Effect of Physical and Chemical Factors in Production of Alkaline Protease Enzyme by Bacillus Strains (original) (raw)

Optimisation of Production Condition of Alkaline Protease Using Indigenous Bacillus subtilis from Agricultural Soil

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.

Optimization of Alkaline Protease production from bacteria isolated from soil

Protease producing bacteria were isolated from soil and were identified as Pseudomonas fluorescens, Bacillus subtilus, E.coli and Serratia marscens. Optimization of the fermentation medium for maximum protease production was carried out. The culture conditions like pH, temperature, carbon sources and nitrogen sources were optimized. The Optimum conditions for protease production were found to be 37 o C at pH 10 with Glucose as Carbon Source and Peptone as Nitrogen Source. Soycake and Calcium chloride stimulates the production of protease with 1ml of inoculums size for 48 hrs of incubation period with less concentration of EDTA. Among all studied bacterial isolates, the highest enzyme activity was observed in B. Subtilis. Sonia Sethi et al J. Microbiol. Biotech. Res., 2012, 2 (6):858-865 ______________________________________________________________________________ 859 Available online at www.scholarsresearchlibrary.com

Production of alkaline protease by Bacillus subtilis using solid state fermentation

African Journal of Microbiology Research, 2013

The present study describes the optimization of nutritional and cultural parameters for the production of alkaline protease by Bacillus subtilis under solid state conditions. Among cultural conditions, incubation temperature, incubation period and moisture level of the substrate were optimized and it was found that maximum production of alkaline protease was observed at 37°C and moisture to substrate ratio of 1:1 after 48 h of incubation period. Among different nutritional parameters, the effects of different diluents, carbon and nitrogen sources on the enzyme production were studied. Maximum enzyme production (101.23 U/g) was observed when D 2 {(% w/v) CaCO 3 , 0.05; peptone, 0.1; glucose, 0.1 and yeast extract, 0.1} was used to moisten the substrate. The best carbon source for the production of alkaline protease by B. subtilis was found to be sucrose at a concentration of 1%. Similarly, nutrient broth (1.5%) and diammonium hydrogen phosphate (0.1%) were found to be best organic and inorganic nitrogen sources, respectively. It was also found that the maximum protease (126.8 U/g) was produced when 25% (v/w) inoculum was used to inoculate the fermentation flasks.

Comparative studies of alkaline protease production in solid state fermentation: Tray bioreactor and flask

Pakistan Journal of Biotechnology

In this study, production of protease in solid state fermentation using Bacillus licheniformis was investigated. Fermentation was carried out in batch and semi-bath systems including flask and tray bioreactor. Wheat bran, rice bran and mixtures of both agro-wastes were used as substrate. Maximum enzyme production was obtained using wheat bran as sole substrate with high activity of 798.83, 776.91 and 562.11U/gds for top, middle trays and flask, respectively. Results showed that optimum incubation time of 48h was defined. Enzyme production was evaluated with different incubating temperatures and initial moisture content of solid substrates. Maximum protease activity was observed at 35 and 40˚C for the tray bioreactor and flask, respectively. The obtained results showed that maximum protease production was achieved with substrate initial moisture of 150, 200 and 150% for the flask, top and middle trays, respectively. In addition, the enzyme activity was improved by supplementary substrate such as corn meal as an inducer, which gave protease activities of 678, 830 and 940U/gds, for flask, middle and top trays, respectively.

Response of Surface Optimization for the Enhanced Production of Alkaline Protease Isolated from Bacillus Sp. With Bean Husk as a New Substrate

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...

SCREENING, OPTIMIZATION OF PRODUCTION AND PARTIAL CHARACTERIZATION OF ALKALINE PROTEASE FROM HALOALKALIPHILIC BACILLUS SP

Bacillus strains isolated from the salteren pond (Kakinada) were screened and identified for high alkaline protease activity. The isolates which were positive on skim milk agar (1%) were selected as protease producing strains. Of the ten bacterial isolates screened, isolate S-8 was observed as a potential haloalkaline protease producer and it was identified as Bacillus cereus strain S8 (MTCC NO: 11901) by 16S rRNA gene sequencing, phylogenetic tree analysis and by different biochemical tests. Protease production was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also physical parameters like temperature, incubation time, pH, inoculum size were optimized for the maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed that maximum protease production was obtained in the medium supplemented with Molasses,1%(w/v); Potassium nitrate, 0.75%(w/v); salt solution-5%(v/v) {MgSo 4. 7H 2 O, 0.5%(w/v); KH 2 PO 4, 0.5%(w/v)}; FeSO 4. 7H 2 O, 0.01%(w/v) and CaCO 3, 0.5% respectively. Thus, with selected carbon and nitrogen sources along with 1 % NaCl and 2% inoculum the maximum protease production (205.0 U/ml) was obtained in the period of 72 h incubation at pH-12.0 under 160 rpm when compared to the initial enzyme production (165.0 U/ml). The crude enzyme extract of this strain was also characterized with respect to temperature, pH, incubation period and different concentrations of casein which was used as enzyme substrate. This study shows that the enzyme has wide range of pH stability from 8 to 11 with optimum activity at pH-10.0. It is thermostable with optimum activity at 70°C (392U/ml) with 1h incubation of enzyme with 1% casein as its substrate. From the above investigations it was concluded that the protease production by these microorganisms at wide temperatures and pH ranges could be explored for varied industrial applications.

Optimization of fermenting medium by statistical method for production of alkaline protease by Bacillus licheniformis MZK05M9

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.

Optimization of Protease Enzyme Production Using Bacillus Sp. Isolated from Different Wastes

Joe Botany Research …, 2009

The bacterial isolates including Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus licheniformis isolated from different wastes were screened for protease enzyme production. Among the different sources glucose was found to be best carbon source for all the four Bacillus isolates. Yeast extract was found to be the optimum nitrogen source for protease enzyme production by all the test isolates. A temperature of 50°C was found to be optimum for all the isolates for enzyme production. Soy cake found to be the best substrate for enzyme production among different agro based wastes for all the four test isolates grown under submerged fermentation conditions.

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.

Production, Optimization, Purification, Kinetic analysis and applications of alkaline proteases produced from Bacillus subtilis through solid state fermentation of agricultural byproducts

Kuwait Journal of Science, 2021

Proteases have gained more commercial value to date due to multiple applications in different industrial sectors. Current research was aimed to use the cheaper agricultural waste for optimal protease production. Maximum level of protease production was achieved at 37 °C, incubation period of 24 h, pH 9.0, inoculum size 3%, 1.5 g sucrose as a carbon source and 30% moisture content by using solid-state fermentation. Among the various inorganic and organic nitrogen sources, ammonium nitrate and yeast extract tremendously increased the production of protease. Among metal ions and surfactants tested, Ca2+ and Tween 40 showed the optimal protease production. The purification of protease was carried out by ammonium salt precipitation followed by sephadex G-100 gel filtration chromatography. The purification resulted in 1.3 fold of purified protease with a specific activity of 51.5 and a total yield of 37.5 %. Molecular weight of purified protease was predicted upon SDS-PAGE with a single b...