2.1. Description of study Area
Adequate soil samples were collected from dairy farms and grape farm at three regions of Ethiopia, i.e, Amhara Region (Shewa Robit), Oromia Region (Ziway) and Southern Nations Nationalities People Region of Ethiopia (Halaba). Shewa Robit is located at 100 06' 650" - 090 57'957" N, 0390 54'37"-0390 56'579" E, in North Showa Zone of Amhara Regional State, 225 km North of Addis Ababa, Ethiopia. The agro-ecology of the study site is low-land or Erteb Kola (sub-moist warm) with altitude ranging between 1120 and 1350 m above sea level. The climate data of the study area recorded for the last ten years shows average annual maximum and minimum temperature and precipitation of 32.1, 16.1 oC, and 968 mm, respectively. The most dominant soil type in Shewa Robit is vertisols, which have clay texture with clay content of 56%, sand 10%, silt 34% and a pH value of 8.02 (5).
The second study area, Ziway is located at 7.58° N latitude and 38.43° E longitude in the Southern part of Oromia regional state situated in mid rift valley, 160 Kms South of Addis Ababa. The agro ecologically area is categorized under the semiarid, with minimum mean temperature of 12.7°C and maximum mean temperature of 27.2°C with relative humidity of 60%. The area has an altitude ranging between 1500 and 2000 meter above sea level. The average annual rainfall ranges from 650-750mm and the distribution is highly variable between and within years. Vertisol is the predominant soil type with sand-silt-clay in the portion of 33:48:18; respectively, and has a pH of 7.88 (6).
2.2. Sample collection
Five hundred grams of soil (5-10 cm below the surface ) , were collected aseptically from grape farm and dairy farm areas located at Showa Robit, Ziway and Halaba, Ethiopa in September 2019. Soil samples were sieved (3–4 mm mesh), homogenized and stored at 4 0C at Microbiology laboratory of Biology Department for further use as a protocol described by (7).
2.3. Isolation of Fungi
The fungi were isolated by serial dilution technique as described by (8). Thus, ten grams of each soil samples were mixed with a 90 mL of distilled water and homogenized by agitation for 20 min. They were prepared to appropriate dilutions from which 0.1 mL of each sample suspension was plated on Potato Dextrose Agar (PDA) (Microgen, India) plates containing (0.05 g/L) chloramphenicol. The plates were incubated at 30 0C for 5-7 days to isolate distinctive colonies. Each colony was then re-streaked on the same medium to purity and preserved at 4 oC.
2.4. Primary Screening for protease producing Fungi
Primary screening for protease production was tested using skim-milk agar (Nestle TM, Frankfurt, Germany) medium for the production of the clear zone (9). The detection medium (Skim-Milk Agar, SMA) was prepared using 20 g of skim milk, 20 g of agar-agar each dissolved in 200 mL distilled water and 600 mL of 0.2 M Phosphate buffer (K2HPO4 and KH2PO4, pH 5.0). All the three media components were autoclaved separately to avoid coagulation and charring of milk due to the presence of buffer salts and later mixed under sterile conditions. The plates were then subsequently inoculated with previously purified fungal isolates and incubated at 30 oC for 2 days. The plates were examined for the formation of the clearing zone by flooding them with a solution of 10% Trichloroacetic acid (TCA) or 10% tannic acid. The relative enzyme activity was calculated using the inhibition zone diameter and colony diameter.
REA = CZD / CD
Where; REA: relative enzyme activity, CZD: Clear Zone Diameter, CD: Colony diameter (9:10).
2.5 Secondary screening for Acid protease under Solid-State fermentation
2.5.1. Inoculum preparation
Fungal isolates were grown on potato dextrose agar (PDA) and incubated at 30 oC for 5 days. They were scrapped using 10 mL of sterile distilled water to prepare spore suspension. 1 mL of spore solution (106 spores/mL) was used according to (11).
2.5.2. Medium and cultural conditions for solid-state fermentation for fungi (M1)
For solid-state fermentation, 0.5 mL of spore suspension (106 spores/mL) was transferred into 250 mL Erlenmeyer flasks containing wheat bran (10 g), skim milk powder (2.0 g) and 10 mL of salt solution (g/L: 2.0, KNO3; 0.5, MgSO4·7H2O; 1.0, K2HPO4; 0.439, ZnSO4·7H2O; 1.116, FeSO4·7H2O; 0.203, MnSO4·7H2O and pH 5 (11). The flasks were incubated at 30 °C for 6 days under static conditions.
2.5.3. Medium and cultural conditions for solid-state fermentation for Fungi (M2)
The solid-state substrate was also prepared in 250 mL Erlenmeyer flask containing 10 g of wheat bran (Durum wheat bran) moistened by adding 12 mL of HCl (0.2 M) by mixing thoroughly. Then after, the flasks were autoclaved at 121 0C for 30 min. Then 0.5 mL of (106 spores/ml) suspension were inoculated into SSF media and incubated at 30 0C for 6 days (12).
2.6 Enzyme extraction
The enzyme was extracted according to the method of (13). Thus, the fermented substrates were dispersed in 100 mL of distilled water (1:10 ratio of Bran - solvent w/v), and vigorously shaken on a rotary shaker at 240 rpm at room temperature for 40 min and filtered by cotton cloth. The filtrate was then centrifuged (NF200, year, 2009, SN, 02-4005) at 10000 x g, 4 0C for 10 min. The supernatant was used as a crude enzyme.
2.7. Assay for acid protease activity
Protease activity was assayed according to the method of (14) using hemoglobin as a substrate. Enzyme preparation (0.5 mL), suitably diluted, was mixed with 1 mL of 2% (w/v) hemoglobin in 100 mM glycine-HCl (pH 3.0) and the mixture was incubated in a water bath at 50oC for 10 min . The reaction was terminated by adding 2 mL trichloroacetic acid 5% (w/v). The mixture was allowed to stand at room temperature for 15 min and then centrifuged (NF200, year, 2009, SN, 02-4005) at 10,000×g for 15 min to remove the precipitate. The absorbance of the soluble fraction was measured at 280 nm. A standard curve was generated using tyrosine solutions at 0–50 mg/L. One unit of protease activity was defined as the amount of enzyme required to liberate 1 μg of tyrosine per min under the experimental conditions. Tyrosine standard curve and protease activity of the enzyme was calculated.
2.8. Tyrosine standard curve
Tyrosine standard curve was used to quantify the amount of enzyme produced under SSF. Determination of protein concentration by measuring absorbance at 280 nm (A280) was based on the absorbance of UV light by the aromatic amino acids tryptophan and tyrosine. The measured absorbance of a protein sample solution was used to calculate the concentration by comparison with a calibration curve prepared from measurements with standard protein solutions. This assay was used to quantitate solutions with protein concentrations of 20 to 3000 μg/ml. Protease enzyme produced by fungal isolate under SSF was quantified by using tyrosine standard curve. The absorbance of the soluble fraction was estimated at 280 nm. A standard curve was generated using tyrosine solutions at 0–50 μg /ml. One unit of protease activity was defined as the amount of enzyme required to liberate 1 μg of tyrosine per min under the experimental conditions. According Protease Colorimetric Detection Kit (sigma)
Whereas PA; Protease activity, µTry: µg of tyrosine equivalent released, Vt; Total volume of assay in ml, Vs; Sample volume, T ; Reaction time in water bath, Va; Volume of assay for absorbance.
2.9. Assay for milk-clotting activity
Milk clotting activity was determined according to the method of (15), which is based on the visual evaluation of the appearance of the first clotting flakes, and expressed in terms of Soxhlet units (SU). One Soxhlet unit is defined as the amount of enzyme that clots 1 ml of substrate in 40 min at 35°C. In order to perform the assay, 0.1 ml of the sample was added to a glass test tube containing 1 ml of reconstituted skim milk solution (10 g skim-milk powder dissolved in 100 ml of 0.01 M CaCl2 solution) pre-incubated at 35°C for 10 min. The mixture was mixed well and the clotting time t (s) was measured with a chronometer. The clotting activity was calculated using the following formula: SU = (2400 x 1 x D) / 0.1 x t
Where, D is the dilution factor and t is the clotting time in seconds
2.10. Morphological characterization
The cultural characteristics (colony growth rate, colony texture, colony color, colony size and degree of sporulation) of the fungal isolates were studied by inoculating them on Czapek Dox agar (CDA), Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) as described (16). The micro morphological characteristics of the isolates were observed under the microscope after having prepared them on a slide culture.
2.11. Optimization of cultural conditions and media composition for production of acid protease under SSF
1.1.1. Experimental set-up for preliminary screening for production of acid protease in SSF
The experimental set up for solid-state fermentation was according to (12), with slight modification. The inoculum (0.5 mL of 10 6 spores/mL) was transferred into 250 mL Erlenmeyer flasks containing 10 g of wheat bran (WB) and 2 g of skim-milk powder moistened by 12 mL HCL (0.2 M) / 10 ml of mineral solutions. The flasks were incubated at 30 oC for six days.
2.11.2. Effect of substrate on acid protease production
Screening of the media composition for acid protease production was performed by one-variable-at-a-time approach (17;18). Thus, 0.5 mL of spore suspension (1*106 spore/mL) from potential fungi isolate were inoculated into flask containing ten grams of each substrate (wheat bran, Rice Bran) in 250 Erlenmeyer flasks moistened by 12 mL HCL (0.2M)/salt solution and incubated at 30 0C for 144 h. The crude enzyme was extracted as previously described and assayed for acid protease activity according to (14). After having tested the effect of substrates on enzyme production, the highest enzyme-producing substrate was selected and tested for further optimization.
2.11.3. Effect of incubation time
The effect of incubation time on acid protease production was studied by inoculating the flasks containing the best substrates with 0.5 mL of spore suspension (1*106 spore/mL) and incubated at 30 0C for different time periods ranging from 24 h to 144 h (18). The acid protease activity was monitored as previously described.
2.11.4. Effect of incubation temperature
The fungal spores inoculated into SSF medium in 250 mL Erlenmeyer flask was incubated at a temperature of 20 0C ,25 0C, 30 0C, 35 0C ,40 0C, and 45 0C for 120 h to determine the optimum temperature. Then, the obtained optimum temperature used for further study (18). The acid protease activity was determined as described previously.
2.11.5. Effect of inoculum size
The effect of inoculum size on acid protease production was studied by inoculating 0.2 mL, 0.5 mL, 1 mL, 1.5mL and 2 mL of (3.2*106 spores/mL) spore suspension in to SSF media. Then the inoculated flasks were incubated at optimum temperature for 120 h (18). Acid protease activity was assayed as described previously.
2.11.6. Effect of moisture content
The effect of initial moisture content for enzyme production were tested by moistening substrate with distilled water at different percentages of moisture content; 45%, 50%, 55%, 60%, 65%, and 70% to find out the best percentage for enzyme production (19). The acid protease activity was assayed as described previously.
2.11.7. Effect initial media pH
The effect of initial media pH on acid protease production was optimized by adjusting the SSF medium to pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 using HCL or diluted NaOH (18). The acid protease activity was assayed as described previously.
2.11. Partial purification
2.12. Acetone precipitation
The crude enzyme extract was precipitated with chilled acetone (acetone or 75% acetone). Two volumes of chilled acetone were slowly added to the extract and the precipitate was allowed to settle for 1h at -18 0C to permit complete precipitation. The precipitated protein was then separated by centrifuging at 10000 g and 4 °C for 10 min. The pellet was dried in the open air for 30 min to remove trace amounts of acetone and dissolved in 0.02 M phosphate buffer, pH 6.0 (20).
3.9.2. Ammonium sulfate precipitation
The crude enzyme extract was precipitated with ammonium sulfate according to (21). Accordingly, 300 mL of the crude extract was be added to 1200 ml saturated ammonium sulfate (100%) to precipitate. Then, the enzymatic solution will be decanted for one night at 4 0C and centrifuged at 10000 x g for 10 min at 4 0C and the pellet was suspended in the phosphate buffer (0.02 M; pH 6).
3.10. Enzyme characterization
3.10.1. Determination of optimum pH and its stability
The optimum pH of the enzyme preparation was studied over a pH range of 2.0–7.0 at 50 ◦C using 2% (w/v) hemoglobin as described previously. For studying pH stability, the crude enzyme was incubated in buffers (pH 3 and 3.5, Glycine HCL,4-6 by sodium acetate,6.5 and 7 with potassium phosphate) of different pH values in the range of pH 3.0–7.0 for 1 h at 4◦C. Residual proteolytic activity was then determined under standard assay conditions. The following buffer systems were used: 100 mM glycine-HCl buffer for pH 3.0, 100mM sodium acetate buffer for pH 4.0–6.0, 100mM potassium phosphate buffer for pH 7.0 (17).
3.10.2. Determination of optimum temperature and its stability
To investigate the effect of temperature, proteolytic activity was tested at different temperatures (20, 25, 30,35,40,45,50,55,60 and 65) using hemoglobin as a substrate for 5 min at pH 5.0. Thermal stability was examined by incubating the enzyme for 60min at 30, 40, 50, 60 and 70◦C. Aliquots were withdrawn at desired time intervals to test the remaining activity at optimum conditions of pH and temperature. The non-heated enzyme was considered as the 100% control (17).
3.9. Data Analysis
The data analyses were performed by using SPSS software version 23 (Inc. Cary NC USA). All experiments were carried out in triplicate. Analysis of variance (ANOVA) and means comparisons will be done by Sigma plot version12.