Research Publications (Applied Sciences)
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Item Molecular dynamics simulation of chitinase I from Thermomyces lanuginosus SSBP to ensure optimal activity(Taylor and Fancis Online, 2016-09-22) Khan, Faez Iqbal; Bisetty, Krishna; Gu, Ke-Ren; Singh, Suren; Permaul, Kugen; Hassan, Md. Imtaiyaz; Wei, Dong-QingThe fungal chitinase I obtained from Thermomyces lanuginosus SSBP, a thermophilic deuteromycete, has an optimum growth temperature and pH of 323.15 K and 6.5, respectively. This enzyme plays an important task in the defence mechanism of organisms against chitin-containing parasites by hydrolysing β-1, 4-linkages in chitin. It acts as both anti-fungal and biofouling agents, with some being thermostable and suitable for the industrial applications. Three-dimensional model of chitinase I enzyme was predicted and analysed using various bioinformatics tools. The structure of chitinase I exhibited a well-defined TIM barrel topology with an eight-stranded α/β domain. Structural analysis and folding studies at temperatures ranging from 300 to 375 K using 10 ns molecular dynamics simulations clearly showed the stability of the protein was evenly distributed even at higher temperatures, in accordance with the experimental results. We also carried out a number of 20 ns constant pH molecular dynamics simulations of chitinase I at a pH range 2–6 in a solvent. This work was aimed at establishing the optimum activity and stability profiles of chitinase I. We observed a strong conformational pH dependence of chitinase I and the enzyme retained their characteristic TIM barrel topology at low pH.Item Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications(Springerlink, 2015) Khan, Faez Iqbal; Bisetty, Krishna; Singh, Suren; Permaul, Kugen; Hassan, Md. ImtaiyazChitinases are ubiquitous class of extracellu-lar enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chi-tinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, envi-ronmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanug-inosus produced a large number of chitinases, of which chi-tinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher tempera-ture. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanug-inosus and its industrial application.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports thatchitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.Item Cloning, expression, and molecular dynamics simulations of a xylosidase obtained from Thermomyces lanuginosus(Taylor and Francis Online, 2015-10-19) Gramany, Vashni; Khan, Faez Iqbal; Govender, Algasan; Bisetty, Krishna; Singh, Suren; Permaul, KugenthirenThe aim of this study was to clone, express, and characterize a β-xylosidase (Tlxyn1) from the thermophilic fungus Thermomyces lanuginosus SSBP in Pichia pastoris GS115 as well as analyze optimal activity and stability using computational and experimental methods. The enzyme was constitutively expressed using the GAP promoter and secreted into the medium due to the alpha-mating factor secretion signal present on the expression vector pBGPI. The 1276 bp gene consists of an open reading frame that does not contain introns. A 12% SDS–PAGE gel revealed a major protein band at an estimated molecular mass of 50 kDa which corresponded to zymogram analysis. The three-dimensional structure of β-xylosidase was predicted, and molecular dynamics simulations at different ranges of temperature and pH were performed in order to predict optimal activity and folding energy. The results suggested a strong conformational temperature and pH dependence. The recombinant enzyme exhibited optimal activity at pH 7 and 50°C and retained 80% activity at 50°C, pH 7 for about 45 min. This is the first report of the cloning, functional expression, and simulations study of a β-xylosidase from Thermomyces species in a fungal host.Item PKR-inhibitor binds efficiently with human microtubule affinity-regulating kinase 4(Elsevier, 2015) Naz, Farha; Shahbaaz, Mohd; Khan, Shama; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md. ImtaiyazMAP/microtubule affinity-regulating kinase 4 (MARK4) plays a central role in the cellular physiology, and it is inseparably linked with many human diseases including cancer, diet induced obesity, type2 diabetes and neurodegenerative disorders. Here, we studied the interaction of PKR-inhibitor with two variants of human MARK4. One variant is named as MARK4-F1 which has 59 N-terminal residues along with kinase domain while another variant is MARK4-F2 which has kinase domain only. Molecular-docking, molecular dynamics (MD) simulation and fluorescence-binding studies were undertaken to understand the role of N-terminal 59-residues in the binding of substrate/inhibitors. Molecular docking studies revealed that the PKR-inhibitor binds in the large hydrophobic cavity of the kinase domain of MARK4 through several hydrophobic and hydrogen-bonded interactions. Furthermore, MD simulation showed a stable param-eters for the complexes of both MARK4-F1 and MARK4-F2 to PKR-inhibitor with marginal difference in their binding affinities. A significant decrease in the fluorescence intensity of MARK4 was observed on successive addition of the PKR-inhibitor. Using fluorescence data we have calculated the binding-affinity and the number of binding site of PKR-inhibitor to the MARK4. A significantly high binding affinity was observed for the PKR-inhibitor to the MARK4 variants. However, there is no any significant difference in the binding affinity of PKR-inhibitor to the MARK4 variants was observed, indicating that 59 N-terminal residues of MARK4-F1 are not playing a crucial role in the ligand binding. The present study will pro-vide an insights into designing of new PKR-inhibitor derivative as potent and selective therapeutic agent against many life threatening diseases which are associated with MARK4.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP : Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015-04-08) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports that chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.Item In silico approaches for the identification of virulence candidates amongst hypothetical proteins of Mycoplasma pneumoniae 309(Elsevier, 2015) Shahbaaz, Mohd; Bisetty, Krishna; Ahmad, Faizan; Hassan, Md. ImtaiyazMycoplasma pneumoniae type 2a strain 309 is a simplest known bacterium and is the primary cause of community acquired pneumonia in the children. It mainly causes severe atypical pneumonia as well as several other non-pulmonary manifestations such as neurological, hepatic, hemolytic anemia, cardiacdiseases and polyarthritis. The size of M. pneumoniae genome (Accession number: NC_016807.1) is relatively smaller as compared to other bacteria and contains 707 functional proteins, in which 204 are classified as hypothetical proteins (HPs) because of the unavailability of experimentally validated functions. The functions of the HPs were predicted by integrating a variety of protein classification systems, motif discovery tools as well as methods that are based on characteristic features obtained from the protein sequence and metabolic pathways. The probable functions of 83HPs were predicted successfully. The accuracy of the diverse tools used in the adopted pipeline was evaluated on the basis of statistical techniques of Receiver Operating Characteristic (ROC), which indicated the reliability of the functional predictions. Furthermore, the virulent HPs present in the set of 83 functionally annotated proteins were predicted by using the Bioinformatics tools and the conformational behaviours of the proteins with highest virulence scores were studied by using the molecular dynamics (MD) simulations. This study will facilitate in the better understanding of various drug resistance and pathogenesis mechanisms present in the M. pneumoniae and can be utilized in designing of better therapeutic agents.Item Fabrication of copper nanoparticles decorated multiwalled carbon nanotubes as a high performance electrochemical sensor for the detection of neotame(Elsevier, 2015) Bathinapatla, Ayyappa; Kanchi, Suvardhan; Singh, Parvesh; Sabela, Myalowenkosi Innocent; Bisetty, KrishnaA highly sensitive and novel electrochemical sensor for the detection of neotame using differential pulse voltammetry with a modified glassy carbon electrode is presented. The method was further customized by the fabrication of the electrode surface with copper nanoparticles–ammonium piperidine dithiocar-bamate–mutiwalled carbon nanotubes assimilated with β-cyclodextrin. The multiwalled carbon nano-tubes assimilated with β-cyclodextrin/glassy carbon electrode exhibited catalytic activity towards the oxidation of neotame at a potential of 1.3 V at pH 3.0. The transmission electron microscopy, thermogravimetric analysis, frontier transform infrared spectroscopy and cyclic voltammetry were employed to characterize the electrochemical sensor. The sensitivity and detection limits of the electrode increased two-fold in contrast to the β-CD-MWCNTs/GCE sensor. The developed method was successfully applied for the determination of neotame in food samples, with results similar to those achieved by our modified capillary electrophoresis method with a 96% confidence level.