A new pH indicator dye-based method for rapid and efficient screening of l-asparaginase producing microorganisms.

l-asparaginase is a pharmaceutically and industrially important enzyme as it has potential to treat different cancers and inhibit acrylamide formation in fried and baked food products. In the present study, an attempt was made to screen for new and novel l-asparaginase producers using a widely applied phenol red and bromothymol blue (BTB)1 dye-based plate assay. Screening of four different soil samples for l-asparaginase producers resulted in the isolation of three new potential l-asparaginase producing bacteria. These three strains identified (by 16S rRNA sequencing) as a Pseudomonas resinovorans strain IGS-131, a Bacillus safensis strain IGS-81, and a Glutamicibacter arilaitensis strain ICS-13 with enzyme activities of 10.91 IU/ml, 6.65 IU/ml, and 1.47 IU/ml, respectively. These three strains of bacteria have not been reported as l-asparaginase producers previously. Also, we developed a new pH indicator dye-based plate assay for the screening of l-asparaginase producers after testing eight different pH indicator dyes. This cresol red dye-based method gave a better differentiable zone of hydrolysis and consistent results as compared to previously reported phenol red and BTB-based plate assay. It was also found to be efficient in comparison to all other dyes studied. It produced a bright yellow color at acidic pH (5.5) and turned into a dark red or maroon color when pH was increased (above 7.5). This finding is expected to make screening of all kinds of l-asparaginases more comfortable, rapid, and efficient.

Sonorensin: A new bacteriocin with potential of an anti-biofilm agent and a food biopreservative.

The emergence of antibiotic resistant bacteria has led to exploration of alternative therapeutic agents such as ribosomally synthesized bacterial peptides known as bacteriocins. Biofilms, which are microbial communities that cause serious chronic infections, form environments that enhance antimicrobial resistance. Bacteria in biofilm can be upto thousand times more resistant to antibiotics than the same bacteria circulating in a planktonic state. In this study, sonorensin, predicted to belong to the heterocycloanthracin subfamily of bacteriocins, was found to be effectively killing active and non-multiplying cells of both Gram-positive and Gram-negative bacteria. Sonorensin showed marked inhibition activity against biofilm of Staphylococcus aureus. Fluorescence and electron microscopy suggested that growth inhibition occurred because of increased membrane permeability. Low density polyethylene film coated with sonorensin was found to effectively control the growth of food spoilage bacteria like Listeria monocytogenes and S. aureus. The biopreservative effect of sonorensin coated film showing growth inhibition of spoilage bacteria in chicken meat and tomato samples demonstrated the potential of sonorensin as an alternative to current antibiotics/ preservatives.

Bioprocess development for the production of sonorensin by Bacillus sonorensis MT93 and its application as a food preservative.

Media composition and environmental conditions were optimized using statistical tools, Plackett Burman design and response surface methodology, to maximize the yield of a bacteriocin, named as sonorensin, from a new marine isolate Bacillus sonorensis MT93 showing broad spectrum of antimicrobial activity. Under optimized conditions, MT93 produced 15-fold higher yield of sonorensin compared to that under initial fermentation conditions. As oxygen supply is a critical parameter controlling growth and product formation in aerobic bioprocesses and used as a parameter for bioprocess scale up, the effects of oxygen transfer, in terms of volumetric oxygen transfer coefficient (kLa), on production of sonorensin was investigated using optimized medium composition in a bioreactor. Studies on effectiveness of sonorensin against Staphylococcus aureus and Listeria monocytogenes in fruit juice and as a preservative in pasteurized milk demonstrated its potential as a biopreservative in fruit products and shelf life extender of the pasteurized milk.

Sonorensin: an antimicrobial peptide, belonging to the heterocycloanthracin subfamily of bacteriocins, from a new marine isolate, Bacillus sonorensis MT93.

Marine environments are the greatest fronts of biodiversity, representing a resource of unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, antimicrobial peptides (AMPs) have received great attention recently due to their applications as food preservatives and therapeutic agents. A new marine soil isolate producing an AMP was identified as Bacillus sonorensis based on 16S rRNA gene sequence analysis. It produced an AMP that showed a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The peptide, named sonorensin, was purified to homogeneity using a combination of chromatographic techniques. The intact molecular mass of the purified peptide, 6,274 Da, as revealed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), was in agreement with Tricine-SDS-PAGE analysis. A PCR array of primers was used to identify AMP structural genes, which allowed the successful amplification of the related genes from strain MT93. The putative open reading frame of sonorensin was amplified, cloned into the pET-32a(+) vector, expressed as a thioredoxin (Trx) fusion protein in Escherichia coli, and then purified. Sequence alignment analysis revealed that the bacteriocin being reported could belong to new subfamily of bacteriocins, heterocycloanthracin. The peptide indicated its potential as a biocontrol agent or food antimicrobial agent, due to its antimicrobial activity against bacteria such as Listeria monocytogenes and Staphylococcus aureus. This is the first report of the production, purification, and characterization of wild-type and recombinant bacteriocin by B. sonorensis and the first bacteriocin of the heterocycloanthracin subfamily to be characterized.