The effect of substitution of Phe181 and Phe182 with Ala on activity,substrate specificity and stabilization of substrate at the active site of Bacillus thermocatenulatus lipase |
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| Affiliation: | 1. Sustainable Environment Research Centre, Faculty of Computing Engineering and Science, University of South Wales, Pontypridd CF37 1DL, United Kingdom;2. HyET Hydrogen Efficiency Technologies B.V., Leemansweg 15, 6827 BX Arnhem, The Netherlands;1. State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074, PR China;2. Department of Environmental Sciences and Engineering, School of Environmental Studies, China University of Geosciences, Wuhan 430074, PR China;3. Department of Geography, Faculty of Earth Sciences, China University of Geosciences, Wuhan 430074, PR China;4. UC Davis Stable Isotope Facility, Department of Plant Sciences, One Shields Avenue, Davis, CA 95616, USA;1. Shandong Key Laboratory of Microbiology, College of Plant Protection, Shandong Agricultural University, Taian, Shandong 271000, China;2. State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China;3. School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China;1. Austrian Institute for Nonlinear Studies, Akademiehof, Friedrichstr. 10, 1010 Vienna, Austria;2. Institute for Atomic and Subatomic Physics, Vienna University of Technology, Operng. 9, 1040 Vienna, Austria;1. Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, T1J 4B1, Canada;2. Key Laboratory for Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, Hunan, 410125, China;3. Department of Production Animal Health, Faculty of Veterinary Medicine University of Calgary, Calgary, AB, T2N 4Z6, Canada |
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| Abstract: | Steric hindrance leads to limitation in the access of substrate into the enzyme active site. In order to decrease steric hindrance, two conserved residues, Phe181 and Phe182, in the lid domain of Bacillus thermocatenulatus lipase were substituted with alanine by using site-directed mutagenesis. As a result, three mutant lipases were produced. Circular dichroism (CD) spectroscopy showed that the secondary structure of all lipases is similar to one another. F181A mutation increased the distance between phe181 and catalytic ser114, which is buried in the active site by 3.24 Å. It can be suggested that such an increase in distance may lead to a decrease in steric hindrance. F181A mutation increased overall lipase activity by up to 2.6-fold (4670 U mg−1) toward C8 substrate. It also resulted in optimal lipase activity at 65 °C rather than 55 °C. F182A mutation increased the distance between phe182 and catalytic ser114 by 1.54 Å but failed to induce any significant effect on lipase activity. However, F181A–F182A mutation significantly decreased the activity due to decreased van der Waals interactions between the phenyl group of phenylalanines and the acyl chain of triacylglycerol. These results indicate that presence of one of the two residues, Phe181 or Phe182, is important for stabilizing triacylglycerols in active site. |
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