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Methanol tolerance of a lipase is one of the important factors affecting its esteri cation ability in biodiesel preparation. By B factor indicated prediction of Candida antarctica lipase B (CalB) surface amino acids, 8 sites (Val 139 , Ala 146 , Leu 147 , Pro 218 , Val 286 , Ala 287 , Val 306 , and Gly 307 ) with high B value indicating more exibility were chosen to perform saturation mutagenesis. High-methanol-tolerant variants, CalB-P218W and -V306N, created larger haloes on emulsi ed tributyrin solid plate including 15% (v/v) methanol and showed 19% and 31% higher activity over CalB-WT (wild type), respectively. By modeling, a newly formed hydrogen bond in CalB-V306N and hydrophobic force in CalB-P218W contributing more stability in protein may have resulted in increased methanol tolerance. CalB-P218W and -V306N transesteri ed the soybean oil into biodiesel at 30 °C by 85% and 89% yield, respectively, over 82% by CalB-WT for 24 h reactions. These results may provide a basis for molecular engineering of CalB and expand its applications in fuel industries. The as-developed semi-rational method could be utilized to enhance the stabilities of many other industrial enzymes.

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