Microchip Immobilized Enzyme Reactors for Hydrolysis of Methyl

Apr 12, 2005 - Methylated cellooligosaccharides with degrees of polymerization (DP) between 1 and 6 formed during hydrolysis were analyzed by direct i...
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Anal. Chem. 2005, 77, 3284-3291

Microchip Immobilized Enzyme Reactors for Hydrolysis of Methyl Cellulose Claes Melander,† Dane Momcilovic,‡ Carina Nilsson,† Martin Bengtsson,§ Herje Schagerlo 1 f,| | § † ,† Folke Tjerneld, Thomas Laurell, Curt T. Reimann, and Lo Gorton*

Departments of Analytical Chemistry, Technical Analytical Chemistry, and Biochemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, and Department of Electrical Measurements, LTH, P.O. Box 118, Lund University, Sweden

Microchip immobilized enzyme reactors (µIMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two µIMERs containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the µIMER could be confirmed from the average molar masses and molar mass distributions measured by size exclusion chromatography (SEC) with online multiangle light scattering and refractive index detection. Methylated cellooligosaccharides with degrees of polymerization (DP) between 1 and 6 formed during hydrolysis were analyzed by direct infusion electrospray ionization ion-trap mass spectrometry (ESI-ITMS). Mass spectra of µIMER- and batch-hydrolyzed samples were compared and no significant differences were found, indicating that µIMER hydrolysis was as efficient as conventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved by connecting the µIMER to high-performance liquid chromatography and ESI-ITMS. This online separation reduced the relative intensities of interfering signals and increased the signal-to-noise ratios in MS. The µIMER hydrolysates were also subjected to SEC interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With this technique, oligomers with DP 3-30 could be detected. The hydrolysis by the µIMER was performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for at least 24 h. The µIMER also allowed hydrolysis after 10 days of continuous use. The method presented in this work offers new approaches for the analysis of derivatized cellulose and provides the possibility of convenient online, fast, and more versatile analysis compared with the traditional batch method. Cellulose is a naturally occurring polymer, which is used as raw material in a number of industrial applications, e.g., in the paper, paint, textile, food, and pharmaceutical industries.1,2 It is a * Corresponding author. E-mail: [email protected]. Tel.: +46 46 2227582. Fax: +46 46 2224544. † Department of Analytical Chemistry. ‡ Department of Technical Analytical Chemistry. § Department of Electrical Measurements. | Department of Biochemistry.

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linear polysaccharide composed of anhydroglucose units, linked together with 1-4 β-D-glycosidic bonds. To fulfill the various demands of functionality from industry, the cellulose is often modified by physical, chemical, or enzymatic means. Chemical modification implies the substitution of one or more of the free hydroxyl groups of the glucose monomer on carbon 2, 3, or 6. Enzymatic modification is sometimes performed to change the properties of the cellulose.3,4 The physical and chemical properties of the modified cellulose depend on several factors, such as the modification reaction, the type of substituent, the average molar mass (Mw), the molar mass distribution, the degree of substitution (DS), and the distribution of the substituents along the polymer chain. Since the properties of the modified cellulose are correlated with their technological usefulness, techniques to accurately characterize them are of great importance. One approach to estimate the substituent distribution of a cellulose polymer is to hydrolyze the polymer using either cellulose-hydrolyzing enzymes such as endoglucanases or acids to obtain oligomers that are possible to analyze with various chromatographic and mass spectrometric techniques.5-7 Endoglucanases are enzymes capable of hydrolyzing nonterminal glycosidic bonds (i.e., interior parts) of the cellulose chain.8-11 The ability of an endoglucanase to hydrolyze cellulose will depend on the structure of the active site of the endoglucanase, the type of substituent, and its distribution along the cellulose chain. Different endoglucanases will be more or less hindered by substituents depending on the nature of their active site. The activity of the endoglucanases on different substrates and the sensitivity of enzyme activity to substituent position and distribution are to some extent unknown, and these enzyme characteristics are currently under extensive investigation.8,12,13 However, (1) Batdorf, J. B.; Desmarais, A. J. Chem. Addit., Symp. 1971, 136-141. (2) Batdorf, J. B.; Rossman, J. M. Industrial Gums, 2nd ed.; 1973; pp 695-729. (3) Miettinen-Oinonen, A. VTT Publ. 2004, 550, 1-96. (4) Miettinen-Oinonen, A.; Paloheimo, M.; Lantto, R.; Suominen, P. J. Biotechnol. 2005, 116, 305-317. (5) Richardson, S.; Gorton, L. Anal. Chim. Acta 2003, 497, 27-65. (6) Cohen, A.; Schagerlo¨f, H.; Nilsson, C.; Melander, C.; Tjerneld, F.; Gorton, L. J. Chromatogr., A 2004, 1029, 87-95. (7) Mischnick, P.; Heinrich, J.; Gohdes, M. Papier 1999, 53, 739-743. (8) Karlsson, J.; Momcilovic, D.; Wittgren, B.; Schu ¨ lein, M.; Tjerneld, F.; Brinkmalm, G. Biopolymers 2002, 63, 32-40. (9) Bayer, E. A.; Chanzy, H.; Lamed, R.; Shoham, Y. Curr. Opin. Struct. Biol. 1998, 8, 548-557. (10) Tomme, P.; Warren, R. A.; Gilkes, N. R. Adv. Microb. Physiol. 1995, 37, 1-81. (11) Beguin, P.; Aubert, J. P. FEMS Microbiol. Rev. 1994, 13, 25-58. 10.1021/ac050201r CCC: $30.25

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it is known that unmodified parts of the cellulose chain will be hydrolyzed to a higher degree than the modified parts, where the substituents typically hinder the enzyme. The hydrolysis techniques used today are performed in solution, batchwise, with a mixture of enzyme and substrate.6,8 Due to the high cost of producing and purifying enzymes, the total concentration of the enzyme in the batch is often low during the hydrolysis. The procedure is therefore time-consuming, it is not possible to automate for online analysis, and it is also very difficult to recycle the enzyme. This makes the batch methodology quite inefficient and expensive. To overcome the disadvantages of the manual batch methods, miniaturization and automation are generally good strategies. Silicon micromachining offers several interesting possibilities to achieve the goals of low manufacturing costs and mass production.14,15 Silicon can be made highly porous giving it a high surface area-to-volume ratio. It is also possible to fabricate well-defined structures with high mechanical strength and chemical durability, making it feasible to construct analytical systems that are extremely small. The main interest in downsizing the analytical system is to enhance the performance of the analysis, mainly due to the fact that small dimensions yield very small diffusion paths for the reactive species to travel. In this study, a novel method for hydrolysis of derivatized cellulose has been developed using a microchip immobilized enzyme reactor (µIMER) containing the immobilized endoglucanase from Bacillus agaradhaerens (BaCel 5A). BaCel 5A has a molecular mass of 44 kDa and has been extensively studied with respect to its crystal structure.16,17 According to these studies, the active site of BaCel 5A has five subsites, two on the reducing side of the cleavage site and three on the nonreducing side. Cellobiose is the smallest oligomer that will be produced in any significant amount during hydrolysis, and cellotetraose is the smallest substrate for the enzyme. Although the enzyme has five subsites, only four glucose units are necessary for cleavage.12,16,18 The reactors used in this work, developed according to Laurell et al., were constructed from highly porous silicon.19,20 µIMERs of this type have previously been used for sucrose analysis21 and protein hydrolysis,22 and in the present work, this concept has been expanded to encompass cellulose analysis. The possibility of immobilizing the enzymes gives a much more efficient method, making it possible to use them for a long period of time with only a minor consumption of the enzyme. The usage of the µIMER format also includes the possibility of online connection to chromatographic and MS equipment. The sheer speed of the (12) Melander, C.; Bengtsson, M.; Schagerlo¨f, H.; Tjerneld, F.; Laurell, T.; Gorton, L. In manuscript. (13) Momcilovic, D.; Schagerlo ¨f, H.; Ro¨me, D.; Jo ¨rnte´n-Karlsson, M.; Karlsson, K.-E.; Wittgren, B.; Tjerneld, F.; Wahlund, K.-G.; Brinkmalm, G. Anal. Chem. In press. (14) He, B.; Tait, N.; Regnier, F. Anal. Chem. 1998, 70, 3790-3797. (15) Fintschenko, Y.; Van Den Berg, A. J. Chromatogr., A 1998, 819, 3-12. (16) Davies, G. J.; Dauter, M.; Brzozowski, A. M.; Bjørnvad, M. E.; Andersen, K. V.; Schu ¨ lein, M. Biochemistry 1998, 37, 1926-1932. (17) Varrot, A.; Schu ¨ lein, M.; Davies, G. J. J. Mol. Biol. 2000, 297, 819-828. (18) Davies, G. J.; Mackenzie, L.; Varrot, A.; Dauter, M.; Brzozowski, A. M.; Schu ¨ lein, M.; Withers, S. G. Biochemistry 1998, 37, 11707-11713.:. (19) Laurell, T.; Rosengren, L. Sens. Actuators, B 1994, 19, 614-617. (20) Laurell, T.; Drott, J.; Rosengren, L. Biosens. Bioelectron. 1995, 10, 289299. (21) Lendl, B.; Schindler, R.; Frank, J.; Kellner, R.; Drott, J.; Laurell, T. Anal. Chem. 1997, 69, 2877-2881. (22) Ekstro ¨m, S.; O ¨ nnerfjord, P.; Nilsson, J.; Bengtsson, M.; Laurell, T.; MarkoVarga, G. Anal. Chem. 2000, 72, 286-293.

µIMER format makes it feasible to carry out extensive measurements that are needed to analyze the hydrolysis products that in turn will give information on both the mother polymer and the action of the enzyme and also provides an increased utilization and increased cost efficiency. To our best knowledge, endoglucanases have never before been immobilized on a solid support for hydrolysis of derivatized cellulose. We found that a further advantage of using the µIMER format as in the present work is that this format does not utilize packed material. The open channel structure reduces the pressure in the system. The highly viscous solutions that are used would not be able to be pumped through a packed material, such as controlled pore glass or Sephadex support materials, without affecting the immobilized enzymes. EXPERIMENTAL SECTION Materials and Methods. Polyethylenimine (PEI; linear, MW ) 750 000, 50% w/v aq) was from Aldrich (St. Louis, MO). Glutardialdehyde (GA, 25% v/v grade I), and NaBH3CN were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Succinic acid, tris(hydroxymethyl)aminoethane, concentrated NH3 (aq), HCl (aq), and H2O2 were from Merck (Darmstadt, Germany). BaCel 5A was a kind gift from the late Dr. Martin Schu¨lein (NovoZymes, Bagsvaerd, Denmark). The full-length enzyme was used including the catalytic core and the cellulose binding module.16 The water used in all experiments was purified in a Milli-Q system from Millipore (Bedford, MA). The methyl celluloses (MCs) used in this study were commercial products under the name of Metolose (SM-4, Lot 907540, DS 1.76 (low-mass MC); SM-15, Lot 109614, DS 1.78 (intermediate-mass MC); and SM-1500, Lot 103674, DS 1.80 (high-mass MC)) manufactured by Shin-Etsu (Tokyo, Japan). The 1-TIME 2.5-dihydroxybenzoic acid (DHB) precoated MALDI foils were from LabConnections (Minneapolis, MN). All other chemicals used were of analytical grade. Silicon Microchip Reactors. Microchip reactors (µIMERs) were fabricated in 3 in., 〈110〉, p-type silicon (20-70 Ω cm, Siltronix SA, Geneva, Switzerland), using standard micromachining techniques.23 In short, a 1-µm SiO2 wet oxide was grown and patterned in a standard UV-photolithography process. The SiO2 was used as etch mask in the subsequent anisotropic etching of the parallel channel microreactor: etch solution, 30% KOH (30 mg/100 mL of H2O) at 80 °C. The reactors were etched to a channel depth of 250 µm. To obtain a porous layer on the channel walls, the microchip reactor was anodized in an ethanol (96%)-HF (40% in water) solution mixed (1:1) during illumination. The reactor dimension was 13.1 × 3.2 mm, comprising 42 porous flow channels of approximately 235 µm depth and 25 µm width, giving a total internal surface area of 242 mm2 before fabrication of the porous layer (Figure 1). To ensure sufficient contact with the electrolyte during the anodization process, the wafer backside was previously doped with boron. The current density during anodization was fixed to 50 mA/cm2 for 10 min, to achieve an optimized porosity throughout the channel walls.24 After anodization, the reactors were rinsed several times in ethanol and H2O, removing any remaining HF. After the porous silicon fabrication, all reactors were dried and (23) Drott, J.; Lindstro ¨m, K.; Rosengren, L.; Laurell, T. J. Micromech. Microeng. 1997, 7, 14-23. (24) Bengtsson, M.; Ekstro ¨m, S.; Marko-Varga, G.; Laurell, T. Talanta 2002, 56, 341-353.

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Figure 2. Delivery of the substrate solution to the µIMER performed using a syringe pump. The outlet is connected directly to a loop for online injection to the ESI-ITMS system. Alternatively, the hydrolyzed product is collected in a sample vial. In this work, online analysis of the hydrolyzate has been performed using LC-ESI-ITMS. However, connection to any detection systems with loop injection is possible.

Figure 1. Microchip immobilized enzyme reactor. The reactor is approximately 9 mm long and 3 mm wide. It consists of two reservoirs that are connected by channels wherein the channel walls are highly porous. Due to the high porosity of the walls, a high surface area is obtained.

inspected through an optical microscope and the obtained channel widths were measured. Immobilization of Enzymes on PEI-GA Activated Surface. Prior to the immobilization, the silicon microchips were cleaned in a mixture of 25% NH3, 30% H2O2, and H2O (1:1:5 by volume) followed by cleaning in a mixture of 37% HCl, 30% H2O2, and H2O (1:1:5 by volume) at 100 °C for 5 min per cleaning step. The microchips were then thoroughly rinsed with water. To bind the enzyme, a microchip was placed in 0.2% PEI in 10 mM succinate adjusted to pH 5.5 with 1 M NaOH and stirred at 4 °C overnight. The PEI forms a thin polymer layer of bound amine groups at the surface of the silicon oxide. After rinsing with succinate buffer, the reactor was placed under stirring in 2.5% GA in succinate buffer for 1 h. The GA serves as a cross-linker between the amino groups of the enzyme and the amino groups of PEI. The reactor was rinsed again with succinate buffer and placed for at least 12 h at 4 °C in an enzyme solution (100 µM) containing the BaCel 5A enzyme. To deactivate unreacted GA, the reactor was placed under stirring for 1 h in 0.1 M tris(hydroxymethyl)aminoethane adjusted with 18.5% HCl to pH 5.5. To reduce the bond between the enzyme and the GA, the reactor was placed in 2 mg/mL NaCNBH3 in 10 mM tris(hydroxymethyl)aminoethane at pH 5.5. The enzyme was now immobilized by means of reductive amination. Finally the reactor was rinsed with tris buffer. Enzymatic Hydrolysis. For batch hydrolysis, the high-mass MC at a concentration of 10 g/L was diluted four times in 12.5 mM sodium formiate, was incubated with the enzyme for 72 h with BaCel 5A (1 µM), and then filtered to remove the enzyme and larger derivatized cellulose chains using 5 kDa cutoff Millipore Ultrafree centrifuge filtration tubes (Millipore). The hydrolysates were stored at +4 °C prior to analysis. For µIMER hydrolysis, to lower the viscosity a solution of MC at a concentration of 1 g/L dissolved in 12.5 mM sodium formiate was pumped using a syringe pump (CMA/Microdialysis, Stockholm, Sweden) at 0.5 µL/min through the two µIMERs connected in series. The outlet of the reactors, containing the hydrolysate, was collected in a sample vial or injected directly into a 20 µL sample loop. 3286 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005

ESI-ITMS and LC-ESI-ITMS. Direct infusion electrospray ionization ion-trap mass spectrometry (ESI-ITMS) was performed on an Esquire-LC (Bruker Daltonik, GmbH, Bremen, Germany). Direct infusion of the analyte solution was performed at 3 L/min. Nitrogen was used as drying gas at a flow rate of 3 L/min with a temperature of 350 °C. Nitrogen was also used as nebulizer gas at 7 psi. The following voltages were used: nebulizer end plate 4000 V, end cap 3500 V, and capillary exit 150 V. The mass spectra from the full-scan experiments were averaged over 100 scans. In full-scan mode, the ESI-ITMS was typically set to scan between m/z 100 and 1500. All batch-hydrolyzed samples were filtered prior to injection to remove the enzyme. For comparison, µIMER-hydrolyzed samples were filtered with the same filter but at an analyte concentration of 1 g/L. For the online method, the outlet of two series-connected µIMERs was connected to a Rheodyne valve with a 20 µL loop (Figure 2). The same settings of the mass spectrometer were used except for nitrogen, which was used as drying gas where the flow was 7 L/min and the nebulizer at 30 psi. Trap drive was set to 47.4, optimizing instrumental sensitivity at m/z ∼700. Carrier liquid was MilliQ water at a flow rate of 0.5 mL/min. After injection, the sample was separated on a TSK GMPWXL size exclusion precolumn (40 mm × 7.8 mm2) (TosoHaas Bioseparation Specialists, Stuttgart, Germany) and then injected into the ESI interface. SEC/MALDI-TOF-MS. Size exclusion chromatography interfaced with matrix-assisted laser desorption/ionization time-offlight mass spectrometry (SEC/MALDI-TOFMS) was performed using an LC-Transform Series 500 interface (Round Rock, TX) and a PerSeptive Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Framingham, MA) according to methodology presented by Momcilovic et al. and used with some modifications.25 In this setup, the SEC effluent is sprayed from a heated steel capillary, by assistance of a sheath gas, onto a MALDI foil that is coated with DHB. The matrix coating is briefly wetted by the solvent so that the analyte can be incorporated into the matrix crystals. The SEC column was either a TSKgel G 3000 PW (30 cm × 7.5 mm i.d., TosoHaas) or a TSK GMPWXL mixedbed column (30 cm × 7.8 mm i.d., TosoHaas). The µIMERhydrolyzed MC (at a concentration of 0.6 g/L in 12.5 mM sodium formiate (pH 5.5) buffer) collected from the two series-connected µIMERs was injected without any pretreatment. The movement rate of the sample stage that supported the MALDI target in the LC-Transform interface was 4 mm/min. (25) Momcilovic, D.; Wittgren, B.; Wahlund, K.-G.; Brinkmalm, G. Rapid Commun. Mass Spectrom. 2005, 19, 947-954.

MALDI-TOF mass spectra were acquired in both linear and reflector modes. The instrument settings for the reflector mode acquisitions have been described elsewhere.25 For the linear mode acquisitions, an acceleration voltage of 25 kV was used. The laser intensity was held slightly above threshold, and the lag time was 400 ns plus instrument offset. The guide wire voltage was set to 0.05% of the acceleration voltage. Mass spectra were accumulated for 100-300 laser shots. SEC-MALS-RI. The hydrolyzed samples were analyzed by SEC coupled to multiangle light scattering and refractive index (SEC-MALS-RI) detection. The separation was carried out using a TSKgel GMPWXL column (30 cm × 7.8 mm i.d., TosoHaas). The pump in use was a Shimadzu LC-10AD liquid chromatography pump (Shimadzu Corp., Tokyo, Japan), and the degasser was a Shimadzu DGU-14A. Injection of the polymer solution was carried out by a Waters 717 plus autosampler. MALS and RI detection was performed utilizing DAWN EOS MALS and Optilab RI detectors (Wyatt Technology Corp., Santa Barbara, CA), respectively. A solution consisting of 10 mM NaCl + 0.02% NaN3 was used as mobile phase at a flow rate of 0.5 mL/min, and the sample volume was 100 µL. The analyte concentration was 0.75 g/L, and double injections were made. A 0.05 µm VM Millipore filter was placed between the pump and the injector. A Sartorius CA 0.45 µm filter (Go¨ttingen, Germany) was placed after the column to improve the MALS signal. Astra for Windows version 4.73.04 was used for the data evaluation. RESULTS AND DISCUSSION By using µIMERs, the possibility of performing hydrolysis with online analysis, e.g., MS, is provided. In batch experiments, hydrolysis has been performed completely offline and the enzyme had to be removed by filtration. In this work, the µIMER was connected directly to LC-MS, where separation of unwanted components could easily be performed. SEC has previously been used for the determination of the average molar mass and the molar mass distribution of enzymatic hydrolysates of starch and cellulose derivatives.26-28 If SEC is used together with RI and MALS detection, determination of the molar masses of the hydrolysis products can give information about the enzyme action on the derivatized polysaccharide without any standards.29-31 MALDI-TOF-MS has been employed in a variety of applications for carbohydrate analysis.32 As with many types of mass spectrometry, MALDI-TOF-MS can provide valuable information on several aspects of structural analysis, such as determination of sequence, branching, and linkage. One great advantage is that MALDI-TOF-MS can be used to analyze polysaccharides up to 10 000 Da with high mass accuracy. The limitations of MALDITOF-MS in the lower mass range (m/z