Physiological and Microbiological Studies on Production of Alkaline Protease from Locally Isolated Bacillus Subtilis.

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1 Australian Journal of Basic and Applied Sciences, 6(3): , 2012 ISSN Physiological and Microbiological Studies on Production of Alkaline Protease from Locally Isolated Bacillus Subtilis. 1 Mohamed, M.I. Helal, 1 Hassan Amer, 1 Nayera, A.M. Abdelwahed and 2 Madeha, O.I.Ghobashy. 1 Chemistry of Natural and Microbial Products Dept., National Research Center, Dokki, Cairo, Egypt. 2 Microbiology Department, Faculty of Science, Ain Shams University. Abstract: In this study, we have shown the microbial alkaline protease production by using isolated Bacillus subtilis. Bacillus subtilis which produces an extracellular alkaline protease was isolated from meet surface purchased from local Egyptian markets. Maximum enzyme activity was achieved when the bacterium was grown on corn steep liquor (2.0 %, w/v) instead of soybean meal (2 %) followed by casein hydrolysate (2%, w/v) and 12 % cane sugar molasses as a carbon source at ph 10.0 and 37ºC over 24 h incubation period (maximum enzyme production at 48 hr.). The enzyme has an optimum ph of around 10.0 and maintained its stability over a broad ph range between 5.0 to Its optimum temperature is around 37 C, and exhibited a stability of up to 50 ºC. Kew words: Physiological and microbiological, production of alkaline protease, locally isolated Bacillus subtilis INTRODUCTION Proteases are important class of enzymes, which constitute >65% of the total industrial enzyme market. Proteases are used for various industrial applications, such as laundry detergents, leather preparation, protein recovery and meat tenderization (Johnvesly et al., 1 and Gupta et al., 2a ). Considering the commercial importance of proteases, there were several attempts to study and maximize protease production and economize them in detergents (Chauhan and Goupta 4). Proteases catalyze the cleavage of peptide bonds in other proteins, are the class of enzymes having tremendous applications in both physiological and commercial fields. Alkaline proteases are produced by a wide range of microorganisms including bacteria, molds, yeasts and also mammalian tissues. Of all the alkalophilic microorganisms that have been screened for the use in various industrial applications, members of the genus Bacillus were found to be predominant and a prolific source of alkaline protease (Chauhan and Goupta 4 and Joo et al., 4). Some of the Gram negative bacteria producing alkaline proteases were identified as Pseudomonas spp. (Joo et al., 4, Puri et al., 2, Tari et al., 6) and Vibrio spp. (Gupta et al., 2a and Beg et al., 2). Many of the fungi have also been reported to produce alkaline proteases (Ferrero et al., 1996) and they have found application in detergents (Beg et al., 3). Yeast are also reported to produce alkaline proteases (Singh et al., 4 and Gupta et al., 2b). Very few reports exist on the alkaline protease producing alkalophilic actinomycetes (Krishna et al., 5). Media components were found to have great influence on extracellular protease production and are different for each microorganism. Therefore, the required constituents and their concentrations have to be optimized (Puri et al., 2, Tari et al., 6, Gupta et al., 2a, Beg et al., 2 and Beg et al., 3). Industrial fermentation is moving away from traditional and largely empirical operation towards knowledge based and better controlled process (Singh et al., 4). A number of optimization techniques could be used for this purpose. Proteases are classified into acid, neutral and alkaline proteases on the basis of ph range in which their activities are optimum (Sandhya et al., 5). Among all proteases, alkaline proteases are primarily used as detergent additives (Gupta et al., 2b, Sandhya et al., 5 and Krik et al. 2). Thirty to forty % of the production cost of industrial enzymes is estimated to be accounted for by the cost of the growth medium. Considering this fact, the use of cost-effective growth medium for the production of alkaline proteases from an alkalophilic Bacillus sp. is especially important, because these enzymes account for approximately 25% of world-wide enzyme consumption (Gessesse 1997). The aim of the present study was to identify the culture conditions that supported alkaline protease production by the strain Bacillus subtilis as locally isolated strain using inexpensive materials, and some properties of the enzyme produced were determined. 1. Microorganism And Culture Maintenance: MATERIALS AND METHODS Corresponding Auther: Mohamed, M.I. Helal, Chemistry of Natural and Microbial Products Dept., National Research Center, Dokki, Cairo, Egypt 193

2 The microorganism used in this study was isolated from surface of meet samples, screened using a nutrient agar plates and later in alkaline broth (Eftekhar et al., 3). It was identified as Bacillus subtilis according to morphological and biochemical tests according to Bergy s manual (Eftekhar et al., 3). Stock cultures of the isolate were stored in 20% glycerol at 70 C. Prior to each experiment, the bacterium was subcultured from the frozen stocks onto alkaline agar (ph 10.5) containing (g l 1 ): glucose, 10; peptone, 5; yeast extract, 5; KH 2 PO 4, 1; MgSO 4 7H 2 O, 0.2; Na 2 CO 3, 10 and agar, 15 (Takami H. et al., 1989). Alkaline broth was used as the basal medium for preliminary studies of the bacterial growth and protease production. 2- Gelatin Clearing Zone Technique: The protease enzyme activity was determined by El-Safey and Ammar, (2) briefly, according to gelatin clearing zone (GCZ) technique. In this assay, soluble gelatin (1 % w/v) was emulsified and supplemented with (1.5 % w/v) Bacto-agar, ph was adjusted as required with proper buffer (e.g. phosphate buffer at ph 7.0) cups were made (3 cups optimal) in each plate. Equal amounts (0.1 ml suitable) of extracted enzyme (or enzyme solution) to be assayed were introduced into each cup. The plates were incubated at 35 ºC for 24 h., at the end of incubation time, the plates were flooded with previously prepared Mercuric chloride (HgCl) in HCl solution (HgCl, 15g and 20 ml of 6N HCl completed to 100 ml with distilled water), and the mean diameters of recorded clearing zones were calculated. Then expressed in terms of units/ml using a special standard curve constructed for such a purpose. 3. Inoculum Preparation: For enzyme production, bacterial cells from a 24 h aged culture were inoculated into 250 ml Erlenmeyer flasks containing 50 ml of sterile inoculation medium. The composition of the inoculum's medium was the same as experimental tests with constant amounts of glucose, yeast extract and KNO 6 as 10, 10 and 5 gl 1, respectively. The cultures were grown at 37 C on a rotary shaker at 150 rpm for 24 h. After reaching an optical density of about 2.5 at 600 nm, 3% (v/v) of the culture was used to the inoculation of the production flasks. 4. Growth Medium: According to Viccaro, (1973), the alkaline medium (ph 10.0) used for protease production contained (g %)(w/v): glucose, 6 ; soybean meal, 2.0, calcium chloride, 0.04, magnesium chloride, 0.02, sodium dihydrogen phosphate, 0.09 and disodium hydrogen phosphates, After autoclaving and cooling the medium, 10 ml of a trace element solution containing (g l 1 ): Na 3 C 6 H 5 O 7, 10; (NH4) 6 MO 7 O 24, 0.1; FeSO 4 7H 2 O, 2; CuSO 4 5H 2 O, 0.2; ZnCl 2, 0.2, was added to one liter of the medium. The operating conditions were maintained at a temperature of 37 C and agitation of 150 rpm on the shaking incubator for 48 hrs. 5. Protease Assay: The proteolytic activity of the enzyme was quantitatively measured by a modified method of Joo et al. 2. Substrate solution was consisted of 1% casein in 0.1M Tris HCl buffer at ph 9.0. The assay mixture consisted of 450 μl of substrate solution and 50 μl of enzyme solution (culture supernatant) suitably diluted with 0.1M Tris HCl buffer (ph 9.0). The mixture was incubated at 35 C for 10 min and the reaction was terminated by adding 500 μl of 10% trichloroacetic acid (TCA) followed by centrifugation at 5000 g for 15 min to remove the precipitate. Protease activity was determined as the amount of released tyrosine from the supernatants at 275 nm. One unit of enzyme activity was defined as the amount of the enzyme resulting in the release of 1 μg of tyrosine per min. per ml. at 37 C under the reaction conditions. 6- Different Factors Affecting Protease Production By The Bacterial Isolates: The study was carried out at 250 ml conical flask each containing 25 ml growth medium. The flasks were inoculated by 0.5 ml of inoculum prepared and incubated at specific temperature in a rotary shaker (180 rpm). At the end of fermentation period, the culture medium was centrifuged to obtain the culture filtrate that was used as the enzyme source. In all experiments, the biomass dry weight, final ph and protease production were monitored. 6-1 Environmental Factors: 6-1-a- Effect of different incubation period at 24, 48, 72 and 96 h. 6-1-d- Effect of different ph values as 6, 7, 8, 9, 10 and b- Effect of different incubation temperature as 20, 25, 30, 35, 37 and 45 C. 6-1-c- Effect of different inoculum size. 194

3 6-2 Nutritional Factors: 6-2-a- Effect of different carbon sources including D-fructose, D-galctose, sucrose, maltose, lactose, and cane sugar molasses instead of D-glucose. 6-2-b- Effect of different nitrogen sources including urea, peptone, casein hydrolysate, yeast extract, beef extract and corn steep liquor instead of soy bean meal. 6-2-c- Effect of salt elimination including elimination of each salt separately., total dry weight and enzyme activity must be detected after each step. The specific activity was detemined by substracting the enzyme activity over total protein in the filtrate.(joo et al., 2). Results: 1- Isolation Of Bacterial Isolates From Different Meat Samples: A total of 5 meat samples were collected from different markets in Egypt (2 samples from El zaytone market, 2 samples from Kobri elkopa market and 1 sample from Elmarg market). Screening onto nutrient agar medium resulted in isolation of 20 bacterial isolates 80 % of them belonging to genus Bacillus. The selected 20 isolates were identified according to Hensyl (1994) among them 16 belonging to Bacillus sp., 3 isolates were belonging to Serratia marcescens and one Stapohylococcus sp. 2- Prelimenary Screaning For The Proteolytic Activity Of The Bacterial Isolates: The proteolytic activity of the isolates was determined on gelatin agar plates by streak-line inoculation of the pure fresh slants of the isolates. The plates were incubated at 37 C for 24 h. followed by flooding with acidic mercuric chloride solution. A clear zone was recorded on both sides of the inoculating line the zone 10 mm considered as high productivity and that of diameter 7 mm considered as low productivity. Also, qualitative estimation of protease productivity by the selected high protease producing isolates through further and further screening. The protease productivity of the high protease producing isolates which comprised 16 Bacillus isolates, 3 Serratia sp. and 1 Staphylococcus sp. were determined qualitatively by further screening using the previous techniques. The results shown in table (1) with different ph values as 6, 7, 8 and 9 prepared by buffered gelatin agar medium. The largest production of protease enzyme was recorded at ph 9 more than that of ph 6, 7 and 8 by the isolates number 11. Table 1: Prelimenary screening of all bacterial isolates on different ph ranges on gelatin agar medium Isolate No. Clear zone (mm) ph 6 ph 7 ph 8 ph Identification Of The Selected Isolates: After selecting the most producible isolates of alkaline protease on different phs gelatin medium, these isolates were identified according to Hensyl (1994). According to the results obtained, the isolate no. 11 could be identified as Bacillus subtilis and it was the most potent isolate in protease production. 195

4 4- Effect Of Incubation Periods: By using different incubation period at 24, 48, 72 and 96 h., figure (1) clearly illustrate that the protease production by B. subtilis being affected by time of incubation, it reached a maximum at 48 h. which was 370 U/ml and the minimal production at 24 h was 209 U/ml Total Dry Weight (g/l) Incubation period (hours) Fig. 1: Effect of incubation period on the production of alkaline protease production by B. subtilis 5- Effect of initial ph: The enzyme activity was greatly affected by the hydrogen ion concentration of the media. Seven levels were chosen for studying their effects on enzyme production ph 5, 6, 7, 8, 9, 10 and 11. All environmental and nutritional factors were fixed. Figure (2) showed that the ph 9, 10 and 11 were the most favorable ph ranges for the maximum production of enzyme than that of 5, 6, 7 and 8 under the same experiment. The maximum production of enzyme after 2 days at ph 10 being 368 U/ml followed by ph 11 which was 324 U/ml for B. subtilis. 6- Effect Of Incubation Temperature: The effect of different incubation temperature on protease production by B. subtilis was carried out by incubating the inoculated flasks at 20, 25, 30, 37 and 45 C on rotating shaker (150 rpm) for 2 days. At the end of incubation period of each temperature, the dry weight, final ph and protease productivity were determined and recorded in figure (3). Data recorded show that the protease activity was affected markedly by the incubation temperature, the enzyme activity was maximum at 37 C (368 U/ml) but at low temperature (20 C) the activity decreased by about 79.1 % (77 U/ml). 196

5 Total Dry Weight (g/l) Initial ph Fig. 2: Effect of different phs on the protease production by B. subtilis Total Dry Weight (g/l) Incubation temperature Fig. 3: Effect of incubation temperature on protease production by B. subtilis. 7- Effect of inoculum size: This experiment was designated to study the effect of inoculum size on the production of alkaline protease (table 2). All other culture conditions were fixed to the best findings and use different inoculum size as (3, 6, 9, 12, 15, 18, 21, 24 or 27) x 10 8 CFU/ml. It was found that the best inoculum size was 18 x

6 Table 2: Effect of inoculum size on alkaline protease production by B. subtilis. Inoculum size x 10 8 cell/ml Dry weight (mg/ml) Enzyme activity(u/ml) Effect Of Different Carbon Sources: At this study different carbon sources were used instead of 6 % glucose in the enzyme production medium as 6 % fructose, 12 % sucrose, 12 % maltose, 6 % lactose, 12 % galactose and 12 % cane sugar molasses (i.e. 6 % monosaccharide and 12 % disaccharides). The data shown in figure (4) clearly shown that the best sole carbon source was glucose followed by sucrose, maltose and fructose as 368, 360, 308 and 301 U/ml respectively. On other hand, using cane sugar molasses as carbon source gave the best protease production (371 U/ml) at the same medium and conditions. So, figure (5) show the using of different concentrations of sugar cane molasses which proved that the best concentration of sugar cane molasses was 12 % with fixed condition. Since sugar cane molasses was the most favorable carbon source for the enzyme production, it was necessary to study the effect of its concentration on the enzyme activity, from the results, concentration 12% of cane sugar molasses is the best one followed by 18 % concentration to produce protease enzyme with dry mass 3.0 mg/ml (Fig. 5) Total dry weight(g/l) glucose fructose sucrose maltose lactose galactose Sugar cane molasses -- 0 Fig. 4: Effect of different carbon sources on alkaline protease production by Bacillus subtilis. 9- Effect Of Different Nitrogen Sources: Seven nitrogen sources were used instead of soy bean meal (as 2 % N ) in the enzyme production medium, these nitrogen sources were; urea, peptone, casein hydrolysate, gelatin, yeast extract, beef extract and corn steep liquor (on equivalent N-basis). Figure (6) show that the best nitrogen source was corn steep liquor (401 U/ml) followed by casein hydrolysate which gave almost the same protease activity (380 U/ml) as control soy bean meal (381 U/ml) while the lesser one was urea (324 U/ml). Data also proved the direct proportionality between proteolytic activity and the dry weight biomass i.e.; the dry weight biomass increased with alkaline protease increasing. 198

7 Total Dry Weight (g/l) Sugar cane molasses concentration (%) Fig. 5: Effect of different concentrations of sugar cane molasses on alkaline protease production by Bacillus subtilis Total dry weight(g/l) Soy bean meal (control) Urea Peptone Casein hydrolysate Gelatine Yeast extract Beef extract Corn steep liqour -- 0 Fig. 6: Effect of different nitrogen sources on alkaline protease production. Different concentrations of corn steep liquor were also tested (figure 7), corn steep liquor 15 % concentration gave the best alkaline protease production by the locally isolated Bacillus subtilis as well as 2 % concentration (figure 6) and then 2% concentration will be used as the best concentration of corn steep liquor for alkaline protease production from the studied isolate. 199

8 Total Dry Weight (g/l) Corn steep liquor concentration % Fig. 7: Effect of different concentration of corn steep liquor on alkaline protease production by Bacillus subtilis. 10- Effect of salt elimination: The experiment was illustrated by table (3), it was clearly shown that the protease activity resulted from original medium was 368 U/ml. and the medium after elimination of all salts except CaCl 2 was 362 U/ml, while the elimination of Na 2 HPO 4 is very effective and the enzyme production is very low (239 U/ml). From previous results, the presence of CaCl 2 only in the medium was the most efficient one (362 U/ml) followed by MgCl 2 (351 U/ml) followed by Na 2 HPO 4 (337 U/ml) and finally NaH 2 PO 4 (330 U/ml). On other hand elimination of all salts from the medium will decrease the enzyme activity from 368 U/ml of original medium to 283 U/ml. from these results all salts were important to be present in the medium together with concentrations as shown on growth medium. Table 3: Effect of different salt elimination from protease production medium Salt in the medium Biomass Dry weight Enzyme activity CaCl 2 MgCl 2 Na 2HPO 4 NaH 2PO 4 (mg/ml) (U/ml) Effect Of Modified Medium On Alkaline Protease Production: From previous data we can conclude that the best ph range is 10, temperature 37 ºC, 18 x 10 8 inoculum size, 12 % sugar cane molasses as carbon source and 2% corn steep liquor as nitrogen source. Table 4 clearly show that the comparison between the basal and modified protease production media occurred and recorded that the modified medium is relatively more producer for protease production (393 U/ml) than the basal medium (368 U/ml). Table 4: Effect of basal and modified medium on protease production at 37 ºC and ph 10.0 by B. subtilis Incubation period (hours) Dry weight (mg/ml) Basal Modified Basal Modified Basal Modified

9 Discussion: Proteases are a unique class of enzymes, they posses both degradative and synthetic properties. There is a renewed interest in proteases as targets for developing theurapeutic agents against relentlessly spreading of total disease such as cancer, malaria and AIDS (Mala et al., 1998 and Maria et al., 1999). Protease produced by bacteria are of main interest for many workers (Ammar et al., 1991a, b and c; Ammar et al., 1995 and 1999; Abd Rahman et al., 1998 and Mabrouk et al., 1999). In this study 5 meat samples, collected from different localities in Egypt, 20 isolates of aerobic were recorded. Each of these isolates was primarily screened for its ability to produce protease enzyme using gelatin clearing zone technique. Results of screening technique revealed that Gram positive bacilli bacteria were characterized by high productivity of protease enzyme than the rest of other isolates. It might be though that the high productivity of these isolates were relatively wide range of ph was due to the presence of more than one type of protease enzyme or a combination of many proteases or isoenzymes (Abd Rahman et al., 1998). In this study, the production of protease enzyme from the high productive Bacillus sp. isolate showed maximum activity and stability within ph ranged from 9.0 to Results clearly show a direct correlation between the biomass of cells and proteolytic activities of the isolates. This correlation was also reported by Fujiwara & Yamamoto (1987), Ammar et al. (1991a, b and c) and Ibrahim (1991). Environmental conditions play an important role in the microbial role in the microbial growth and enzyme productivity (Ammar et al., 1991 a & b; Abd Rahman et al., 1998 and Mala et al., 1998). In the present study the influence of various factors on the proteases production was studied carefully as follow. The effect of initial ph on the growth and protease productivity by the isolate B. subtilis was tested and results revealed that the maximum increase in the enzyme production and the biomass dry weight was attained at ph 10. These results agreed with those obtained by Fujiwara (1986) and Fujiwara & Yamamoto (1987) who produced alkaline protease using initial ph range from 10.0 to Ibrahim (1991) obtained an alkaline protease using glycin-naoh at ph 10.6 with maximum enzyme activity. Others used phosphate buffer at ph 8.5 to 10.0 as the best buffer for maximum production of alkaline protease(s) from B. subtilis (Kembhavi et al., 1993). Using a definite inoculum size of the bacterial isolates was also tested, maximum production of protease enzyme obtained due to the addition of 18 X 10 8 cell/ml for B. subtilis. During the course of fermentation of protease(s) production by B. subtilis, it was observed after 48 h incubation (Fujiwara & Yamamoto 1987, Ibrahim et al and Ammar et al., 1991 a, b and c). The present study showed that the maximum protease production by the all bacterial isolate within temperature range from C and an increase in protease production was observed at 37 C and it was agree with Fujiwara et al. (1993) and Fujiwara & Yamamoto (1987). Nutritional factors greatly affected the production of alkaline protease enzyme, supplementation of the basal protease production medium with different carbon sources by replacing glucose as a sole carbon source by 6 carbon sources including sugar cane molasses as a byproduct. The biomass dry weight gained about 35 % increase than that of other carbon sources as well as protease production, this may be due to that the sugar cane molasses contains proteins, minerals and vitamins in addition to carbohydrates in great amounts and also growth promoting agents (Ammar et al., 1998). By using different concentration of cane sugar molasses from 6 to 30 %, it was found that by increasing the concentration of the molasses from 6 to 18 % the enzyme activities and biomass dry weight increased. The other nutritional factor was nitrogen source, 7 different nitrogen sources were used in addition to soy bean meal ; data obtained gave a strong evidence about the strong stimulatory effect of the nitrogen sources. The most suitable nitrogen source was corn steep liquor which gave the highest activity of protease activity (401 U/ml) for B. subtilis. Also, data showed a direct proportionality between proteolytic activity and dry weight biomass and independence of final ph. Different nitrogen sources were reported by many other investigators for maximum protease production. Sampath and Chandrakasan (1998) proved that soybean meal of 2 % concentration gave optimum expression of extracellular protease production by bacteria and Streptomyces sp. Ellaiah and Srinivasulu (1996), observed that high level of protease detected in presence of gelatin and peptone as nitrogen sources. The presence of the salts in the medium were essential for high protease production, while the data from table 4 proved that the modified medium is relatively alkaline protease enhancer than the basal medium this is may be due to the molasses and the corn steep liquor contains many desirable and important constituents as vitamins, carbohydrates, proteins and important amino acids. Conclusion: From previous data we can use modified medium (which consists of; sugar cane molasses, 12%, corn steep liquor, 2 %, ph, 10, incubation temperature, 37 C, inoculum size 18 x 10 8, fermentation medium salts as basal medium and incubation period 48 hr. with shaking at 150 rpm.) for alkaline protease production instead of basal 201

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