Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation

• Published in: 3 Biotech
• Main Authors: Tan, Zhongbiao, Li, Xiangqian, Shi, Hao, Yin, Xiulian, Zhu, Xiaoyan, Bilal, Muhammad, Onchari, Mary Mongina
• Format: Text
• Language: English
• Published: Springer International Publishing 2021
• Subjects: Original Article; Antarc*; Antarctica
• Online Access: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8695645/; http://www.ncbi.nlm.nih.gov/pubmed/35036270; https://doi.org/10.1007/s13205-021-03095-x

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 (Val(139), Ala(146), Leu(147), Pro(218), Val(286), Ala(287), Val(306), and Gly(307)) 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.