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

Diana Hooi-Ean Ch’Ng,Kumar Sudesh

doi:10.1186/2191-0855-3-22

Abstract

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.

Highlights

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters produced as carbon storage by some bacteria
Two-step cultivation was practiced by using D. acidovorans for the biosynthesis of copolymer
The NM consisted of 0.37 g K2HPO4, 0.58 g KH2PO4, 0.1 mM MgSO4 · 7H2O supplemented with 0.1 ml trace elements (TE) solution. 2.78 g FeSO4 · 7H2O, 1.98 g MnCl2 · 4H2O, 2.81 g CoSO4 · 7H2O, 1.67 g CaCl2 · 2H2O, 0.17 g CuCl2 · 2H2O and 0.29 g ZnSO4 · 7H2O were included in the TE solution. 1,4-butanediol was autoclaved separately and added aseptically to the nitrogen-free MM (1% v/v) as the sole carbon source to promote synthesis of the copolymer

Summary

Introduction

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-4hydroxybutyrate) [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)

Methods

Results

Conclusion