Cloning and Characterization of the Polyhydroxybutyrate Depolymerase Gene of
            <i>Pseudomonas stutzeri</i>
            and Analysis of the Function of Substrate-Binding Domains

Ohura, Takeshi; Kasuya, Kazuhiko; Doi, Yoshiharu

doi:10.1128/aem.65.1.189-197.1999

Cited by 69 publications

(50 citation statements)

References 54 publications

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“…The binding isotherm acquired at 25°C reveals a maximum loading capacity of 20 Ϯ 2 g PhaF/mg PHB and a dissociation constant of 0.5 Ϯ 0.1 mg/ml (18.3 M) ( Table 1). These values are similar to those from other PHA-binding proteins (37).…”

Section: Resultssupporting

confidence: 88%

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Bello-Gil

Maestro

Fonseca

et al. 2018

Appl Environ Microbiol

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Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate in the cytoplasm of certain bacteria. One promising biotechnological application utilizes these biopolymers as supports for protein immobilization. Here, the PHA-binding domain of the KT2440 PhaF phasin (BioF polypeptide) was investigated as an affinity tag for the functionalization of poly-3-hydroxybutyrate (PHB) particles with recombinant proteins, namely, full-length PhaF and two fusion proteins tagged to BioF (BioF-C-LytA and BioF-β-galactosidase, containing the choline-binding module C-LytA and the β-galactosidase enzyme, respectively). The protein-biopolyester interaction was strong and stable at a wide range of pHs and temperatures, and the bound protein was highly protected from self-degradation, while the binding strength could be modulated by coating with amphiphilic compounds. Finally, BioF-β-galactosidase displayed very stable enzymatic activity after several continuous activity-plus-washing cycles when immobilized in a minibioreactor. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials. Our results confirm the biotechnological potential of the BioF affinity tag as a versatile tool for functionalizing PHA supports with recombinant proteins, leading to novel bioactive materials. The wide substrate range of the BioF tag presumably enables protein immobilization of virtually all natural PHAs as well as blends, copolymers, or artificial chemically modified derivatives with novel physicochemical properties. Moreover, the strength of protein adsorption may be easily modulated by varying the coating of the support, providing new perspectives for the engineering of bioactive materials that require a tight control of protein loading.

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Section: Resultssupporting

confidence: 88%

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Bello-Gil

Maestro

Fonseca

et al. 2018

Appl Environ Microbiol

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“…The PHB depolymerases of several Gramnegative PHB-degrading bacteria, such as Alcaligenes faecalis AE122 (Kita et al, 1997), Comamonas sp. (Jendrossek et al, 1995a), Pseudomonas lemoignei (Jendrossek et al, 1993(Jendrossek et al, , 1995b, and Pseudomonas putida (Ohura et al, 1999), have been extensively studied, whereas a PHB depolymerase gene of a Gram-positive bacterium has been cloned only from Streptomyces exfoliates K10 so far (Klingbeil et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase ofBacillus megateriumN-18-25-9

Takaku

Kimoto

Kodaira

et al. 2006

FEMS Microbiology Letters

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A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.

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“…An E. coli-based recombinant protein production system was selected, based on its relative success to produce rPhaZs (Briese et al, 1994;Jendrossek, Frisse, et al, 1995;Jendrossek et al, 1993;Kasuya et al, 2003;Kita et al, 1997;Ohura et al, 1999;Takaku et al, 2006;Takeda et al, 2000;Wang et al, 2012) (Table 2). Since PhaZ Cte was classified as insoluble (predicted solubility score 0.503), and the other PhaZs had scores (0.657-0.765) near the PROSO II threshold for solubility of 0.6, insolubility was considered a potential drawback for production of rPhaZs.…”

Section: Selection Of Expression Approachmentioning

confidence: 99%

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases

et al. 2020

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Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His‐tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta‐gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate‐based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

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Cloning and Characterization of the Polyhydroxybutyrate Depolymerase Gene of Pseudomonas stutzeri and Analysis of the Function of Substrate-Binding Domains

Cited by 69 publications

References 54 publications

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase ofBacillus megateriumN-18-25-9

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases

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