Polycaprolactone (PCL), an important polymer due to its strong mechanical properties, biodegradability, and miscibility with a number of other polymers (Kim and Rhee 2003, Labet and Thielemans 2009), has been investigated for its degradation in terrestrial and aquatic environments. A number of PCL-degrading bacterial and fungal strains of Alcaligenes, Clostridium, Aspergillus, Penicillium, Fusarium, and Streptomyces have been isolated (Tokiwa et al. 1976, Benedict et al. 1983a, 1983b, Tokiwa et al. 2009). In this study, we isolated a PCL-degrading bacterium from soil and identified it as Streptomyces thermoviolaceus subsp. thermoviolaceus based on its morphology, growth characteristics, and 16S rRNA gene sequence. This isolate was designated 76T-2. It grew well and formed clear zones on PCL emulsified agar plate at 45-50°C in one day, indicating that it is a fast-growing thermophile. Its PCL degradation ability was confirmed by demonstrating that the culture supernatant contained PCL-degrading enzymes.
Two PCL depolymerases of approximately 55 and 25 kDa in size were detected. Although several extracellular PCL depolymerases have been isolaed (Murphy et al. 1996, Oda et al. 1997, Li et al. 2012), few of them have been fully characterized. Therefore, there is no previous report on the size range of bacterial or fungal PCL depolymerases. To our knowledge, this is the first report describing the characterization of a purified PCL depolymerase from a thermophilic Streptomyces. Based on its N-terminal amino acid sequence and activity against chitin, we consider the 25 kDa enzyme a chitinase with PCL-degrading activity.
PCL can be degraded by two types of esterases: One is lipase, and the other is cutinase. One example of lipase with PCL-degrading activity is the lipase from Alcaligenes faecalis (Oda et al. 1997). Cutinase is a serine hydrolase which breaks ester bonds in cutin. Fusarium is one example of bacteria producing such enzyme (Murphy et al. 1996). Chitin is a polymer of β-1,4-N-acetylglucosamine in insects, crustaceans, fungi, and algae. The finding that the 25-kDa PCL depolymerase from 76T-2 had chitinase activity is novel as no other PCL depolymerases have been found to have such activity. Therefore, the 25-kDa enzyme isolated from 76T-2 may be a novel type of PCL depolymerase. Since isolate 76T-2 does not form clear zones on PHB-containing agar plate and there is no report that PCL depolymerases have PHA degrading activity, it is likely that the PCL depolymerases of 76T-2 do not degrade PHA.
Zymographic studies revealed another PCL depolymerase with a molecular mass of 55 kDa, and the purified 55-kDa protein was able to degrade PCL. Although numerous attemps were made, we were unable to determine the N-terminal amino acid sequence of this protein. Since the molecular mass of the Fusarium PCL depolymerase is 25 kDa and that of lipases are larger (30 kDa) (Murphy et al. 1996), we focused our study on the 25-kDa PCL depolymerase.
Four chitinases (Chi40, Chi35, Chi30 and Chi25) have been identified in Streptomyces thermoviolaceus OPC-520 (Tsujibo et al. 1993). Based on amino acid sequence similarity of their catalytic domains, Chi40 and Chi30 are considered as members of family 18, and Chi35 and Chi25 are classified as family 19 of glycosyl hydrolases (Tsujibo et al. 2000b). Chi40 is the major chitinase of Streptomyces thermoviolaceus OPC-520 secreted into the culture medium. Since Chi25 and Chi35 are produced in very low levels, they are postulated to function as antifungal agents rather than as major enzymes to digest chitin (Tsujibo et al. 2000a). Genes encode for Chi25 and Chi35 have been cloned and found to be arranged in tandem. The deduced amino acid sequences of the catalytic domains of Chi25 and Chi35 are highly similar to each other. In addition to the catalytic domain, Chi35 has a N-terminal domain for polysaccharide binding (Tsujibo et al. 2000b) and sequences similar to certain domains of bacterial polymer-degrading enzymes such as xylanase I of S. thermoviolaceus OPC-520 (Tsujibo et al. 1997).
Although chitinases from many Streptomyces species have been extensively studied with respect to their structure, function, and regulation of gene expression (Saito et al. 1999, Kim et al. 2003, Saito et al. 2003, Okazaki et al. 2004, Yano et al. 2008, Hoang et al. 2011), few studies have been conducted on the degradation of biodegradable plastic by chitinases. In this study, we found that the 25-kDa PCL-degrading enzyme is homologous to the chitinase (Chi25) of Streptomyces thermoviolaceus OPC-520 (GenBank accession number AB016843) (Tsujibo et al. 2000a, 2000b). Further study of the 25-kDa PCL-degrading enzyme from 76T-2 will advance our knowledge about the PCL depolymerases of S. thermoviolaceus.