Heterologous expression of polyhydroxyalkanoate depolymerase from Thermobifida sp. in Pichia pastoris and catalytic analysis by surface plasmon resonance
| Autor: | Thidarat Wangkam, Toemsak Srikhirin, Lily Eurwilaichitr, Ratama Daduang, Chitwadee Phithakrotchanakoon, Arinthip Thamchaipenet, Verawat Champreda |
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| Rok vydání: | 2008 |
| Předmět: |
Molecular Sequence Data
Gene Expression Applied Microbiology and Biotechnology Esterase Polyhydroxyalkanoates Catalysis Pichia law.invention Pichia pastoris Bacterial Proteins law Actinomycetales Amino Acid Sequence Surface plasmon resonance Cloning Molecular chemistry.chemical_classification Molecular mass biology General Medicine Surface Plasmon Resonance biology.organism_classification Enzyme chemistry Biochemistry Recombinant DNA Heterologous expression Carboxylic Ester Hydrolases Sequence Alignment Biotechnology |
| Zdroj: | Applied microbiology and biotechnology. 82(1) |
| ISSN: | 1432-0614 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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