Bacillus velezensis Identification and Recombinant Expression, Purification and Characterization of Its Alpha-Amylase

Amylases account for about 30% of the global market of industrial enzymes, and the current amylases cannot fully meet industrial needs. This study aimed to identify a high α-amylase producing bacterium WangLB, to clone its α-amylase coding gene, and to characterize the α-amylase. Results showed that WangLB belonged to Bacillus velezensis whose α-amylase gene was 1980 bp coding 659 amino acids designated as BvAmylase. BvAmylase was a hydrophilic stable protein with a signal peptide and a theoretical pI of 5.49. The relative molecular weight of BvAmylase was 72.35 kDa, and was verified by SDS-PAGE. Its modeled structure displayed that it was a monomer composed of three domains. Its optimum temperature and pH were 70 °C and pH 6.0, respectively. It also showed high activity in a wide range of temperatures (40–75 °C) and a relatively narrow pH (5.0–7.0). It was a Ca2+-independent enzyme, whose α-amylase activity was increased by Co2+, Tween 20, and Triton X-100, and severely decreased by SDS. The Km and the Vmax of BvAmylase were 3.43 ± 0.53 and 434.19 ± 28.57 U/mg. In conclusion, the α-amylase producing bacterium WangLB was identified, and one of its α-amylases was characterized, which will be a candidate enzyme for industrial applications.

Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis

The cell envelope proteinase (CEP) of Lactococcus lactis is a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains of L. lactis are typically auxotrophic for several amino acids and in order to grow to high cell densities in milk they need an extracellular protease. The structure of the entire CEP enzyme is difficult to determine experimentally due to the large size and due to the attachment to the cell surface. We here describe the use of a combination of structure prediction tools to create a structural model for the entire CEP enzyme of Lactococcus lactis. The model has implications for how the bacterium interacts with casein micelles during growth in milk, and it has implications regarding the energetics of the proteolytic system. Our model for the CEP indicates that the catalytic triad is activated through a structural change caused by interaction with the substrate. The CEP of L. lactis might become a useful model for the mode of action for enzymes belonging to the large class of S8 proteinases with a PA (protease associated) domain and a downstream fibronectin like domain.

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.

Molecular Modeling of Chemosensory Protein 3 from Spodoptera litura and Its Binding Property with Plant Defensive Metabolites

Chemosensory perception in insects involves a broad set of chemosensory proteins (CSPs) that identify the bouquet of chemical compounds present in the external environment and regulate specific behaviors. The current study is focused on the Spodoptera litura (Fabricius) chemosensory-related protein, SlitCSP3, a midgut-expressed CSP, which demonstrates differential gene expression upon different diet intake. There is an intriguing possibility that SlitCSP3 can perceive food-derived chemical signals and modulate insect feeding behavior. We predicted the three-dimensional structure of SlitCSP3 and subsequently performed an accelerated molecular dynamics (aMD) simulation of the best-modeled structure. SlitCSP3 structure has six α-helices arranged as a prism and a hydrophobic binding pocket predominated by leucine and isoleucine. We analyzed the interaction of selected host plant metabolites with the modeled structure of SlitCSP3. Out of two predicted binding pockets in SlitCSP3, the plant-derived defensive metabolites 2-b-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA), 6-Methoxy-2–benzoxazolinone (MBOA), and nicotine were found to interact preferably to the hydrophobic site 1, compared to site 2. The current study provides the potential role of CSPs in recognizing food-derived chemical signals, host-plant specialization, and adaptation to the varied ecosystem. Our work opens new perspectives in designing novel pest-management strategies. It can be further used in the development of CSP-based advanced biosensors.

Computational Guided Drug Repurposing for Targeting 2'-O-Ribose Methyltransferase of SARS-CoV-2

<p>The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards global health emergency. Currently no proper medicine or effective treatment strategies are available, therefore repurposing may play an important role in overcoming the situation. The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2’OMTase) which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2’OMTase of SARS-CoV-2 was analysed and its structure was modeled by comparative modeling approach and validated. The modeled structure displayed the conserved characteristic fold of class I MTase family. The library of 3000 drugs was screened against the active site of 2’OMTase. The docking analysis displayed that the active site of 2’OMTase accommodates an array of drugs which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids and other drugs. The results suggested that these drugs may be used potential inhibitors for 2’OMTase for combating the SARS-CoV-2 infection.</p>

Computational Guided Drug Repurposing for Targeting 2'-O-Ribose Methyltransferase of SARS-CoV-2

<p>The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards global health emergency. Currently no proper medicine or effective treatment strategies are available, therefore repurposing may play an important role in overcoming the situation. The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2’OMTase) which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2’OMTase of SARS-CoV-2 was analysed and its structure was modeled by comparative modeling approach and validated. The modeled structure displayed the conserved characteristic fold of class I MTase family. The library of 3000 drugs was screened against the active site of 2’OMTase. The docking analysis displayed that the active site of 2’OMTase accommodates an array of drugs which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids and other drugs. The results suggested that these drugs may be used potential inhibitors for 2’OMTase for combating the SARS-CoV-2 infection.</p>

Behavior of Full and Partially Infilled Reinforced Concrete Frame Subjected to Horizontal Loading

In this research work, six storey, three bay prototype structure were designed for strong earth quake load and bottom storey, interior bay was considered. A 1/3rd scaled three reinforced concrete (RC) frames of bare, partially infilled and full infilled RC frames was selected to check the behavior of full and partially infilled RC frames subjected to horizontal loading. The effect of lateral (horizontal) loads on any reinforced concrete frame that might arise as a result of seismic loads was investigated. The RC frames were scaled down to a modeled structure that was tested in a loading frame of capacity 20 tons. Bare frame with just columns and beams, masonry infill with openings and masonry infill without openings RC frames were the 3 different specimens that were made for testing after 28 days of curing. Lateral loading was applied by loading frame. The results showed that the full infill frame took maximum load as expected with least deflections, the bare frame took the least load and the infill frame with opening resisted little more load than bare frame but less than the infill frame without opening. However, at any given point of loading the deflection was highest for the bare frame and least for the full infill frame, which suggested that frames with infill performed better as compared to the bare frame.

Ceres’ partial differentiation: undifferentiated crust mixing with a water-rich mantle

Aims. We model thermal evolution and water-rock differentiation of small ice-rock objects that accreted at different heliocentric distances, while also considering migration into the asteroid belt for Ceres. We investigate how water-rock separation and various cooling processes influence Ceres’ structure and its thermal conditions at present. We also draw conclusions about the presence of liquids and the possibility of cryovolcanism. Methods. We calculated energy balance in bodies heated by radioactive decay and compaction-driven water-rock separation in a three-component dust-water/ice-empty pores mixture, while also taking into consideration second-order processes, such as accretional heating, hydrothermal circulation, and ocean or ice convection. Calculations were performed for varying accretion duration, final size, surface temperature, and dust/ice ratio to survey the range of possible internal states for precursors of Ceres. Subsequently, the evolution of Ceres was considered in five sets of simulated models, covering different accretion and evolution orbits and dust/ice ratios. Results. We find that Ceres’ precursors in the inner solar system could have been both wet and dry, while in the Kuiper belt, they retain the bulk of their water content. For plausible accretion scenarios, a thick primordial crust may be retained over several Gyr, following a slow differentiation within a few hundreds of Myr, assuming an absence of destabilizing impacts. The resulting thermal conditions at present allow for various salt solutions at depths of ≲10 km. The warmest present subsurface is obtained for an accretion in the Kuiper belt and migration to the present orbit. Conclusions. Our results indicate that Ceres’ material could have been aqueously altered on small precursors. The modeled structure of Ceres suggests that a liquid layer could still be present between the crust and the core, which is consistent with Dawn observations and, thus, suggests accretion in the Kuiper belt. While the crust stability calculations indicate crust retention, the convection analysis and interior evolution imply that the crust could still be evolving.

Homology Modeling and Structural Dynamics of the Glucose Oxidase

Glucose oxidase from Aspergillus niger IPBCC.08.610 (GOD_IPBCC) is a locally sourced flavoenzyme from Indonesia that can potentially be developed in a variety of industrial processes. Although this enzyme has a high activity in catalyzing the redox reactions, the use of this enzyme was still limited to be applied as glucose biosensor. Using information from the amino acid sequences, a computational structure of GOD_IPBCC was therefore designed by homology modeling method using two homologous structures of GOD from protein data bank (1CF3 and 5NIT) as the templates. The quality of the resulting structures was evaluated geometrically for selection of the best model, and subsequently, 50 ns of MD simulations were carried out for the selected model as well as the corresponding template. Results obtained from the validation analysis showed that the 1CF3 template-built structure was selected as the best reliable model. The structural comparison exhibited that the best-modeled structure consisted of two functional domains and three catalytic residues similarly to the corresponding experimental structure. The overall dynamic behavior of the 50 ns of the structure was structurally stable and comparable with that of the positive control both from globally and locally observations. Implications of these stable nature within the best-modeled structure unfold the possibilities in search of notable residues and their roles to enhance enzyme thermostability.