Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 ,-anomeric bonding of Nacetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-0-N-acetyl-D-galactosaminide (26% of the activity of pNP-,B-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a D-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10-4 to 6 x 10-4 M, while those for the pNP derivatives were 4 x 10-3 to 8 x 10-3 M. The optimum temperature of the enzyme was 75°C, and it retained 100 and 28% reactivity after heating at 60 and 80°C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5.

We developed a simple colorimetric method which can separately determine paraquat and diquat utilizing automatic reduction property of diquat under existence of tetramethylammonium hydroxide. In theˆrst step, diquat in the sample was automatically reduced by adding tetramethylammonium hydroxide, and UV/VIS spectrum (l max ＝430 nm) and absorbance at 430 nm were measured for conˆrma-tion and determination of diquat. In the second step, paraquat in the sample was also reduced by adding sodium hydrosulˆte, and UV/VIS spectrum (l max ＝396 nm, 600 nm.) and absorbance at 600 nm were measured for conˆrmation and determination of paraquat. The calibration curves were linear in the concentration range from 1 100 ppm for both compounds. The herbicide containing both paraquat and diquat, Preeglox L , showed typical UV/VIS spectra of each compound, and there was a good correlation between herbicide concentration and paraquat and diquat concentrations. The established method was successfully applied to forensic samples, gastric content and heeltap solution.