Production, purification, and characterization of a novel cold-active superoxide dismutase from the Antarctic strain Aspergillus glaucus 363.
The Antarctic fungal strain Aspergillus glaucus 363 produces cold-active (CA) Cu/Zn-superoxide dismutase (SOD). The strain contains at least one gene encoding Cu/Zn-SOD that exhibited high homology with the corresponding gene of other Aspergillus species. To our knowledge, this is the first nucleotide sequence of a CA Cu/Zn-SOD gene in fungi. An effective laboratory technology for A. glaucus SOD production in 3 L bioreactors was developed on the basis of transient cold-shock treatment. The temperature downshift to 10 °C caused 1.4-fold increase of specific SOD activity compared to unstressed culture. Maximum enzyme productivity was 64 × 10(3) U kg(-1) h(-1). Two SOD isoenzymes (Cu/Zn-SODI and Cu/Zn-SODII) were purified to electrophoretic homogeneity. The specific activity of the major isoenzyme, Cu/Zn-SODII, after Q-Sepharose chromatography was 4000 U mg(-1). The molecular mass of SODI (38 159 Da) and of SODII (15 835 Da) was determined by electrospray quadropole time-of-flight (ESI-Q-TOF) mass spectrometry and dynamic light scattering (DLS). The presence of Cu and Zn were confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The N-terminal amino acid sequence of Cu/Zn-SODII revealed a high degree of structural homology with Cu/Zn-SOD from other fungi, including Aspergillus species.
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Radoslav
R
Abrashev
Laboratory of Biochemistry, Centre for Protein Engineering, University of Liège, B-4000 Liège, Belgium.
Georges
G
Feller
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Nedelina
N
Kostadinova
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Ekaterina
E
Krumova
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Zlatka
Z
Alexieva
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Maria
M
Gerginova
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Boryana
B
Spasova
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Jeni
J
Miteva-Staleva
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria.
Spassen
S
Vassilev
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgaria. Electronic address: mariange@microbio.bas.bg.
Maria
M
Angelova
D000864:Antarctic Regions; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects; D003080:Cold Temperature; D017124:Conserved Sequence; D003300:Copper / Q000032:analysis; D013058:Mass Spectrometry; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015032:Zinc / Q000032:analysis
D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc
Antarctic fungi; Biosynthesis; Cold-active SOD; Cu/Zn-SOD gene sequence; Enzyme characterization