RESUMO
A lipolytic mesophilic fungus which produces lipase extracellularly was isolated from soil. Based on ITS1-5.8S-ITS4 region sequences of ribosomal RNA, it was concluded that the isolate JK-1 belongs to genus Rhizopus and clades with Rhizopus oryzae. The present paper reports the screening, isolation, identification, and optimization of fermentation conditions for the production of lipase (EC 3.1.1.3). Culture conditions were optimized, and the highest lipase production was observed in basal medium with corn steep liquor as nitrogen source and glucose as carbon source. Maximum lipase production was observed at 72 h, which is about 870 U/ml. Optimization of fermentation conditions resulted in 16-fold enhancement in enzyme production.
Assuntos
Proteínas de Bactérias , Biotecnologia/métodos , Lipase , Rhizopus/enzimologia , Microbiologia do Solo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Meios de Cultura , DNA Bacteriano/análise , Fermentação , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lipase/biossíntese , Lipase/metabolismo , Nitrogênio/metabolismo , Filogenia , Rhizopus/classificação , Rhizopus/genética , Rhizopus/isolamento & purificação , Análise de Sequência de DNA , Temperatura , Zea mays/metabolismoRESUMO
A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India. This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis were 9.0 and 60 degrees C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K(M) and k(cat) values of 3.08 microM and 6.32 x 10(2) s(-1), respectively, for glycodeoxycholic acid. The NH(2)-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases. To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source.
