Optimization of Protease Production by Bacillus mojavensis A21 on Chickpea and Faba Bean (original) (raw)
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International Journal of Molecular and Clinical Microbiology, 2013
Article history: Received 2 June 2013 Accepted 17 November 2013 Available online 1 December 2013 Optimization of the fermentation medium for maximum alkaline protease production was carried out. Fifteen positive isolates were examined for their extent of alkaline protease production. The most potent producer was identified as Bacillus sp. The solid substrate screening showed that the combination of wheat straw and bean husk was the best one. The initial screening by using Plackett-Burman's design demonstrated that among the tested factors, casein, ammonium sulphate and pepton as the nitrogen sources and glucose, lactose and sucrose as carbon sources, glucose and casein significantly (P < 0.05) enhanced the protease production in combinatory solid state fermentation (SSF). Further optimization of protease production by Bacillus sp. strain k7 on different factors such as incubation time (84 h), inoculums size (64%), initial moisture content (97%), buffer volume (4.9%) in SSF by...
Journal of Bioscience and Bioengineering, 2010
Media composition and culture conditions for surfactant stable alkaline protease production by Bacillus mojavensis A21 were optimized using two statistical methods. Plackett-Burman design was applied to find the optimal ingredients and conditions to improve yields. Response surface methodology (RSM), including central composite design, was used to determine the optimal concentrations and conditions. The results indicated that several components, including hulled grain of wheat (HGW), sardinella peptone (SP), NaCl, CaCl 2 , MgSO 4 , K 2 HPO 4 , KH 2 PO 4 , agitation, culture temperature and initial medium pH, had significant effects on production. The statistical model was constructed via central composite design (CCD) using four selected variables (HGW, NaCl, KH 2 PO 4 and K 2 HPO 4 ). Under the proposed optimized conditions, the protease experimental yield (1860.63 U/mL) closely matched the yield predicted by the statistical model (1838.60 U/mL) with R 2 = 0.98. An overall 14.0-fold increase in protease production was achieved using the optimized medium (HGW 30.0 g/L, SP 1.0 g/L, NaCl 2.0 g/L, KH 2 PO 4 1.0 g/L, K 2 HPO 4 0.3 g/L, CaCl 2 2.0 g/L, MgSO 4 1.0 g/L and pH 9.0, compared with the unoptimized basal medium (starch 10.0 g/L, yeast extract 2.0 g/L, KH 2 PO 4 0.1 g/L, K 2 HPO 4 0.1 g/L, CaCl 2 0.5 g/L and pH 8.0; 137 U/mL). A successful and significant improvement (14-fold) in the production of protease by the A21 strain was accomplished using cheap carbon and nitrogen substrates (HGW and SP), which may result in a significant reduction in the cost of medium constituents.
Optimization of alkaline protease production from Bacillus sp. by response surface methodology
Current microbiology, 2002
Optimization of alkaline protease production by Streptomyces ambofaciens NRRL 2420 in free and immobilized form was investigated using submerged fermentation technique. The optimum conditions for maximum alkaline protease production 342 unit mL −1 were 30°C at pH 8.5 and incubation time 96 h in free cell cultures using starch 20 g L −1 as carbon source and yeast extract 5 g L −1 as nitrogen source. The incubation time for the best yield of 344 unit mL −1 was reduced to 72 h under the optimized fermentation conditions by immobilized cells adsorbed on synthetic cotton fibers. Data obtained during 5 reusable cycles showed higher levels of enzyme in shorter time duration. Immobilization of Streptomyces ambofaciens NRRL 2420 on synthetic cotton fiber permit repeated reuse of the cells under the optimized fermentation conditions.
Proteases is family of enzymes and it has crucial role due to their physiological roles and very valuable commercial applications. Alkaline protease are produced by Bacillus species are particular importance because of their thermal stability and stability at different pH values. This study aimed to investigate the effect of physical and chemical factors in production of alkaline protease enzyme fermentation by members of the genus Bacillus. In this study, alkaline protease enzyme production were evaluated in submerged fermentation by Bacillus strains which were isolated from alkaline soils of Guilan province. Factors incubation were optimized such as time, pH, amount of inoculation and ammonium sulfate in alkaline protease enzyme production whit using response surface methodology (RSM) in culture. The maximum enzymatic activity was observed in incubation time of 36 hours, pH=9, inoculation amount of 15% (V) and ammonium sulfate 1.5% (W/V). Factors had significant effect on the production of alkaline protease enzyme such as pH and ammonium sulfate.
Optimization of alkaline protease production by Bacillus sp. using Taguchi methodology
… and biotechnology, 2005
Optimization of alkaline protease production by Streptomyces ambofaciens NRRL 2420 in free and immobilized form was investigated using submerged fermentation technique. The optimum conditions for maximum alkaline protease production 342 unit mL −1 were 30°C at pH 8.5 and incubation time 96 h in free cell cultures using starch 20 g L −1 as carbon source and yeast extract 5 g L −1 as nitrogen source. The incubation time for the best yield of 344 unit mL −1 was reduced to 72 h under the optimized fermentation conditions by immobilized cells adsorbed on synthetic cotton fibers. Data obtained during 5 reusable cycles showed higher levels of enzyme in shorter time duration. Immobilization of Streptomyces ambofaciens NRRL 2420 on synthetic cotton fiber permit repeated reuse of the cells under the optimized fermentation conditions.
Statistical optimization of conditions for protease production from Bacillus sp.
Acta Biologica Szegediensis, 2010
Response surface methodology was employed for the optimization of different nutritional and physical parameters for the production of protease by a soil isolated Bacillus strain in submerged fermentation. Initial screening of production parameters as carbon (glucose) and nitrogen source (soybean) were optimized together with four variables K 2 HPO 4 , NaCl, MgSO 4 .7H 2 O, CaCl 2. 2H 2 O and four physical parameters including agitation, inoculum size, pH and time was performed using Plackett Burman design and the variables with statistically significant effect on the protease production identified. These variables were selected for further optimization studies using central composite design in RSM. The protease activity under unoptimized conditions was 330 U/ml. Under the final optimized conditions, the predicted response for protease production was 449 U/ml, and the observed validated experimental value was 577 U/ml. The statistical optimization by response surface methodology resulted in about two fold increase in the production of the enzyme by the selected bacterial strain Acta Biol Szeged 54(2):135-141 (2010) KEY WORDS central composite design protease optimization Placket Burman design production media response surface methodology
2014
Three microbial cultures Bacillus subtilis DSM 1970, Bacillus subtilis GCU-8 and Bacillus licheniformis DSM 1969 were screened for protease production by casein agar plate method. Among these Bacillus subtilis GCU-8 was found to be the most potent protease producer in wide pH range (5.0 to 8.0). Fermentation conditions were optimized for the production of alkaline protease using two statistical tools: Placket Burmen Model for linear regression study and Response Surface Model for interactive effects of significant factors on production. The alkaline protease was optimally produced after 48 hours of incubation at 37 o C in fermentation media containing equal amounts of substrates (soybean meal and wheat bran, 7.5 g), MgSO4.7H2O, 0.10 g and yeast extract 0.55 g. The protease was purified to homogeneity by salt precipitation, ionexchange chromatography and size exclusion chromatography. The homogeneity and molecular weights were checked by SDS-PAGE. The protease was 45 KDa protein, pre...
Biological Sciences - PJSIR
In this study, proteases have been isolated from agricultural soil samples and then cultured by shake flask method. The growth of the Bacillus subtilis has been confirmed by microbiological test on the agar plate and skim milk agar in rough, raised and irregular colonies. The yield of the alkaline protease has been optimised by varying the main factors i.e., nitrogen source (peptone, yeast extract, beef extract, casein, ammonium carbonate and urea), carbon source (sucrose, fructose, mannose, lactose, glucose, maltose and starch), incubation period (12, 24, 36, 48, 72, 84 and 96 h), temperature (35, 40, 45, 50, 55, and 60 °C) and salts (potassium sulphate, magnesium sulphate, calcium sulphate and manganese sulphate). The results revealed that the maximum enzyme production was obtained using casein and minimum activity was obtained using urea as a nitrogen source. Similarly, other factors have shown significant effect on the activity of the enzyme.
The production of alkaline protease by Bacillus licheniformis (MTCC 1483) and Bacillus subtilis of indigenous isolates using cheapest sources-groundnut cake (N 2 Source) and wheat bran (C source) was studied. The proteolytic activity was found maximum at 72 hr of the culture for both the bacterial types but the pH of the medium for maximal enzyme production varied as 9 for Bacillus licheniformis and as 8 for Bacillus subtilis. Thus investigations of this study reveal the possibility of employing cheapest nutrient source for microbial proteases that can be used on a commercial scale.
To optimize the fermentation medium for the production of alkaline protease by Bacillus licheniformis MZK05M9 (BlM9) molasses as a carbon source, soybean meal as a nitrogen source, and the salts NaCl, MgSO 4 .7H 2 O, and K 2 HPO 4 were selected by Plackett-Burman approach. The response surface methodology based on central composite design revealed that the optimum values for the tested variables were found as (% w/v) molasses (0.92%), soybean meal (0.79%), NaCl (0.125%), MgSO 4 (0.125%), and K 2 HPO 4 (0.59%) with the protease activity 761 U/ml predicted by statistical software Minitab Version 17. The experimental value was found as 765 U/ml. The granular size of soybean meal 4.7 mm supported the enzyme production 5% higher than that of the mixed sizes between 6 and 4 mm. Fermentation in 7 l bioreactor exhibited the enzyme activity 1020 U/ml after 28 h.