Research Article: Densitometry based microassay for the determination of lipase depolymerizing activity on polyhydroxyalkanoate

Date Published: May 8, 2013

Publisher: Springer

Author(s): Diana Hooi-Ean Ch’ng, Kumar Sudesh.


A novel method for the assay of polyhydroxyalkanoate (PHA)-degrading ability of triacylglycerol lipases was developed. By applying the natural affinity of lipases towards hydrophobic interfaces, a sensitive and rapid densitometry analysis for the evaluation of hydrolytic activity of lipase droplets towards PHA-coated surface was successfully carried out. We found that 12 out of 14 tested lipases which are of fungal, bacterial and animal origin were able to hydrolyze P(3HB-co-92 mol% 4HB) thin film. The patterns and opacity of the hydrolysis spots of lipases on PHA films allowed easy comparison of PHA-hydrolytic strength of lipases. Lipase from the bacterium Chromobacterium viscosum exhibited the highest PHA-degrading activity. The hydrolytic activity of lipases on water insoluble PHA, emulsified p-nitrophenyl laurate and olive oil were also compared and interestingly some lipases showed better activity when PHA was used as a substrate.

Partial Text

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters produced as carbon storage by some bacteria. The most common type of PHA is polyhydroxybutyrate (PHB), which consists of 3-hydroxybutyrate (3HB) monomer units. Besides 3HB, more than 140 different types of monomers have been discovered as the monomer constituents of PHAs (
Steinbüchel 2005
). Out of the many types of monomers discovered to date, 4-hydroxybutyrate (4HB) provides interesting properties to PHAs. The incorporation of 4HB in a polymer chain containing 3HB results in the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer. The higher the 4HB monomer composition in the copolymer, the more stretchable is the resulting polymer (
Nakamura et al. 1992

In the present study, 12 out of 14 lipases tested showed positive signs of polymer degradation by using the novel microassay without the addition of emulsifiers or other supplements (Table 2). A small droplet of concentrated lipase solution was sufficient to show very convincing evidence of polymer degradation (opaque marks) in a matter of minutes. The work of Timmins and coworkers (
Timmins et al. 1997
) further supports the idea of increasing opacity of transparent polymer film by the action of depolymerase enzymes. This may be due to the enzyme acting first on the amorphous part of the film, which leaves out the crystalline portion which is more opaque (
Kumagai et al. 1992
Sudesh et al. 2000
). It may also be simply due to the corrosion of the smooth surface of the polymer film by the action of enzymes, which affected the light scattering property of the film, causing it to lose transparency.

DHEC and KS have filed for patent application for the method to assay PHA-degrading activity of triacylglycerol lipases described in this paper.

DHEC and KS participated in the design of the experiment. DHEC performed all the experiments. DHEC and KS analyzed the data. DHEC wrote the manuscript. KS edited the manuscript. All authors read and approved the final manuscript.




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