Cloning, purification, and characterization of a thermophilic ribulokinase from Anoxybacillus kestanbolensis AC26Sari

Müslüm TOKGÖZ; Kadriye İNAN; Ali Osman BELDÜZ; Öznur GEDİKLİ; Sabriye ÇANAKÇI

doi:https://doi.org/10.3906/biy-1401-92

Article Details

Cloning, purification, and characterization of a thermophilic ribulokinase from Anoxybacillus kestanbolensis AC26Sari

Authors:

Müslüm TOKGÖZ | Kadriye İNAN | Ali Osman BELDÜZ | Öznur GEDİKLİ | Sabriye ÇANAKÇI

Publisher:

TUBITAK

Main Topics:

Life sciences

Journal:

Turkish Journal of Biology 2014 Vol.38 Issue 5, pp.633-639

Document Type:

Article

Publication Date:

4/2014

Document Language:

English

Document ID (Org No.):

2000812129

Frequency:

Bimonthly

eISSN:

1303-6092

pISSN:

1300-0152

DOI:

https://doi.org/10.3906/biy-1401-92

Keywords:

Abstract:

 

The gene encoding ribulokinase araB from Anoxybacillus kestanbolensis AC26Sari was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21 under the control of isopropyl-b-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as AC26RK, was purified with the MagneHis Protein Purification System. The molecular mass of the native protein, as determined by SDS-PAGE, was about 61 kDa. AC26RK was active throughout a broad pH (pH 5.0-10.0) and temperature (50-75 °C) range, and it had an optimum pH of 9.0 and optimum temperature of 60 °C. The enzyme displayed about 90%-100% of its original activities after a 30-min incubation at a pH interval of 5.0-10.0. The enzyme exhibited a high level of D-ribulose activity with apparent Km, Vmax, and Kcat values of 0.94 mM, 3.197 U/mg, and 3.31 s-1, respectively. AC26RK activity was strongly inhibited by Zn2+ but increased by Mg2+. The effects of some chemicals on the ribulokinase activity revealed that Anoxybacillus kestanbolensis AC26Sari does not need metallic cations for its activity. In this paper, we describe for the first time the cloning and characterization of a thermophilic ribulokinase from thermophilic bacteria.

 

The gene encoding ribulokinase araB from Anoxybacillus kestanbolensis AC26Sari was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21 under the control of isopropyl-b-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as AC26RK, was purified with the MagneHis Protein Purification System. The molecular mass of the native protein, as determined by SDS-PAGE, was about 61 kDa. AC26RK was active throughout a broad pH (pH 5.0-10.0) and temperature (50-75 °C) range, and it had an optimum pH of 9.0 and optimum temperature of 60 °C. The enzyme displayed about 90%-100% of its original activities after a 30-min incubation at a pH interval of 5.0-10.0. The enzyme exhibited a high level of D-ribulose activity with apparent Km, Vmax, and Kcat values of 0.94 mM, 3.197 U/mg, and 3.31 s-1, respectively. AC26RK activity was strongly inhibited by Zn2+ but increased by Mg2+. The effects of some chemicals on the ribulokinase activity revealed that Anoxybacillus kestanbolensis AC26Sari does not need metallic cations for its activity. In this paper, we describe for the first time the cloning and characterization of a thermophilic ribulokinase from thermophilic bacteria.

Article Details

Cloning, purification, and characterization of a thermophilic ribulokinase from Anoxybacillus kestanbolensis AC26Sari

Authors:

Müslüm TOKGÖZ | Kadriye İNAN | Ali Osman BELDÜZ | Öznur GEDİKLİ | Sabriye ÇANAKÇI

Publisher:

TUBITAK

Main Topics:

Life sciences

Journal:

Turkish Journal of Biology 2014 Vol.38 Issue 5, pp.633-639

Document Type:

Article

Publication Date:

4/2014

Document Language:

English

Document ID (Org No.):

2000812129

Frequency:

Bimonthly

eISSN:

1303-6092

pISSN:

1300-0152

DOI:

https://doi.org/10.3906/biy-1401-92

Anoxybacillus

ribulokinase

thermophilic

expression

Issues

Article Details

Cloning, purification, and characterization of a thermophilic ribulokinase from Anoxybacillus kestanbolensis AC26Sari

Authors: Müslüm TOKGÖZ | Kadriye İNAN | Ali Osman BELDÜZ | Öznur GEDİKLİ | Sabriye ÇANAKÇI

Publisher: TUBITAK

Main Topics: Life sciences

Journal: Turkish Journal of Biology 2014 Vol.38 Issue 5, pp.633-639

Document Type: Article

Publication Date: 4/2014

Document Language: English

Document ID (Org No.): 2000812129

Frequency: Bimonthly

eISSN: 1303-6092

pISSN: 1300-0152

DOI: https://doi.org/10.3906/biy-1401-92

Anoxybacillus

ribulokinase

thermophilic

expression

Issues

Authors:

Müslüm TOKGÖZ | Kadriye İNAN | Ali Osman BELDÜZ | Öznur GEDİKLİ | Sabriye ÇANAKÇI

Publisher:

TUBITAK

Main Topics:

Life sciences

Journal:

Turkish Journal of Biology 2014 Vol.38 Issue 5, pp.633-639

Document Type:

Article

Publication Date:

4/2014

Document Language:

English

Document ID (Org No.):

2000812129

Frequency:

Bimonthly

eISSN:

1303-6092

pISSN:

1300-0152

DOI:

https://doi.org/10.3906/biy-1401-92