Statistical optimization and sequential scale-up of α-galactosidase production by Actinoplanes utahensis B1 from shake flask to pilot scale.

α-Galactosidase (α-GAL) is a class of hydrolase that releases galactose from galacto-oligosaccharides and synthetic substrates such as pNPG. In this study, the production of α-GAL by Actinoplanes utahensis B1 in submerged fermentation was enhanced by using statistical methods. The effects of temperature, pH, and inoculum percentage on enzyme secretion were optimized using BBD of RSM. The optimized process was scaled up from the shake flask to the laboratory scale (5 L) and to pilot scale (30 L) using KLa based scale-up strategy. By using BBD, a maximum yield of 62.5 U/mL was obtained at a temperature of 28 °C, a pH of 6.9, and an inoculum of 6.4%. Scale-up was performed successfully and achieved a yield of 74.4 U/mL and 76.8 U/mL in laboratory scale and pilot scale fermenters. The TOST was performed to validate the scale-up strategy and the results showed a confidence level of 95% for both scales indicating the perfect execution of scale-up procedure. Through the implementation of BBD and scale-up strategy, the overall enzyme yield has been significantly increased to 76%. This is the first article to explore the scale-up of α-GAL from the A. utahensis B1 strain and provide valuable insights for industrial applications.

Antioxidant potential and optimization of production of extracellular polysaccharide by Acinetobacter indicus M6.

BACKGROUND: Extracellular polysaccharides (ECPs) produced by biofilm-producing marine bacterium have great applications in biotechnology, pharmaceutical, food engineering, bioremediation, and bio-hydrometallurgy industries. The ECP-producing strain was identified as Acinetobacter indicus M6 species by 16S rDNA analysis. The polymer produced by the isolate was quantified and purified and chemically analyzed, and antioxidant activities have been studied. The face-centered central composite design (FCCCD) was used to design the model. RESULTS: The results have clearly shown that the ECP was found to be endowed with significant antioxidative activities. The ECP showed 59% of hydroxyl radical scavenging activity at a concentration of 500 µg/mL, superoxide radical scavenging activity (72.4%) at a concentration of 300 µg/mL, and DPPH˙ radical scavenging activity (72.2%) at a concentration of 500 µg/mL, respectively. Further, HPLC and GC-MS results showed that the isolated ECP was a heteropolymer composed of glucose as a major monomer, and mannose and glucosamine were minor monomers. Furthermore, the production of ECP by Acinetobacter indicus M6 was increased through optimization of nutritional variables, namely, glucose, yeast extract, and MgSO4 by "Response Surface Methodology". Moreover the production of ECP reached to 2.21 g/L after the optimization of nutritional variables. The designed model is statistically significant and is indicated by the R2 value of 0.99. The optimized medium improved the production of ECP and is two folds higher in comparison with the basal medium. CONCLUSIONS: Acinetobacter indicus M6 bacterium produces a novel and unique extracellular heteropolysaccharide with highly efficient antioxidant activity. GC-MS analyses elucidated the presence of quite uncommon (1→4)-linked glucose, (1→4)-linked mannose, and (→4)-GlcN-(1→) glycosidic linkages in the backbone. The optimized medium improved the production of ECP and is two folds higher in comparison with the basal medium. The newly optimized medium could be used as a promising alternative for the overproduction of ECP.

Screening of phytochemical compounds of Tinospora cordifolia for their inhibitory activity on SARS-CoV-2: an in silico study.

In the present study, we explored phytochemical constituents of Tinospora cordifolia in terms of its binding affinity targeting the active site pocket of the main protease (3CL pro) of SARS-CoV-2 using molecular docking study and assessed the stability of top docking complex of tinosponone and 3CL pro using molecular dynamics simulations with GROMACS 2020.2 version. Out of 11 curated screened compounds, we found the significant docking score for tinosponone, xanosporic acid, cardiofolioside B, tembetarine and berberine in Tinospora cordifolia. Based on the findings of the docking study, it was confirmed that tinosponone is the potent inhibitor of main protease of SARS-CoV-2 with the best binding affinity of -7.7 kcal/mol. Further, ADME along with toxicity analysis was studied to predict the pharmacokinetics and drug-likeness properties of five top hits compounds. The molecular dynamics simulation analysis confirmed the stability of tinosponone and 3CL pro complex with a random mean square deviation (RMSD) value of 0.1 nm. The computer-aided drug design approach proved that the compound tinosponone from T. cordifolia is a potent inhibitor of 3CL main protease of SARS-CoV-2. Further, the in vitro and in vivo-based testing will be required to confirm its inhibitory effect on SARS-CoV-2.Communicated by Ramaswamy H. Sarma.