Microbiology

ABSTRACT
Objective: Mutagenic/genotoxic impurities in the clinically approved drugs have been a major concern for the pharmaceutical industry. Nilotinib (N), which is an approved drug of chronic leukemia, has a number of impurities (nilotinib impurity [NI]-3, NI-5, and NI-12). For drugs, either semi empirical or quantum mechanical (QM) or topological molecular descriptors (MDs) have been popular for QSAR studies. However, details of MDs for
impurities are yet to be established. Thus, the objective of the study has been to compute QM-based MDs for impurities of N and to compare them with that of approved drugs to identify MDs of the former in relation to their known genotoxic/mutagenic properties.

Methods: Impurities are optimized by B3LYP/6-311G (d,p) level of theory and ionization potential (IP), electron affinity (EA), and other MDs are
determined. Further, non-linear optical (NLO) descriptors such as dipole moment (DM) and polarizability (α) are also determined.

Results: Impurities of N show much deviation of IP, EA, MD, α, and other properties from the reported mean values of approved drugs. Unlike NI-5 and NI-12, NI-3 shows increase in DM (~double) and α properties, which may point to its higher interactivity with cellular targets (like DNA/ RNA/protein), might be due to additional substituents, π-conjugation, and planarity in its structure. The latter seems to be due to compensation of oppositely sensed dihedral properties of the structure of NI-3.

Conclusion: The study identifies QM-based differential MDs for impurities of N, which seems to have a relationship with their genotoxicity/ mutagenicity properties. Similar studies can be done for other such systems.

Background: Kaempferol (K), Quercetin (Q), Myricetin (M) are flavonols, a class of flavonoid, that are accumulated in different parts of plants. While plants produce these secondary metabolites for their selfprotection, human make use of these molecules for medicinal, therapeutic, commercial and food purposes. While studies reveal that these molecules, being in the root-exudates of legume plants, play crucial roles for the i] establishment of cross-nodulating groups in symbiosis and ii] maintenance of aseptic ecochemistry of rhizosphere, understanding of differential molecular properties remain to be worked out. Present study involves the extraction and comparison of electronic and other properties among K, Q and M.

Materials and Methods: We use B3LYP/6-311G (d, p) level of theory of GAUSSIAN 09, to extract differential molecular properties such as EHOMO, ELUMO, band-gap, electrophilicity (γ), chemical potential
(c), chemical hardness (η) , kinetic stability etc, thermodynamic stability and dipole polarizability of flavonols (i.e. K, Q and M).

Results: The ground state optimized structures are used to extract electronic, thermodynamic, and polarizability properties. Although, bond length, bond angle are almost similar, dihedral angle between AC and B rings of Q differs from that of K and M. ELUMO, ω, I, η, IP and band gap follow the order as K>M>Q, which is reversed for σ and EHOMO. It, therefore, seems the orientation of rings, OH-substituent in them and
their intra molecular stabilization act as determinant for these properties. Thermodynamic stability of flavonols follow M>Q>K, which is due to additional OH substitutions in rings of these molecules. Polarizability (α), anisotropy of polarizability (Δα), β, γ|| and γ_|_ show the similar trends in that Q has the highest with K and M have comparable but lower values.

Conclusion: The study highlights differential electronic, thermodynamic and non-linear optical (NLO) properties of flavonols, which seems to have relation with their chemical reactivity against cellular targets in relation to biological nitrogen fixation.

Global minimal structure of protein/enzyme is energetically compromised that maintains an intricate balance between the rigidity and the flexibility. Such a state makes it interactive to its ligand molecules. Although protein data bank files (PDB) may have achieved the state, in many situations minimization has been crucial to overcome unwanted steric clashes, and other conformational strains. It is more so, when orthologous PDB structures that are intended in a given study, show variations in resolution, R-factor, shell-water contents, loop characteristics etc. Here, a fully automated procedure of minimization would be highly useful. AUTOMINv1.0 is such an automation of minimization that runs on any number of structure files with any number of chains in them along with the inclusion of selective/non-selective shell-waters interacting with polar and or non-polar atom-types of protein. Comparison of the mean binaryitems of salt-bridges of minimized and un-minimized structures (chains > 100) of nucleoside diphosphate kinase from mimi virus shows dramatic improvements in the earlier. Again, the mean steric clashes of 2AZ3.pdb are reduced upon minimization. Remarkably, the observed steric clashes between shell-waters and atom-types of protein are seen to be removed upon minimization. Taken together, AUTOMINv1.0 is an automation of minimization that finds applications in structural bioinformatics.

We analyzed the water-ferredoxin interaction in mesophilic (moderate temperature) algae (PDB ID: 1AWD) and halophilic (salt-tolerant) archaea (PDB ID: 1DOI) using POWAIND version 2.0 (a protein-water interactions calculation program). It is found that the shell water (SW) is 2.5 fold greater in halophilic ferredoxin than mesophilic ferredoxin. Water-ferredoxin interactions in the core and cavity are the signature of stability. The normalized frequency of such interactions is less in halophilic relative to mesophilic ferredoxin and the halophilic signature for stability by such interactions is negligible. However, the surface dominated with such interactions seems to be important for ferredoxin and oxido-reductase recognition.

Keywords: Biochemistry Bioinformatics Biotechnology Microbiology Molecular biology Proteins Tannase Pathogenic and non-pathogenic bacteria Salt bridge Microenvironment Order and disorder forming residues A B S T R A C T Tannase is an inducible industrially important enzyme, produced by several microorganisms. A large number of bacteria have reported as tannase producers; however, some of them are pathogenic in nature. Therefore, it is quite uncertain whether the application of these tannase enzymes from such pathogenic bacteria is suitable for industries and human welfare. Till date, there is no clear evidence regarding which group of bacteria (non-pathogenic or pathogenic) is better suited for their application in the edge of industries with particular reference to the food industry. The present study is following the findings of the above queries. In this study, a large number of tannase protein sequences have been retrieved from the databases, including both non-pathogenic and path-ogenic bacterial species. Physiochemical and evolutionary properties of those sequences have been evaluated. Results have shown that non-pathogenic bacterial tannase possesses a high number of acidic and basic amino acid residues as compared to their pathogenic counterparts. The acidic and basic amino acid residues of tannase provide unique microenvironment to it. In the other hand, the numbers of disorder forming residues are higher in tannase sequences of pathogenic bacteria. The study of tannase microenvironment leads in the formation of salt bridges, which finally favoring the stability and proper functioning of tannase. This is the first report of such observation on tannase enzyme using in silico approach. Study of the microenvironment concept will be helpful in protein engineering.

Purpose Chicken feather protein hydrolysate (CFPH) has drawn a significant attention as a component/type of biofertilizer in recent years, because of the beneficial impact on the growth of the plant. The current study aims to evaluate the potential influence of the combination of CFPH with vermicompost (VC) on growth-promotion and yield improvement in tomato plants. Method Feather degrading bacteria were isolated and characterized using 16s-rDNA sequencing, and assessed for biochemical reactions, growth-promoting attributes and keratinase activity. The medium used for feather degradation studies consisted of 0.75% (w/v) of raw feather, with 1% (v/v) of inoculum at 37°C, pH 7.5 and at 120 rpm. A field study was done by randomized block design (RBD) with five treatments in tomato. Results Keratinolytic and feather degrading bacteria isolated and used in this study were identified as Bacillus cereus PKID1 with accession number MT158702. The bacterium gave the highest keratinase activity of 80±0.28 U/ml. The CFPH showed the potential to promote remarkably the germination % of tomato (84.13), rice (87.24), onion (84.13), chilli (84.13), chickpea (73.24) seeds; field experiment significantly increased plant growth and yield compared with control. Conclusion The principal component analysis of the field experiment as a result of tomato plant-growth, the order of best treatment efficacy for improvement of parameter estimates was as follows: CFPH and VC > CFPH > VC > recommended dose of fertilizers (RDF) > control. Thus, the application of CFPH with VC could improve the productivity of crops and decrease the use of chemical fertilizers.

Halophilic proteins have greater abundance of acidic over basic residues in sequence. In structure, the surface is decorated by negative charges, with lower content of Lysine. Using sequence BLOCKs and 3D model of malate dehydrogenase from halophilic archaea (Halobacterium salinarum; hsMDH) and X-ray structure from mesophilic bacteria (E. coli; ecMDH), we show that not only acidic and basic residues have higher mean relative abundance (MRA) and thus, impart higher polarity to the sequences, but also show their presence in the surface of the structure of hsMDH relative to its mesophilic counterpart. These observations may indicate that both the acidic and the basic residues have a concerted role in the stability of hsMDH. Analysis on salt bridges from hsMDH and ecMDH show that in the former, salt bridges are highly intricate, newly engineered and global in nature. Although, these salt bridges are abundant in hsMDH, in the active site the design remains unperturbed. In high salt where hydrophobic force is weak, these salt bridges seem to play a major role in the haloadaptation of the tertiary structure of hsMDH. This is the first report of such an observation.

Protein is the most exposed biomolecule in the aqueous environment of the cell. Its structure maintains a delicate balance between the rigidity and the flexibility that imparts binding specificity to its substrate/ligand, etc. Intramolecular interactions of polar and non-polar groups of amino acid residues and intermolecular weak interactions between these groups and shell-waters may contribute to the overall stability of the tertiary structure. However, the question as to what are the dynamics of interactions of shell-water with respect to weak forces and atom-groups of protein (AGP), requires systematic investigations. In this end, we have developed a procedure POWAINDv1.0 that analyzes interactions of crystallographic shell-waters (CSH) in residues and AGP specific manner. The shell-water and AGP specific bridge-interactions are also extracted. Further, the program analyzes favorable and unfavorable nature of each interaction based on the actual and 75% of the sum of van der Waals (vdW) radii of interacting atoms. The EXCEL-outputs are useful in understanding the profile for AGP-CSH interactions and contribution of each component in AGP. Taken together, the program provides intricate details on CSHprotein interactions and finds application in the structural Bioinformatics.

Psychrophilic microorganisms have with success inhabited all extremely cold environments from the deep ocean to freezing mountains. They have maintained their physiological activity from-20°C to +10°C temperatures. But generally, protein goes cold denaturation in these temperatures. So, how can they manage to survive in these adverse conditions? It has been reported that some extremophilic organisms have engineered their protein to get stability in extreme conditions [1-2]. Are the same things applied in the case of psychrophiles? This study will help to get those answers. Pseudoalteromonas haloplanktis is an Antarctic marine gram-negative rod-shaped bacterium that operates its biological activity in a very adverse environment [3]. This Antarctic bacterium has a rapid growth rate (doubling time of 4 h at 4°C) [4]. Alpha-amylase helps in the hydrolysis of 1,4-alpha glucosidic linkages in polysaccharides of psychrophiles [5]. In this present study, psychrophilic alpha-amylase was compared with mesophilic alpha-amylase to check their structural properties by which they have stabilized their proteins in that extreme environment. Amino acids i.e., the building blocks of protein, are generally four types-hydrophobic/unpolar, charged-polar, uncharged-polar, and aromatic-polar [6-7]. Non-covalent interactions occur based on the physical characters of amino acids. Many types of non-covalent interactions present are responsible for protein stabilization [8]. Intra-protein interactions like aromatic-aromatic interactions, aromatic-sulfur interactions, cation-pi interactions, and salt bridges have been reported in extremophilic protein [9] and viral protein stabilization [10]. It has been reported that salt bridges played a crucial role in extremophilic protein stabilization [11-14].

Non-specific electrostatics is crucial for structure and stability; recently, it has been argued that psychrophilic proteins may also utilize specific electrostatic interactions. Do psychrophilic proteins increase the number of salt bridges for cold adaptation? Are there any changes that occur in their sequence, which helps them to adapt in an extreme environment? Do intra-protein interactions affect their stability? Is there any special type of intra-protein interaction present in psychrophilic protein structure? This study will give all those answers. Sequences (n ~ 100) and structures of psychrophilic isocitrate dehydrogenase and mesophilic isocitrate dehydrogenase extracted from databases. Sequences had been analyzed in BLOCK and non-BLOCK format. The sequences of psychrophiles and mesophiles create two separate clades. The number of charged and uncharged polar residues is very much high in psychrophilic proteins. The formation of long network aromatic-aromatic interactions and network aromatic-sulfur interactions are very crucial for psychrophilic protein stability. Identification of these types of interactions is also a novelty of this study. Favorable mutation of charged residues with high-energy contributions affects the protein stability. This study will help in protein engineering.

Hyper thermophilic archaea not only tolerate high temperature but also operate its biochemical machineries, normally under these conditions. However, the structural signatures in proteins that answer for the hyper thermo-stability relative to its mesophilic homologue remains poorly understood. We present comparative analyses of sequences, structures and salt-bridges of prolyl-oligopeptidase from Pyrococcus furiosus (pfPOP-PDB ID: 5T88) and human (huPOP-PDB ID: 3DDU). A similar level of hydrophobic and hydrophilic residues in pfPOP and huPOP is observed. A low level of interactions between shell-waters and atom-types in pfPOP indicated hyper thermophilic features are negligible. Salt-bridge-forming-residues (sbfrs) are high in pfPOP's core and surface (pfPOP). Increased sbfrs largely indicate specific-electrostatic is important for thermo stability in pfPOP. Four classes of sbfrs are found namely positionally non-conservative (PNCS), conservative (PCS), unchanged (PU) and interchanged (PIC) type of substitutions. PNCS-sbfrs constitutes 28% and it is associated with the topology of pfPOP at high temperature. PCS helps to increase the salt-bridge population. It is also found that PU maintains similar salt-bridges at the active site and other binding sites while PIC abolishes mesophilic patterns.

Thermophilic proteins function at high temperature, unlike mesophilic proteins. Thermo-stability of these proteins is due to the unique buried and networked salt-bridge (BNSB). However, it is known that the desolvation cost of BNSB is too high compared to other favorable energy terms. Nonetheless, it is known that stability is provided generally by hydrophobic isosteres without the need for BNSB. We show in this analysis that the BNSB is the optimal evolutionary design of salt-bridge to offset desolvation cost efficiently. Hence, thermophilic proteins with a high level of BNSB provide thermo-stability.

Tannase is an inducible industrially important enzyme, produced by several microorganisms. A large number of bacteria have reported as tannase producers; however, some of them are pathogenic in nature. Therefore, it is quite uncertain whether the application of these tannase enzymes from such pathogenic bacteria is suitable for industries and human welfare. Till date, there is no clear evidence regarding which group of bacteria (nonpathogenic or pathogenic) is better suited for their application in the edge of industries with particular reference to the food industry. The present study is following the findings of the above queries. In this study, a large number of tannase protein sequences have been retrieved from the databases, including both non-pathogenic and pathogenic bacterial species. Physiochemical and evolutionary properties of those sequences have been evaluated. Results have shown that non-pathogenic bacterial tannase possesses a high number of acidic and basic amino acid residues as compared to their pathogenic counterparts. The acidic and basic amino acid residues of tannase provide unique microenvironment to it. In the other hand, the numbers of disorder forming residues are higher in tannase sequences of pathogenic bacteria. The study of tannase microenvironment leads in the formation of salt bridges, which finally favoring the stability and proper functioning of tannase. This is the first report of such observation on tannase enzyme using in silico approach. Study of the microenvironment concept will be helpful in protein engineering.

Residues in allelic positions, in the local segment of aligned sequences of proteins show wide variations. Here, we describe PROPAB that computes the propensity tables for helix, strand and coil types from multiple 3D structure files following ab initio statistical procedure. It also classifies them in range specific and chain specific manners. It further computes percentage composition and physicochemical properties along with residues propensities. It also prepares FASTA files for different segments (helix, strand and coil) in the exact order that they follow in the sequence. Representative analyses on orthologous (homologous across species) proteins demonstrate wide segmental variations of physicochemical properties. Such variations provide insights to relate the adaptation of these proteins in a given functional constraint under diverse environmental conditions. Thus, the program finds applications in the structural and evolutionary analysis of proteins. PROPAB is freely availab...

We analyzed the water-ferredoxin interaction in mesophilic (moderate temperature) algae (PDB ID: 1AWD) and halophilic (salt-tolerant) archaea (PDB ID: 1DOI) using POWAIND version 2.0 (a protein-water interactions calculation program). It is found that the shell water (SW) is 2.5 fold greater in halophilic ferredoxin than mesophilic ferredoxin. Water-ferredoxin interactions in the core and cavity are the signature of stability. The normalized frequency of such interactions is less in halophilic relative to mesophilic ferredoxin and the halophilic signature for stability by such interactions is negligible. However, the surface dominated with such interactions seems to be important for ferredoxin and oxido-reductase recognition.

Global minimal structure of protein/enzyme is energetically compromised that maintains an intricate balance between the rigidity and the flexibility. Such a state makes it interactive to its ligand molecules. Although protein data bank files (PDB) may have achieved the state, in many situations minimization has been crucial to overcome unwanted steric clashes, and other conformational strains. It is more so, when orthologous PDB structures that are intended in a given study, show variations in resolution, R-factor, shell-water contents, loop characteristics etc. Here, a fully automated procedure of minimization would be highly useful. AUTOMINv1.0 is such an automation of minimization that runs on any number of structure files with any number of chains in them along with the inclusion of selective/non-selective shell-waters interacting with polar and or non-polar atom-types of protein. Comparison of the mean binaryitems of salt-bridges of minimized and un-minimized structures (chains > 100) of nucleoside diphosphate kinase from mimi virus shows dramatic improvements in the earlier. Again, the mean steric clashes of 2AZ3.pdb are reduced upon minimization. Remarkably, the observed steric clashes between shell-waters and atom-types of protein are seen to be removed upon minimization. Taken together, AUTOMINv1.0 is an automation of minimization that finds applications in structural bioinformatics.

The current nightmare for the whole world is COVID-19. The occurrence of concentrated pneumonia cases in Wuhan city, Hubei province of China was first reported on December 30, 2019. SARS-CoV first discloses in 2002, but not outspread worldwide. After 18 years, in 2020, it reemerges and outspread worldwide as SARS-CoV-2 (COVID 19), as the most treacherous virus creating disease in the world. Is it possible to create a favorable evolution within this (18 years) short time? If possible, then what are those properties or factors that are changed in SARS-CoV-2 to make it undefeated? What are the fundamental differences between SARS-CoV-2 and SARS? This study will find all those queries. Here, we took 4 types of protein sequences from SARS-CoV-2 and SARS are retrieved from the database to check their physicochemical and structural properties. Results showed that charged residues are playing a pivotal role in SARS-CoV-2 evolution. Those charged residues also contribute to helix stabilizati...

Introduction- Now a day’s colorectal cancer (CRC) is one of the most deadly cancers in the world. The objective of this investigation was to evaluate the protective effect of gallic acid and pyrogallol in colorectal cancer. Previous reports suggest that there is an association present between some tannase producing bacteria and colorectal cancer. Tannase hydrolyze tannic acid into gallic acid and pyrogallol. Are those compounds have any therapeutic effect on colorectal cancer? This study will help to find those quarries. Methods-The remedial effect of gallic acid and pyrogallol was studied by descriptor properties and molecular docking methods. 100 CRC causing protein structures were docked in this investigation. Results- Lipinski Rule of Five and other descriptor properties of those compounds have showed their nontoxic and therapeutic nature. Molecular docking studies have showed highest score -38.22 KJ/Mol with gallic acid and -33.6 KJ/Mol with pyrogallol. Conclusion- This is the ...

Purpose Chicken feather protein hydrolysate (CFPH) has drawn a significant attention as a component/type of biofertilizer in recent years, because of the beneficial impact on the growth of the plant. The current study aims to evaluate the potential influence of the combination of CFPH with vermicompost (VC) on growth-promotion and yield improvement in tomato plants. Method Feather degrading bacteria were isolated and characterized using 16s-rDNA sequencing, and assessed for biochemical reactions, growth-promoting attributes and keratinase activity. The medium used for feather degradation studies consisted of 0.75% (w/v) of raw feather, with 1% (v/v) of inoculum at 37°C, pH 7.5 and at 120 rpm. A field study was done by randomized block design (RBD) with five treatments in tomato. Results Keratinolytic and feather degrading bacteria isolated and used in this study were identified as Bacillus cereus PKID1 with accession number MT158702. The bacterium gave the highest keratinase activit...