Muhammad Javed

Invertase from Hyper Producer Strain of Aspergillus niger: Physiochemical Properties, Thermodynamics and Active Site Residues Heat of Ionization

Here we report for the first time heat of ionization of invertase (E.C.3.2.1.26) active site residues from hyper-producer strain of Aspergillus niger (34.1 U ml(-1)), along with its physiochemical properties, kinetics and thermodynamics of stability-function. The Invertase showed great potential for industry as being highly efficient (k(cat) = 24167 s(-1) at 65 degrees C, pH 5.0) and stable (half life= 12 h at 56 degrees C).

Improvement of Aspergillus oryzae for hyperproduction of endoglucanase: expression cloning of cmc-1 gene of Aspergillus aculeatus

FI-Carboxymethylcellulase (cmc1; family 12) is one of the endoglucanases of Aspergillus aculeatus and consists of single polypeptide chain of 221 amino acids. The cmc1 gene was expressed in Aspergillus oryzae niaD300 (niaD−) under promoter 8142. The plasmid pCMG14 carrying the cmc1 gene at PstI site was used as a source of the gene (920 bp) and Aspergillus oryzae was successfully transformed by the plasmid pNAN-cmc1 (harboring cmc1 gene). The plasmid was integrated in Aspergillus oryzae niaD300 genome at niaD locus and the transformed fungus constitutively produced very high amounts of endoglucanases when grown on glucose, maltose, soluble starch and wheat bran.

In-Silico Determination of Insecticidal Potential of Vip3Aa-Cry1Ac Fusion Protein Against Lepidopteran Targets Using Molecular Docking.

Study and research of Bt (Bacillus thuringiensis) transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac) insecticidal protein and vegetative insecticidal protein (Vip3Aa) have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN) and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, and Spodoptera litura) revealed that the Ser290, Ser293, Leu337, Thr340, and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

Screening and design of anti-diabetic compounds sourced from the leaves of neem (Azadirachta indica)

Diabetes Mellitus is affecting people of all age groups worldwide. Many synthetic medicines available for type 2 diabetes mellitus in the market. However, there is a strong requirement for the development of better anti-diabetes compounds sourced especially from natural sources like medicinal plants. The extracts from the leaves of neem (Azadirachta indica) is traditionally known to have anti-diabetes properties. Therefore, there is an increased interest to identify potential compounds identified from neem leaf extracts showing predicted binding property with the known diabetes mellitus type 2 protein enzyme target phosphoenol-pyruvate carboxykinase(PEPCK). The structure data for compounds found in the leaf extract of neem was screened against PEPCK using molecular docking simulation and screening techniques. Results show that the compound 3-Deacetyl-3-cinnamoyl-azadirachtin possesses best binding properties with PEPCK. This observation finds application for further consideration in in vitro and in vivo validation.

Designing and screening of universal drug from neem (Azadirachta indica) and standard drug chemicals against influenza virus nucleoprotein

BackgroundDifferent strains of influenza virus are affecting a large number of people worldwide. Many synthetic antiviral medicines are available for influenza virus in the market. But still there is a need for the development of universal drugs against these strains of influenza virus.MethodsFor this purpose conserved residues within the influenza virus nucleoprotein have been retrieved. The drugs, previously known to have antiviral properties, were screened to identify the best candidate universal drug against Influenza virus strains. Compounds from leaf extracts of neem, were also screened to identify the natural drugs without side effects.ResultMolecular docking identified three potential compounds (Nimbaflavone, Rutin, and Hyperoside) having perfect binding with reported conserved residues (ASP302, SER50) of influenza virus nucleoprotein that is involved in the binding of drugs. Further analysis showed Hyperoside as a universal drug against various influenza strains. Some chemical drugs were also evaluated through screening against nucleoprotein. The results showed six drugs (OMS, CBX, LGH, Naproxen, BMS-883559, and BMS-885838) which were interacting with same conserved residues (ASP302, TYR52, SER50, GLY288, SER376, and ARG99) as were found in the case of neem phytochemicals. Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein.ConclusionThe compound Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. So these compounds have been identified for their potential against influenza strains to be utilized as a universal drug.

CRISPR-Cas System: History and Prospects as a Genome Editing Tool in Microorganisms

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR or more precisely CRISPR-Cas) system has proven to be a highly efficient and simple tool for achieving site-specific genome modifications in comparison to Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). The discovery of bacterial defense system that uses RNA-guided DNA cleaving enzymes for producing double-strand breaks along CRISPR has provided an exciting alternative to ZFNs and TALENs for gene editing & regulation, as the CRISPR-associated (Cas) proteins remain the same for different gene targets and only the short sequence of the guide RNA needs to be changed to redirect the site-specific cleavage. Therefore, in recent years the CRISPR-Cas system has emerged as a revolutionary engineering tool for carrying out precise and controlled genetic modifications in many microbes such as Escherichia coli, Staphylococcus aureus, Lactobacillus reuteri, Clostridium beijerinckii, Streptococcus pneumonia, and Saccharomyces cerevisiae. Though, concerns about CRISPR-Cas effectiveness in interlinked gene modifications and off-target effects need to be addressed. Nevertheless, it holds a great potential to speed up the pace of gene function discovery by interacting with previously intractable organisms and by raising the extent of genetic screens. Therefore, the potential applications of this system in microbial adaptive immune system, genome editing, gene regulations, functional genomics & biosynthesis along ethical issues, and possible harmful effects have been reviewed.

Modelling and simulation of mutant alleles of breast cancer metastasis suppressor 1 (BRMS1) gene

Computational tools occupy the prime position in the analysis of large volume of post-genomic data. These tools have advantage over the wet lab experiments in terms of high coverage, cost and time. Breast cancer is the most common cancer in females worldwide. It is a genetically heterogeneous disorder and many genes are involved in the pathway of the disease. Mutations in metastasis suppressor gene are the major cause of the disease. In this study, the effects of mutations in breast cancer metastasis suppressor 1gene upon protein structure and function were examined by means of computational tools and information from databases.This study can be useful to predict the potential effect of every allelic variant, devise new biological experiments and to interpret and predict the patho-physiological impact of new mutations or non-synonymous polymorphisms.

Lead Toxicity in Cereals and Its Management Strategies: a Critical Review

Cereal grains such as wheat, rice, and maize are widely consumed as a staple food worldwide. Lead (Pb) is one of the non-essential trace elements and its toxicity in crops especially cereals is a widespread problem. The present review highlighted Pb toxicity in cereal and management strategies to reduce its uptake in plants. Lead toxicity reduced the cereal growth, photosynthesis, nutritional value, yield, and grain quality. The response of cereals to excess varies with plant species, levels of Pb in soil, and growth conditions. Reducing Pb bioavailability in the soil is a viable approach due to its non-degradability either by microbes, chemicals, or other means. Cultivation of low Pb-accumulating cultivars may reduce the risk of Pb toxicity in plants and humans via the food chain. Use of plant growth regulators, microbes, organic, and inorganic amendments might be promising techniques for further decreasing Pb contents in shoot and grains. Soil amendments along with selecting low Pb-accumulating cultivars might be a feasible approach to get cereal grains with low Pb concentrations. Furthermore, most of the studies have been conducted under controlled conditions either in hydroponic or pots and less is known about the effects of Pb management approaches under ambient field conditions.

Physiochemical and Thermodynamic Characterization of Highly Active Mutated Aspergillus niger β-glucosidase for Lignocellulose Hydrolysis

BACKGROUND Cellulose represents a major source of fermentable sugars in lignocellulosic biomass and a combined action of hydrolytic enzymes (exoglucanases , endoglucanases and β-glucosidases) is required to effectively convert cellulose to glucose that can be fermented to bio-ethanol. However, in-order to make the production of bio-ethanol an economically feasible process, the costs of the enzymes to be used for hydrolysis of the raw material need to be reduced and an increase in specific activity or production efficiency of cellulases is required. Among the cellulases, β-glucosidase not only hydrolyzes cellobiose to glucose but it also reduces the cellobiose inhibition, resulting in efficient functioning of endo- and exo-glucanases. Therefore, in the current study kinetic and thermodynamic characteristics of highly active β-glucosidase from randomly mutated Aspergillus niger NIBGE-06 have been evaluated for its industrial applications. OBJECTIVE The main objective of this study was the identification of mutations and determination of their effect on the physiochemical, kinetic and thermodynamic characteristics of β-glucosidase activity and stability. METHODS Pure cultures of Aspergillus niger NIBGE and its 2-Deoxy-D-glucose resistant γ-rays mutant Aspergillus niger NIBGE-06 were grown on Vogels medium containing wheat bran (3% w/v), at 30±1 °C for 96-108 h. Crude enzymes from both strains were subjected to ammonium sulfate precipitation and column chromatography on Fast Protein Liquid Chromatography (FPLC) system. The purified β-glucosidases from both fungal sources were characterized for their native and subunit molecular mass through FPLC and SDS-PAGE, respectively. The purified enzymes were then comparatively characterized for their optimum temperature, activation energy (Ea), temperature quotient (Q10), Optimum pH, Heat of ionization (ΔHI) of active site residues , Michaelis-Menten constants (Vmax, Km, kcat and kcat/Km) and thermodynamics of irreversible inactivation through various enzyme assays. The genomic DNA from both fungal strains was also extracted by SDS-method and full length β- glucosidase genes (bgl) were amplified through PCR. The PCR products were cloned in TA cloning vector followed by the sequencing of potentially full length clones using the commercial services of Macrogen, Korea. The in silico analyses of the sequences thus obtained were also performed using various online tools such as blastn, blastp, GeneWise, SignalP, Inter- ProScan. RESULTS The extracellular β-glucosidases (BGL) from both fungal sources were purified to homogeneity level by ammonium sulfate precipitation and FPLC system. The BGLs from both strains were dimeric in nature, with subunit and native molecular masses of 130 kDa and 252 kDa, respectively. The comparative analysis of nucleotides of bgl genes revealed 8 point mutations. Significant improvement was observed in the kinetic properties of the mutant BGL relative to the wild type enzyme. Arrhenius plot for energy of activation (Ea) showed a biphasic trend and ES-complex formation required Ea of 50 and 42 kJ mol-1 by BGL from parent and mutant, respectively. The pKa1 and pKa2 of the active site residues were 3.4 & 5.5 and 3.2 & 5.6, respectively. The heat of ionization for the acidic limb (ΔHI-AL) and the basic limb (ΔHI-BL) of BGL from both strains were equal to 56 & 41 and 71 & 45 kJ mol-1, respectively. Kinetic constants of cellobiose hydrolysis for BGL from both strains were determined as follows: kcat = 2,589 and 4,135 s-1, Km = 0.24 and 0.26 mM cellobiose, kcat/Km = 10,872 and 15,712 s-1 mM-1 cellobiose, respectively. Thermodynamic parameters for cellobiose hydrolysis also suggested that mutant BGL is more efficient compared to the parent enzyme. Comparative analysis of Ea(d), ΔH* and ΔG* for irreversible thermostability indicated that the thermostabilization of mutant enzyme was due to higher functional energy (free energy), which enabled the enzyme to resist against unfolding of its transition state. CONCLUSION Physiochemical and thermodynamic characterization of extracellular β-glucosidases (BGL) from 2-Deoxy-Dglucose resistant mutant derivative of A. niger showed that mutagenesis did not greatly affect the physiochemical properties of the BGL enzyme, like temperature optima, pH optima and molecular mass, while the catalytic efficiency for cellobiose hydrolysis was significantly improved (High kcat and kcat/Km). Furthermore, the mutant BGL was more thermostable than the parent enzyme. This shows that random mutagenesis has changed the BGL structural gene, resulting in improvement within its stability- function characteristics. Hence, directed evolution or random mutagenesis with careful selection can result in the engineering of highly efficient enzymes for intended industrial applications.

Zinc oxide nanoparticles alter the wheat physiological response and reduce the cadmium uptake by plants

An experiment was performed to explore the interactive impacts of zinc oxide nanoparticles (ZnO NPs) and cadmium (Cd) on growth, yield, antioxidant enzymes, Cd and zinc (Zn) concentrations in wheat (Triticum aestivum). The ZnO NPs were applied both in Cd-contaminated soil and foliar spray (in separate studies) on wheat at different intervals and plants were harvested after physiological maturity. Results depicted that ZnO NPs enhanced the growth, photosynthesis, and grain yield, whereas Cd and Zn concentrations decreased and increased respectively in wheat shoots, roots and grains. The Cd concentrations in the grains were decreased by 30-77%, and 16-78% with foliar and soil application of NPs as compared to the control, respectively. The ZnO NPs reduced the electrolyte leakage while increased SOD and POD activities in leaves of wheat. It can be concluded that ZnO NPs (levels used in the study) could effectively reduce the toxicity and concentration of Cd in wheat whereas increase the Zn concentration in wheat. Thus, ZnO NPs might be helpful in decreasing Cd and increasing Zn biofortification in cereals which might be effective to reduce the hidden hunger in humans owing the deficiency of Zn in cereals.