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

Abstract

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.

Enabling technology and core theory of synthetic biology

“…Considerable progress has been made with the creation of "smart libraries" (Qu et al, 2020). These approaches have been reported to successfully improve enzyme properties such as thermostability, catalytic activity, and enantioselectivity (Fan et al, 2022;Tan et al, 2022;Tong et al, 2022). The combination of enzyme engineering and systems metabolic engineering has also significantly increased the metabolic flux of target products (Qian et al, 2019;Yang et al, 2020).…”

Artificial Intelligence Aided Lipase Production and Engineering for Enzymatic Performance Improvement

“…strain from a high-salinity and high-alkalinity environment to produce thermoalkaline lipase . Tan et al improved the tolerance of Candida antarctica lipase B to methanol for biodiesel preparation . Adequate thermostability, resistance to harmful organic solvents, and especially the absence of unwanted aggregation after development are prerequisites for practical (industrial) applications in organic chemistry and biotechnology.…”

“…To better improve enzyme activity, it is essential to identify the amino acid sites directly related to enzyme activity. Many methods, e.g., the fluorescence organophosphorus ester method and B value method, , have been successfully utilized to find active sites more conveniently. In the mature structure of the enzyme, the active site is generally located in the “gap”, and amino acid residues at the site may be neighboring while being far away in the primary structure due to the folding required by the tertiary structure, which may lead to inaccuracies and errors in active-site forecasting.…”