Lid Swapping On The Lipase A From Burkholderia Sp. ZYB002 Shifted The Chimeras' Substrate Specificity

To explore the role of lid domain in substrate specificity, the lid domain of LipA from Burkholderia sp. ZYB002 was substituted for the coreesponding domain from Ophiostoma piceae cholesterol esterase ?OPE?, Candida rugosa lipase Lip2 and C. rugosa lipase Lip3 respectively. Five LipA mutants were constructed, purified and characterized. The main results can be summarized as follows:Based on the known 3D structural information from Burkholderia cepacia lipase LipA ?PDB accession number:3LIP?, the three-dimensional structure model of Burkholderia sp. ZYB002 lipase LipA was constructed, and then optimized using the YASARA software. The lid structure from lipase LipA was substituted for the corresponding domain from OPE, Lip2 and Lip3, which result in five mutants, including BCL-LOPE1, BCL-LOPE2, BCL-LLip2, BCL-LLip31 and BCL-LLip32, respectively. The above five lipase variants was constructed using overlap extension PCR technique, and then verified by sequencing. The homologous lipase LipA/LipB complex was obtained using His Trap^TM FF affinity column and Hi Trap DEAE FF ion exchange column, while other four homologous lipase variants LipA/LipB complexs were obtained using His Trap^TM FF affinity column and Hi Trap^TM Phenyl FF ?low sub? hydrophobic column. The K_m value and Vmax value for 4-nitrophenyl laurate hydrolysis were assayed.The results showed that:the K_m value for the variant BCL-LOPE2 and lipase LipA is 0.14 ± 0.03 mmo/L and 0.17±0.03 mmol/L, respectively. Other variants showed weaker affinity towards substrate than the wild-type, which corresponded to the higher K_m value of the variants. The V_max valus of all four variants were decreased.