NDLI: Heterologous expression of polyhydroxyalkanoate depolymerase from Thermobifida sp. in Pichia pastoris and catalytic analysis by surface plasmon resonance

Content Provider	Springer Nature Link
Author	Phithakrotchanakoon, Chitwadee Daduang, Ratama Thamchaipenet, Arinthip Wangkam, Thidarat Srikhirin, Toemsak Eurwilaichitr, Lily Champreda, Verawat
Copyright Year	2008
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 V max and K m of 3.63 ± 0.16 μmol min−1 mg−1 and 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−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.
Starting Page	131
Ending Page	140
Page Count	10
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	82
Issue Number	1
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2008-11-12
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Thermofida sp. Pichia pastoris Polyhydroxyalkanoate Polyhydroxyalkanoate depolymerase Surface plasmon resonance Atomic force microscopy Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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