Production, Optimization and Characterization of an Acid Protease From Filamentous Fungi by Solid-state Fermentation


 Acid proteases represent an important group of enzymes, extensively used in food and beverage industries. There are a diversification of food industries and thus an increasing demand for biocatalysts capable of adapting the industrial extreme environments. These demands can be covered by a plant and animal proteases; however there is a shortage to meet the present industrial demands. This necessitates the search for an alternative acid protease sources from fungi. The fungal isolates were recovered from grape and dairy farm soil using potato-dextrose Agar. The fungi were further screened for protease production based on the hydrolysis of clear-zone on skim-milk agar media. The potential fungi were then subjected to secondary screening under solid-state fermentation. After primary and secondary screening, the potential fungus (isolate Z1BL1) was identified to the genus level by combination of macroscopic and microscopic morphological study. The growth condition and media composition for potential fungal isolate (Z1BL1) was further optimized under solid-state fermentation. The crude enzyme produced from isolate Z1BL1 was characterized after partial purification by acetone and ammonium sulphate precipitation. A total of 9 fungal isolates were showed protease production in primary and secondary screening, however 1 potential isolate (Z1BL1) was selected for further study based on its protease activity. The potential fungus, isolate Z1BL1 was identified to the genus Aspergillus based on their morphological features. The optimization of media composition and growth conditions for acid protease production from Z1BL1 were slightly increased the protease activity. The acetone precipitated enzyme exhibited the maximum activity at 50 0C and pH 5 with stability at pH 4-6 and temperature 40-60 0C. Thus based on the above findings, the acid protease produced from Aspergillus was shown suitable enzyme characteristics required in industry and could be a candidate to be applicable in food industry after further purification by high resolution techniques.

Most of the aspartic proteases (Aps) show the best activity at low pH (pH 3 to 4) and have isoelectric points in the pH range of 3 to 4.5. They are inhibited by a hexapeptide from Streptomyces that contains two statin residues called pepstatin. Aspartic proteases are also sensitive to diazoacetyl-DL-norleucine methyl ester (DAN) and 1, 2-epoxy-3-(p-nitrophenoxy) propane (EPNP) in the presence of copper ions.
Microbial acid proteases exhibit speci city against aromatic or bulky amino acid residues on both sides of the peptide bond, which is similar to pepsin, but their action is less stringent than that of pepsin (4). Acid protease has a wide range of application in various industries to make a change in product taste, texture, and appearance and in waste recovery. Besides this, they have extensive applications in food industry, beverage industry, and pharmaceutical industry (4). The increasing importance of these enzymes and their numerous applications in different industries made us to investigate acid protease production from a new source. The isolation and characterization of new promising strains are possible ways to increase the yield of such enzymes. The aim of the present study was to isolate the fungi from the soil, identi cation of the culture, screen the protease producing culture, optimize of cultural conditions for the production of acid protease and partial characterization of the enzyme.

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 agroecology 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 o C, 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).

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 0 C at Microbiology laboratory of Biology Department for further use as a protocol described by (7).

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 0 C for 5-7 days to isolate distinctive colonies. Each colony was then re-streaked on the same medium to purity and preserved at 4 o C.

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 (K 2 HPO4 and KH 2 PO4, 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 puri ed fungal isolates and incubated at 30 o C for 2 days. The plates were examined for the formation of the clearing zone by ooding 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.
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 o C for 5 days. They were scrapped using 10 mL of sterile distilled water to prepare spore suspension. 1 mL of spore solution (10 6 spores/mL) was used according to (11).

Medium and cultural conditions for solid-state fermentation for fungi (M1)
For solid-state fermentation, 0.5 mL of spore suspension (10 6 spores/mL) was transferred into 250 mL Erlenmeyer asks containing wheat bran (10 g), skim milk powder (2.0 g) and 10 mL of salt solution (g/L: The solid-state substrate was also prepared in 250 mL Erlenmeyer ask 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 asks were autoclaved at 121 0 C for 30 min. Then 0.5 mL of (10 6 spores/ml) suspension were inoculated into SSF media and incubated at 30 0 C for 6 days (12).

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 ltered by cotton cloth. The ltrate was then centrifuged (NF200, year, 2009, SN, 02-4005) at 10000 x g, 4 0 C for 10 min. The supernatant was used as a crude enzyme.

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 50 o C 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 de ned 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.

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 quanti ed 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 de ned 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.

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 rst clotting akes, and expressed in terms of Soxhlet units (SU). One Soxhlet unit is de ned 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 Where, D is the dilution factor and t is the clotting time in seconds

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 modi cation. The inoculum (0.5 mL of 10 6 spores/mL) was transferred into 250 mL Erlenmeyer asks 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 asks were incubated at 30 o C for six days.

Effect of substrate on acid protease production
Screening of the media composition for acid protease production was performed by one-variable-at-atime approach (17;18). Thus, 0.5 mL of spore suspension (1*10 6 spore/mL) from potential fungi isolate were inoculated into ask containing ten grams of each substrate (wheat bran, Rice Bran) in 250 Erlenmeyer asks moistened by 12 mL HCL (0.2M)/salt solution and incubated at 30 0 C 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.

Effect of incubation time
The effect of incubation time on acid protease production was studied by inoculating the asks containing the best substrates with 0.5 mL of spore suspension (1*10 6 spore/mL) and incubated at 30 0 C for different time periods ranging from 24 h to 144 h (18). The acid protease activity was monitored as previously described.

Effect of incubation temperature
The fungal spores inoculated into SSF medium in 250 mL Erlenmeyer ask was incubated at a temperature of 20 0 C ,25 0 C, 30 0 C, 35 0 C ,40 0 C, and 45 0 C 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.

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*10 6 spores/mL) spore suspension in to SSF media. Then the inoculated asks 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 nd out the best percentage for enzyme production (19). The acid protease activity was assayed as described previously.

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 puri cation 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 0 C 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).

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 0 C and centrifuged at 10000 x g for 10 min at 4 0 C and the pellet was suspended in the phosphate buffer (0.02 M; pH 6). 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).

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).

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.  (Table 1). Among the isolates, the highest REA were recorded by isolate H1W1 (0.8) and Z1BL1 (0.74) ( Table 1). The spore concentration of all the 12 isolates was determined using the Neubauer chamber and was in the range between 1.2 x 10 6 and 4.4 x10 8 spores/mL (Table 1).

Secondary screening of Acid protease under Solid-State fermentation (SSF)
All the twelve isolates that showed a relative protease activity (REA=CZD/CD) ≥0.4 were subjected for further screening in solid-state fermentation. Out of the twelve isolates, 9 isolates showed an acid protease activity under SSF ( Table 2). The highest protease activity (PA) (78.2 U/mL) was recorded from isolate Z1BL1 whereas the lowest PA (31.7U/mL) was noticed from isolate Z1Y2 under SSF. Hence, isolate Z1BL1 that showed the highest acid protease activity was selected for further study.

Morphological characterization of potential fungi isolates
The potential fungal isolate were identi ed to the genus level based on species descriptions (16 ; 22) ( Table 3). The potential fungi isolate Z1BL1 was identi ed and categorized under the genus Aspergillus whereas isolate Z1BL1 based micro morphological characteristics. In the present study, two solid substrates (wheat bran and rice bran) moistened by acid/mineral salt solution were used for acid protease production. All the substrates treated by acid and mineral solution were produced signi cant protease activity. However, the maximum protease activity (107.4 U/mL) was obtained from the potential isolates (Z1BL1) using wheat bran treated with minerals solution whereas the least protease activity (48.5 (U/mL) was recorded using rice bran treated with mineral solution (Fig 2).

Effect of incubation period
The effect of incubation time on the production of acid protease activity was shown in Table 4. The highest protease activity (95.2 U/mL) was recorded at 120 h. Further increase the incubation temperature reduced the enzyme activity.

Effect of moisture content
The effect of moisture content on acid protease production was shown Fig. 3. Accordingly, the maximum enzyme (87.4 U/ml) production was obtained at 50% moisture content where the lowest (44.7 U/mL) was recorded at a moisture content of 65%.

The effect of incubation temperature
In the present study, the highest protease activity (106.1 U/mL) was obtained when SSF carried out at a temperature of 30 °C, while the lowest protease activity was achieved at 45 °C (Fig. 4).

Effect of initial media pH
The optimum pH for acid protease production by fungi isolates Z 1 BL 1 was found at pH 4.6. The protease activity was sharply decreased on both side of pH 4.5 (Fig 5).

Effect of inoculum size
The effect of inoculum size on acid protease production by isolate Z1Bl1 was shown in g 6. The maximum protease activity was obtained at inoculum size of 1 mL (3.2*10 6 ) whereas the lowest activity was recorded at 0.2 mL (3.2*10 5 ).

Enzyme characterization
The crude enzyme was partially puri ed by acetone and ammonium sulfate precipitation and the partially puri ed enzyme was used for characterization. Partial puri cation of the crude enzyme extract from isolate Z1BL1using chilled acetone and 100% (NH 4 ) 2 SO 4 was increased by the ratio of MCA/PA while reducing the MCA and PA ( Table 5). The enzymes were also tested for milk-clotting activity and the ratio of MCA/PA was calculated. The highest ratio was recorded from acetone precipitated enzyme. Thus, based on the protease activity and ratio (MCA/PA) recorded from the enzymes, acetone precipitated enzymes was subjected for further enzyme characterization. As shown in gure 4.8, the crude enzyme precipitated with chilled acetone showed signi cant protease activity at a temperature ranges from 40°C to 60°C. However, the highest activity was recorded at a temperature of 50°C. The enzyme activity gradually increased with increasing temperature, followed by a steep decrease at temperatures above 50°C (Fig. 7). The enzyme retained 79% of its activity up on its exposure at a temperature between 40 °C-60 °C (Fig.8) for 1 h.

Effect of pH on enzyme activity
In the present study, the optimum pH for the activity of acetone precipitated enzyme was obtained at pH 5.0 which is highest protease activity 97.1 U/ml as shown (Fig 9).

pH stability of acetone precipitated enzyme
The pH stability test for acetone precipitated enzyme from isolate Z 1 BL 1 was stable from pH 4-6 as shown in (Fig.10). The enzyme retained 83% of its activity up on its exposure at a pH ranges from 4 to 6.
The activity of the enzyme at optimum pH was taken as 100%.

Discussion
In the current study, 12 (38%) fungi isolates were shown clear-zone hydrolysis on Skim-milk Agar media during primary screening. Casein or skimmed milk agar plate assays allow qualitative determinations of protease activity, the hydrolysis zone produced on the agar could be related to the amount of protease produced by the fungus (9 23 24 ). The fungi isolates produced a clear zone diameter and relative enzyme activity between 6.7 mm to 21.3 mm and 0.40 to 0.80 in 48 hour respectively. Similar to the present study, substantial clear zone been hydrolysis were reported by Mucor sp. (25) and Aspergillusniger FFB1 (19) skim-milk agar and agar media, respectively. Whereas the clear zone diameter (8mm-19.25 mm) and relative enzyme activity (1.07-2.09) recorded in 48 hours from varies lamentous fungi using skim milk agar media were higher than this study (10). The variation in inhibition zone diameter and relative enzyme activity could be attributed to the low media pH used in this study.
Out of the 12 isolates subjected to secondary screening, 9 isolates were shown signi cant protease activity (between 31.7 U/mL and 78.2 U/mL) under SSF. This could be due to lamentous fungi preferred SSF for production of enzymes (18). Similar protease activities were recorded by Aspergillus species (26)(27)(28)(29) and Mucor species under SSF.
The potential fungal isolate Z1BL1 that exhibited the highest protease activity in SSF was identi ed and categorized under genus Aspergillus based on macroscopic and microscopic characteristics (Table 4.1).
In comparison to the present study, lamentous fungi isolates collected from Petaling Jaya region, Malaysia identi ed into different species under the genus Aspergillus (10). In other study, (30) reported that identi cation of lamentous fungi in to genus Aspergillus based on morphological feature.
The selection of best substrate for enzyme production in a SSF process governed by several factors, mainly related with cost and availability of the substrate material, and thus may involve screening of agricultural waste products (31). In the present study, the suitability of two substrates (wheat bran and rice bran) moisted with acid/mineral solution was veri ed for protease production in SSF. The highest enzyme activity was obtained from wheat bran moisted with mineral solution. The variation in protease activity between the solid substrates could be due to differences in particle size which is partly related to porosity even though the particle size of each substrate was not determined (32). In other study, wheat bran is considered as the best substrate for production of acid protease from A. oryzae MTCC 5341 (33), other Aspergillus species (19;34) and Mucor species (35).
In this study, the solid state fermentation carried out at 30 0 C was the most suitable temperature for acid protease production with protease activity of (106.1 U/mL) (Fig. 4.5) and any uctuation from this optimum temperatures signi cantly reduced the enzyme activity. The production of enzyme directly related to the biomass of isolate fungi so this implies optimum growth temperature for isolate Z 1 BL 1 was 30°C. Similar to the current study, optimum acid protease production by Aspergillus spp. was obtained at 30 0 C (27-29; 33). In other study, (37) also reported maximum milk-clotting proteases production by Amylomyces rouxii, Mucor pusillus and Mucor J20 at 30 °C. A maximum acid protease activity was also recorded from A. oryzae HG76 at 30 0 C and showed signi cant decrease when the temperature got higher or lower (34).
The protease activity was started from 24 h and reached maximum value (95.2 U/mL) at 120 h incubation period (Table 4.3). Further incubation signi cantly reduced the enzyme production. The reduction in enzyme yield after optimum incubation period was probably due to depletion of nutrients available to microbial growth (11). The maximum enzyme activity recorded from M. circinelloides, A.
oryzae MTCC 5341 and Aspergillus sp on 5 th day of fermentation time under SSF was comparable with the present study (11;33;38).
The optimum media pH for acid protease production by isolate Z1BL1 was found at pH 4.5 (Fig.4.6).
Further increasing of the media pH signi cantly reduced enzyme production and this implies that fungi prefer an acidic media pH for inducing acid protease enzyme. Similar observation was reported by (39) on production of aspartic protease from Mucor mucedo. In other study, maximum acid protease activity from Rhizopus oligosporus HIS13 (40) and Aspergillus species (28) under SSF was also obtained at initial media pH 5. Generally, changes of pH during SSF procedure are almost never controlled, except the initial pH of the substrate which is adjusted before inoculation (29).
Size of inoculum is an important biological factor that determines biomass production during fermentation. A highly concentrated inoculum may produce excessive biomass leading to the rapid depletion of nutrients needed for rapid growth of the culture and production of metabolites, while a lower inoculum density may give insu cient biomass inducing low yields of products (29 41 42). Thus determining an appropriate inoculum size in SSF is vital for optimum biomass growth and hence maximum enzyme production. In the present study, the highest protease activity (107.4 U/mL) of the crude enzyme produced from Z1BL was obtained using an inoculum size of 1mL (3.2 x 10 6 spores/mL) ( Fig. 4.7). Correspondingly, maximum protease production from A. oryzae NRRL 1808 (41) and Mucor circinelloides (11) and was reported at 1mL inoculum size.
The decrease in enzyme activity that has been observed with higher inoculum size could be due to the shortage of the nutrients available for the larger biomass and faster growth of the culture. Hence, a balance between the proliferating biomass and available material is vital for maximum enzyme production (11).
Moisture content is one of a signi cant factor in enzyme production in solid state fermentation (43).
Since microbial growth and product formation occurs at or near the surface particle with optimized water level that controls the water activity (aW) (11). In this study, the highest protease activity was observed from crude enzyme produced at 50% moisture content (Fig 4.4). A similar ndings were reported at 55% moisture content for maximum milk-clotting enzyme production from Aspergillus spp under SSF (26 44).The decrease in enzyme production at lower moisture content could be due to the non-availability of nutrients. On the other hand, the high moisture content of the fermentation media decreased the porosity and low oxygen transfer that may affect enzyme production (45).
Characterization of protease is an important practice to determine the optimum temperature and pH of the enzyme for its application. In this study, the crude enzyme precipitated using acetone was used for characterization. The partially puri ed enzyme showed maximum activity at temperature 50 •C on and stable at temperature between 40°C -60 °C. The thermo stability of the enzyme showed in this study, corresponds with the potential applications of the enzyme in food industries such as baking, brewing, diary (2 28), was also reported the highest activity of acid protease from Aspergillus oryzae MTCC 5341 at 55° C and its stability between 40 to 57 °C. The acid protease most active at pH 5.0 and protease activity decreased signi cantly below and above pH 5.0. The enzyme was active between pH 4 up to 6 as shown (Fig. 4.10). This suggests that the enzyme is active at acidic pH and appropriate for food industry and beverage industry (4). Similarly, (46), reported the highest activity of Acid protease from Aspergillus speciesresult at pH 5. Optimum pH values between 3.0 and 5.5 have been reported for protease activities of other fungi, such as Penicillium camembertii, pH 3.5 (47) and Rhizopus oryzae, pH 5.5 (48). The optimum pH of A. niger I1 protease was lower than that of A. niger NRRL 1785 protease which exhibited an optimum at pH 4.0 (49). Thus, the stability of the enzyme in present study corresponds with pH used processing food in food and beverage industries (28).

Conclusions
Based on the results from this study, we can make a conclusion as follows.
Filamentous fungi isolated from diary and grape farm soil have a potential to produce acid protease.
Production of acid protease enzyme from lamentous fungi under SSF was good strategy. From primary and secondary screening, isolate Z1BL1 was selected as a potential fungus for acid protease production.
The potential isolate Z1BL1 was successfully identi ed and categorized under genus Aspergillus using macroscopic and microscopic features. Optimization of media composition and growth condition slightly increased the acid protease production by isolate Z1BL1.The characterization study of the enzyme con rms that the enzyme works best at pH 5 and 50 0 C. Thus based on the above ndings, the acid protease produced in the current study is a candidate to be applicable in food industry.

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