Yeast Isolation
Samples were serially diluted in sterile distilled water and plated out using the pour plate method. The media used was Yeast Extract Agar (Oxoid). The media was prepared and sterilized according to Manufacturer’s specification. The media was supplemented with glucose and streptomycin to enhance yeast growth and to suppress the growth of bacteria respectively. All plates were incubated at 30°C for about 3 to 5 days. Yeast cells were further purified by streaking on fresh plates in order to obtain a pure colony. The pure colonies were examined and identified microscopically. The pure cultures were then stored in Yeast Extract broth and glycerol at 4°C for further use (Barnett 2003)
Screening for mannanase producing yeasts
The isolates were screened for mannanase producing ability by inoculating them in a sterile medium containing 1% Gum Arabic, 0.1% yeast extract, 0.1% peptone, 0.1% NH4NO3, 0.14% KH2PO4, 0.02% MgCl2, 1% Congo Red and 3% Agar (Rattanasuk and Ketudat-Cairns 2009). The plates were incubated at 30ºC for 24 hours, 48 hours and 72 hours. The mannanase activity of each isolate was measured based on the ratio of the clearing zone (dark blue-black colouration) formed. The colonies with highest clear zone were collected and maintained as frozen stocks in the presence of 20% glycerol (Adebayo-Tayo et al. 2013).
Secondary mannanase screening
The Mannanase Screening Medium (MSM) containing in g/l: Peptone 0.1, yeast extract 0.1, MgCl2 0.02, KH2PO4 0.14, NH4NO3 0.1, and Gum Arabic 1.0, Distilled water 1 liter and pH 5.5 was used. 100 ml of the sterile mannanase production medium was inoculated with 0.5 ml of the isolate after using Mac Farland Standard and incubated for 48 hrs. After incubation, the fermentation medium was harvested by centrifugation at 4000rpm for 30 minutes at 4°C. The supernatant was used to assay for mannanase activity (Adebayo-Tayo et al. 2013).
Molecular identification of the tolerant strain
DNA Extraction Protocol
The extraction of the yeast genomic DNA for molecular analysis was carried out according to the method of Arnold et al. (2011). Colonies of yeast isolates were picked from a Yeast Extract Agar (YEA) plate and was suspended in 100 μL 200 mM LiOAc 1% SDS solution, and incubated at 70°C for 15 min. After incubation, 300 μL 96% ethanol was added and the samples were mixed by brief vortexing. The DNA was collected by centrifugation at 15,000× g for 3 minutes. Precipitated DNA was dissolved in 100 μL Tris-EDTA (TE) buffer. The cell debris was spun down by brief centrifugation (15,000× g for 1 min), and 1 μL supernatant was used for PCR.
Mannanase production in submerged fermentation
The Mannanase Production Medium (MPM) containing in g/l: Bacteriological peptone 0.1, yeast extract 0.1, MgCl2 0.02, KH2PO4 0.14, NH4NO3 0.1, and locust bean gum (LBG) 1.0, Distilled water 1 liter and pH 5.5 was used. 100 ml of the sterile mannanase production medium was inoculated with 0.5 ml of the isolate and incubated for 24 hrs. After incubation, the fermentation medium was harvested by centrifugation at 4000rpm for 30 minutes at 4°C. The supernatant was used to assay for mannanase activity (Adebayo-Tayo et al. 2013).
Mannanase assay and protein determination
The mannanase activity was assayed by measuring the reducing sugars using dinitrosalicylic acid (DNSA) method (Miller 1959). An assay mixture containing 0.5 ml of the culture supernatant and 0.5 ml of 0.5% (w/v) Locust bean gum (LBG) in 0.05M phosphate buffer at pH 5.5.0 was incubated at 50°C for 30 min. After incubation, 1 ml of reagent was added and boiled for 5 – 15 min. The developed red brown color was measured at 575 nm. The amount of reducing sugar released was determined by the dinitrosalicylic acid (DNSA) method (Miller 1959). One unit of mannanase activity was defined as amount of enzyme producing 1 micromole of mannose per minute under the experimental conditions. The amount of protein produced in the basal medium was also determined by the method of Lowry et al 1951 using Bovine Serum Albumin (BSA) as the standard.
Optimization of the cultural parameters in submerged fermentation
The basal medium was optimized with various factors that influence the mannanase production. The various physicochemical parameters of fermentation were optimized which included; temperature, pH, incubation time, incubation temperature, carbon sources and nitrogen sources.
Effect of Temperature
The effect of temperature on mannanase production using submerged fermentation was carried out at different temperatures. This was done between the range of (25, 30, 35, 40, and 45) °C (Adebayo-Tayo et al. 2013; Oskay and Yalcin 2014).
Effect of pH
The effect of pH on enzyme production was also carried out. pH values ranging from ( 4, 5,6,7 and 8) was used to investigate the effect of pH on the production of mannanase. Acetate, citrate and phosphate buffer were the buffers of choice used in this experiment (Adebayo-Tayo et al. 2013; Oskay and Yalcin 2014).
Effect of Incubation time
The production enzyme for mannanase were harvested at different incubation time (24, 48, and 72) hours and assayed to determine the best time that supports enzyme production (Adebayo-Tayo et al. 2013; Oskay and Yalcin 2014).
Effect of nitrogen sources
To investigate the effect of the different nitrogen sources on mannanase production, the only nitrogen sources in the basal medium which was peptone and yeast extract were replaced by different nitrogen sources such as Urea, casein, ammonium sulphate, ammonium chloride and yeast extract/peptone(1:1) at1% (w/v) concentrations (Adebayo-Tayo et al. 2013; Oskay and Yalcin 2014).
Effect of carbon sources
The effect of various carbon sources such as glucose, fructose, sucrose, maltose and mannitol at 1% (w/v) concentrations was also carried out in addition to gum arabic which was the sole source of carbon in the basal medium (Adebayo-Tayo et al. 2013; Oskay and Yalcin 2014).
Purification of enzyme
Ammonium Sulphate (NH4SO2) Precipitation
After determining the percentage saturation of ammonium sulphate salts that gave the highest activity, the equivalent amount of salt for 1 litre of crude enzyme was added. The salt was allowed to dissolve completely and the mixture was allowed to stand for 30 hrs at 4ºC. It was then centrifuged at 4000 rpm for 30minutes. The pellets were collected and stored in a cool place for further studies (Khairnar et al. 2009).
Dialysis
Dialysis tubes stored in 90% ethanol were used. The tubes were rinsed thoroughly with distilled water and finally with 0.05M phosphate buffer in order to remove traces of ethanol. An amount of the precipitated enzyme was poured into the dialysis tubes and placed in a beaker containing 0.05 M phosphate buffer. The beaker was placed on a magnetic stirrer which allows for a homogenous environment. The dialysis was carried out according to Dixon and Webb (1964) for 12 hours and the buffer is changed after 6 hours which allows for the exchange of low molecular weight substances and left over ammonium sulphate salts that may interfere with the activity. After dialysis, mannanase activity was measured in each fraction applying DNS method: 0.5 ml of dialysed partially purified enzyme was added to 0.5 mL gum arabic 1% (w/v) in 0.05M phosphate buffer (pH 6.0) separately. Test tubes were covered and incubated for 5mins at 65°C in a water bath. Then, 1 mL DNS reagent was added to each tube to stop the reaction and placed in boiling water bath for 5mins. After cooling the samples in a cold water bath, the absorbance was read at 540 nm (Khairnar et al. 2009).
Gel-filtration chromatography
Mannanase was purified to homogeneity by the gel-filtration chromatography. The dialysed enzyme was loaded on the gel permeation column (Sephadex G-25). All the eluted fractions were estimated for enzyme activity and absorbance was read at 540nm and 575nm respectively. The fractions showing the highest enzyme activity were pooled and assayed for protein content. The specific activity of purified enzyme fractions was compared to that of crude enzyme and fold purification was calculated (Sharma et al. 2005).
Characterization of partially purified mannanase enzyme
Effect of pH Change on Mannanase Activity
The effect of pH on enzyme activity was determined using 0.05M sodium acetate buffer pH 3.5 – 5.5, phosphate buffer pH 6.0 – 7.5 and Tris-HCl buffer pH 8.0 – 10.0 at intervals of 1.0. 0.1% gum arabic solution was prepared by dissolving 0.1g gum arabic in 100ml of 0.05M of the respective buffers separately. Also 0.5 ml of the partially purified enzyme was added to 0.5 ml of each of the buffers. Then ultimately, 0.5ml of the enzyme-buffer solution was mixed with 0.5ml gum arabic solution at the corresponding pH for mannanase assay as described previously (Adebayo-Tayo et al. 2013; Oskay and Yalcin 2014).
Effect of Temperature Change on Mannanase Activity
The optimum temperature was determined by incubating the enzymes with gum arabic solution at 35- 50ºC interval of 5ºCfor 1hour and at the pH with the highest activity. The activity was then determined as described previously (Adebayo-Tayo et al. 2013; Oskay and Yalcin 2014).
Effect of Substrate Concentration on Mannanase Activity
The effect of substrate concentration on the activity of mannanase was determined by incubating the enzyme with 0.5 up to 2.0mg/ml gum arabic at an interval of 0.5 using the buffer at the pH with highest activity and the temperature at which highest activity was determined (Adebayo-Tayo et al.,2013; Oskay and Yalcin 2014).
Km and Vmax were determined by incubating the enzyme with varying concentrations of substrate. The results were plotted as a graph of rate of reaction (v) against concentration of substrate[S].
The relationship is defined by the Michaelis-Menten equation:
v = Vmax / (1 + (Km/[S])
Thermal Stability of Mannanase
For thermal stability, the partially purified enzyme was pre-incubated for 1h at various temperatures (50, 60, 70, 80, 90)˚C before enzyme assay, and promptly cooled on ice and residual activity was determined under standard assay conditions.
Statistical Analysis
Each of the 𝛽-mannanase activity experiment was done in triplicate and expressed as mean ±SD (standard deviation) using SPSS and analysis of variance (ANOVA) was used to compare various treatment groups.