The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono-and diglyceride specific lipase |
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Authors: | Yamaguchi S; Takeuchi K; Mase T; Oikawa K; McMullen T; Derewenda U; McElhaney RN; Kay CM; Derewenda ZS |
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Affiliation: | Tsukuba Research Laboratories, Amano Pharmaceutical Co. Ltd Tsukuba, Japan
2Department of Biochemistry, MRC Group in Protein Structure and Function, University of Alberta Edmonton, Alberta, Canada
3Department of Molecular Physiology and Biological Physics, University of Virginia Charlottesville, VA, USA |
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Abstract: | The extracellular lipase from Penicillium camembertii has uniquesubstrate specificity restricted to mono- and diglycerides.The enzyme is a member of a homologous family of lipases fromfilamentous fungi. Four of these proteins, from the fungi Rhizomucormiehei, Humicola lanuginosa, Rhizopus delemar and P.camembertii,have had their structures elucidated by X-ray crystallography.In spite of pronounced sequence similarities the enzymes exhibitsignificant differences. For example, the thermo-stability ofthe P.camembertii lipase is considerably lower than that ofthe H.lanuginosa enzyme. Since only the P.camembertii enzymelacks the characteristic long disulfide bridge, correspondingto Cys22Cys268 in the H.lanuginosa lipase, we have engineeredthis disulfide into the former enzyme in the hope of obtaininga significantly more stable fold. The properties of the doublemutant (Y22C and G269C) were assessed by a variety of biophysicaltechniques. The extra disulfide link was found to increase themelting temperature of the protein from 51 to 63°C. However,no difference is observed under reducing conditions, indicatingan intrinsic instability of the new disulfide. The optimal temperaturefor catalytic activity decreased by 10°C and the optimumpH was shifted by 0.7 units to more acidic. |
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Keywords: | enzyme engineering/ protein stability/ site-specific/ mutagenesis/ thermostabi 1 ity |
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