Halidohydrolase activities of the bacterial strains isolated from pesticide-free environment

English. Whole and partial fragments of hal/IVa were used as probes to detect the presence of 2-haloacid halidohydrolase activities on bacterial strains isolated from an area which was free from pesticide contamination. A 3.5 kb EcoRI band which was homologous with hd/IVa could be detected on strains S4, S9 and R4B. A PCR-amplified DNA fragment of 636 bp was subcloned downstream of a tac promoter, resulting in a low level constitutive expression of dehalogenase. Further confirmation of the enzyme with the activity-silver strain electrophoresis and rabbit-anti-HdIIVa antibody revealed that the enzymes from the recombinant S4 and S9 whereas type II halidohydrolase was detected on strain R4B. Optimum halidohydrolase activities were well in the range of pH 8 to pH 10 and between 30 deg. C and 35 deg. C. Specificity assay demonstrated that all strains were active towards D-2MCPA, L-2MCPA and DL-2MCPA

Indonesian. Fragmen DNA lengkap dan partial dari hd/IVa (Pseudomonas capacia MBAA4) digunakan sebagai probe untuk melacak adanya aktivitas 2-haloacid halidohydrolase dari bakteri tanah yang diisolasi dari daerah yang belum tercemar pestisida. Pita EcoRI sepanjang 3.5 kb yang homolog dengan gen hd/IVa terdeteksi pada galur-galur S4, S9 dan R4b. Sub-kloning dari fragmen DNA berukuran 636 bp hasil amplifikasi dengan PCR pada daerah hilir promoter tac menghasilkan ekspresi dehalogenase tingkat rendah. Konfirmasi dengan rekombinan ini mempunyai karakteristik yang sama dengan galur asli. Halidohydrolase tipe IVa ditemukan pada galur S4 dan S9 sedangkan halidohydrolase tipe II terdeteksi pada galur R4b. Aktifitas optimum halidohydrolase berada pada rentang pH 8-10, dan rentang temperatur 30 derajat C sampai 35 derajat C. Assay spesifisitas substrat menunjukkan bahwa semua galur bakteri aktif terhadap D-2MCPA. L-2MCPA dan DL-2MCPA