Purification and characterization of an extracellular chitinase from antagonistic Streptomyces violaceusniger.
The actinomycetes Streptomyces violaceusniger showed strong antagonistic activity against various tested wood rotting fungi. An extracellular chitinase, produced by antagonistic S. violaceusniger MTCC 3959, was purified as follows: ammonium sulfate precipitation, chitin affinity and chromatographic separation of Q Sepharose. The molecular mass of the purified chitinase was estimated as 56.5 kDa by SDS-PAGE. Chitinase was optimally active at pH of 5.0 and 50 °C. It retained almost 100% activity at pH 5.0 and also had high thermal tolerance at 50 °C. Enzyme activity was inhibited by Hg(2+) and Ag(+) cations, but was neither substantially inhibited by K(+) cation nor by chelating agent EDTA. The apparent Km and Vmax at 37 °C were 0.1426 mM and 6.6 U/mg, respectively using pNP-(GlcNAc)2 as substrate. The 56.5 kDa chitinase of strain MTCC 3959 represented an exo-type activity. The purified chitinase was further identified by MALDI-TOF. The results of peptide mass fingerprinting showed that 10 tryptic peptides of the chitinase were identical to the chitinase C from Streptomyces albus J1074 (GenBank Accession No. gi|239982330). The sequence of N-terminal amino acid (AA) of the chitinase was determined to be G-D-G-T-G-P-G-P-G-P.
University School of Biotechnology, Guru Gobind Singh Indraprastha University, Dwarka, New Delhi-110 075, India.
Anand
A
Nagpure
Rajinder K
RK
Gupta
D000595:Amino Acid Sequence; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D002686:Chitin; D002688:Chitinases