Microchip immobilized enzyme reactors for hydrolysis of methyl cellulose.
Microchip immobilized enzyme reactors (microIMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two microIMERs containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the microIMER 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 microIMER- and batch-hydrolyzed samples were compared and no significant differences were found, indicating that microIMER hydrolysis was as efficient as conventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved by connecting the microIMER 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 microIMER 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 microIMER was performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for at least 24 h. The microIMER 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.
Melander
Claes
C
Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Momcilovic
Dane
D
Nilsson
Carina
C
Bengtsson
Martin
M
Schagerlöf
Herje
H
Tjerneld
Folke
F
Laurell
Thomas
T
Reimann
Curt T
CT
Gorton
Lo
L
D001407:Bacillus / Q000201:enzymology; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D006868:Hydrolysis; D008747:Methylcellulose / Q000737:chemistry / Q000378:metabolism*; D046208:Microchip Analytical Procedures; D008970:Molecular Weight; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D004800:Enzymes, Immobilized; D008747:Methylcellulose; D002480:Cellulase