Quantitative In Vitro Microdialysis Sampling Coupled to HPAEC-PAD for the Monitoring of Mannan-Degrading Enzymes
Loading...
Links to Files
Permanent Link
Collections
Author/Creator
Author/Creator ORCID
Date
2017-01-01
Type of Work
Department
Chemistry & Biochemistry
Program
Chemistry
Citation of Original Publication
Rights
This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Access limited to the UMBC community. Item may possibly be obtained via Interlibrary Loan through a local library, pending author/copyright holder's permission.
Access limited to the UMBC community. Item may possibly be obtained via Interlibrary Loan through a local library, pending author/copyright holder's permission.
Subjects
Abstract
In vitro microdialysis sampling coupled to high performance anion exchange chromatography and pulsed amperometric detection (MD-HPAEC-PAD) is a powerful tool for carbohydrate analysis in complex matrices. Microdialysis is a sampling technique that employs a semi-permeable membrane that achieves on-line sample cleanup and in situ sampling in both in vivo and in vitro applications. The selectivity of this sampling technique is due to the molecular weight cut-off of the probe, where freely diffusing species in a sample may be transported along a concentration gradient across the membrane into a continuous perfusion flow. The collected dialysate can be assayed to develop a dynamic concentration versus time profile of the system under study. Microdialysis sampling overcomes problems associated with traditional bioprocess monitoring by eliminating the need for sample removal, enzymatic reaction quenching, and filtration steps, while also minimizing sample handling and contamination issues associated with bioprocess sampling. Improvement in the quantitative aspects of on-line microdialysis was accomplished using a computer-programmable pump designed for precision fluid delivery with flow rates in the nano- and microliter per minute range. A second pump was added in-line to the setup to achieve on-line dilution, which compensates for long acquisition times associated with slow perfusate flow rates and dead volume prior to the injection valve. Technical improvements to the on-line in vitro MD-HPAEC-PAD setup were implemented to develop an assay for assessing the efficiencies of ?-mannanase enzymes used in industrial processes. A model digestion assay was developed to validate the technical improvements of in vitro microdialysis sampling as a quantitative assessment of ?-mannanase enzymatic reactions. The development of an assay to measure the efficiency of ?-mannanase enzymes will impact industrial applications by offering on-line sampling and sample cleanup when assessing manufacturing performance. This research provides a fingerprinting tool to accurately compare the breakdown of mannan by various ?-mannanase enzymes. Overall, this research further extends microdialysis to be a powerful analytical tool for monitoring bioprocesses by allowing for on-line analysis and quantitative assessment of enzymatic efficiencies in complex samples.