Fresh sweet whey was utilized exclusively as a substrate for the production of extracellular proteolytic biocatalyst by a ubiquitous fungus Rhizopus oryzae. In pure whey medium only mineral salts were added as (g/L): MnSO4.H2O (0.01%), FeSO4.H2O (0.01%), CaCl2 (0.05%) at pH 6. Fermentation process parameters were studied for optimum production of protease. All the experimental work was done in Erlenmeyer flask and then in airlift bioreactor. Rhizopus oryzae produced 700.5 PU/ml by shake flask culture at 120 rpm for 168 hr and incubation at 35°C. Strain was further improved by ultraviolet radiation and ethyl methane sulfonate. Crude enzyme from wild, UV treated and chemically treated Rhizopus oryzae was partially characterized and compared. Ethyl Methane Sulfonate (EMS) treated mutant produced highest units of protease (1242.2 PU/ml) when compared with wild type and UV treated fungal strains. EMS mutant was evaluated for production in lab scale airlift bioreactor. Two fold higher proteolytic units were obtained from EMS mutant (1509.5 PU/ml) than wild strain (700.5 PU/ml) under optimum fermentation conditions in the bioreactor.
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Key words: Whey, Rhizopus oryzae, Ethyl Methane Sulfonate, Mutagenesis, Airlift bioreactor.
Whey is a milk serum obtained after coagulation of casein during cheese manufacturing or from butter extraction process. It contains 4-5% lactose, 0.8-1.0% proteins and other minerals and vitamins. Whey is a major by-product of dairy industry in Pakistan. Its direct disposal into sewerage water causes a threat of water pollution. Due to its high biochemical oxygen demand (BOD), its disposal is a costly and time consuming process (Ashraf et al. 2008). So various value added products are being produced by a cheaper substrate.
Proteases have acquired more importance due to their diverse use as an industrial enzyme. Proteases are protein digesting enzymes and represent one of the three largest groups of industrial enzymes. These biocatalysts carry out the enzyme modification, regulation of metabolism, gene expression, blood coagulation, pathogenesity and release of hormones within the body. In industrial applications these are involved in textile finishing, detergent making, leather dehairing and pharmaceutical industries. More recently proteases are also used in lens cleaning solutions and extraction of oils from plants replacing hazardous organic chemicals like hexane and chloroform (Sumantha et al. 2006).
A cosmopolitan filamentous fungus Rhizopus oryzae is being used commercially to produce an assortment of products such as lactic acid, amylolytic enzymes, lipases and variety of proteases.(Ikasari and Mitchell, 1996; 1998 and Sumantha et al., 2006). In this study alkaline protease production has been carried out under submerged fermentation conditions using Rhizopus oryzae. The potential of whey as a substrate for proteolytic enzyme was investigated under submerged state conditions.
Materials and Methods
Sweet whey employed throughout this study as fermentation media was an industrial waste obtained from Halla milk factory in Pakistan.
Microorganism and maintenance of culture
Rhizopus oryzae obtained from Culture Collection Bank of University of the Punjab was cultivated and maintained on Potato Dextrose Agar (PDA) slants at 35°C.
Inoculum was prepared by dispersing spores in sterile normal saline solution containing 0.1% Tween 80 from three days old slant. Sterilized inoculation loop was used to transfer spores from culture slant to normal saline. Culture was diluted to 107-108 spores /ml. Then 2% of this spore inoculum was used to inoculate the fermentation medium for protease production.
Whey was filtered to remove suspended particles and different salts were added as follows (g/L); MnSO4.H2O (1), FeSO4.H2O (1), CaCl2 (5). Initial medium pH was set to 6.0 and sterilized at 121°C for 20 minutes. Inoculum was added as 2% v/v and fermentation was carried out for seven days (168hr) in shaking water bath at 120 rpm. All the experiments were performed in duplicate.
Asssay for proteolytic enzyme
Protease activity was measured by modified Anson method (Yang and Huang 1994). The reaction mixture containing 1 ml of crude enzyme extract and 2 ml of casein solution (1%) in Tris-HCL buffer (0.1 M, pH 8.0) was incubated at 37°C for 20 min. Reaction was arrested by the addition of 3 ml trichloroacetic acid (10%). Absorbance of the clear supernatant was recorded at 280nm using tyrosine as a standard. One unit of enzyme activity was defined as the amount enzyme liberating 1µg tyrosine per ml under the assay conditions.
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Total soluble protein content was measured by the method of Lowry et al. (1951) using Bovine Serum Albumin (BSA) as a standard and was expressed as mg protein per ml of fermented medium.
Total sugar measurement
method developed by Suzane et al. (2002) was used for the measurement of total carbohydrate content. To 1 ml crude enzyme filtrate, 1 ml phenol (5%) was added and calibrated at 25 °C for 5 minutes. 5 ml H2SO4 (concentrated) was added slowly in the reaction mixture and allowed to develop color at room temperature for 15 min. Absorbance was measured at 470nm and total sugar was expressed as mg/ml.
Optimization of Fermentation characteristics of the strain
A number of fermentation parameters for Rhizopus oryzae strain were optimized for hyper production of protease on dairy industrial waste. Single process parameter was optimized at a time and subsequently integrated into the next parameter for its evaluation. All the experiments were performed in duplicate mode and their analysis was done by the quantitative estimation of the enzyme activities. These fermentation parameters include the optimization of agitation speed, fermentation time, initial pH and fermentation temperature.
To obtain the hyper producing protease isolate, UV and Chemical mutagenesis was performed. After mutagenesis, hyper producing mutant was isolated by fermentation performed under optimum conditions studied before.
Spore suspension prepared in distilled water was serially diluted to get the spore size of 7 x 108/ml. 1 ml was taken in Petri dish and subjected to UV irradiation for 0, 15, 30, 60 and 90 minutes and put in dark for at least 30 minutes. PDA was poured in irradiated spores and mixed well for even distribution of spores in PDA. Best mutant was selected by enzyme activity after fermentation. Experiment was repeated till the best mutant was obtained (data not shown).
1 ml of 108 spores/ml spore suspension was taken in eppendorfs and centrifuged. Pellet was dissolved in 1 ml (4µl/ml) of Ethyl Methane Sulfonate (EMS). Spores were exposed to EMS for 0, 15, 30, 60, 90 and 120 minutes. After exposure spore were washed twice with sodium thisulphate and once with distilled water and poured on PDA plates and mixed well. Putative mutant was selected on the basis of protease activity after fermentation. Experiment was repeated for several times to obtain the best mutant (data not shown).
Partial characterization of crude protease
Partial characterization of crude protease was performed to study the influence of different parameters on activity of enzyme.
Phosphate buffer (7), Tris-HCl buffer (8, 9) and Glycine-NaOH (10, 11) were used to examine the optimum pH of enzyme. Experiment was performed in duplicate.
To find out the best time for maximum enzyme activity, it was incubated for varying time periods at 10, 15, 20, 25 and 30 minutes and enzyme activity was recorded at 280nm.
To determine the optimum temperature of proteolytic activity, effect of different temperatures (31, 34, 37, 40, 43 and 46) was observed.
Effect of metal ions
Effect of modulators and inhibitors of enzyme was studied by using a variety of metal ions viz, Ca2+, Fe2+, Mg2+, Zn2+ and Mn2+. 0.1 M solution of every metal ion was prepared and preincubated with crude enzyme for 20 minutes. Then enzyme assay was performed by taking 2ml (1%) substrate and 1ml enzyme and incubated for 30 min. Trichloroacetic acid was used to stop the reaction. Then absorbance was recorded at 280nm.
Results and Discussion
Optimization of shaking speed
Different morphological forms of fungus were observed under agitated conditions from suspended mycelial to pellet formation. Protease activity was peaked at 120 rpm. (659 PU/ml) although no distinguished pellet formation was observed rather mycelial clumps were formed. Various other factors contribute to the morphological form of the filamentous fungi. These factors include the inoculum spore count, metal ion, pH and finally the agitation speed. At 120 rpm it might be due to horizontal agitation of the flasks and the addition of metal ions in the media which facilitate the fast growth of the organism and ultimately mycelial clump formation. Present study reveals the fact that maximum protease yield is observed in mycelial clump form. Negative effect of metal ions on pellet formation is supported in a study by Liao et al. (2006). Although reports have shown the production of fumaric acid and lactic acid by Rhizopus oryzae is highest in pellet form but insufficient data is available to associate the maximum protease formation by this strain in pellet form.
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Rhizpus oryzae was incubated for 192 hr to study its optimum fermentation time in whey medium. The biomass growth begins to stop after 72 hr, then enters the stationary phase and begins to decline after 120 hr while maximum proteolytic activity (675 PU/ml) was observed at 168 hr when the strain is in death phase and then a sharp decline in enzymatic activity is observed (Fig. 1). Correlation of dry cell weight (DCW) to protease activity reveals the fact that production of the enzyme in submerged fermentation is non-growth associated and is maximum in death phase. It might be the fact that proteases are released in maximum quantity to the fermentation media in nutrient starvation and biomass degradation phase. Similar results are also observed for Aspergillus oryzae where product level increases even after starvation of substrate and biomass. Macroscopic morphology of the organism and hydrodynamic forces also influence the product formation (Kelly et al. 2002).
Fig.1. Effect of optimization of time period of fermentation on proteolytic activity.
A pH range 4-8 was employed to observe the effect of pH in fermentation. Initial pH 6.0 was the best for enhanced proteolytic activity. A slightly lower or higher value of pH affected the growth of the organism and so the reduced proteolytic activity. In another study carried out by Agrawal et al. (2004) protease production by a fungus was carried out at initial pH 7.0 and the protease was alkaline in nature.
to be corrected
Fig.2. Optimization of fermentation pH and proteolytic activity
Rhizopus oryzae being a mesophilic culture was very sensitive to temperature changes. Enzyme activity was peaked at 35°C as shown in fig. 3. At higher temperature cell growth decreased and the proteolytic activity also decreased.
to be corrected
Fig.3. Effect of temperature on proteolytic activity and cell biomass
Optimization of fermentation parameters didn't reveal any significant increase in proteolytic activity (659PU/ml to 700PU/ml) by the parent strain of Rhizous oryzae at 35°C, 120rpm and at pH 6.0. For hyper production of proteolytic enzyme, parent culture was improved by physical (UV) and chemical mutagenesis (Ethyl Methane Sulfonate). Various isolates were initially screened on the basis of plate clearing zone on casein agar plates. One Putative mutant from each treatment (UV, EMS) was then selected by qualitative and quantitative assay of enzymatic activity after fermentation of all screened mutant on casein agar plate (data not shown).
Treatment with UV radiation was performed for 15, 30, 60 and 90 minute. One isolate from each treatment time was screened for proteolytic activity, qualitatively and quantitatively. Enzyme assay of the mutants revealed that mutant after 30 minute treatment with UV was best among all yielding 1359 PU/ml proteolytic activity.
Among 6 isolates from EMS treatment from each treatment time (15, 30, 60, 90, 120 and 150), one isolate (ROAC5) after 120 minutes treatment produced the highest proteolytic yield. On Casein agar plate the diameter of the holo produced by this mutant was also highest among all others (35mm). Quantitative assay revealed a two fold increase in proteolytic activity (1449 PU/ml) by treatment with EMS.
Culture improvement with both UV and EMS showed almost comparable results with ROAU2 and ROAC5 producing 1359 PU/ml and 1449 PU/ml respectively.
Partial Characterization of protease
Crude enzyme filtrate from wild strain and two mutant strains ROAU2 and ROAC5 were partially characterized and their activities were compared. Protease from wild strain was active at slightly alkaline (pH 8.0) but both mutants showed strongly alkaline behavior and proteolytic activity of the mutants ROAU2 and ROAC5 was higher at pH 11 and 10 respectively. It shows that the protease produced by this strain of Rhizopus oryzae is alkaline in nature and is very unstable at any other pH unit. Genckal and Tari (2005) in their study carried out serine alkaline protease production which was also active at pH 10.
Optimization of incubation time
Optimization parameter did not show any distinct change. Maximum activity of the enzyme was observed after 20 minutes of incubation for mutants and wild type protease.
Proteases from both mutants were active in the range from 30 to 43°C.
Chemical and UV mutants both showed an increase in proteolytic activity at 43 °C as compared to protease from wild strain which is active at 40 °C. This result is consistent with the study by Olajyuigbe and Ajele (2005). So proteases from fungi can be used at industrial scale due to their thermotolerant properties.
Effect of metal ions on protease activity
The effect of metal ions showed that alkaline protease was not a metalloprotease but ZnCl2 and FeSO4 severely affected the activity of enzymes and acted as enzyme inhibitors (Fig.4). Relative activity of enzymes from wild and mutant strains was calculated using different metal ions. Only Mg++ did not affect the activity of the three proteases. A neutral metalloprotease activated by Mn2+ for activity and inhibited by FeSO4 and MgSO4 and chelating agents was described by Sumantha et al. (2006).
Fig.4. Comparison of metal ions effect on enzyme activity of Wild, UV-treated(AU1) and EMS-treated (AC1) fungal strains.
Airlift fermentor study
The best mutant obtained by EMS treatment after 120min treatment was subjected to production in 1L airlift fermentor. The Mutant strain of Rhizopus oryzae produced two fold increase in proteolytic activity than wild strain. While in comparison, 700PU/ml enzyme units were obtained from wild Rhizopus oryzae but EMS treated strain produced 1509.5 PU/ml in airlift fermentor. Plate assay of enzyme from wild, EMS treated mutant and fermenter study is shown in Fig.5.
Fig.5. A comparison of activities of wild and chemical mutant with fermenter produced protease.