An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1. Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor. Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme.Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction. The amount of Cr(V) species formed was about 10 times larger than that of the nonenzymatic reduction. These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(HI) (14) found NAD(P)H-dependent Cr(VI)-reducing activity in the cell-free supernatant fluids from Pseudomonas putida. Das and Chandra (6) have reported that Cr(VI) was reduced to Cr(III) in the presence of NAD(P)H in the cell extract of Streptomyces species. A membrane-associated chromate reduction system was also reported. Ohtake and coworkers (20) and Wang and coworkers (26)(27)(28) have shown that Cr(VI) was reduced to Cr(III) under anaerobic conditions by Enterobacter cloacae and its reduction was caused by the respiratory chain system of the cell membrane. We considered that these bacterial Cr(VI) reduction systems contribute to its detoxification by Cr(VI)-resistant bacteria. However, the Cr(VI) reductase has not yet been purified, and the details of the reaction mechanism are still unclear.P. ambigua G-1 was able to grow in medium containing up to 20 mM K2CrO4 (13). The Cr(VI)-reducing activity (11) of this strain was more thermostable than that of other pseudomonads (14). The Cr(VI)-sensitive mutants of this strain lost the reducing activity (12). These results indicate that this strain is a good source to investigate the Cr(VI) reduction mechanism which contributes to Cr(VI) detoxication.In the present paper, we report the purification of NAD(P)H-dependent Cr(VI) reductases of P. ambigua G-1 and the analysis of the catalytic mechanism of Cr(VI) reduction by the enzymes. Preparation of Cr(VI) reductase. Cells grown in 4 liters of L broth at 37°C were harvested at the late logarithmic phase, washed twice with 20 mM Tris-HCl, pH 7.2, suspended in 100 ml of the same buffer, and disrupted by sonication. After removal of the unbroken cells by centrifugation (1,500 x g, 30 min), the supernatant fluid was heat treated at 60°C for 2 min and centrifuged (1,500 x g, 30 min). The supernatant fluid was separated on a DE32 column (3.5 by 6 cm; Whatman Ltd., Maidstone, England) previously equilibrated with buffer A (50 mM glucose in 20 mM Tris-HCl, pH 7.2), and proteins were eluted with 0.4 M NaCl in buffer A at a flow rate of 100 ml/h. The fractions carrying the Cr(VI)-reducing activity were concentrated and separated by Cu2" chelate affinity chromatography using an AF-chelate Toyopearl 650M column (2.6 by 4 cm; Toso Ltd., Tokyo, Japan) which was sequentially equilibrated with 200 PM CUSO4 solution and buffer B (0.5 M NaCl in buf...

Effective biological pretreatment method for enhancing cellulase performance was investigated. Two alkali lignin-degrading bacteria were isolated from forest soils in Japan and named CS-1 and CS-2. 16S rDNA sequence analysis indicated that CS-1 and CS-2 were Bacillus sp. Strains CS-1 and CS-2 displayed alkali lignin degradation capability. With initial concentrations of 0.05-2.0 g L(-1), at least 61% alkali lignin could be degraded within 48 h. High laccase activities were observed in crude enzyme extracts from the isolated strains. This result indicated that alkali lignin degradation was correlated with laccase activities. Judging from the net yields of sugars after enzymatic hydrolysis, the most effective pretreatment method for enhancing cellulase performance was a two-step processing procedure (pretreatment using Bacillus sp. CS-1 followed by lactic acid bacteria) at 68.6%. These results suggest that the two-step pretreatment procedure is effective at accelerating cellulase performance.

The effect of culture conditions on xylitol production rate was investigated using Candida tropicalis IFO 0618. From the variance analysis of xylitol production rate, it was found that initial yeast extract concentration was highly significant (99%), while the interaction between D-xylose concentration and aeration rate was significant (95%). These results show the importance of initial yeast extract concentration and of the balance between D-xylose concentration and aeration in the production of xylitol. It was also clearly shown that C. tropicalis needed more yeast extract concentration for efficient xylitol production than for its growth. In order to enhance xylitol production rate, culture conditions were optimized by the Box-Wilson method. In this respect, initial D-xylose concentration, yeast extract concentration, and K(L)a were chosen as the independent factors in 2(3)-factorial experimental design. As the result of experiments, a maximum xylitol production rate of 2.67 g/L . h was obtained when initial D-xylose concentration and yeast extract concentration were 172.0 and 21.0 g/L, respectively, and K(L)a was 451.50 h(-1) by 90% oxygen gas.

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The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His-tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp-285-to-Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.z 2000 Federation of European Biochemical Societies.