Abstract
Methanol tolerance of lipase is one of the important factors affecting its esterification ability in biodiesel preparation. By B factor indicated prediction of Candida antarctica lipase B (CalB) surface amino acids, eight sites (Val139, Ala146, Leu147, Pro218, Val286, Ala287, Val306, and Gly307) with high B value indicating more flexibility were chosen to perform saturation mutagenesis. High-methanol-tolerant variants, CalB-P218W and -V306N, created larger haloes on emulsified tributyrin solid plate including 15% (v/v) methanol and showed 19% and 31% higher activity over wild-type CalB (CalB-WT), 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 transesterified 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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This work was supported by the Huai'an Science and Technology (Guiding) Project (No. HABZ201706), the Open Project of Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration (No. JPELBCPL2014003), the Natural Science Research Project of Huaiyin Institute of Technology (No. 15HGZ007), and the Major Projects of Natural Science Research of Jiangsu Province Colleges and Universities (No. 18KJA530002).
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ZT performed the experiments, wrote the original manuscript, and reviewed the manuscript. XL checked the original data. HS performed the data analysis. XY conceived and designed the experiments. XZ contributed analysis tools. MB reviewed the manuscript. MMO reviewed the manuscript. All authors read and approved the final manuscript.
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Tan, Z., Li, X., Shi, H. et al. Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation. 3 Biotech 12, 22 (2022). https://doi.org/10.1007/s13205-021-03095-x
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DOI: https://doi.org/10.1007/s13205-021-03095-x