Publication: Heterologous expression of polyhydroxyalkanoate depolymerase from Thermobifida sp. in Pichia pastoris and catalytic analysis by surface plasmon resonance
Issued Date
2009-02-01
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ISSN
01757598
Other identifier(s)
2-s2.0-58549091878
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Mahidol University
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SCOPUS
Bibliographic Citation
Applied Microbiology and Biotechnology. Vol.82, No.1 (2009), 131-140
Suggested Citation
Chitwadee Phithakrotchanakoon, Ratama Daduang, Arinthip Thamchaipenet, Thidarat Wangkam, Toemsak Srikhirin, Lily Eurwilaichitr, Verawat Champreda Heterologous expression of polyhydroxyalkanoate depolymerase from Thermobifida sp. in Pichia pastoris and catalytic analysis by surface plasmon resonance. Applied Microbiology and Biotechnology. Vol.82, No.1 (2009), 131-140. doi:10.1007/s00253-008-1754-1 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/27290
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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 His6-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 Vmaxand Kmof 3.63∈±∈0.16 μmol min-1mg-1and 0.79∈±∈0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50-55°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-2h-1for 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. © 2008 Springer-Verlag.