Deleting the Ig-Like Domain of Alicyclobacillus acidocaldarius Endoglucanase Cel9A Causes a Simultaneous Increase in the Activity and Stability
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Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.
KeywordsEndoglucanase Cel9A Ig-like domain Catalytic efficiency Thermal stability Enzyme rigidity Calcium
Alicyclobacillus acidocaldarius endoglucanase Cel9A
AaCel9A without Ig-like domain
carbohydrate binding domain
We thank Professor S. Moréra (from Laboratoired’Enzymologie et BiochimieStructurales (LEBS), CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France) for the gift of pDEST17-AaCel9A. The authors express their gratitude to the research council of Azarbaijan Shahid Madani University for the financial support during the course of this project.
Compliance with Ethical Standards
Conflict of interest
The authors declare that there are no conflicts of interest.
- 9.Tomme, P., Driver, D. P., Amandoron, E. A., Miller, R. C, Jr, Antony, R., Warren, J., & Kilburn, D. G. (1995). Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain. Journal of Bacteriology, 177, 4356–4363.Google Scholar
- 14.Kataeva, I. A., Seidel, R. D, 3rd, Shah, A., West, L. T., Li, X. L., & Ljungdahl, L. G. (2002). The fibronectin type 3-like repeat from the Clostridium thermocellum cellobiohydrolase CbhA promotes hydrolysis of cellulose by modifying its surface. Applied and Environment Microbiology, 68, 4292–4300.CrossRefGoogle Scholar
- 15.Kataeva, I. A., Uversky, V. N., Brewer, J. M., Schubot, F., Rose, J. P., Wang, B. C., & Ljungdahl, L. G. (2004). Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA. Protein Engineering, Design and Selection, 17, 759–769.CrossRefGoogle Scholar
- 18.Verjans, P., Dornez, E., Segers, M., Van Campenhout, S., Bernaerts, K., Belien, T., et al. (2010). Truncated derivatives of a multidomain thermophilic glycosyl hydrolase family 10 xylanase from Thermotoga maritima reveal structure related activity profiles and substrate hydrolysis patterns. Journal of Biotechnology, 145, 160–167.CrossRefGoogle Scholar
- 22.Eckert, K., Zielinski, F., Lo Leggio, L., & Schneider, E. (2002). Gene cloning, sequencing, and characterization of a family 9 endoglucanase (CelA) with an unusual pattern of activity from the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009. Applied Microbiology and Biotechnology, 60, 428–436.CrossRefGoogle Scholar
- 28.Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.Google Scholar
- 29.Han, Q., Liu, N., Robinson, H., Cao, L., Qian, C., Wang, Q., et al. (2013). Biochemical characterization and crystal structure of a GH10 xylanase from termite gut bacteria reveal a novel structural feature and significance of its bacterial Ig-like domain. Biotechnology and Bioengineering, 110, 3093–3103.CrossRefGoogle Scholar
- 34.Bork, P., Holm, L., & Sander, C. (1994). The immunoglobulin fold. Structural classification, sequence patterns and common core. Journal of Molecular Biology, 242, 309–320.Google Scholar
- 41.Pingali, S. V., O’Neill, H. M., McGaughey, J., Urban, V. S., Rempe, C. S., Petridis, L., et al. (2011). Small angle neutron scattering reveals pH-dependent conformational changes in Trichoderma reesei cellobiohydrolase I: implications for enzymatic activity. Journal of Biological Chemistry, 286, 32801–32809.CrossRefGoogle Scholar