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 bovine serum albumin 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.

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KCN and ascorbic acid showed competitive inhibition patterns with Ki, values of 0.032 and 0.2 7 mM, respectively. Uncompetitive inhibition patterns were obtained with sodium azide, L-cysteine and NaCl with Kii values of 3.3 mM, 0.12 mMand 0.3 M, respectively. A noncompetitive inhibition pattern was obtained for thiourea with 0.067 mM for Kis and 0.59 mM for Ki. Cu2+ increased the activity about 2.5 fold at or above 40 p M and K' decreased the enzyme activity about 33% at 0.4 M. Other metal ions did not have any effects on the activity. Two p K values of 5.8 and 8.0 were obtainedfrom V,,profile and two pK values of 5.9 and 8.1 from VmJKm prople. The data suggest that cysteine is likely to be involved in catalysis and histidine in binding. Data from chemical modification show that cysteine was completely inactivated at I. 74 mM o-methylisourea, and histidine and tryptophan were modified at much higher concentrations of diethylpyrocarbonate and N-bromosuccinimide, respectively. It is suggested that the protonated cysteine works as a general base, tryptophan as a substrate binding residue and histidine as a oxygen binding residue.

Polyphenol oxidase (Isozyme I) from potato was extracted and purified with ammonium sulfate, cation-exchange (Bio-Rad Bio-Scale S2) and Sephadex G-I 00 column chromatography. The enzyme was purified 11.8 fold resulting in a spectfic activity of 250.3 unitdmg. Optimum pH of the enzyme was 6.6. Optimum temperature of the enzyme was 40C and its half-life was 0.8 min at 70C. The K, for catechol, pyrogallol, 4-methyl catechol, caffeic acid and L-DOPA were 4. I I mM, 0.61 mM, 0.78 mM, 0.50 mM and 32 mM, respectively. However, monophenols such as tyrosine, p-cresol and I-naphtol did not show any activity. Data for V,JKm which represents catalytic efficiency show that 4-methyl catechol has the highest value. The molecular weight of the active enzyme was 86,000 Da, composed of two identical subunits. The number of Cu2' ions bound was found to be 2 per enzyme molecule. dihydroxy-L-phenylalanine:oxygen oxidoreductase; EC 1.14.18.1) catalyzed oxidation of mono-and diphenols to o-quinones as shown in Scheme I. Polyphenol oxidase is a mixed function oxidase that catalyzes both the ortho hydroxylation of monophenols to o-diphenols (cresolase activity) and the further oxidation o-diphenols to o-quinones (catecholase activity). The o-quinones 'Corresponding author: ykcho@sarim.changwon.ac.kr 589 200 210 220 230 240 250 260 Elution Volume (ml) FIG. 8. MOLECULAR WElGHT DETERMlNATlON OF ISOZYME-I FROM SEPHADEX G-150 CHROMATOGRAPHY 1 : P-galactosidase (1 16 kDa), 2: rabbit phosphorylase (97.5 kDa), 3: bovine serum albumin (66.2 kDa), 4: ovalbumin (45 kDa), 5: carbonic anhydrase (31 kDa)

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 and 50°C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 1.42 and 231 h at 40°C, respectively. The improved stability of EC-TR can be explained by covalent linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40°C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of 1 week. In the same condition, CA-TR showed poor performance from the beginning and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

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