Solubilization of organic sludge by thermophilic aerobic bacteria as a pretreatment for anaerobic digestion was investigated. The thermophilic bacteria which solubilized organic sludge were isolated from the thermophilic aerobic digestion reactor. The bacterium type SPT2-1 could grow at pH ranging from 5.0 to 8.5 with optimal temperature at 60-70 degrees C. In batch experiments, 25-30% of volatile suspended solids (VSS) in the pre-heated sludge were solubilized on inoculating with the isolated bacteria although little was solubilized without inoculation. The isolated bacteria appeared to secret the extracellular enzymes including proteases and amylases. In continuous flow experiments, sludge solubilization rate (VSS removal) was around 40% under aerobic as well as microaerobic conditions. No accumulation of volatile fatty acids in the treated sludge was observed under aerobic conditions while significant amounts of them were accumulated under microaerobic conditions. Production of biogas on anaerobic digestion of the microaerobically-pretreated sludge was increased by 1.5 when compared with the sludge without pretreatment.

Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYPlAl) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pCFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNCOl under the control of the cauliflower mosaic virus 35.5 promoter and nopaline synthase gene terminator. l h e fused enzyme cDNA was integrated into tobacco genomes by Agrobaderium infection techniques. In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction. l h e microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo[a]pyrene than in the control plant. The transgenic plants also showed resistance to the herbicide chlortoluron.Microsomal Cyt P450 monooxygenases are composed of P450 and NADPH-Cyt P450 oxidoreductase (EC 1.6.2.4), and catalyze oxidative metabolism of a wide variety of endogenous and exogenous compounds. The catalytic versatility of P450 monooxygenase in mammals is derived from the multiplicity of P450 enzymes as well as the overlapping substrate specificity of each P450 species. Over 221 P450 genes have been identified in animals, plants, and microorganisms and have been classified into 36 gene families on the basis of sequential similarity (Nelson et al., 1993).We have established the yeast expression systems for microsomal P450 species Yabusaki et al., 1991) and constructed chimeric P450 enzymes among different P450 species and fused enzyme variants between rat CYPlAl and rat reductase (Murakami et al., 1987;Yabusaki et al., 1988), bovine CYP17A and yeast reductase , and bovine CYP2lB and yeast reductase . The fused enzymes were catalytically self-sufficient and exhibited high specific monooxygenase activities.P450 monooxygenase in higher plants is involved in not only the biosynthesis of a variety of secondary metabolites but also the metabolism of xenobiotics including herbicides (Durst, 1991). Oxidative metabolism mediated by P450 is particularly important for detoxification and selective toxicity Corresponding author; fax 81-78-871-3617.of herbicides in a variety of crop species. However, molecular studies on plant P450 species involved in the metabolism of herbicides have not been reported yet. Saito et al. (1991) reported that the transgenic tobacco plants expressing rabbit CYP2C14 showed phenotypical changes and rapid senescence but no monooxygenase activities derived from CYP2C14.In this study we describe expression of a genetically engineered fused enzyme between rat CYPlAl (EC 1.14.99.8) and yeast reductase in tobacco plants. Transgenic tobacco plants were analyzed for integration of cDNA into genomes, production of mRNA and protein, and monooxygenase activities. In addition, these transgenic plants were assayed for resistance to the herbicide chlortoluron. MATERIALS AND METHODS Chemicals and Biochemicals

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The human gene for CYP2B6, a cytochrome P450 monooxygenase that inactivates xenobiotic chemicals, was introduced into Oryza sativa cv. Nipponbare by Agrobacterium-mediated transformation. At germination, R(1) seeds of transgenic rice plants expressing CYP2B6 (CYP2B6 rice) showed a high tolerance to 5 microM metolachlor, a preemergence herbicide that is degraded by CYP2B6. Thin-layer chromatography after culture with (14)C-labeled metolachlor revealed that the amounts of residual metolachlor decreased in plant tissues and the medium of CYP2B6 rice faster than those of untransformed Nipponbare. CYP2B6 rice plants were able to grow in the presence of 13 out of 17 herbicides: five chloroacetamides and mefenacet, pyributicarb, amiprofos-methyl, trifluralin, pendimethalin, norflurazon, and chlorotoluron. These herbicides differ in their modes of action and chemical structures. Transgenic rice expressing a xenobiotic-degrading human CYP2B6, which has broad substrate specificity, should be good not only for developing herbicide tolerant rice but also for reducing the environmental impact of agrochemicals.