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Feruloyl esterases, effects of glycosylation on activity, stability and immobilization

Cyrielle Bonzom (Institutionen för biologi och bioteknik, Industriell bioteknik) ; Sun-Li Chong (Institutionen för biologi och bioteknik, Industriell bioteknik) ; Silvia Hüttner (Institutionen för biologi och bioteknik, Industriell bioteknik) ; Laura Iancu ; Lisbeth Olsson (Institutionen för biologi och bioteknik, Industriell bioteknik)
12th Carbohydrate Bioengineering Meeting (2017)
[Konferensbidrag, poster]

Feruloyl esterases (FAE; EC 3.1.1.73) are a subclass of carboxylic ester hydrolases. They catalyze the hydrolysis of ester linkages in plant cell walls materials releasing ferulic acid and other hydroxycinnamic acids and therefore have been classified in the CAZy database [1] in the carbohydrate esterase (CE) family. Currently, all FAEs in the CAZY database belong to the CE1 family. Immobilization is a powerful tool to allow enzyme reuse and therefore decrease their cost in the overall process. Immobilization on solid carriers can be achieved by different approaches, one of them is by physical adsorption. This technique relies mainly on the surface charges of the support and enzyme. Mesoporous silica particles (MPS) are porous supports made of silica which pore size can be tuned from 2 to 50 nm depending on the synthesis conditions [2]. Because they are negatively charged, silica particles allow for rapid enzyme immobilization by physical adsorption. Furthermore, the diameter of their pore size can be tuned to match the one of the enzyme used and greatly reduces leaching after immobilization. Depending on the production host of the enzyme, the glycosylation extent and/or pattern can vary from none in Escherichia coli to hyper-glycosylation in Pichia pastoris. Glycosylation has been shown to alter activity and stability of enzymes [3]. Our hypothesis was that glycosylation would influence the immobilization behavior of the studied FAE in MPS as well as its activity. In this study, the same enzyme MtFae1a from the fungus Myceliophtora thermophila has been expressed in three different host organisms: E. coli, M. thermophila and P. pastoris to achieve different degrees of glycosylation. The proteins were then purified and biochemically characterized. Different behaviors were observed depending on the production host of the enzymes, for instance in terms of specific activity and temperature optima. Furthermore, glycosylation affected the immobilization capacity of the enzymes on MPS, as well as the activity of immobilized FAEs. [1] B.L. Cantarel, P.M. Coutinho, C. Rancurel, T. Bernard, V. Lombard, B. Henrissat, The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics, Nucleic Acids Res. 37 (2009) D233–238. doi:10.1093/nar/gkn663. [2] D. Zhao, J. Feng, Q. Huo, N. Melosh, G.H. Fredrickson, B.F. Chmelka, G.D. Stucky, Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores, Science. 279 (1998) 548–552. doi:10.1126/science.279.5350.548. [3] A. Basso, P. Braiuca, S. Cantone, C. Ebert, P. Linda, P. Spizzo, P. Caimi, U. Hanefeld, G. Degrassi, L. Gardossi, In Silico Analysis of Enzyme Surface and Glycosylation Effect as a Tool for Efficient Covalent Immobilisation of CalB and PGA on Sepabeads®, Adv. Synth. Catal. 349 (2007) 877–886. doi:10.1002/adsc.200600337.



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Denna post skapades 2018-01-12.
CPL Pubid: 254450