Chitin-binding domain based immobilization of D-hydantoinase.
Chitin-binding domain (ChBD) of chitinase A1 from Bacillus circulans WL-12 comprises 45 amino acids and exhibits remarkably high specificity to chitin (Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., Shirakawa, M., Watanabe, T., 2000. Expression and characterization of the chitin-binding domain of chintinase A1 from B. circulans WL-12. J. Bacteriol. 182, 3045-3054.). To investigate the feasibility of exploiting ChBD as affinity tags to confine enzymes of interest on chitin, ChBD fused to the C-terminus of the gene encoding D-hydantoinase was constructed. Subsequent expression of the hybrid protein in Escherichia coli gave a soluble fraction accounting for 8% of total cell protein content. Direct adsorption of the ChBD-fused D-hydantoinase on chitin beads was carried out, and SDS-PAGE analysis showed that the linkage between the fusion protein and the affinity matrix was highly specific, substantially stable, and reversible. As compared to its free counterpart, the immobilized D-hydantoinase exhibited higher tolerance to heat and gained a half life of 270 h at 45 degrees C. In addition, the shelf life (defined as 50% of initial activity remained) of the immobilized enzyme stored at 4 degrees C was found to reach 65 days. Furthermore, D-hydantoinase immobilized on chitin could be reused for 15 times to achieve the conversion yield exceeding 90%. Overall, it illustrates the great usefulness of ChBD for enzyme immobilization.
Chern
Jong-Tzer
JT
Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung, Taiwan.
Chao
Yun-Peng
YP
D000327:Adsorption; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000493:pharmacokinetics*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002686:Chitin / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D006207:Half-Life; D006358:Hot Temperature; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000493:pharmacokinetics; D013379:Substrate Specificity; D014170:Transformation, Genetic
D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D002686:Chitin; D000581:Amidohydrolases; C020047:dihydropyrimidinase