title: Heterologous expression of polyhydroxyalkanoate depolymerase from Thermobifida sp. in Pichia pastoris and catalytic analysis by surface plasmon resonance.
abstract: A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His(6)-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline-serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V(max) and K(m) of 3.63 +/- 0.16 micromol min(-1) mg(-1) and 0.79 +/- 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50-55 degrees C and pH 7-8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm(-2) h(-1) for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.
authors.affiliation: Enzyme Technology Laboratory, Bioresource Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Paholyothin Road, Klong Luang, Pathumthani, 12120, Thailand.
authors.forename: Chitwadee
authors.initials: C
authors.lastname: Phithakrotchanakoon
authors.forename: Ratama
authors.initials: R
authors.lastname: Daduang
authors.forename: Arinthip
authors.initials: A
authors.lastname: Thamchaipenet
authors.forename: Thidarat
authors.initials: T
authors.lastname: Wangkam
authors.forename: Toemsak
authors.initials: T
authors.lastname: Srikhirin
authors.forename: Lily
authors.initials: L
authors.lastname: Eurwilaichitr
authors.forename: Verawat
authors.initials: V
authors.lastname: Champreda
mesh_terms: D000192:Actinomycetales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase