Gene Cloning, Overproduction, and Characterization of Thermolabile Alkaline Phosphatase from a Psychrotrophic Bacterium

Suzuki, Yutaka; Mizutani, Yoichi; Tsuji, T; Ohtani, Naoto; Takano, Kazufumi; Haruki, Mitsuru; Morikawa, Masaaki; Kanaya, Shigenori

doi:10.1271/bbb.69.364

Cited by 22 publications

(25 citation statements)

References 41 publications

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“…(Murakawa et al 2002) and Shewanella sp. SIB1 (Suzuki et al 2005), and the moderately halophilic Halomonas sp. 593 (Ishibashi et al 2005) have been purified and biochemically studied.…”

Section: Discussionmentioning

confidence: 99%

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Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

et al. 2014

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The psychrophilic marine bacterium, Cobetia marina, recovered from the mantle tissue of the marine mussel, Crenomytilus grayanus, which contained a gene encoding alkaline phosphatase (AP) with apparent biotechnology advantages. The enzyme was found to be more efficient than its counterparts and showed k cat value 10-to 100-fold higher than those of all known commercial APs. The enzyme did not require the presence of exogenous divalent cations and dimeric state of its molecule for activity. The recombinant enzyme (CmAP) production and purification were optimized with a final recovery of 2 mg of the homogenous protein from 1 L of the transgenic Escherichia coli Rosetta(DE3)/Pho40 cells culture. CmAP displayed a half-life of 16 min at 45°C and 27 min at 40°C in the presence of 2 mM EDTA, thus suggesting its relative thermostability in comparison with the known cold-adapted analogues. A high concentration of EDTA in the incubation mixture did not appreciably inhibit CmAP. The enzyme was stable in a wide range of pH (6.0-11.0). CmAP exhibited its highest activity at the reaction temperature of 40-50°C and pH 9.5-10.3. The structural features of CmAP could be the reason for the increase in its stability and catalytic turnover. We have modeled the CmAP 3D structure on the base of the high-quality experimental structure of the close homologue Vibrio sp. AP (VAP) and mutated essential residues predicted to break Mg 2+ bonds in CmAP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in CmAP could be attributed to the adapted mutualistic lifestyle in oceanic waters.

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“…(Murakawa et al 2002) and Shewanella sp. SIB1 (Suzuki et al 2005), and the moderately halophilic Halomonas sp. 593 (Ishibashi et al 2005) have been purified and biochemically studied.…”

Section: Discussionmentioning

confidence: 99%

“…(Murakawa et al 2002), Shewanella sp. SIB1 (Suzuki et al 2005), Holomonas sp. 593 (Ishibashi et al 2005) and C. marina KMM 296 (Plisova et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

et al. 2014

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“…These variations in ALP activity which are consequences of adjustments in K m or V max values or both, suggest that pH/activity relationship of ALP may be a factor in the intracellular regulation of its activity. Enzyme activity/temperature linked properties reported elsewhere (Suzuki et al, 2005;Lee et al 2007;Copeland et al 1985;Aberomand et al 2008) defined the so-called enzyme "temperature optimum" of ALP activity. The present report suggests that at experimental temperature of about 45°C, ALP extract of L. townsendii exhibited maximum conformational flexibility to accommondate the substrate and enhanced conformational changes required for maximum catalysis.…”

Section: Discussionmentioning

confidence: 99%

Kinetic studies of alkaline phosphatase extracted from rabbit (Lepus townsendii) liver

Njoku¹,

Chikezie²,

Kaoje³

2011

Afr. J. Biotechnol.

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“…When recombinant APase from antarctic strain TAB 5 or the psychrophilic Shewanella sp. SIB1 was expressed in E. coli, the signal peptide of the protein with APase activity had been cleaved (Rina et al 2000;Suzuki et al 2005). The signal peptide for CmAPase played a key role in stimulation of enzyme conformation and activity.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…(Murakawa et al 2002) and Shewanella sp. SIB1 (Suzuki et al 2005), the aerobic hyperthermophilic archaeon Aeropyrum pernix (Helianti et al 2007) and the hyperthermophilic archaeon Pyrococcus abyssi (Zappa et al 2001) have been investigated. The activities of these enzymes are in the range 427-3,707 U/mg.…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina

Nasu

Ichiyanagi²,

Gomi³

2011

Biotechnol Lett

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Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55-60 kDa, as determined by SDS-PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.

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Gene Cloning, Overproduction, and Characterization of Thermolabile Alkaline Phosphatase from a Psychrotrophic Bacterium

Cited by 22 publications

References 41 publications

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

Recombinant Production and Characterization of a Highly Active Alkaline Phosphatase from Marine Bacterium Cobetia marina

Kinetic studies of alkaline phosphatase extracted from rabbit (Lepus townsendii) liver

Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina

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