The present study aimed at the isolation, screening and partial purification of beta-mannanase from fungi isolated from soil and water samples collected from Ilaje Lake, Ondo state, Nigeria. The associated fungi were isolated and counted by standard microbiological methods. Partial purification of crude mannanase was done by standard biochemical methods. Quantitatively, mannanase production was performed in mineral salt medium into which Locust Bean Gum (LBG) had been incorporated as the sole carbon source. Enzyme activity was determined by dinitrosalicylic acid (DNSA) method, while protein content was evaluated by Lowry's method. The highest fungal counts were recorded for water sample collected from Ilaje Lake with 3.4×10 8 sfu/mL. The organisms encountered include Aspergillus flavus, Rhizopus stolonifer, A. fumigatus, A. niger, R. japonicus, Penicillum italicum, Fusarium solani and Candida albicans. All the fungal isolates encountered from these sources showed varied degrees of mannanase activities. The highest specific mannanase activity was recorded for isolate 4B1, while the lowest value was obtained with isolate 1B2. Purification of crude mannanase from A. niger was carried out by ammonium sulphate precipitation and gel filtration . Fractionation of ammonium sulphate precipitated mannanase from A. niger on Sephadex G-200 produced two activity peaks. In this investigation, fungal isolates evaluated for mannanase production from this source gave appreciable mannanase activity and this could be applied in many industrial processes.
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Materials and Methods
Sources of SampleWater and soil samples were collected from Ilaje Lake, Ondo State, Nigeria in sterile bottles. The samples were transferred to the laboratory and were used as sources for the isolation of mannanase-producing fungi [9,10].
Isolation and identification of mannanase-producing fungi from soil and water samplesThe total fungal counts from the samples were determined using the pour plate method on potato dextrose agar. The samples were serially diluted and 1ml of an appropriate dilution was used to inoculate the plate in duplicate. The plates were incubated at 28 ± 2˚C for 72hrs, after which the total colony count was determined as previously described [11]. At the end of incubation, the colonies were sub-cultured from the mixed cultures and identified on the bases of cultural characters (colour, shape of colony, surface and reverse pigmentation and texture of the colony) as well as microscopic structure (septate or nonseptate hyphae, structure of hyphae and conidia) [12].
Screening of mannanase-producing fungi in submerged state fermentationMedium composition described by Mandles & Weber [13] modified by Arotupin & Olaniyi [10] was used for submerged fermentation (static condition). The basal medium contained (g/L): LBG 10g, peptone 2g, yeast extract 2g, NaNO 3 2 g, KH 2 PO 4 1 g, MgSO 4 .7H 2 O 0.5 g, KCl 0.5 g and FeSO 4 .7H 2 O traces. The pH of the media was adjusted to 6.8 with pH meter (Denver Instrument, Model 20 pH/ Co...