Microchip Immobilized Enzyme Reactors for Hydrolysis of Methyl Cellulose

摘要

Microchip immobilized enzyme reactors (muIMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two muIMERS containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the muIMER 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 electrosprayionization ion-trsp mass spectrometry (ESI-ITMS). Mass spectra of muIMER- and batch-hydrolyzed samples werecompared and no significant differences were found, indicating that muIMER hydrolysis was as efficient asconventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved byconnecting the muIMER to high-performance liquid chromatography and ESI-ITMS. This online separation reduced the relative intensities of niterfering signals and increased the signal-to-noise ratios in MS. The muIMERhydrolysates were also subjected to SEC interfaced with matrix-assisted laser desorption/ionzation time-of-flightmass spectrometry. With this technique, oligomers with DP 3-30 could be detected. The hydrolysis by the muIMERwas performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for atleast 24 h. The muIMER also allowed hydrolysis after 10 days of continuous use. The method presented in thiswork 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.