Pseudomonas alcaligenes脂肪酶（LipA）純化與特性分析
Journal
臺灣農業化學與食品科學
Journal Volume
57
Journal Issue
4
Pages
165-172
Date Issued
2019
Author(s)
林佳慧(Chia-Hui Lin)
Abstract
Pseudomonas alcaligenes, which was isolated from the environments, could secret extracellular lipases and grown on tributyrin agar plate with surrounded transparent zone. By the bioinformatics analysis, the amino-acid sequences of its LipA were referred to the phylogenetic tree analysis. The LipA belongs to lipase family 1, which needs a lipase-specific foldase (LifB) to assist an active folding of itself. In this study, uping the dual active promoter plasmid, lipA+-ha and lifB+ genes were in series cloned into pACYC-duet-1. Through a coincident expression of LipA and LifB by IPTG induction, the soluble LipA protein could be purified and detected by Western blot analysis. By amino-acid sequence alignment, an active site, Seri 11, Asp257 and His279, were revealed in LipA. Ser 111 was in a conserved position, GHSHG and a catalytic triad site was formed with the other two amino acids. In the degradation assay, LipA preferred to degrade short chain p-nitrophenyl esters. Especially, LipA degraded pNPC2 (p-nitrophenyl acetate) efficiently. Therefore, LipA belongs to the esterase family (3.1.1.1). The optimal temperatures for LipA activity are wide-range; it maintains a higher activity between 30°C and 70°C. Specifically, it had the highest activity between 4Q°C~ 50°C. At pH 8, the enzyme activity was also optimal. LipA retains its stability at pH 8 and 20°C. In addition, LipA could tolerate most of the metal ions used in the assays. However, its enzymatic activity was inhibited by Zn2+. In contrast, its enzymatic activity was elevated by nonionic-surfactants, Brij 35, but it was inhibited by ionic-surfactant, SDS. Similarly, the enzymatic activity of LipA was inhibited by most organic solvents, such as isopropanol.
Publisher
臺灣農業化學會&臺灣食品科學技術學會
Type
journal article