Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1

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Abstract

Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and by-products of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GC–MS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH4)2SO4 fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDS–PAGE), but was confirmed by matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N3−). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca2+, Mg2+, Na+, and K+. The kinetic parameters, Vmax and Km, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 × 10−1 μmol min−1 and 6.73 × 10−1 mM, respectively.

Highlights

Streptomyces sp. IN1 produced a thermoalkanophilic P(3HB-co-3HV) esterase. ► The esterase had molecular weight 62 kDa, optimum activity at 80°C and pH 12. ► Hydrophobic regions, disulphide bonds, but not metalions, are required for activity.

Introduction

There is increasing interest in the identification of novel polyhydroxyalkanoate (PHA)-degrading microorganisms because of their biotechnological potential as sources of extracellular esterases. These enzymes could play a significant role in the degradation of industrial pollutants and natural materials such as PHA (Lenz and Marchessault, 2005). Degradation of extracellular PHA and the use of its by-products as a source of carbon and energy is controlled by the secretion of specific extracellular PHA depolymerase(s). These may be specific for medium-chain-length PHA or short-chain-length (scl) PHA, such as poly (3-hydroxybutyrate), P(3HB), or its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV) (Ha and Cho, 2002, Kim et al., 2002, Gonçalves and Martins-Franchetti, 2009).

Although significant strides have been made in extracellular PHA depolymerase research, the majority of studies have focused on Gram-negative bacteria having optimum depolymerase activity ≤65 °C and pH ≤10. Notable exceptions were observed for the depolymerase from Comamonas testosteroni ATSU with an optimum temperature at 70 °C (although activity was not retained at this temperature) (Kasuya et al., 1994), and Paucimonas lemoignei with an optimum pH of 12 (Handrick et al., 2001). Fewer reports have addressed the isolation and characterization of depolymerases, particularly thermophilic and/or alkanophilic depolymerases, from Gram-positive bacteria, especially Streptomyces sp., despite their abundance in soil (Klingbeil et al., 1996, Marbrouk and Sabry, 2001, Kim et al., 2003, Calabia and Tokiwa, 2006). However, none of these depolymerases had molecular mass ≥50 kDa or optimum pH and temperature above 10 and 60 °C, respectively.

In our previous report, the copolymer P(3HB-co-3HV) was produced from saponified Jatropha curcas oil (Allen et al., 2010). Here, we report on the biodegradation of P(3HB-co-3HV) by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1.

Section snippets

Isolation of PHA-degrading bacteria

Soil was collected, dried to constant weight at 30 °C, and serially diluted using 0.1% proteose peptone. Inocula were plated onto PHA latex medium (PHALM) and incubated under aerobic and anaerobic conditions at 30 °C over 2–10 days or until zones of clearing were visible. PHA degraders were then sub-cultured onto 0.4% (w/v) PHALM, and those that exhibited rapid degradation of the substrate were used for further analyses. Sterilized soil was used as a control.

Growth medium (PHA latex medium)

A 0.4% PHALM (w/v) was prepared by

Isolation of PHA-degrading bacteria

PHA-degrading bacteria were isolated based on clear zones of hydrolysis on PHALM plates (Fig. 1, Fig. 2). Although only five degraders were isolated from the soil sample under aerobic conditions, only one, isolate IN1, showed rapid degradation of the substrate over 10 days. Therefore, this isolate was used for all analyses. PHA degraders were not isolated under anaerobic conditions or in the control (autoclaved soil sample).

Phenotypic characterization of isolate

The isolate was a Gram-positive rod, non-motile, catalase positive,

Discussion

In the current study, a novel thermoalkanophilic P(3HB-co-3HV) esterase from the soil bacterium Streptomyces sp. IN1 (GenBank accession JF268582) was isolated and partially characterized. Presumptive identification was made in accordance with the methods as outlined by the International Streptomyces Project (ISP). Growth responses for Streptomyces sp. IN1 in PHA latex broth and agar (PHA, 0.4%, w/v) were obtained by interpolating optical densities (OD) at 650 nm or zone of clearing (mm) versus

Acknowledgments

We would like to express our sincere gratitude to Dr. Oumar Diall from the University of Mali for the gift of the Jatropha curcas oil. Some aspects of this work were supported through the Howard Hughes Core Laboratory at Howard University.

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