Characterization Of The Depolymerizing Activity Of Commercial Lipases And Animal Organ Extracts Using A Simple Polyhydroxyalkanoate-Based Microassay
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Date
2015-09
Authors
Mok, Pei Shze
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Abstract
Lipase is an enzyme that is widely used in different applications and it is important to screen its activity before application. However, most of the available lipase activity assays require toxic chemicals and tedious preparation. Therefore, a solid substrate known as poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is used and it can be degraded by lipases. This substrate is biodegradable and does not require toxic chemical. In this study, 2.0 ± 0 g/L of cells consisting 28 ± 2 % of PHA with 92 ± 1 mol % of 4HB were produced through biosynthesis. The stability of P(3HB-co-4HB) films was evaluated by storing them in different locations, in vacuum oven, desiccators and bench for 15 months. The film was found to be the most stable in desiccators containing silica gel. The depolymerizing activity of different known commercial lipases and animal organ extracts using P(3HB-co-92 mol% 4HB) as substrate was investigated via microassay. The depolymerizing activity of lipases on P(3HB-co-4HB) was higher in range of pH 6 to 8 and temperature above 30 °C but lower than 50 °C. Addition of metal ions and detergents in different concentrations had variable effects on the depolymerizing activity of commercial lipases. For instance, 1 mM of calcium ions stimulated depolymerizing activity of lipase from porcine pancreas and Rhizopus arrhizus but inhibited the activity of other lipases. For lipase from Candida rugosa, 0.1 % of Triton X-100 inhibited its depolymerizing activity while 0.1 % of SDS, Tween 20 and Tween 80 stimulated its activity. Besides commercial lipases, the crude extracts of different organs from mice and chicken were also screened for the presence of depolymerizing activity. The duodenum and pancreas from both mice and chicken showed depolymerizing activity on the P(3HB-co-4HB) film. This for the first time has produced a direct evidence for the involvement of enzymes in the degradation of this PHA in animals. Lipase is the most likely enzyme from pancreas that was involved in the degradation.
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