Effect of different sulphate sources on alkaline protease production 

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... meal, 10; K 2 HPO 4 , 3; KH 2 PO 4 , 1; at pH-7 (Oberoi et al., 2001) was inoculated with 2% fresh culture (A 550 nm ≈ 0.2). The inoculated medium was incubated at 37 o C and 180 rpm. The culture was centrifuged at 10.000 × g for 10 min at 4 o C. The cell pellet was discarded and the supernatant was used for assay of protease activity. Protease activity was determined by a modified meth- od of Folin and Ciocalteu (Folin and Ciocalteu, 1927). 200 μl of the protease broth was added to the reaction mixture, containing 0.65% (wv -1 ) casein in 800 μl of 50 mM in phosphate buffer (pH 9). The mixture was incu- bated at 75 o C for 10 min. The reaction was stopped by the addition of 1 ml of 5% (wv -1 ) trichloroacetic acid (TCA), followed by centrifugation at 10.000× g for 15 min. The supernatant were analyzed by the Folin-Ciocalteu reagent. One unit of protease activity was defined as the amount of enzyme that liberated 1μg tyrosine per min per ml of protease broth. Selection of carbon source was done by one variable at a time method where in the production medium, starch was replaced by different carbon sources viz. D-mannitol, glycerol, maltose, lactose, sucrose, glucose, mannose and fructose. All carbon sources were used at a final concentra- tion of 1% Carbon (wv -1 ) (Puri et al., 2002a). Selection of nitrogen source was done by one variable at a time method where various organic and inorganic nitrogen sources were added to the fermentation medium at a final concentra- tion of 0.5% Nitrogen (wv -1 ) (Puri et al., 2002). To study the effect of different nitrogen sources on protease produc- tion, soybean meal was replaced by other organic as well as inorganic nitrogen sources such as peptone, casamino acid, gelatine, tryptone, urea, yeast extract, sodium nitrate, ammonium nitrate, ammonium chloride, ammonium sul- phate and potassium nitrate. Selection of sulphate source was carried out by one variable at a time method where various metal sulphates sources such as magnesium sul- phate, ferrous sulphate and calcium sulphate were added to the fermentation medium at a final concentration of 1gl -1 . Protease yield was determined after 96 h of incuba- tion at 37 o C and 180 rpm. Simulated media was prepared by incorporating starch, soybean meal and magnesium sulphate as 1% Car- bon (wv -1 ), 0.5% Nitrogen (wv -1 ) and 1 gl -1 and protease production was compared with basal media after 96 h of incubation at 37 o C and 180 rpm. Maximum alkaline protease production was realised when the medium comprised of starch, soybean meal and magnesium sulphate as carbon, nitrogen and sulphate sources respectively. Maximum extracellular alkaline pro- tease production was obtained in presence of starch, as a complex carbon source, producing 135.23±1.72 (U) which is the maximum yield in comparison with the other carbon sources (shown in Fig. 1). The protease production by oth- er carbon sources were as follows: lactose (119.94±4.31), maltose (117.88±3.92), D-mannitol, (106.50±4.71), sucrose (98.55±2.61), glycerol (62.27±4.31), fructose (39.82±2.78); mannose (38.54±3.07) and glucose (32.36 ±3.39); which seemingly have less influence on protease production when compared to that of starch. These results were in accordance to previous reports for Bacillus sp . (Fer- rero et al. , 1996, Gusek et al. , 1988, Hubner et al. , 1993, Puri et al. , 2002b). Repressive effect on protease synthesis was observed in this experiment when other carbon sourc- es were used. Catabolite repression may be the most likely reason for this lagging effect (Kumar et al. , 1999, Priest, 1977). It was previously established that a catabolite con- trol protein (CcpA) was responsible for this regulatory mechanisms which transduced signal for the repression in protease synthesis (Tobisch et al. , 1999). Maximum extracellular alkaline protease production was obtained in presence of soybean meal, as a complex nitrogen source, producing 134.74±1.77 (U) which is the maximum yield in comparison with the other nitrogen sources (shown in Fig. 2). The protease production by oth- er nitrogen sources are as follows: peptone (131.90±4.18), gelatine (89.34±2.67), sodium nitrate (80.12±2.50), po- tassium nitrate (79.53±6.33), yeast extract (54.82±2.17), ammonium chloride (45.90±3.18), casamino acid (41.78±4.44), tryptone (33.73±2.45), ammonium nitrate (15.89±3.98), ammonium sulphate (12.66±2.70) and urea (11.38±1.77), which seemingly have less influence on protease production when compared to that of soybean meal. These results are in accordance to previous reports for Bacillus sp. (Ferrero et al. , 1996 ; Gupta et al. , 2002 ; Hubner et al. , 1993 ; Joo et al. , 2002 ; Puri et al., 2002) and this can be explained by the mechanism of feedback inhi- bition (Malathi and Chakraborty, 1991). Magnesium sulphate, when used as the sulphate source, produced 137.69±4.57 (U) of protease whereas ferrous sulphate produced 129.25±5.71 U and calcium sulphate 126.80±8.53 (U) of protease (Fig. 3). Magnesium ion was found to be more effective in protease production than other metal ions. The depletion in magnesium ion results in decreased rate of enzyme production that ultimately af- fects the mechanism of protease synthesis (Hanlon et al., 1982). A significant improvement (4.42-fold) in the alkaline protease production (shown in Fig. 4) by Bacillus licheni- formis NCIM-2042 was found in simulated media than basal media. The present study focused on selection of suitable car- bon, nitrogen and sulphate sources as a media component for maximal alkaline protease production through micro- bial fermentation. It was observed that such study was of utmost important for obtaining higher degree of alkaline protease production as well as reduction of operating cost of the process. Selection of the suitable carbon, nitrogen and sulphate sources were done through classical method involving one variable at a time which is user friendly. Fur- ther experiments on this protease towards optimization of media componants are currently under ...