Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion.

A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Reliable fabrication method of transferable micron scale metal pattern for poly(dimethylsiloxane) metallization.

We have developed a reliable fabrication method of forming micron scale metal patterns on poly(dimethylsiloxane) (PDMS) using a pattern transfer process. A metal stack layer consisting of Au-Ti-Au layers, providing a weak but reliable adhesion, was deposited on a silicon wafer. The metal stack layer was then transferred to a PDMS substrate using serial and selective etching. We demonstrate that features as small as 2 microm were reliably transferred on to the PDMS substrate for use as interconnects and electrodes for biosensors and flexible electronics application.

Microwave-assisted pretreatment of cellulose in ionic liquid for accelerated enzymatic hydrolysis.

For increasing cellulose accessibility to the enzymatic attack, the pretreatment is a necessary step to alter some structural characteristics of cellulosic materials. As a new pretreatment method, microwave irradiation on cellulose dissolution pretreatment with ionic liquids (ILs) was investigated in this study. Microwave irradiation not only enhanced the solubility of cellulose in ILs but also significantly decreased the degree of polymerization of regenerated cellulose after IL dissolution pretreatment, resulting in significant improvement of cellulose hydrolysis. The rate of enzymatic hydrolysis of cotton cellulose was increased by at least 12-fold after IL dissolution pretreatment at 110 °C and by 50-fold after IL dissolution pretreatment with microwave irradiation. Our results demonstrate that cellulose pretreatment with ILs and microwave irradiation is a potential alternative method for the pretreatment of cellulosic materials.

Bi-level optimizing control of a simulated moving bed process with nonlinear adsorption isotherms.

A bi-level optimizing control scheme originally proposed for a simulated moving bed (SMB) with linear isotherms has been extended to an SMB with nonlinear isotherms. Cyclic steady state optimization is performed in the upper level to determine the optimum switching period and time-varying feed/desorbent flow rates, and repetitive model predictive control is run in the lower level for purity regulation, taking the decision variables from the upper level as feed-forward information. Experimental as well as numerical study for an SMB process separating a high-concentration mixture of aqueous L-ribose and L-arabinose solutions showed that the proposed scheme performs satisfactorily against various disturbances. In contrast, an alternative scheme based on an SMB model with linear isotherms showed a limitation in the control performance; this scheme was apt to fail in purity regulation.

Production of lactic acid from paper sludge by simultaneous saccharification and fermentation.

Production of lactic acid from paper sludge has been performed by simultaneous saccharification and fermentation (SSF). The SSF process design was based on experimental data obtained from cellulose hydrolysis and fermentation. The SSF process was employed to avoid an excessively dense solution when the sludge content of the feed was higher than 15%; this is one of several benefits of SSF. The enzyme system used for hydrolysis of paper sludge for production of glucose was optimized. CMCase and beta-glucosidase with activities of 2.5 and 10 U mL(-1), respectively, were found to be optimum for hydrolyzing 5% sludge. In batch SSF 16 g L(-1) lactic acid was produced from 5% paper sludge with an yield of 80%. Paper sludge which served as a feed seemed to have a buffering effect during SSF, probably because of the inorganic ash component in the sludge. The final product concentration by SSF was observed to be limited by the cellulose content of the system, which can probably be resolved by intermittent feeding of the paper sludge. SSF of paper sludge fed in batch mode, with intermittent feeding, produced lactic acid at 162 g L(-1), with a yield of 74% and a productivity of 1.4 g L(-1) h(-1). The lactic acid production performance of the modified bioreactor improved after removal of indigestible solid materials from the upper compartment, which enabled the feed of paper sludge to be increased. A mathematical model is described which predicts glucose and subsequent lactic acid production on the basis of the rate expressions of each step of the SSF process. Saccharification kinetics were determined by experiments on enzymatic cellulose hydrolysis, by use of a Michaelis-Menten equation; growth kinetics of L. rhamnosus were determined by use of a Monod expression which incorporated lactic acid inhibition. The kinetic model is expected to predict the performance of the SSF process. For further use of the lactic acid, i.e. polylactic acid, it must be recovered and purified. Results from application of the simulated moving-bed (SMB) process for separation of lactic acid and acetic acid are given, as are several methods of lactic acid purification.

Capillary electrochromatography and preconcentration of neutral compounds on poly(dimethylsiloxane) microchips.

Capillary electrochromatography (CEC) and preconcentration of neutral compounds have been realized on poly(dimethylsiloxane) (PDMS) microchips. The channels are coated with polyelectrolyte multilayers to avoid absorption of hydrophobic analytes into PDMS. The structures of a microchip include an injector and a bead chamber with integrated frits, where the particles of the stationary phase are completely retained. Dimensions of the frit structures are 25 micro mx20 micro m, and the space between the structures is 3 micro m. A neutral compound, BODIPY, that is strongly absorbed into native PDMS, is successfully and selectively retained on octadecylsilane-coated silica beads in the bead chamber with a concentration enhancement of up to 100 times and eluted with elution buffer solution containing 70% acetonitrile. Preconcentrations and CEC separations of coumarins have been conducted with the same device and achieved complete separations in less than 50 s.