학술논문
Structural and Functional analyses of novel esterases from Lactobacillus acidophilus, Lactococcus garvieae and Leuconostoc citreum
Document Type
Dissertation/ Thesis
Author
Source
Subject
Language
English
Abstract
Lactic acid bacteria (LAB), which are involved in the fermentation of vegetables, meats, or dairy products, are widely used for the productions of small organic molecules and bioactive peptides. Here, a new member of novel acetylesterase (LaAcE) from Lactobacillus acidophilus NCFM was identified, functionally characterized, immobilized, and site-directed mutated for biotechnological applications. Enzymatic properties of LaAcE were investigated using biochemical and biophysical methods including native PAGE, acetic acid release, biochemical assay, enzyme kinetics, and spectroscopic methods. Interestingly, LaAcE exhibited the ability to act on a broad range of substrates including glucose pentaacetate, glyceryl tributyrate, fish oil, fermentation-related compounds. Furthermore, cross-linked enzyme aggregates of LaAcE (CLEA-LaAcE) showed good recycling ability and high thermal stability compared to free LaAcE. A structural model of LaAcE was used to guide mutational analysis of hydrophobic substrate-binding region that is comprosed of Leu156, Phe164, and Val204. Three mutants of L156A, L156A/F164A, L156A/V204A were generated and investigated to elucidate the the roles of hydrophobic residues in substrate specificity. This work provides valuable insight on the properties of LaAcE, and LaAcE can be used as a model enzyme of acetylesterase in lactic acid bacteria, which make LaAcE a great candidate for industrial applications. Key words: LaAcE, acetylesterase, Lactobacillus acidophilus