Swati Mohapatra

In Silico Designing of an Industrially Sustainable Carbonic Anhydrase Using Molecular Dynamics Simulation

Carbonic anhydrase (CA) is a family of metalloenzymes that has the potential to sequestrate carbon dioxide (CO2) from the environment and reduce pollution. The goal of this study is to apply protein engineering to develop a modified CA enzyme that has both higher stability and activity and hence could be used for industrial purposes. In the current study, we have developed an in silico method to understand the molecular basis behind the stability of CA. We have performed comparative molecular dynamics simulation of two homologous α-CA, one of thermophilic origin (Sulfurihydrogenibium sp.) and its mesophilic counterpart (Neisseria gonorrhoeae), for 100 ns each at 300, 350, 400, and 500 K. Comparing the trajectories of two proteins using different stability-determining factors, we have designed a highly thermostable version of mesophilic α-CA by introducing three mutations (S44R, S139E, and K168R). The designed mutant α-CA maintains conformational stability at high temperatures. This study shows the potential to develop industrially stable variants of enzymes while maintaining high activity.

Structural and thermal characterization of PHAs produced by Lysinibacillus sp. through submerged fermentation process

In this investigation an attempt has been made to characterize and identify Lysinibacillus sp. 3HHX by 16S-rDNA sequencing. The bacterium exhibited occurrence of PHAs granules on an average 11±1 per cell of 1.0μm length and breadth 0.72μm, revealed from TEM studies. Under optimized condition, 4.006gm/L of PHAs was extracted using hypochlorite digestion and multi-solvent extraction process. PhaC gene of ∼540bp and higher PHA synthase activity was detected at 48h of cultivation. The extracted PHAs was structurally characterized by GC-MS and 1H NMR reported to be P(3HB-co-3HDD-co-3HTD) and amorphous in nature with 112°C melting point, -11.0°C glass transition point and 114.76°C decomposition temperature detected by DSC & TGA respectively. The C/O of biopolymer disc was 1:65 as revealed from C1s and O1s spectra of XPS, that was completely biodegradable within 30 days. This biopolymer was observed to be non-cytotoxic to NIH 3T3 mouse fibroblast cells. The report is of its kind in establishing the abilities of Lysinibacillus sp. 3HHX for non-growth associated PHA co-polymer production. Moreover the biocompatible and biodegradable nature of the biopolymer conferred to its substantial biomedical applications.

Bacillus and biopolymer: Prospects and challenges

The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.

Putative protein VC0395_0300 from Vibrio cholerae is a diguanylate cyclase with a role in biofilm formation

The hallmark of the lifecycle of Vibrio cholerae is its ability to switch between two lifestyles - the sessile, non-pathogenic form and the motile, infectious form in human hosts. One of these changes is in the formation of surface biofilms, when in sessile aquatic habitats. The cell-cell interactions within a V. cholerae biofilm are stabilized by the production of an exopolysachharide (EPS) matrix, which in turn is regulated by the ubiquitous secondary messenger, cyclic di-GMP (c-di-GMP), synthesized by proteins containing GGD(/E)EF domains in all prokaryotic systems. Here, we report the functional role of the VC0395_0300 protein (Sebox3) encoded by the chromosome I of V. cholerae, with a GGEEF signature sequence, in the formation of surface biofilms. In our study, we have shown that Escherichia coli containing the full-length Sebox3 displays enhanced biofilm forming ability with cellulose production as quantified and visualized by multiple assays, most notably using FEG-SEM. This has also been corroborated with the lack of motility of host containing Sebox3 in semi-solid media. Searching for the reasons for this biofilm formation, we have demonstrated in vitro that Sebox3 can synthesize c-di-GMP from GTP. The homology derived model of Sebox3 displayed significant conservation of the GGD(/E)EF architecture as well. Hence, we propose that the putative protein VC0395_0300 from V. cholerae is a diguanylate cyclase which has an active role in biofilm formation.