Production of Alkaline Protease Using Industrial Waste Effluent as Low-cost Fermentation Substrate

Alkaline proteases are the most important groups of commercial enzymes, which have been broadly used in industrial processes. In this study, Bacillus sp. PTCC 1538 was selected as a biological agent to produce alkaline protease. Enzyme production under submerge fermentation using industrial waste effluent was investigated. Since the costs of the raw material plays an important role in the cost of enzyme production, corn steep liquor (CSL) was selected as a low-cost substrate to reduce the cost of enzyme production. Various carbon sources were used as the auxiliary substrates to enhance enzyme production. Results showed that maximum enzyme activity was obtained when wheat bran was used as an auxiliary substrate. Optimal media composition and growth conditions for alkaline protease production were defined. The optimum conditions were found to be pH 8, incubation temperature of 37 °C, CSL inoculum size of 5 v/v %, yeast extract and wheat bran concentrations of 2 and 6 g/l, respectively. CaCl2 was used as an activator to enhance proteolytic activity of the enzyme. Under optimum condition, enzyme activity of 100.7 U/ml was obtained at CaCl2 concentration of 1.5 g/l.


INTRODUCTION 1
Proteases represent one of the most important groups of enzymes, which have found a wide application in various industries such as pharmaceutical, detergents, X-ray films, animal feed stocks and food [1][2][3][4][5][6]. Proteases are classified into alkaline, acidic and neutral proteases according to the optimum pH of their activities [7-10]. Alkaline proteases are generally used as detergent additives and they represent 60% of the total worldwid e enzymes sales [11,12]. Alkaline proteases show high enzymatic activity under harsh conditions such as alkaline pH and high temperature in the presence of various chemical agents [13].
Microbial proteases are the most significant in compare to proteases obtained from plants or animal organs [14][15][16]. Generally, proteases are commercially produced by microorganisms that generate a considerable *Corresponding Author Email: najafpour8@gmail.com (G. D. Najafpour) amount of extracellular product. For commercial production of alkaline protease, microbial cells should ensure high yield using low cost methods [17]. Alkalin e proteases are derived from a wide range of microorganisms such as yeasts, molds and bacteria. Bacillus species are the most important microorganisms which have been commercially used for the production alkaline proteases [18][19][20]. Bacillus sp. are generally aerobic and gram positive microorganisms that grow well in simple media. These species are able to produce hydrolytic enzymes such as glucanase, mannanase and protease using various media [11,21].
Microbial proteases can be produced using submerged and solid-state fermentation processes [22,23]. It has been reported that about 90% of the commercial enzymes are produced by the submerged fermentation processes. Submerged method is the best fermentation processes for the bacteria because it ensures high moisture content for bacterial cell growth. The advantages of submerged fermentation processes are consistent enzyme production and simple purification of products [24][25][26].
The extracellular protease production is significantly affected by process conditions such as incubation time, pH, temperature and inoculum size, and especially media composition [27,28]. In addition, in submerged fermentation up to 40% of the cost of enzyme production is due to the media component and substrate [29,30]; therefore, optimization of the medium composition is essential in microbial protease production [3].
Culture composition significantly affect enzyme production. The source of carbon and nitrogen plays an important role in the production of enzymes. According to the literature, both nature and the concentration of carbon and nitrogen sources are essential nutritional factors that regulate enzyme production; therefore, selecting the appropriate carbon and nitrogen sources in the enzyme production process is critical stage of experimental work [31,32].
Production of protease from low cost feed stocks or agricultural byproducts as substrate is one of the common method for reducing cost of the production [33]. Low cost sources of carbon and nitrogen play an important role in commercialization of the production processes [34]. Corn steep liquor (CSL) is a byproduct of corn wet-millin g . CSL is an important constituent of some growth media. CSL is a source of elemental nutrients such as nitrogen, reducing sugars, minerals, proteins, enzymes, organic acids and vitamins. In the chemical industry, CSL as a low cost substrate is widely used in fermentation processes [35][36][37].
The aim of this work is to investigate an inexpensive agro-based feed stocks for production of protease under submerged fermentation. CSL, a waste material of corn wet-milling, was selected as a low cost substrate for protease production using Bacillus sp.. Various carbon and nitrogen sources were evaluated to enhance enzyme production. Media composition and growth conditions of Bacillus sp. for alkaline protease production were optimized.

Microorganism and culture maintenance
Bacillus sp. PTCC 1538 was supplied by Iranian Research Organization for Scientific and Technology (IROST). The bacterial strain isolated from soil known as an antifungal and native strain. This microorganism was cultivated in a solution containing (g/l): glucose 4, yeast extract 2, peptone 2, potassium dihydrogen phosphate 1, and magnesium sulfate 0.2. For the solid media preparation, 2% agar was added to this formulation and stocks were maintained at 4 °C. The prepared seed culture was incubated at 37 ℃, 110 rpm for duration of 24h. The culture was regenerated every 2-3 weeks on a fresh plate from the stock culture. Ability of production protease by Bacillus sp. was investigated with cultivation of Bacillus sp. on casein agar plate containing (g/l): peptone 5.0, beef extract 5, NaCl 5, casein 10 and bacteriological agar 12. The appearance of clear zone showed that Bacillus sp. can produce protease enzyme.

Materials
In this research the CSL was collected from Glucosan Company (Alborz industrial town, Qazvin). The waste effluent collected from Glucosan Company was characterized. Table 1 summarizes waste effluent characterization. Total solid 7%, choline 5.6 mg/kg , niacin 0.16 mg/kg, histidine 2.8%, isoleucine 3.6%, leucine 4.7%, lysine 2.5%, phenylalanine 4.4%, tyros ine 3.4% and valine 5.8%. Wheat bran was locally supplied, grinded and used as carbon source. Waste materials were washed with tap water followed by distilled water to remove the adhered surface dust particles. Folin -Ciocalteu's phenol solution was purchased from Sigma -Aldrich (USA). All other chemicals were purchased from Merck (Darmstadt, Germany).

Inoculum preparation and protease production
The inoculum was prepared by adding a loop full of pure culture into 50 ml of sterile medium containing (g/l): glucose 4, yeast extract 2, peptone 2, KH2PO4 1, MgSO4.H2O 0.2 and incubated at 37 °C, 130 rpm for 24 h. A 3% inoculum from the culture was added to mediu m containing: KH2PO4 1(g/l), K2HPO4 0.5 (g/l), MgSO4.7H2O 0.2 (g/l) and CSL 30% (w/w). This volume of CSL was determined with preliminary experiments and used as a carbon and nitrogen source. After 72 h of incubation at 37 °C under shaking condition (130 rpm), the culture was harvested and protease activity was measured. The media were sterilized by autoclaving at 121 °C and 15 psig for 20 min.

Assay of protease activity
The protease activity was determined by the method that suggested by Sigma-Aldrich (St. Louis, MO, USA) based on scientific literature [38,39]. According to this procedure, 5 ml of 0.65% (w/v) casein dissolved in the potassium phosphate buffer (pH 7.5, 50 mM), then placed in a water bath at 37 °C for 5 min until equilibriu m condition was achieved. In the next step, 1 ml of enzyme solution was added to this mixture and allowed the enzymatic reaction to proceed for 10 min. The reaction was stopped by addition of 5 ml of 110 mM trichloroacetic acid solution. After 30 min the entire mixture was centrifuged at 8000 rpm for 10 min . Supernatant in the amount of 2 ml was mixed with 5 ml of 500 mM Na2CO3 and 1 ml of Folin-Ciocalteu's phenol solution and then kept at water bath at 37 °C for 30 min . The optical density of the solutions was determined with respect to the blank samples at 660 nm. One unit of protease activity was defined as the amount of enzyme required to release 1µg of tyrosine per ml per min under standard assay conditions.

Effect of carbon sources
Various simple and complex carbon sources including glucose, fructose, lactose, starch, wheat bran, grain of wheat and bagasse were used as a supplement source of carbon (5 g/l). The enzyme activity was monitored after 96 h incubation at 37 °C in an incubator-shaker operating at 130 rpm.

Effect of nitrogen sources
Organic nitrogen sources including yeast extract, peptone and casein; and inorganic nitrogen sources such as ammonium chloride, ammonium nitrate, sodium nitrate and potassium nitrate were used. The respective nitrogen sources were added in the production medium (2 g/l). The enzyme activity was monitored after 96 h incubation at 37 °C, under shaking conditions of 130 rpm.

Effect of CaCl2
The effect of salt on protease secretion by variation of CaCl2 concentrations (0.5-2 g/l) at constant pH of 8 in production medium was investigated. Enzyme activity was quantified after incubation for 72 h at 37 °C under shaking at 150 rpm.

Growth kinetics and protease production
In order to study growth kinetics of microorganism, the inoculum was introduced to optimized and regular media. Samples were aseptically taken and cell density was measured at 600 nm. Also, the enzyme activity of samples was determined.

Effect of additional carbon sources
Several carbon sources such as glucose, fructose, lactose, starch, wheat bran, grain of wheat and bagasse were supplemented to culture medium at 5 g/l level. Table 2 shows the effect of various carbon sources on enzyme production. Bacillus sp. produced a high level of protease in the presence of complex organic carbon sources. The maximu m levels of proteolytic activities were obtained for starch and wheat bran (47.54 and 46.43 U/ ml respectively). The scope of present study was to identify desired composition of low cost media. Wheat bran, which is agro-industrial waste, was preferred over starch due to its high cost. Similar results were obtained by Meena et al. [15]. Simple carbon sources like fructose, lactose and glucose resulted in low activity protease production, 21.49, 31.22 and 27.20 U/ml, respectively. The obtained results in this work were consistent with the literature as complex carbon sources are much better than simple carbon sources [3, 24, 40,41].
In order to define optimum concentration of carbon source enzyme production was investigated by supplementation of several wheat bran concentrations (2-10 g/l). Maximum production (50.81U/ml) was obtained with 6 g/l wheat bran concentration. Further increase in this carbon source adversely affected protease production by Bacillus sp. It appears that the high sugar content in production medium due to waste had an inhibitory effect on the protease production.

Effect of nitrogen supplementati on
Alkaline protease production is significantly dependent on the nitrogen sources available in the medium. Nitrogen is an essential element which participates in the structure of proteins, nucleic acids, amino acids and other cell components of microorganisms [9, 17,41]. The nitrogen sources also function as inducers of enzyme production [41]. The organic nitrogen sources used in our study supported protease production, while the maximu m effect on production was achieved with yeast extract (Table 3).
The effect of yeast extract on the enzyme production was further investigated at different concentrations (1-5 g/l). Maximum production was obtained with 2 g/l of yeast extract. Furthermore, inorganic nitrogen sources proved less favorable towards enzyme secretion. There are several reports on the repressive role of organic nitrogen sources, for instance excessive amino acid and ammonium ions in alkaline protease production [42][43][44].
Fermentation was carried out with optimized conditions to investigate the effect of carbon and nitrogen sources on the alkaline protease production by Bacillu s strain. As shown in Figure 1, protease production increased with increasing incubation time from 24 to 72 h and then decreased at 96 h. At incubation time of 72 h, the protease production was more than 75.75 U/ml and three-fold increase in protease activity was observed. The time required for maximu m protease production depends on the nature of microorganism, medium composition and cultural conditions of fermentation process. An increase in incubation time resulted in an increase in protease secretion and consequently the yield of protease production increases. The decrease in enzyme yield after the optimum incubation time might be explained because of auto-degradation of proteases, catabolic repression, cell growth inhibition and also because of nutrient depletion.

Effect of pH
Culture pH is one of the key parameters affecting alkalin e protease production by microorganisms. In fermentation process, many biochemical and enzymatic reactions, and also components transfer through the cell membranes intensely depends on the extracellular pH. As shown in Figure 2, high enzyme activity was observed at alkalin e pH ranges from 7 to 9. Maximum enzyme production (77.56 U/ml) was obtained at pH of 8 ( Figure 2).

Effect of inoculum size
The optimization of inoculum cell density is quite important, as high inoculum density can reduce lag phase and increase enzyme production due to competition for the availability of nutrients. In a similar manner, low cell density can result in a mitigation of enzyme secretion, owing to a drop in cell populations [45]. Inoculum size for this study was selected according to literature in the range of 2 to 10% [46][47][48][49]. It is apparent that protease production increased steadily with increasing the inoculum size, until a magnitude was reached at which Activity (U/ml) Figure 2. Effect of initial pH of medium on protease production enzyme productivity achieved maximu m levels (inoculum size of 5%, v/v) ( Figure 3); this concentration became probably a threshold changing the pathways of cell growth towards enzyme synthesis. Inoculation with inoculum size of 10% may increases the old and inactive cells in the production medium and causes the protease production to decrease. In addition, low inoculum sizes of 2 and 3 % causes long lag phase of microbial growth which leads to decrease in enzyme productivities.

Effect of incubation temperature
In Fermentation process, the cell growth rate and metabolic activities of the microorganism are highly dependent on the incubation temperature. The effect of temperature (30 to 45 °C) on the production of protease was investigated.
The optimum temperature of incubation was found to be 37 °C that resulted in protease production of 90.1 U/ ml (Figure 4). A comparison of the alkaline Bacillus strains producing alkaline proteases revealed that most of the alkaline Bacillus strains were of mesophilic type with optimum temperature range of 30-37 °C [8, 11, 13].

Effect of CaCl2 concentration
In order to stabilize the protease secreted by Bacillus sp., CaCl2 was used in the fermentation media. CaCl2 also maintain the molecular conformation and enhances the enzyme activity. Figure 5 shows the effect of CaCl2 concentration on protease production. The positive effect of CaCl2 at 1.5 g/l concentration was pronounced with respect to specific protease activities. The higher concentration of CaCl2 (2 g/l) had an inhibitory effect on enzyme production and decreased the enzyme activity to 65.26 U/ml.

Activity and stability of alkaline protease
The effect of different pH values (7-13) on the activity of produced protease was studied. The relative activity (%) was calculated as the ratio of the enzyme activity to the maximu m activity. Protease from Bacillus sp. PTCC 1538 showed proper enzymatic activity at pH lower than 11. Maximum protease activity was obtained at pH 8 ( Figure 6a). Alkaline protease retained 82% of its activity at pH 11. The effect of temperature from 30 to 70 °C on the protease activity was investigated at pH 8. As shown in Figure 6b maximu m protease activity was achieved at 50 °C. The thermal stability of the protease produced with and without CaCl2 was determined at pH 8 and different temperatures from 30 to 60 °C for 30 min. Results indicate that CaCl2 Enhanced the thermal stability of alkaline protease (Figure 7).

Growth study and protease production
The growth kinetics of Bacillus sp. for production of alkaline protease under unoptimized and optimized conditions were investigated. Figure 8 represents protease activity and cell dry weight of Bacillus sp. with respect to the incubation time. Medium compositions and cultural conditions of the optimized and unoptimized were KH2PO4 1g/l, K2HPO4 0.5 g/l, MgSO4.7H2O 0.2 g/l, waste 30% (w/w), wheat bran 6 g/l, yeast extract 2 g/l, Activity (U/ml) Figure 5. Effect of CaCl2 concentration on protease production CaCl2 1.5 g/l, initial pH of 8, 37 ºC and 150 rpm, and KH2PO4 1 g/l, K2HPO4 0.5 g/l, MgSO4.7H2O 0.2 g/l, waste 30% (w/w), initial pH of 7, 37 ºC and 130 rpm, respectively. For optimized condition protease activitywas sharply increased during the exponential growth phase and then the activity reached to 100.7 U/ ml after 72 h.  (1) and optimized (2) medium, and protease production in initial (3) and optimized (4) medium

CONCLUSION
Due to an increase in feedstock costs for production of alkaline protease enzymes, this work was carried out to optimize a variety of fermentation parameters, including medium compositions and incubation conditions, for maximu m alkaline protease production. The optimal medium composition and cultural conditions for highly extracellular alkaline protease production were KH2PO4 1g/l, K2HPO4 0.5 g/l, MgSO4.7H2O 0.2 g/l, waste 30% (w/w), wheat bran 6 g/l, yeast extract 2 g/l, CaCl2 1.5 g/l and initial pH of 8, respectively. Selected operating parameters for extracellular production were incubation temperature of 37 °C and agitation rate at 150 rpm.
Results showed that addition of CaCl2 as an activator significantly enhanced enzymatic activity and thermal stability of alkaline protease.