Biotechnology and Bioprocess Engineering 2022; 27(2): 234-243  
Substrate-binding Site Engineering of Candida antarctica Lipase B to Improve Selectivity for Synthesis of 1-monoacyl-sn-glycerols
Ji-Min Woo, Young-Seo Kang, Sun-Mee Lee, Seongsoon Park, and Jin-Byung Park
Ji-Min Woo, Young-Seo Kang, Jin-Byung Park*
Department of Food Science & Engineering, Ewha Womans University, Seoul 03760, Korea
Tel: +82-2-3277-4509; Fax: +82-2-3277-4213
E-mail: jbpark06@ewha.ac.kr
Sun-Mee Lee
Department of Food and Nutrition, Daejeon University, Daejeon 34520, Korea
Seongsoon Park*
Department of Chemistry, Center for NanoBio Applied Technology, Sungshin Women’s University, Seoul 01133, Korea
Tel: +82-2-920-7646; Fax: +82-2-920-2047
E-mail: spark@sungshin.ac.kr
Received: June 2, 2021; Revised: August 19, 2021; Accepted: August 22, 2021; Published online: April 30, 2022.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Lipases are extensively used for regiospecific esterification of polyols with fatty acids. However, side reactions generating byproducts limited the enzymes for industrial applications. Here, we have engineered the substrate-binding site of Candida antarctica lipase B (CALB) to improve selectivity for monoacylation of glycerol using medium chain fatty acids (e.g., nonanoic acid) as acyl donors. The enzyme engineering was based on the substrate-binding region of a lipase from Penicillium camemberti (i.e., Lipase G), which showed very high selectivity for monoacylation of glycerol with medium chain fatty acids (e.g., decanoic acid) but low reaction rates. One of the CALB variants (e.g., CALB_A282E/I285F), which was designed to have a narrow substrate binding region, has exhibited ca. 2-fold greater selectivity for the synthesis of 1-monoacyl-sn-glycerol with nnonanoic acid. The double mutant allowed the production of 1-nonanoyl-glycerol to a concentration of 2.27 M in glycerol to a reaction rate of 1.0 M/h. This study will contribute to the use of lipases for regiospecific esterification of polyols with carboxylic acids.
Keywords: monoacyl glycerol, glycerol, medium chain fatty acids, Candida antarctica lipase B


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