Enzymatic degradation of polyhydroxyalkanoate using lipase from Bacillus subtilis

Abstract

Polyhydroxyalkanoates (PHAs) are an important class of biodegradable polymers synthesized by a few bacteria under nutrient-limiting conditions. In this study, the lipase-catalysed degradation of PHA synthesized by Enterobacter sp. was monitored. For this, the lipase-encoding gene from Bacillus subtilis DI2 was PCR-amplified, cloned into a T vector system and sequenced. It was expressed in Escherichia coli DH5a cells, the recombinant enzyme was purified 24.25-fold, and its molecular weight was determined to be around 28 kDa. When PHA biodegradation studies were carried out with this enzyme, gel permeation chromatography showed 21.3 and 28.3 % molecular weight decrease and weight loss, respectively. Further, scanning electron micrographs revealed alterations in polymer surface morphology. Changes in molecular vibrations were noticed in the FTIR spectra. When the chemical shifts in NMR spectra were studied, a steep reduction in area under the peak at 1.57 ppm was observed. In the heating range of 30-930°C employed during thermogravimetry analysis, the degraded sample showed a total of 45.82 % weight loss, as against 18.89 % for the native sample. The melting temperature (T m ) of the polymer was also brought down from 126.22 to 118.18°C, as inferred from differential scanning calorimetry. Lipase-catalysed chain scission reactions could thus be used to generate low molecular weight functional biopolymers with wide-ranging pharmaceutical applications, such as in sustained drug release.

Influence of Depolymerases and Lipases on the Degradation of Polyhydroxyalkanoates Determined in Langmuir Degradation Studies

“…[ 32 ] In the past, few lipases from eukaryotic and prokaryotic origin were shown capable of degrading PHB and PHAs consisting of an ω‐hydroxyalkanoic acid such as poly(6‐hydroxyhexanoate), poly(4‐hydroxybutyrate) or their blends. [ 22,45,49–51 ] However, only minor changes for β‐PHAs polymers were found in most studies; for instance 7% and 3% weight loss for PHB‐ co ‐12%‐HHx and PHB, respectively, after 50 days incubation in PBS plus lipase 100 T (Novozymes), [ 13 ] which is not significant when compared to results on pure hydrolysis of PHB in PBS. [ 28 ] Based on our results, we support the idea that PHAs composed of β‐hydroxyalkanoic acid repeating units are not degraded by common microbial lipases, chosen based on previous reports in the field.…”

Enabling electrospinning of medium-chain length polyhydroxyalkanoates (PHAs) by blending with short-chain length PHAs

“…The lipase-encoding gene was expressed in Escherichia coli cells and the recombinant lipase was purified for PHA degradation studies. After 72 h of incubation at 40 °C, it showed 21.3% and 28.3% molecular weight decrease and weight loss, respectively [88].…”

“…As lipase concentrations increased from 0.5 to 2.5 mg/mL, more holes were produced on the film (Fig 2-6) [87]. Bacillus subtilis DI2, a lipolytic strain, has been used for lipase-catalysed degradation of PHA [88].…”

Enzymatic Hydrolysis of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Scaffolds

“…On the other hand, PHAs are very prospective materials for medical applications requiring the stability and slow decomposition of materials. However, PHAs can be non-specifically degraded by some ubiquitous hydrolases such as lipases, which are omnipresent enzymes in the human body environment [24]. The course of PHA degradation depends on several parameters.…”