EGI, EGII, etc.), which cleave randomly the internal p-l,4-glycosidic bonds on the cellulose chain along its length to produce free chain ends that will be acted upon by exoglucanases. Most cellulases also contain both P-l,4-glycosidase, which hydrolyze cellobiose units to glucose monomers, and various hemicellulases, which may have side chain cleaving capabilities. For complete hydrolysis of cellulosic material the synergistic combination of endoglucanase, exoglucanase and P-l,4-glycosidase is required. Most cellulolytic enzymes are composed of two functionally distinct domains. The cellulose-binding domain is responsible for the close association of the enzyme with solid cellulose through strong binding to crystalline cellulose. The catalytic domain is responsible for catalyzing cellulose hydrolysis through an acid mechanism.

Cellulosic Substrates Interactions of cellulases with cellulose play an important role in determining the efficiency of the enzymatic hydrolysis. Therefore, an understanding of enzyme interactions with the substrates is of great importance in processing wood and cellulosic fibers. The rate of enzymatic hydrolysis and its yield are dependent on the adsorption of enzyme onto the substrate surface. Cellulose fibers are heterogeneous, porous substrates with both external and internal surfaces. The external surface area is determined by the shape and size of the cellulosic fibers, while the internal surface area depends on their capillary structure. The accessibility of cellulose to the cellulase is also controlled by the physicochemical properties of the substrate, the multiplicity of the cellulase complexes, and reaction parameters including those associated with mass transport and temperature. Furthermore, cellulose fibers contain both amorphous and crystalline regions. Crystalline regions are considered to be more difficult to degrade than amorphous regions and therefore significantly affect the rate and extent of enzyme hydrolysis. Various kinetic models have been developed to describe the hydrolysis rate of cellulase. The models are based on parameters such as the amount of adsorbed enzyme on the cellulosic surface, the structural characteristics of the substrate including pore size distribution, crystallinity index and specific surface area, and cellulase-cellulose adsorption rates.


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Enzyme Activity There are two basic approaches to determine enzyme/cellulase activity. The first is based on the measurement of the individual activities of component enzymes and the second one is based on the activity of the total complex. Measurements of individual activities vary according to enzyme nature, namely, endoglucanases, exocellobiohydrolase and exoglucanases (7/). In the case of endoglucanases, activity is usually measured with any of the following techniques: i) Viscometric method. It measures the endoglucanase activity according to changes in viscosity with time, as the enzyme degrades cellulose. It is basically simple but requires complex mathematical analysis in order to express the results in absolute units. ii) The carboxymethylcellulose (CMC)-plate-clearing assay. Endoglucanase activity can be detected on agar plates; zymogram overlay gels, etc. by staining or precipitation of C M C present in the solid gel matrix. iii) Measurement of reducing sugars. Reducing sugar released from C M C is often used jointly with measurements of changes of C M C viscosity in order to determine endoglucanase activity. Released sugars can be detected colorimetrically and the activity expressed in terms of moles of glucosidic bonds cleaved. iv) Measurement of the chromophore and fluorescent groups released from modified C M C . This is based on chemically modified forms of cellulose that release soluble colored or fluorescent groups when their glucosidic bonds are hydrolyzed. v) Chromatography. High performance liquid chromatography (HPLC) can provide valuable insight into the activities of components of cellulase complex, as transient cellooligosaccharide products can be detected, provided a means of stopping the hydrolysis at various stages is available. Separated hydrolysis products then could be detected photometrically. Exoglucanases cleave cellobiose units from the non-reducing ends of cellulose or cellooligosaccharide molecules. Actually there is not a widely accepted method for measuring exoglucanases activity in cellulase complexes. When β-glucosidases are present then cellobiose is only a transitory product. A graphical method for determining exocellobiohydrolase activity is reported in the literature (P). Selective determination of exocello-biohydrolase activity in a mixture of cellulolytic enzymes using synthetic substrates is based on the selective cleavage of the agluconic bond and not on the holosidic or heterosidic bond (72-75). Exoglucosidases cleave glucose units successively from the non-reducing end of the glucan. A single enzyme of this type, acting alone, could only attack a single terminal residue. Different approaches have been used to measure exoglucosidases activity. One of the methods is based on the different


482 susceptibilities of cellotetraose and cellobiose to hydrolysis (14,15,16,17). A second method uses glucono-lactone, which is a potent inhibitor of β-glucosidase but has no effect on exo-P-l,4-glucosidase (18). A third method is based on the study of the rate of formation of D-glucose in the presence of various concentrations of added β-glucosidase (10). A fourth alternative uses as substrate a substituted cellodextrin supplemented with β-glucosidase. This substrate is susceptible to further hydrolysis by exoglucosidase from Trichoderma viride whereas endoglucanase, exocellobiohydrolase or β-glucosidase would not attack it (19).


Total Cellulase Complex Activity Measurement "Total cellulase" assay methods measure the combined activities of the complex of cellulolytic enzymes towards an appropriate substrate, typically filter paper or powdered crystalline cellulose. Some other substrates, such as carboxymethylcellulose (CMC), trinitrophenyl-CMC (TNP-CMC) and cellulose azure, may be attacked by exo-acting cellulases as well as by endocellulases, and therefore provide some indication of their combined activities. However, hydrolysis of native crystalline cellulose is a much more stringent measure of the activity of a "complete" cellulase complex (9). Total cellulase activity can be measured by the residual cellulose by weight (20) or by determination of the sugars produced from insoluble cellulose. Such methods normally employ a colorimetric method to measure reducing sugars released. Substrates commonly used are filter paper or microcrystalline wood alpha-cellulose. Overall, the mechanism of adsorption of most cellulase components onto cellulose is still unknown as well as the role of the different structural domains in cellulase action and their effects on substrates. Furthermore, with complex mixtures it can be difficult to understand how to dose enzymes to provide the correct level of activity. Often dosage rates are arrived at empirically based on a supposedly constant activity. In this investigation we propose the use of a piezoelectric technique, the Quartz Crystal Microbalance (QCM) to monitor, in situ and in real time the catalytic activity of cellulases on model substrates. This approach will allow unveiling the complex mechanism involved in enzymatic degradation of cellulosic materials.

Experimental Materials and Methods The enzyme used in this work was a commercially-available multicomponent cellulase (Rocksoft™ A C E p150, Dyadic Int., FL). Sodium


483 bicarbonate, sodium hydroxide, hydrochloric acid and acetic acid (all from Fisher Scientific), sodium acetate trihydrate (Fluka) and tris (hydroxymethyl) aminomethane (Acros) were used in the preparation of the buffer solutions of p H 4.5, 7 and 10. Water was obtained from a Milli-Q® Gradient system (resistivity >18ΜΩ cm).


Cellulose Thin Films and Related Methods Cellulose thin films were used as models for cellulose. The process for preparing these surfaces (21) starts with either silicon wafers or Q C M - D (Quartz Crystal Microbalance with Dissipation Monitoring) electrodes which are cleaned by standard chemical treatment and UV-ozone plasma. The Q C M - D electrodes consisted of quartz crystals (Q-sense) coated with a conductive gold layer and a top 50-nm silica layer. A cellulose solution was prepared by dissolving micro-crystalline cellulose ( M C C ) in 50 %wt water/N-Methylmorpholine-N-oxide ( N M M O ) at 115 °C. Dimethyl Sulfoxide (DMSO) was added to adjust the concentration of cellulose (0.05%) in the mixture. The cellulose solution was then spin coated (spin coater from Laurell Technologies model WS-400A-6NPP) on the substrates (silica wafer or Q C M electrodes) at 5000 rpm for 40 seconds. The substrate was removed from the spin coater and placed in a milli-Q water bath to precipitate the cellulose. The cellulose-coated substrate was then washed thoroughly with milli-Q water, dried in a vacuum oven at 40 °C and stored at room temperature in a clean chamber for further use. Cellulose Characterization Ellipsometry, X-ray Photoelectron Spectroscopy (XPS), and Atomic Force Microscope ( A F M ) were used to characterize the cellulose films. X-ray Photoelectron Spectroscopy (XPS) was performed on bare silicon, polyvinyl amine (PVAm) and cellulose-coated wafers to quantify the respective chemical composition. This was accomplished by using a RIBER LAS-3000 X P S system with M g Κα (1253.6 eV) as X-ray source. The thin cellulose films were characterized in terms of material distribution, surface roughness and topography using a Q-Scope 250 A F M model (Quesant Inst. Corp.), before and after enzyme treatment. The scans were performed in dried conditions using N S C 16 standard wavemode S i N tips with a force constant of 40 N/m (according to the manufacturer) and a typical resonant frequency of 170±20kHz. Finally, a Rudolph single-wavelength ellipsometer was used to determine the thickness of cellulose films on the surface of the silicon wafers (the refractive index of cellulose film was taken as 1.56). 3

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484 Enzyme/Cellulose Surface Interactions Quartz Crystal Microbalance A Quartz Crystal Microbalance with Dissipation monitoring, Q C M - D (Qsense D-300, Sweden) was used to study enzyme binding and activity on cellulose thin films deposited on quartz/gold electrodes which are coated with a 50 nm S i 0 layer. Temperature was controlled within ± 0.02 °C of the respective set point via a Peltier element. Q C M - D consists of a thin plate of a piezoelectric quartz crystal, sandwiched between a pair of electrodes. It measures simultaneously changes in resonance frequency, f, and dissipation, D (the fractional and viscoelastic energy losses in the system), due to adsorption on a crystal surface. / is measured before disconnecting the driving oscillator, and D is obtained by disconnecting the driving field and recording the damped oscillating signal as its vibration amplitude decays exponentially. Mechanical stress causes electric polarization in a piezoelectric material. The converse effect refers to the deformation of the same material by applying an electric field. Therefore, when an A C voltage is applied over the electrodes the crystal can be made to oscillate. Resonance is obtained when the thickness of the plate is an odd integer, n, of half wavelengths of the induced wave, η being an integer since the applied potential over the electrodes is always in anti-phase. If any material is adsorbed onto the crystal, it can be treated as an equivalent mass change of the crystal itself. The increase in mass, Am, induces a proportional shift in frequency, / There exists a linear relationship between m and/as described by Sauerbrey equation (22):


2

^ - - ^ ν ^ _-P v *f q

q

=

CA/

[1]

where p and v are the specific density and the shear wave velocity in quartz respectively; t is the thickness of the quartz crystal, and f the fundamental resonance frequency (when « =1). For the crystal used in these measurements the constant C has a value of 17.8 ng cm Ηζτ'. The relation is valid when the following conditions are fulfilled: (i) the adsorbed mass is distributed evenly over the crystal, (ii) Am is much smaller than the mass of the crystal itself (

Measurement of Cellulase Activity with Piezoelectric Resonators

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