Purification and Characterization of a Novel Thermo-Alkali-Stable Catalase from Thermus brockianus
A novel thermo-alkali-stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three-step procedure that resulted in 65-fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 C and a pH range from 6 to 10, with optimum activity occurring at 90 C and pH 8. At pH 8, the enzyme was extremely stable at elevated temperatures with half-lives of 330 h at 80 C and 3 h at 90 C. The enzyme also demonstrated excellent stability at 70 C and alkaline pH with measured half-lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6-coordinate low spin state rather than the typical 5-coordinate high spin state. A Km of 35.5 mM and a Vmax of 20.3 mM/min·mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3-amino-1,2,4-triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o-dianisidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase-peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.
- Research Organization:
- Idaho National Lab. (INL), Idaho Falls, ID (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- DE-AC07-99ID-13727
- OSTI ID:
- 911993
- Report Number(s):
- INEEL/JOU-02-01430; JOBAAY; TRN: US200801%%438
- Journal Information:
- Journal of Bacteriology, Vol. 19, Issue 4; ISSN 0021-9193
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ABSORPTION
BLEACHING
CATALASE
CYANIDES
ENZYMES
FILTRATION
HEME
HIGH SPIN STATES
HYDROGEN PEROXIDE
IRON
PEROXIDASES
PROTEINS
PURIFICATION
PYRIDINE
SPIN
STABILITY
SUBSTRATES
Novel Thermo-Alkali-Stable Catalase
Thermus brockianus