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