PHB depolymerases

The thickness of polymer films dropped from 65 to 22 pm (32% of initial thickness) during incubation. The scanning electron microscopic examination showed that the surface of the PHB film after enzymatic degradation was apparently blemished by the action of PHB depolymerase, while no change was observed inside the film. Moreover, the molecular weight of PHB remained almost unchanged after enzymatic hydrolysis the of PHB decreased from 768 to 669 kDa or unchanged (22 kDa) [25-26]. 

The extensive literature data on enzymatic degradation of PHB by specific PHB depolymerases was collected in detail in review of Sudesh, Abe, and Doi [45]. We would like to summarize some the most important data. However, at first it is necessary to note that PHB depolymerase is... 

Spyros, A. Kimmich, R. Briese, B. H. and Jendrossek, D. IH NMR Imaging Study of Enzymatic Degradation in Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Evidence for Preferential Degradation of the Amorphous Phase by PHB Depolymerase B Irom Pseudomonas lemoignei. Macromole. 1997,... 

Calabia BP, Tokiwa Y (2006) A novel PHB depolymerase from a thermophilic Streptomyces sp. Biotechnol Lett 28 383-388... 

Kasuya K-i, Inoue Y, Doi Y (1996) Adsorption kinetics of bacterial PHB depolymerase on the surface of polyhydroxyalkanoate films. Int J Biol Macrom 19 35 0 Kasuya K-i, Ohura T, Masuda K, Doi Y (1999) Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases. Int J Biol Macromol 24 329-336 Kato M, M. HJB, Kang CK, Fukui T, Doi Y (1996) Production of a novel copolyester of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids by Pseudomonas sp. 61-3 from sugars Appl Microbiol Biotechnol 45 363-370 Kaushik N, Kumar K, Kumar S, Kaushik N, Roy S (2007) Genetic variability ruid divergence studies in seed traits and oil content of Jatropha (Jatropha curcas L.) accessions. Biomass Bioenerg 31 497-502... 

Shin M, Yoshimoto H, Vacanti JP (2004) In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold. Tissue Eng 10 33-41 Shinomiya M, Iwata T, Doi Y (1998) The adsorption of substrate-binding domain of PHB depolymerases to the surface of poly(3-hydroxybutyric acid). Int J Biol Macromol 22 129-135 Sim YC, Sudesh K (Unpublished). Annual report 2009 (2009) http //www.simedaiby.com/ downloads/pdfs/SDB/Annual Report/Sime Darby AR2009.pdf. Accessed online. Accessed Oct 1 2010... 

Lee and coworkers [53] demonstrated that PHB could be efficiently produced in vivo by manipulating the environmental conditions. In studying Alcaligenes latus, for example, it was found that providing a more acidic environment, on the order of pH to 3-4, induced a high activity level of intracellular PHB depolymerase. Ren and coworkers [54] placed PHA-containing P. putida cells in phosphate buffer at different pHs and determined that a more basic pH, i.e., pH 11, was the most efficient. 

Another basic issue to be considered was represented by the influence of PHB on the biodegradability of PVA in the presence of specific PVA-degrading microorganims, by considering that biodegradation of the polyester and of the polyhydroxylated polymer is strictly mediated by PHB-depolymerase and PVA-oxidase specific enzymes, respectively. In this connection, the biodegradability of both a graft copolymer and a cast blend was ascertained in the presence of a select bacterial culture able to utilize PVA. 

Focarete, M.L., Ceccorulli, G., Scandola, M., and Kowalczuk, M., 1998, Further evidence of crystallinity-induced biodegradation of synthetic atactic poly(3-hydfoxybutyrate) by PHB-depolymerase A from Pseudomonas lemoignei. Blends of atactic poly(3-hydroxybutyrate) with ciystalline polyesters. Macromolecules 31 8485-8492. 

Due to the differences between the physical structures of the intracellular native granules and extracellular denatured PHA, intracellular PhaZ is unable to hydrolyse extracellular PHA, and extracellular PhaZ cannot hydrolyse intracellular PHA [14]. The enzymatic degradation of P(3HB) is known as a heterogeneous reaction because the polyhydroxybutyrate (PHB) depolymerase is water-soluble, whereas the P(3HB) polymer is water-insoluble. Therefore, the enzymatic degradation of P(3HB) involves two steps, namely adsorption and hydrolysis. During adsorption, the enzyme is attached to the surface of P(3HB) via the binding domain of the polymer. This is followed by hydrolysis of the polymer chain at the active site of the enzyme [5]. 

Qiua Z, Ikeharab T, Nishi T (2003) Melting behaviour of poly(butylene succinate) in miscible blends with poly(ethylene oxide). Polymer 44 3095-3099 Romen F, Reinhardt S, Jendrossek D (2(X)4) Thermotolerant poly(3-hydioxybutyrate)- degrading bacteria from hot compost and characterization of the PHB depolymerase of Schlegelella sp. KB la. Arch Microbiol 182 157-164... 

Fig. 12 Scanning electron micrographs of P(3HB) fiber before (a) and after (b) partial enzymatic degradation in an aqueous solution of PHB depolymerase purified from Ralstonia pickettii T1 at 37°C. c Intensity profiles of equatorial lines in X-ray fiber diagrams before and after partial enzymatic degradation, a and P indicate the reflections derived from a- and p-structure crystals respectively, d Enzymatic degradation behavior of P(3HB) fibers with two kinds of molecular conformations a- and P-structure. (Reprinted with permission from Iwata et al. 2006. Copyright 2006, American Chemical Society)...

More recently, we succeeded in revealing the crystal structure of PHB depolymerase from Penicillium funiculosum (Hisano et al. 2006). The trimer substrate of (/ )-3-hydroxybutyrate with a planar zigzag conformation was perfectly bound in a crevice of the active site. This result suggests that the P-structure (planar zigzag conformation) is degraded faster than the a-structure (2/1 heUx conformation), which was obtained from the enzymatic degradation of P(3HB) fibers with two kinds of molecular conformations. These results indicate that the rate of enzymatic degradation can be controlled by the molecular conformations of polymers. 

The mature form of the fungal poly[(/ )-3-hydroxybutyrate] (PHB) depolymerase from Penicillium fimiculosum (Fig. 2) (Hisano et al. 2006 Kasuya et al. 2007) is a... 

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