Search

Alkaline protease has tremendous applications in different industries, and to fulfil industrial and commercial enzyme requirements, overproduction and optimization of production medium are prerequisites. Protease production by Bacillus amyloliquefaciens SP1, isolated from apple tree rhizosphere was studied. Plackett Burmann design and response surface methodology was used to optimize medium for alkaline protease production. Higher levels of protease activity were observed in presence of disaccharides owing to diauxic growth and inorganic nitrogen sources proved to be less favourable. After screening through one-factor-at-a time experiments and Plackett Burmann design, casein, yeast extract, maltose and KH2PO4 were identified as the most significant variables. Statistical design based three-dimensional (3-D) and contour plots were generated to understand the relationship between enzyme activity and medium variables. The protease production was found to increase from 1730 to 3630 µg/ml/min in optimized medium i.e. approx. 2.1 fold increased compared with original medium. The study assumes significance in the ability of bacterium to survive in wide variety of salts and agricultural wastes and yield optimum levels of extracellular protease. [ABSTRACT FROM AUTHOR]

An extracellular alkaline protease producing B. amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth-promoting activities. B. amyloliquefaciens SP1 protease was immobilized using various concentrations of calcium alginate, agar and polyacrylamide to determine the optimum concentration for formation of the beads. Enzyme activity before immobilization (at 60 °C, pH 8.0 for 5 min) was 3580 µg/ml/min. The results of immobilization with various matrices revealed that 3 % calcium alginate (2829.92 µg/ml/min), 2 % agar (2600 µg/ml/min) and 10 % polyacrylamide (5698.99 µg/ml/min) were optimum concentrations for stable bead formation. Immobilized enzyme reusability results indicated that calcium alginate, agar and polyacrylamide beads retained 25.63, 22.05 and 34.04 % activity in their fifth repeated cycle, respectively. In cell immobilization technique, the free movement of microorganisms is restricted in the process, and a semi-continuous system of fermentation can be used. In the present work, this technique has been used for alkaline protease production using different matrices. Polyacrylamide (10 %) was found with the highest total alkaline protease titer, i.e., 24,847 µg/ml/min semi-continuously for 18 days as compared to agar (total enzyme titer: 5800 in 10 days) and calcium alginate (total enzyme titer: 13,010 in 15 days). This present study reported that polyacrylamide (10 %) among different matrices has maximum potential of immobilization of B. amyloliquefaciens SP1 and its detergent stable alkaline protease with effective application in bloodstain removal. [ABSTRACT FROM AUTHOR]

An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69 U/mg reported 24.14% growth inhibition of F. oxysporum . However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15 mM) to purified enzyme. An 1149 bp nucleotide sequence of protease gene encoded 382 amino acids of 43 kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis . The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH 8.0 and 60 °C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry. [ABSTRACT FROM AUTHOR]