Bioprospecting of serratiopeptidase-producing bacteria from different sources.

Anti-inflammatory enzymes have wide applications in the pharmaceutical industry. The objective of this study was to find new and efficient strains for the commercial production of serratiopeptidase enzyme. Vast number of samples were processed for the isolation of potent strains. The experimental treatment includes processing of twenty soil samples, silkworm gut, and sugarcane stem. The total protein and protease activity was estimated by Lowry's method and casein hydrolysis. The HRBC stabilization assay was performed for finding the anti-inflammatory potential of all strains. The serratiopeptidase production was confirmed by HPLC with the standard. Molecular characterization of selected potent strains was done by 16S rDNA and confirmed the taxonomy. The one step rapid purification of serratiopeptidase was performed by Ultra three phase partitioning method. The clot lysis potential of the Serratia marcescens VS56 was observed by modified Holmstorm method. The results of the study revealed that among the 60 strains, 12 strains were protease-positive on skim milk agar plates and showed significant protease activity. All 12 strains were screened for serratiopeptidase using high-performance liquid chromatography (HPLC) and VS56, VS10, VS12 and VS18 showed a similar retention time (4.66 ± 0.10 min) with standard. The selected potent strain, Serratia marcescens VS56 showed a proteolytic activity of 21.30 units/mL and produced a total protein of 102 mg/mL. The HRBC suspension results also showed a percentage of 94.6 ± 1.00 protection, which was compared to the standard diclofenac. The clot lysis potential of Serratia marcescens VS56 was 53% in 4 h. Furthermore, the molecular weight of the protein was identified to confirm the presence of serratiopeptidase. The study hence contributed successfully to isolating, screening, and identifying a potent producer for serratiopeptidase from an environmental source. This inherent advantage of the strain will undoubtedly contribute much to the coco comm commercial production of serratiopeptidase in the near future.

Molecular and therapeutic insights of rapamycin: a multi-faceted drug from Streptomyces hygroscopicus.

The advancement in pharmaceutical research has led to the discovery and development of new combinatorial life-saving drugs. Rapamycin is a macrolide compound produced from Streptomyces hygroscopicus. Rapamycin and its derivatives are one of the promising sources of drug with broad spectrum applications in the medical field. In recent times, rapamycin has gained significant attention as of its activity against cytokine storm in COVID-19 patients. Rapamycin and its derivatives have more potency when compared to other prevailing drugs. Initially, it has been used exclusively as an anti-fungal drug. Currently rapamycin has been widely used as an immunosuppressant. Rapamycin is a multifaceted drug; it has anti-cancer, anti-viral and anti-aging potentials. Rapamycin has its specific action on mTOR signaling pathway. mTOR has been identified as a key regulator of different pathways. There will be an increased demand for rapamycin, because it has lesser adverse effects when compared to steroids. Currently researchers are focused on the production of effective rapamycin derivatives to combat the growing demand of this wonder drug. The main focus of the current review is to explore the origin, development, molecular mechanistic action, and the current therapeutic aspects of rapamycin. Also, this review article revealed the potential of rapamycin and the progress of rapamycin research. This helps in understanding the exact potency of the drug and could facilitate further studies that could fill in the existing knowledge gaps. The study also gathers significant data pertaining to the gene clusters and biosynthetic pathways involved in the synthesis and production of this multi-faceted drug. In addition, an insight into the mechanism of action of the drug and important derivatives of rapamycin has been expounded. The fillings of the current review, aids in understanding the underlying molecular mechanism, strain improvement, optimization and production of rapamycin derivatives.

A statistical optimization by response surface methodology for the enhanced production of riboflavin from Lactobacillus plantarum-HDS27: A strain isolated from bovine milk.

In the present study, Lactobacillus plantarum-HDS27 strain isolated from bovine milk was used for the enhanced production of riboflavin. Production medium was optimized by one factor at a time with different parameters. Statistical optimization by Response surface methodology (RSM), central composite design was used to optimize variables such as pH, temperature, glucose, and yeast extract. The present study reveals the maximum riboflavin production by one factor at a time was obtained under the culture conditions; glucose, yeast extract, pH 6, the temperature at 40°C, and 3% of inoculum size. In RSM, analysis of variance for the responses was calculated. Among the tested variables, pH, yeast extract, and temperature showed significant impact on riboflavin production. Maximum amount of yeast extract in production medium resulted in increased riboflavin production. The riboflavin production after 24 h with the optimal condition was found to be 12.33 mg/L. It was found proximate to the expected value (12.29 mg/L) achieved by the RSM model. The yield of riboflavin was increased to 3.66-fold after 24 h with the optimized parameters. The current research, emphasizes that the Lactobacillus plantarum-HDS27 could be an excellent strain for the large-scale industrial production of riboflavin.

Bioactivity of Marine Streptomyces sp. VITJS4: Interactions of Cytotoxic Phthalate Derivatives with Human Topoisomerase II α: An In Silico Molecular Docking Analysis.

Despite clinical advances in antimicrobial and anticancer therapy, there is an urge for the search of new bioactive compounds. In the present study, previously isolated Streptomyces sp. VITJS4 strain (NCIM No. 5574) (ACC No: JQ234978.1) crude extract tested for antibacterial activity showed a broad spectrum at the concentration of 20 mg/mL against pathogens. The antioxidant potential tested at 0.5 mg/mL concentration exhibited reducing power activity with a maximum of 90 % inhibition. The anticancer property by MTT assay on HeLa and HepG2 cells showed cytotoxic effect with IC50 of 50 µg/mL each. The DNA fragmentation pattern observed in both HeLa and HepG2 cell indicated laddering pattern at 40 µg/mL concentration. GC-MS analysis revealed that the significant peak corresponding at m/z 149 (M+) was identified as phthalate derivatives. The extract was further separated by HPLC with their retention times (t r) at 6.294 min. The above-obtained results were also supported by molecular docking studies which provide an insight into ligand binding to the active site of the receptor. The in silico docking studies revealed better binding affinity with a binding energy of -5.87 kJ mol-1 of the ligand toward topoisomerase II α.

Potential application of immobilized streptokinase extracted from Streptococcus equinus VIT_VB2.

Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8 ± 1.06%) when compared with entrapment with agarose gel (55.6 ± 2.17%) and cross-linked gelatin formaldehyde gel (71.0 ± 1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68 ± 1.33 and 1121.9 ± 1.2 U mL-1, respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67 ± 2.64% and 76.16 ± 2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15 min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.

Exploring the in vitro thrombolytic potential of streptokinase-producing ß-hemolytic Streptococci isolated from bovine milk.

The aim of this study was to isolate and characterize streptokinase-producing ß-hemolytic Streptococcus sp. from bovine milk. A total of 50 milk samples were collected randomly from different breeds of cow and goat (Vellore, Tamil Nadu, India). The samples were characterized and screened for streptokinase-producing isolates using microbial and biochemical analysis. About 97 colonies were isolated from milk samples showing hemolytic patterns of α (19.6%), ß (24.7%) and γ (55.6 %). Out of 20ß-hemolytic isolates, only 6 colonies (VB2, VB3, VB8, VB14, VB16, and VB17) were identified as ß-hemolytic Streptococci as potent producers of streptokinase. VB2 and VB14 showed the greatest streptokinase activities of 265 U mL(-1) and 225 U mL(-1), respectively. Based on biochemical and molecular characterization, the potent isolates VB2 and VB14 were identified and confirmed as S. equinus and S. agalactiae, respectively. The identified strains were named Streptococcus equinus VIT_VB2 (GenBank accession no. JX406835) and Streptococcus agalactiae VITVS5 (GenBank accession No. KF186620) The strains isolated from bovine milk provide a variance in the fibrinolytic activity on blood clots. The current study has demonstrated that the isolation of streptokinase producers from bovine milk, and the production of streptokinase from novel strain, enhanced the fibrinolytic activity. This study is the first to report that Streptococcus equinus produces streptokinase.

Immobilization of ß-galactosidase from Lactobacillus plantarum HF571129 on ZnO nanoparticles: characterization and lactose hydrolysis.

ß-Galactosidase from Lactobacillus plantarum HF571129 was immobilized on zinc oxide nanoparticles (ZnO NPs) using adsorption and cross-linking technique. Immobilized ß-galactosidase showed broad-spectrum pH optima at pH 5-7.5 and temperature 50-60 °C. Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) showed that ß-galactosidase successfully immobilized onto supports. Due to the limited diffusion of high molecular weight substrate, K m of immobilized enzyme slightly increased from 6.64 to 10.22 mM, while V max increased from 147.5 to 192.4 µmol min(-1) mg(-1) as compared to the soluble enzyme. The cross-linked adsorbed enzyme retained 90 % activity after 1-month storage, while the native enzyme showed only 74 % activity under similar incubation conditions. The cross-linked ß-galactosidase showed activity until the seventh cycle and maintained 88.02 % activity even after the third cycle. The activation energy of thermal deactivation from immobilized biocatalyst was 24.33 kcal/mol with a half-life of 130.78 min at 35 °C. The rate of lactose hydrolysis for batch and packed bed was found to be 0.023 and 0.04 min(-1).

Strain improvement and optimization studies for enhanced production of erythromycin in bagasse based medium using Saccharopolyspora erythraea MTCC 1103.

In the present study, Saccharopolyspora erythraea MTCC 1103 was used for the enhanced production of erythromycin. To enhance the yield of erythromycin, effects of various parameters such as bagasse concentration, organic nitrogen source, inorganic nitrogen source, pH and temperature were analysed. It was found that bagasse can be used as an alternate carbon source in erythromycin production medium. Erythromycin production in the new formulation of bagasse based medium was found to be 512 mg/L which was 28 % higher than glucose based medium. Strain improvement was done by random UV-mutagenesis. When compared to wild type strain, mutant strain showed 40 % higher yield in production medium. Erythromycin potency assay and HPLC analysis were performed to confirm the presence of erythromycin in the partially purified samples. These optimized conditions could be used for the commercial production of this unique antibiotic which gave significant industrial perspectives.

In vitro thrombolytic activity of purified streptokinase extracted from Streptococcus equinus VIT_VB2 isolated from bovine milk.

Streptokinase (SK) is an extracellular enzyme secreted by various strains of ß-hemolytic Streptococci. The main focus of the current study is to evaluate the in vitro thrombolytic activity of purified SK extracted from Streptococcus equinus VIT_VB2 (Accession no. JX406835) isolated from milk sample. The growth rate of S. equinus VIT_VB2 strain was studied with pH and biomass content which has positive significant effect on enzyme yield. A temperature of 10 °C and pH of 6 was found to be optimum for maximum SK activity. The specific activity of the purified SK produced by VIT_VB2 strain was found to be 6,585 IU mg(-1). The molecular mass of the enzyme was determined as 47 kDa by SDS-PAGE. In vitro thrombolytic activity of purified SK was determined using synthetic chromogenic substrate S-2251, the activity of the purified enzyme was found to be 6,330 ± 2.2 IU. The purity of SK was compared with standard SK by HPLC. This is the first report which reveals the SK activity of S. equinus isolated from milk sample.

Development of a novel malarial vaccine design: a hypothetical approach.

P. falciparum is highly virulent in nature because of its ability to modify the infected host red blood cells, adherence to the vascular endothelium and changes in antigenicity at different stages. Also slow migration time in the dermal and endothelial cells leads to decreased immune response. To overcome the problems, there is a need to design a vaccine which increases the migration time of the parasite, enhances the immune response, enables recognition of surface antigens and causes minimal clinical infection as a side-effect. An ITI-based (Infection-Treatment Immunization) vaccine development strategy is to be adopted to develop this novel vaccine. This will include administration of a liquid solution of purified, non-attenuated sporozoites from an infected female Anopheles mosquito, AS02A adjuvant and chlorate (a metabolic inhibitor of sulfation that decreases the extent of GAG sulfation). To control infection, a drug-cover of artemisinin will be administered as a part of the vaccination strategy along with a specific protease inhibitor MRT12113 which prevents RBC rupture and reinvasion by the parasite. This vaccine will intend to increase the overall migration time of the parasite in blood which is otherwise approximately 30 minutes, resulting in an overall enhanced immune response. It also intends to reduce parasite invasion in cells and their consequent rupture thus preventing the clinical condition-malaria.

Studies on growth kinetics of Serratia marcescens VITSD2 and optimization of fermentation conditions for serratiopeptidase production.

Serratia is one of the most important groups of bacteria which produces proteolytic enzymes effectively and known to possess anti- inflammatory properties. The main focus of the current study was to optimize the culture conditions of Serratia marcescens VITSD2 for the mass production of serratiopeptidase. Effect of various nutritional and environmental factors were analysed and optimized. Among the different carbon and nitrogen sources tested, mannose and soya bean meal was found to be the best with enzyme activity of 1391 units /mL and 1800 U/mL respectively. The enzyme showed an optimum activity of 1668 U/mL at pH-8 and 1500 U/mL at 25ºC. Maximum peptidase production during fermentation was obtained after 24 h incubation with 1% inoculum in the medium at 25ºC and yielded 1668 U/mL. Lysine stimulated the production of peptidase and the yield obtained was 2410U/mL. Growth curve analysis was done. Maximum serratiopeptidase production was detected after 24 h incubation with 2155 units/mL and cell density of 2.4g/100mL. Hence the observation of the present study clearly indicates that the yield of Serratiopeptidase was found to be maximum by varying the cultural conditions.

Studies on heavy metal removal efficiency and antibacterial activity of chitosan prepared from shrimp shell waste.

Chitosan, a natural biopolymer composed of a linear polysaccharide of α (1-4)-linked 2-amino 2-deoxy ß-D glucopyranose was synthesized by deacetylation of chitin, which is one of the major structural elements, that forms the exoskeleton of crustacean shrimps. The present study was undertaken to prepare chitosan from shrimp shell waste. The physiochemical properties like degree of deacetylation (74.82 %), ash content (2.28 %), and yield (17 %) of prepared chitosan indicated that that shrimp shell waste is a good source of chitosan. Functional property like water-binding capacity (1,136 %) and fat-binding capacity (772 %) of prepared chitosan are in total concurrence with commercially available chitosan. Fourier Transform Infra Red spectrum shows characteristic peaks of amide at 1,629.85 cm-1 and hydroxyl at 3,450.65 cm-1. X-ray diffraction pattern was employed to characterize the crystallinity of prepared chitosan and it indicated two characteristic peaks at 10° and 20° at (2θ). Scanning electron microscopy analysis was performed to determine the surface morphology. Heavy metal removal efficiency of prepared chitosan was determined using atomic absorption spectrophotometer. Chitosan was found to be effective in removing metal ions Cu(II), Zn(II), Fe(II) and Cr(IV) from industrial effluent. Antibacterial activity of the prepared chitosan was also determined against Xanthomonas sp. isolated from leaves affected with citrus canker.