The genotypic diversity and lipase production of some thermophilic bacilli from different genera

Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.

Optimization and purification of thermostable lipase produced by Aspergillus ustus grown on soy-bean litters

1. Lipase enzyme was produced from A. ustus under optimized cultural conditions [C/N ratio was 0.4/1% i. e: the weight of waste material, soy-bean litters, equivalent to 0.4% lipid is 6.48%, and peptone, 1% for 3 days at pH 6.98 in presence of 1 mg/ml FeCl[3] under shake conditions]. 2. The enzyme was extracted and purified by precipitation with [NH[4]][2] SO[4] then by chromatography on DEAE-Sephadex A[50] followed by Gel-filtration on Sephadex G[100]. Recovery was 27.67%. 3. The enzyme was clearly distinguished from lipases previously characterized, On using SDS-PAGE, the final fraction splits into two bands with electrophoretic mobility 0.24 and 0.56, indicating that the molecule consists of two peptide chains with molecular weight 34.670 and 66.70 Daltons with PI 7.1. km and V[max] values were 5.8 g/100ml and 31.2 U/mg protein, respectively. The maximum activity occurred at 37°C and pH 7.5 for 2.5 hrs. The enzyme has thermostability properties. 4. The enzyme protein is rich in essential amino acids as compared with FAO and it seems to be related to the same group of porcine pancreatic lipase