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

Phithakrotchanakoon, Chitwadee; Daduang, Ratama; Thamchaipenet, Arinthip; Wangkam, Thidarat; Srikhirin, Toemsak; Eurwilaichitr, Lily; Champreda, Verawat
February 2009
Applied Microbiology & Biotechnology;Feb2009, Vol. 82 Issue 1, p131
Academic Journal
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.


Related Articles

  • Development of an open-tubular trypsin reactor for on-line digestion of proteins. Stigter, E. C. A.; De Jong, G. J.; Van Bennekom, W. P. // Analytical & Bioanalytical Chemistry;Nov2007, Vol. 389 Issue 6, p1967 

    A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was...

  • Distribution and Selection of Poly-3-Hydroxybutyrate Production Capacity in Methanotrophic Proteobacteria. Pieja, Allison; Rostkowski, Katherine; Criddle, Craig // Microbial Ecology;Oct2011, Vol. 62 Issue 3, p564 

    Methanotrophs are known to produce poly-3-hydroxybutyrate (PHB), but there is conflicting evidence in the literature as to which genera produce the polymer. We screened type I and II proteobacterial methanotrophs that use the ribulose monophosphate and serine pathways for carbon assimilation,...

  • Alkaline-Resistance Model of Subtilisin ALP I, a Novel Alkaline Subtilisin. Maeda, Hiroshi; Mizutani, Osamu; Yamagata, Youhei; Ichishima, Eiji; Nakajima, Tasuku // Journal of Biochemistry;2001, Vol. 129 Issue 5, p675 

    The alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of...

  • Determination of Mark-Houwink Parameters and Absolute Molecular Weight of Medium-Chain-Length Poly(3-Hydroxyalkanoates). Nerkar, Manoj; Ramsay, Juliana; Ramsay, Bruce; Kontopoulou, Marianna; Hutchinson, Robin // Journal of Polymers & the Environment;Mar2013, Vol. 21 Issue 1, p24 

    Absolute molecular weight distributions were determined for different medium-chain-length poly(3-hydroxyalkanoates) (MCL PHAs) with predominantly 3-hydroxyoctanoate (PHO), 3-hydroxynonanoate (PHN) or 3-hydroxydodecanoate content. This is the first study to estimate the Mark-Houwink constants of...

  • Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.). Van Riet, Liesbet; Nagaraj, Vinay; Van den Ende, Wim; Clerens, Stefan; Wiemken, Andres; Van Laere, André // Journal of Experimental Botany;Jan2006, Vol. 57 Issue 1, p213 

    Fructans, β2-1 and/or β2-6 linked polymers of fructose, are produced by fructosyltransferases (FTs) from sucrose. They are important storage carbohydrates in many plants. Fructan reserves, widely distributed in plants, are believed to be mobilized via fructan exohydrolases (FEHs). The...

  • Xylanase of the micromycete Rhizopus var. microsporus 595: Preparation, structural and functional characteristics, and application. Shuvaeva, G. P.; Sysoeva, M. G. // Applied Biochemistry & Microbiology;Oct2010, Vol. 46 Issue 6, p641 

    Procedures for the production of endo-1,4-β-xylanase have been developed. An active producer- Rhizopus var. microsporus BKMF-595-has been chosen, and the conditions of surface and submerged cultivation, as well as the composition of the culture medium for this strain, have been optimized to...

  • Characterization of an acid-labile, thermostable β-glycosidase from Thermoplasma acidophilum. Hye-Jung Kim; Ah-Reum Park; Jung-Kul Lee; Deok-Kun Oh // Biotechnology Letters;Sep2009, Vol. 31 Issue 9, p1457 

    A recombinant putative β-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg−1. The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90°C. The enzyme was unstable below pH 6.0: at pH 6 its...

  • Properties of hexahistidine-tagged organophosphate hydrolase. Votchitseva, Yu. A.; Efremenko, E. N.; Aliev, T. K.; Varfolomeyev, S. D. // Biochemistry (00062979);Feb2006, Vol. 71 Issue 2, p167 

    The catalytic properties of organophosphate hydrolase (OPH) containing a hexahistidine tag His6 (His6-OPH) and purified to 98% homogeneity were investigated. The pH optimum of enzymatic activity and isoelectric point of His6-OPH, which were shown to be 10.5 and 8.5, respectively, are shifted to...

  • Synthesisof fructose laurate esters catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst in a non-aqueous system. Jin, Zi; Liang, Shuli; Zhang, Xiuqin; Han, Shuangyan; Ren, Changqiong; Lin, Ying; Zheng, Suiping // Biotechnology & Bioprocess Engineering;Apr2013, Vol. 18 Issue 2, p365 

    Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics