Journal
PROCESS BIOCHEMISTRY
Volume 50, Issue 8, Pages 1218-1223Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2015.04.022
Keywords
Penicillium expansum; Lipase; Lid hinge; Mutagenesis
Categories
Funding
- National Natural Science Foundation of China [81072616]
- Natural Science Foundation of Fujian Province [2015J01129]
- Department of Education, Fujian Province, China [JB 13013, JK2014008]
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Saturation mutagenesis at sites displaying the highest B factors in the lid and the hinge regions of Penicilliurn expansum lipase (PEL) has been employed to improve the efficiency of the lipase in biocatalysis. Replacements of amino acid on beneficial mutants were identified as T66L/D7ON, T66V/D7ON, E83K, E83H and E83N. In substrate specificity assays, T66L/D7ON was significantly more active than wild-type PEL on substrates with medium and long chain lengths. In addition this mutant also displayed a 136.4-fold increase in activity on p-nitrophenyl palmitate. Remarkably, E83K lacked interfacial activation while it was observed in wild-type PEL and the other mutants. Insight into the relation between the mutations and enzymatic properties was gained by modeling and docking studies. All these mutants showed an enhanced catalytic activity, indicating their potential in further application. Therefore, these results indicate the amino acid composition of the lid hinge region plays an extremely important role in the interfacial activation, activity and substrate specificity of PEL. Moreover, the results in this work provide a new clue for selecting critical amino acid residues for the enzyme design. (C) 2015 Elsevier Ltd. All rights reserved.
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