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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 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa 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 pH8.0 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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]

Alkaline protease from Bacillus amyloliquefaciens SP1 has been characterized in detail for its ecofriendly application of release of silver particles from gelatin layers of used X-ray films. It exhibited optimum activity at broad temperature range and maximum at 60°C under alkaline pH environment (8-12). Thermal inactivation of the crude enzyme followed first order kinetics. The half-life of the enzyme at 50, 60 and 65°C was 70, 15 and 12.6 min, respectively and the denaturation energy was 114.87 kJ/mol. Enzyme retained 53.83 and 108.33% of its initial activity after heating for 15 min at pH 8.0 and temperature 60°C, in presence and absence of 10 mM MnSO4, respectively. Enzymatic decomposition of gelatin layers was enhanced by increase of enzyme concentration from 38 to 3630 µg/ml/min, at 60°C and pH 8.0. This study reported the shortest time of 1.30 min at 3630 µg/ml/min and 4 : 30 min at 74 µg/ml/min of enzyme concentration for hydrolysis of gelatin layers. Keeping in mind that, nowadays recycling is needed and imperative, this is the first study to report that after addition of Mn2+ ions, thermal stability of enzyme increased and it could be effectively reused for 8 cycles as compared with enzyme without protective agents which also increase its yield of silver recovery i.e. 19.56 ± 0.78% by eight times. [ABSTRACT FROM AUTHOR]

A thermostable extracellular alkaline protease producing Bacillus amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth promoting activities. Strain SP1 was purified to 6.48-fold using four-step purification protocol and characterized in detail for its robustness and ecofriendly application in leather and detergent industries. Structural analysis revealed that the protease was monomeric and had a molecular weight of 43 kDa. It exhibited optimum activity at 60°C in alkaline environment (pH 8.0) and stable in the presence of surfactants and oxidizing agents. Enzyme was thermostable at 50°C and retained more than 70% activity after 30 min incubation. It has shown stain removal property and dehairing of goat skin without chemical assistance and hydrolyzing fibrous proteins. This protease showed Km of 0.125 mg ml−1 and Vmax of 12820 μg ml−1 indicating its excellent affinity and catalytic role. Thermal inactivation of the pure enzyme followed first-order kinetics. The half life of the pure enzyme at 50, 60, and 65°C was 77, 19.80, and 13.33 min, respectively. The activation energy was 37.19 KJ mol−1. The results suggest that the B. amyloliquefaciens SP1 has a potential application in different industries. [ABSTRACT FROM AUTHOR]