We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Pseudomonas alcaligenes脂肪 (LipA)純化與特性分析.
- Authors
林 佳 慧; 吳 蕙 芬
- 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, using 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, Ser111, 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℃ and 70℃. Specifically, it had the highest activity between 40℃~ 50℃. At pH 8, the enzyme activity was also optimal. LipA retains its stability at pH 8 and 20℃. 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.
- Publication
Taiwanese Journal of Agricultural Chemistry & Food Science, 2019, Vol 57, Issue 4, p165
- ISSN
1605-2471
- Publication type
Academic Journal
- DOI
10.6578/TJACFS.201908_57(4).0001