Part:BBa_K3051005
Bacillus Subtilis Lipase
Comparative Enzyme Activity Assay
Figure 1. Relative Comparaison Lipase Activity Assay. Candida Antarctica Lipase A, Bacillus Subtilis Lipase and Compost Lipase enzyme activity were examined using p-Nitrophenol octanoate as a substrate.
Figure 1 compares enzyme activity of Candida Antarctica Lipase A BBa_K3051001, Bacillus Subtilis Lipase BBa_K3051005 and Compost Lipase BBa_K3051421. The lipase parts were tested and characterized using a p nitrophenol octanoate assay. The enzyme activity was determined by measuring change in light absorbance at a wavelength of 400nm, which directly correlated to the concentration of p-Nitrophenol (pNP), caused by lipolysis of p-Nitrophenol octanoate (pNPO) substrate. The data from the pNPO lipolysis assay defined the enzyme activity.
Background
Bacillus Subtilis Lipase A (BSAL) is a lipase from Bacillus Subtilis XF-1; identified during a metagenomic analysis of restaurant wastewater containing high levels of FOGS (fat oil and grease)[1]. The organism appears to express a suitability to thrive in high lipid environments, although there is not yet data to support these assumptions. The lipase contains no secretion tag, and does not express noticeable toxicity when added to E.Coli BL21. The enzyme is not secreted, but cells containing the lipase demonstrated a positive lipase activity in a p-Nitrophenol test. The p-Nitrophenol octanoate (pNPO) substrate undergoes lipolysis via BSAL enzyme action to become p-Nitrophenol (pNP), causing increased absorption of light at a wavelength of 400nm. In order for the lipase activity to be observed the cells had to be lysed using a sonicator. Supernatants of cell cultures were not sufficient in providing lipase activity.
The Bacillus Subtilis lipase has a kDa of 24.1, based on the amino acid sequence and has an expected molar absorption coefficent of 24410 M-1 cm-1 (values found through the ProtoPam ExPaSy tool).
Protein Kinetics
Figure 1. BSAL Kinetics Characterisation via Lineweaver-Burk Plot.Bacillus Subtilis lipase has a BSAL has a Km of 0.349mM and a Vmax of 0.00486 mM/min at pH 7 using p-nitrophenol octanoate as a substrate.
The Km and Vmax of the lipase were tested and characterized using a p nitrophenol octanoate assay.
Structure and domains of BSAL
Figure 2. Simulated 3D structure.Structure of BSAL simulated using Phyre 2 modelling.
Sources
1. Sutar, V.P. and Kurhekar, J.V., 2017. ISOLATION AND CHARACTERIZATION OF LIPASE PRODUCING BACTERIA FROM RESTAURANT WASTE WATER.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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