Characterization of chimeric and mutated isocitrate lyases of a mesophilic nitrogen-fixing bacterium, <i>Azotobacter vinelandii</i>, and a psychrophilic bacterium, <i>Colwellia maris</i>

Hayashi, Tomofumi; Matsuzaki, Wataru; Takada, Yasuhiro

doi:10.1080/09168451.2014.882744

Cited by 6 publications

(4 citation statements)

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“…A successful approach for such task consists of the use of chimeric enzymes, in which a gene encoding for a given psychrophilic protein is divided into several regions that are then replaced by similar regions from a mesophilic homolog (Yoneta et al, 2004 ; Watanabe et al, 2005 ). This strategy allows not only to confirm that in some cases the entire protein does not necessarily need to be flexible to achieve high catalytic activity in at low temperatures, but also to identify which protein regions are responsible for the increased flexibility (Yoneta et al, 2004 ), which can be further combined with mutational analysis to identify the key residues responsible for cold-adaptation (Hayashi et al, 2014 ). These experiments allowed the identification of the C-terminal region of the cold-adapted isocitrate dehydronease from Colwellia maris as responsible for its psychrophilic characteristics (Yoneta et al, 2004 ).…”

Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

et al. 2016

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Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25 • C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

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Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

et al. 2016

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“…Western blot analysis was carried out as reported previously 24) except for using rabbit antibodies against the purified IDH-I and IDH-II of C. maris 25) diluted to 1:30,000 and 1:50,000, respectively.…”

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confidence: 99%

Characterization of NADP+-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium, Colwellia psychrerythraea strain 34H

Suzuki

Takada

2016

Bioscience, Biotechnology, and Biochemistry

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2 NADP + -dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned 5 and overexpressed in Escherichia coli, and the three IDH proteins were purified.Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzyme while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for growth of this Stratagene) and pTrcHisB (Invitrogen) were used as vectors, respectively. Preparation of crude extract of C. psychrerythraea 34H. C. 20psychrerythraea 34H cultivated up to the late exponential phase (for about 21 h)at 10 o C were harvested and washed twice with a sonication buffer (20 mM potassium phosphate buffer (pH 7.5), containing 0.5 M NaCl, 2 mM MgCl 2 and 10 mM 2-mercaptoethanol). Cells suspended in the sonication buffer were disrupted by twelve times sonication for 0.5 min with intervals of 1 min on ice. Western blot analysis. SDS-PAGE 23) of crude extract of the C.psychrerythraea 34H cells was carried out on 10% polyacrylamide gel at 120 V. 5The proteins on the gel were then transferred onto a polyvinylidene fluoride membrane (Immobilon-P, Millipore) at 4 o C, according to the manufacturer's instructions. Western blot analysis was carried out as reported previously 24) except for using rabbit antibodies against the purified IDH-I and IDH-II of C. maris 25) diluted to 1:30,000 and 1:50,000, respectively. 10Cloning of the IDH isozyme genes. Genomic DNA of C.psychrerythraea 34H was isolated and purified with FastPure DNA Kit (TaKaRa). For the cloning of IDH isozyme genes, genomic PCR was carried out as described below. The forward primers, IIa-around-f, IIb-around-f and +2393 and +2417 from that of icdIIb and between +1423 and +1450 from that of icdI, respectively ( conferring the N-terminal (His) 6 -tag on the expressed proteins, to obtain plasmids pHisCp34IDH-IIa, pHisCp34IDH-IIb and pHisCp34H-I, respectively.Precise insertion of these genes into the vector was certified by the nucleotide sequencing as described above. solution was concentrated and then dialyzed as described previously. 27) AllHis-tagged recombinant IDHs were stored at -30 o C.Enzyme assay. The IDH activity was assayed as reported previously.11)The reaction mixture (2 mL) contained 33 mM Tris-HCl (pH 7.5), 0.67 mM Results 15Thermal properties of the IDH activity in crude extract of C. psychrerythraea 34H and Western blot analysisIDH activity in crude extract of C. psychrerythraea 34H grown at 10 o C was assayed at various temperatures ( Fig. 2A). IDH in the crude extract showed respectively. These results indicate that at least both of the two type IDH isozymes are present in the crude extract of this ba...

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“…Protein bands on the gel after electrophoresis were then transferred to a polyvinylidene difluoride membrane (Immobilon‐P; Millipore). Western blot analysis was carried out as described previously [23] except for using rabbit antibodies against the purified dimeric IDH‐I of C. maris [1] and the purified monomeric IDH of V. parahaemolyticus [7] and goat anti‐rabbit IgG (H + L), horseradish peroxidase conjugate (Life Technologies) as a secondary antibody.…”

Section: Methodsmentioning

confidence: 99%

NADP⁺‐dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A

Komura

Takada

2021

J Basic Microbiol

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The genes encoding dimeric and monomeric isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, strain 13A (13AIDH‐D and 13AIDH‐M, respectively), were cloned and sequenced. The deduced amino acid sequences of these two IDHs showed high degrees of identity with those of bacteria of genus Psychrobacter. Analysis of the 16S ribosomal RNA gene of the strain 13A revealed that this bacterium is classified to genus Psychrobacter. The optimum temperatures for activities of 13AIDH‐D and 13AIDH‐M were 55°C and 45°C, respectively, indicating that they are mesophilic. On the contrary, 13AIDH‐D maintained 90% of its maximum activity after incubation for 10 min at 50°C, while the 13AIDH‐M activity was completely lost under the same condition. In addition, 13AIDH‐D showed much higher specific activity than 13AIDH‐M. From northern and western blot analyses, the 13AIDH‐D gene was found to be not transcribed under the growth conditions tested in this study. However, the catalytic ability of the mesophilic 13AIDH‐M was concluded to be enough to sustain the growth of strain 13A at low temperatures. Therefore, a novel pattern of the contribution of IDH isozymes in cold‐living bacteria to their growth at low temperatures was confirmed in strain 13A.

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Characterization of chimeric and mutated isocitrate lyases of a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris

Abstract: Characterization of chimeric and mutated isocitrate lyases of a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris, Bioscience, Biotechnology, and Biochemistry, 78:2,[195][196][197][198][199][200][201]

Cited by 6 publications

References 14 publications

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Characterization of NADP+-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium, Colwellia psychrerythraea strain 34H

NADP⁺‐dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A

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Characterization of chimeric and mutated isocitrate lyases of a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris

Abstract: Characterization of chimeric and mutated isocitrate lyases of a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris, Bioscience, Biotechnology, and Biochemistry, 78:2,[195][196][197][198][199][200][201]

Cited by 6 publications

References 14 publications

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Characterization of NADP+-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium, Colwellia psychrerythraea strain 34H

NADP+‐dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A

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NADP⁺‐dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A