Production and partial purification of beta-mannanase from Aspergillus niger associated with ilaje lake, Ondo state, Nigeria

Beta-mannanase otherwise known as mannan endo-1,4-βmannosidase or 1,4-β-D-mannanase with an enzyme commission (EC) 3.2.1.78 breakdowns β-1,4-mannosidic bonds in the main chain of glucomannans, galactomannans and β-1,4-mannans.1 It transforms the abundant mannan-rich heteromannan, glucomannan and galactomannan substrates into manno-oligosaccharides2 and a small amount of mannose, glucose and galactose.1,3 A number of plants, bacteria, fungi and various invertebrates had been reported to produce β-mannanase.1,3 The mannanase of biological origins have found a variety of applications in different industrial sectors,3 including animal feed formulation, pharmaceutical products preparation, pulp biobleaching,4 as well as pre-treatment of plant biomass for second generation biofuel production.5


Introduction
Beta-mannanase otherwise known as mannan endo-1,4-βmannosidase or 1,4-β-D-mannanase with an enzyme commission (EC) 3.2.1.78 breakdowns β-1,4-mannosidic bonds in the main chain of glucomannans, galactomannans and β-1,4-mannans. 1 It transforms the abundant mannan-rich heteromannan, glucomannan and galactomannan substrates into manno-oligosaccharides 2 and a small amount of mannose, glucose and galactose. 1,3 A number of plants, bacteria, fungi and various invertebrates had been reported to produce β-mannanase. 1,3 The mannanase of biological origins have found a variety of applications in different industrial sectors, 3 including animal feed formulation, pharmaceutical products preparation, pulp biobleaching, 4 as well as pre-treatment of plant biomass for second generation biofuel production. 5 There is a greater demand for biotechnological and industrial stable enzymes with higher catalytic efficiency that can function under harsh environmental conditions. In the recent years, screening and isolation of microorganisms for the production of industrial enzymes of microbial origin with unique and excellence characteristics has become one of the focuses of research in biotechnology. Enzymes required for industrial applications should be stable at high temperature, pH, and presence of salts, solvents, and etcetera. Extracellular enzymes from microbes associated with extreme environments such as halophiles are blessed with higher catalytic power to initiate and sustain metabolic activities under high salt concentrations. Gomes & Steiner 6 and Karan & Khare 7 have been reported that enzymes from extreme environments are expected to be active and stable under more than one extreme condition.
The physicochemical analysis carried out on four different coastal waters from Ilaje, Ondo state, Nigeria revealed that they were slightly acidic with a mean pH of 6.69 and also moderately saline with a mean salinity of 16.68%. 8 Hence, mannanase obtained from bacteria isolated from this source are expected to be halotolerant. Mannanaseproducing microorganisms have been reported from different sources but there has been scanty or no information on mannanase production from bacteria inhabits Ilaje Lake. Hence, enzymes from haloterant bacteria from this location should have an edge in terms of stability. In search of high performance mannolytic organisms; we isolated and screened different fungi from soil and water samples in Ilaje, Ondo State, Nigeria. Therefore, the present study dealt with the isolation and screening of fungal isolates for beta-mannanase production and its partial purification from Aspergillus niger.

Sources of Sample
Water 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 mannanaseproducing fungi. 9,10 Isolation and identification of mannanase-producing fungi from soil and water samples The 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 fermentation
Medium 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 1g,mgSO 4 .7H 2 O 0.5g, KCl 0.5g 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/ Conductivity meter) prior sterilization. Then, 100mL of the liquid medium was dispensed in 250mL Erlenmeyer flask and sterilized at 121°C for 15minutes. The sterile basal medium was inoculated with 2discs of 8mm diameter of the fungal strains from PDA using cork borer. The inoculated media were incubated at 30°C for 5days at static condition. Crude enzyme preparation was obtained by centrifugation at 6000rpm for 10minutes at 4°C using refrigerated ultracentrifuge (Centurion Scientific Limited). The supernatant was used as the crude extracellular enzyme source. Each treatment was carried out in triplicates and the results obtained throughout the work were the mean of at least 3 experiments.

Enzyme assay and protein determination
Mannanase activity was assayed in the reaction mixture comprising of 0.5mL of 1% LBG prepared in 50mM potassium phosphate buffer pH 6.8 and 0.5mL of supernatant at 45 °C for 60minutes (modified method of El-Naggar et al. 14 ). At the end of the incubation period, tubes were removed from the water bath (Lamfield Medical England Model DK-600), and the reaction was terminated by the addition of 2mL of 3, 5-dinitrosalicylic acid (DNSA) reagent per tube. The tubes were incubated for 5minutes in a boiling water bath for colour development and were cooled rapidly. The activity of reaction mixture was measured against a reagent blank at 540nm. Amount of reducing sugar released was determined by the dinitrosalicylic acid reagent (DNS). 15 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 evaluated according to the method of Lowry et al., 16 using Bovine Serum Albumin (BSA) as the standard.

Mannanase purification
Mannanase purification was performed in three stages. In the first stage, the supernatant obtained after centrifugation was precipitated by the addition of ammonium sulphate to achieve 70% ammonium sulphate concentration. 1 The precipitated enzyme was then diluted in phosphate buffer 50mM pH 7.0, and dialyzed in the same buffer. Secondly, the enzyme obtained after dialysis was loaded into ion exchange column chromatography with diethylaminoethyl (DEAE) Sephadex G-200 matrix (20×2.5cm, Pharmacia). The fractions obtained were sequentially washed with ion free water and0.01 M Tris-HCl buffer pH 8.0. The bound protein molecules were eluted with concentration gradient of NaCl. The absorbance of each fraction collected was measured at 280nm with UV spectrophotometer (Lab-Tech Digital) and the activity of mannanase of each fraction was determined. In the third stage, concentrated enzyme from second stage purification step was loaded onto the column chromatography (2.5cm in diameter and 30cm high) which contained Sephadex G-200 (Pharmacia). The concentrated enzyme was eluted with phosphate buffer pH 7.0 at the flow rate of 20mL/h.A fraction of 10mL was collected at interval of 30 mins and the absorbance was taken at 280nm. Fractions with mannanase activity were pooled and concentrated in glycerol solution at 30°C. Table 1 shows the total fungal counts from each of the sources. Water sample had the highest number of fungal population of 3.4x10 8 sfu/ml, while soil sample from Ilaje Lake (2.2×10 8 sfu/g) recorded the least fungal counts.

Mannanase production by fungal isolates from different samples
The quantitative determination of mannan-degrading enzyme from different fungal isolates is shown in Table 4. All the fungal isolates encountered from different sources displayed varied degrees of mannanase activities. The highest mannanase activity and specific activity of 9.37U/mL and 2.59U/mg respectively was produced by fungal isolate 4B1 sourced from water sample, while the lowest values for mannanase activity and specific activity were obtained from 2E3 and 1B2 respectively. Protein content ranged from 3.06mg/ mL to 5.39mg/mL was produced by the fungal isolates, with the highest protein content displayed by isolate IB2 obtained from soil sample. Therefore, fungal isolate 4B1 was selected for purification studies because of its highest mannanase-producing potential.

Elution profile of partially purified mannanase on sephadex G-200
After 25% ammonium sulphate saturation of crude enzyme, the precipitates were dialyzed against 50mM phosphate buffer pH 6.8 for 24hours at 4°C. Dialyzed protein was loaded onto a Sephadex G-200 column which was eluted with the same buffer. The ammonium sulphate-dialysate fraction on Sephadex G-200 tends to produce two activity peaks represented as A and B (Figure 1). Mannanase activity increased appreciably and reached A with an enzyme activity of 98.09μmol/min/mL, while peak B had enzyme activity of 148.12μmol/ min/mL. ILS, soil sample from ilaje lake; ILW, water sample from ilaje lake

Summary of purification procedures
Three step purification procedures were adopted, and a 7.43-fold purification was achieved with specific activity of 48.06μmol/min/ mg (Table 5). Enzyme activity increased from 169.13μmol/min/mL in crude enzyme to 218.46μmol/min/mL after gel filtration. Crude protein and total protein decreased from 26.14 and 3920.46mg/mL to 4.55 and 58.18mg/mL respectively. EA, enzyme activity (μmol/min/mL); CP, crude protein (mg/mL); TP, total protein (mg/mL); TA, total activity (μmol/min/mL); SA, specific activity (μmol/min/mg)

Discussion
Mannanases from Fungi are generally produced into culture medium supplemented with different mannan-rich substrates known as inducers. These include Locust Bean Gum (LBG), guar gum, konjac flour, and copra meal. Also, some microorganisms are capable of secreting considerable volume of extracellular enzymes into their basal media and this property has been adopted for industrial enzyme production.
The high fungal counts from water sample may be due to lack of efficient control measures in the discharge of organic wastes into the water bodies. The excessive addition of organic wastes (nutrients) to water bodies is known to stimulate excessive growth of microorganisms. 10 The fungal isolates obtained from Ilaje lake (water and soil samples) exhibited different mannanase activities in basal media supplemented with LBG as an inducer. The production of mannanase in LBG medium had been reported for Bacillus circulans, Chryseobacterium indologenes, Bacillus sp.mg-33, Bacillus amylolequifaciens 10A1, 4 Bacillus sp., Aspergillus niger, Sclerotium rolfsii, Trichoderma sp. and Scopulariopsis candida. The secretion of mannanases by these isolates on LBG media could be attributed to the ability of their genetic make up to secrete active mannanase coupled with varied diffusion rate. 10 All the tested fungal strains were able to produce extracellular mannanase in submerged state fermentation, although with differences in the rate of enzyme production. These differences might be attributed to the source of isolation and slight variation in their genetic makeup. 10 The variation in protein content generated by each of the strains in submerged state fermentation could be attributed to the production of variety of enzymes (amylases, cellulases, protease and xylanases) in addition to the enzyme been examined in this study. Presumably, the protein from fungal cells and metabolites rich in protein might interfere with mannanase production causing variation in protein contents, since the protein assay could only identify accumulated protein in enzyme production medium. 10 Preliminary investigations in this study revealed that Aspergillus niger gave the highest mannanase activity out of all the fungal isolates encountered from the lake. It was therefore selected for purification studies. Mannanase has been produced by various microorganisms and purified as reported in previous studies. Olaniyi et al., 1 produced and purified mannanase enzyme from Penicillium italicum isolated from yam peel. Madau & Shetati sourced for β-1, 4-mannanase from Scopulariosis candida and purified it while Sumardi isolated Geobacillus stearothermophilus L07 from oil palm shell, screened for mannanase production after which it was purified. Abdel-fattah purified β-mannanase from Aspergillus oryzae. The two major activity peaks obtained from the elution profile could be attributed to the source of isolation coupled with environmental influences. 1

Conclusion
Aspergillus niger isolated from water sample in Ilaje, Ondo state, Nigeria showed a potential to convert substrates containing mannan into simple carbohydrates which could be readily used in many applications such as animal foods and a feed stock for production of prebiotics. It is recommended that the characterization of the purified β-mannanase and molecular study should be carried out on the isolate.