Enzymes have replaced or decreased usage of toxic chemicals for industrial and medical applications leading toward sustainable chemistry. In this study, we report purification and characterization of a biofilm degrading protease secreted by Microbacterium sp. SKS10. The protease was identified as a metalloprotease, Peptidase M16 using mass spectrometry. It showed optimum activity at 60°C, pH 12 and retained its activity in the presence of various salts and organic solvents. The enzyme was able to degrade biofilms efficiently at enzyme concentration lower than other known enzymes such as papain, trypsin and α-amylase. The presence of this protease increased the accessibility of antibiotics inside the biofilm, and was found to be non-cytotoxic toward human epidermoid carcinoma cells (A431) at the effective concentration for biofilm degradation. Thus, this protease may serve as an effective tool for management of biofilms.
Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India) with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.
Background: Plants have been suggested as safest source of therapeutic agents, with multi targeted mode of action and least side effects. Tinospora cordifolia, commonly known as Guduchi in India, is one of the most highly valued herbs in Ayurvedic medicine. It possesses potential anti-cancer, anti-inflammatory, hepatoprotective, anti-diabetic, immune-stimulatory and various other beneficial activities. Purpose: The present study was aimed to investigate the differentiation inducing potential of chloroform and hexane extracts of T. cordifolia using U87MG glioblastoma and IMR-32 neuroblastoma cell lines as model system. Results: Chloroform (Chl-TCE) and hexane (Hex-TCE) extracts significantly reduced the rate of proliferation and induced cell differentiation as evidenced by MTT assay and immunostaining for GFAP and MAP-2 in glioblastoma and neuroblastoma, respectively. Further these extracts increased the expression of stress markers HSP70 and Mortalin and induced senescence. Chloroform and hexane extracts also inhibited the migration of U87MG glioblastoma and IMR-32 neuroblastoma as indicated by wound scratch assay and supported by reduced expression of NCAM. Furthermore these extracts are not toxic to normal cells as they showed no inhibitory effects on primary astrocytic and neuronal cultures. Conclusions: The present study suggests that chloroform and hexane extracts of T. cordifolia retard the rate of proliferation, induce differentiation and inhibit migration of human glioblastomas and neuroblastomas, thus may act as potential phytotherapeutic intervention in treatment of neural cancers.
Enzymes have replaced or decreased usage of toxic chemicals for industrial and medical applications leading towards sustainable chemistry. In this study, we report purification and characterization of a biofilm degrading protease secreted by Microbacterium sp. SKS10. The protease was identified as a metalloprotease, Peptidase M16 using mass spectrometry. It showed optimum activity at 60 °C, pH 12 and retained its activity in the presence of various salts and organic solvents. The enzyme was able to degrade biofilms efficiently at enzyme concentration lower than other known enzymes such as papain, trypsin and -amylase. The presence of this protease increased the accessibility of antibiotics inside the biofilm, and was found to be non-cytotoxic towards human epidermoid carcinoma cells (A431) at the effective concentration for biofilm degradation. Thus, this protease may serve as an effective tool for management of biofilms.
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