• Korean Journal of Microbiology
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• v.39 no.4
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• pp.223-229
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• 2003

The purpose of this work was to perform the characterization of NAD(P)H-nitroreductase isolated from Stenotrophomonas sp. OK-5 capable of degrading 2,4,6-trinitrotoluene (TNT). Initially, NADP(H)-nitroreductase by a series of purification processes including ammonium sulfate precipitation, DEAE-sepharose, andQ-sepharose was prepared. From samples harvested from fraction collector, three different fractions (I, II & III)having the enzyme activity of NAD(P)H-itroreductase were detected. Specific activities of three fractions I, II,and III of NAD(P)H-nitroreductase were determined to approximately 5.06 unit/mg, 4.95 unit/mg and 4.86 unit/mg, and concentrated to 10.5, 9.8, and 8.9-fold compared to crude extract, respectively. Among these three fractions,the fraction I of NAD(P)H-nitroreductase demonstrated the highest specific activity in this experiment. Several factors affecting on the enzyme activity of NAD(P)H-nitroreductase (fractions I, II & III) were investigated.The optimum temperature of all NAD(P)H-nitroreductase (fractions I, II & III) was 30oC, and the optimal pH was approximately 7.5. Metal ions such as Ag+, Cu2+, Hg2+ inhibited approximately 80% enzyme activity of all NAD(P)H-nitroreductase, and the enzyme activities were decreased about 30-40% inhibition in the presence of Mn2+ or Ca2+. However, Fe3+ showed stimulatory effect on the enzyme activity. The molecular weights of NAD(P)H-nitroreductase (fractions I, II & III) were measured about 27 kDa on the SDS-PAGE.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.183-188
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• 2004

Stenotrophomonas sp. OK-5 capable of degrading TNT has been found to have three nitroreductase fractions designated as NTR fractions I, II, and III. NTR in a previous study. This study was attempted to reveal physiological and molecular characteristics of NTR fractions I, II, and III in strain OK-5. Several chemicals (e.g., EDTA, NaCl, dithiothreitol, $\beta$-mercaptoethanol) were tested for their effect on enzyme activity of NTRs, demonstrating that enzyme activities of NTR fractions I, II, and III from OK-5 were inhibited in the presence of $\beta$-mercaptoethanol. Substrate specificity test showed that NTR fractions I, II, and III all have over 70% enzyme activities for nitrobenzene or RDX as a substrate. N-terminal amino acid sequence of NTR fraction I from Stenotrophomonas sp. OK-5 was $^1MSDLLNADAVVQLFRTARDS^20$ and exhibited 70% sequence homology with that of NTR from Xanthomonas campestris. NTR I gene from Stenotrophomonas sp. OK-5 (SmOK5nrI) shared extensive sequence homology in deduced amino acid sequence of PCR product with NTRs from Xanthomonas campestris (81 %), X. axonopodis (75%), Streptomyces avermitilis(30%), whereas they had low homology with that from P. putida KT2440 (pnrB) (16%).