A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

Autor: Nalder TD; Seafood Unit, The New Zealand Institute for Plant & Food Research Limited, 293-297 Akersten Street, Nelson 7010, New Zealand; Centre for Chemistry and Biotechnology, Deakin University, 75 Pigdons Road, Waurn Ponds, Victoria, Australia., Kurtovic I; Seafood Unit, The New Zealand Institute for Plant & Food Research Limited, 293-297 Akersten Street, Nelson 7010, New Zealand. Electronic address: Ivan.Kurtovic@plantandfood.co.nz., Barrow CJ; Centre for Chemistry and Biotechnology, Deakin University, 75 Pigdons Road, Waurn Ponds, Victoria, Australia., Marshall SN; Seafood Unit, The New Zealand Institute for Plant & Food Research Limited, 293-297 Akersten Street, Nelson 7010, New Zealand.
Jazyk: angličtina
Zdroj: Enzyme and microbial technology [Enzyme Microb Technol] 2018 Jun; Vol. 113, pp. 18-23. Date of Electronic Publication: 2018 Feb 17.
DOI: 10.1016/j.enzmictec.2018.02.003
Abstrakt: The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples.
(Copyright © 2018 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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