[Show abstract][Hide abstract] ABSTRACT: Mylabris pustulata (blister beetle) was studied for the enzymes involved in hydrolysis of cellulose. Carboxy methyl cellulose hydrolyzing activity (endo- β–1, 4- D-glucanase) was detected in the salivary glands and fore gut, very little activity was present in the hind gut. The multiple forms of the enzyme activity were detected on zymogram after non-denaturing PAGE. One of the fractions was purified by gel filtration and preparative native PAGE. The purified protein appeared as single band on SDS-PAGE with a molecular weight of 150 kilo Dalton. The characteristics of the enzyme showed two optimum pH values, one acidic and one neutral 2.0 and 7. The optimum temperature for endo-β – D- 1, 4- glucanase was 50 °C. The enzyme was maximum activity against carboxy methyl cellulose. Km and Vmax of the enzyme was determined as 0.6g/l and 0.3, respectively. To our knowledge this is first report on the digestive cellulose hydrolyzing activity of Mylabris pustulata.
Pakistan journal of zoology 01/2011; 42(2):393-401. · 0.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth hormone (GH) variants have been studied for the structure-function relationship of the molecule. The presence of a potential alternate splicing point in mRNA in bGH gene at exon 3, similar to hGH has been reported by workers. Early investigation on the characteristics of the chemistry of 20k oGH showed that the molecule was produced by site-directed mutagenesis by deleting amino acid residues 33-46 and the resultant DNA was expressed in E. coli under the control of lac promoter in pUC based plasmid. The mutant protein remained insoluble and did not refold. To investigate the effect of deletion on the chemistry of the molecule, computational biology tools were employed. The mutant with the deletion of amino acid residues 33-46, was designed and the model was visualized on computer. The structure of 20k bGH was compared with bGH and dissected for hydrogen bonds and hydro-phobicity. Computational biology tools were helpful in elucidating the role of 33-46 amino acid residues domain in the chemistry of the molecule. Furthermore, it was revealed that removal of amino acid residues 33-46 which formed the hydrogen bonds involving Glu 33, Gln 46, Pro 38, Arg 42, Tyr 43,Ala 51, Thr 48, Asn 47, led to the formation of new hydrogen bonds between Thr 33, Tyr 144, Asn 32, Asn 32 and Ser and Asp 153. The removal of the amino acids 33-46 decreased the hydro-phobicity of the first helix of bGH molecule, as compared to 20k hGH, thus altering the solubility of the molecule, confirming the earlier reported results for ovine growth hormone with same deletion.
AFRICAN JOURNAL OF BIOTECHNOLOGY 03/2010; 9(5):711-717. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxya chinensis Thunberg is a very serious paddy pest in Pakistan and is a major cause of rice crop deterioration. Total cellular protein extract was able to degrade plant biomass from sugar cane bagasse, as observed under microscope. Its endoglucanase and xylanase activities were characterized. The endoglucanase activity showed two pH optima 2.0 and 7.0 at 50-60°C. Endo-beta-1,4-glucanase activity enhanced by calcium ions. A reducing agent beta-mercaptoethanol inhibited the enzyme activity, showing the involvement in stabilizing the three-dimensional structure of the molecule. The enzyme product was identified as glucose by thin layer chromatography. For xylanase the pH optimum was 4.2 and 7.0-8.0. CaCl 2 and 2-mercaptoethanol are proven to be activator in lower concentration for xylanase activity.
[Show abstract][Hide abstract] ABSTRACT: Cellulolytic enzymes have immense potential to convert cellulosic biomass into useful products. Tribolium castaneum crude proteins were isolated to screen the cellulolytic activities. The activity was established by substrate-agar plate assay and confirmed by endoglucanase assay. Cellulolytic activity was further purified and characterized using the different chromatographic techniques and electrophoresis. Gel filtration chromatography showed the presence of multiple forms of enzyme activities with different molecular weights. Stability of enzyme activity was investigated at different temperatures and pH. Optimum pH for was found 4.8 at 40oC determined as optimum temperature. Gradually decreasing Enzyme activity remained half at 60oC. Zymography and SDS-PAGE showed the presence of multiple forms of endoglucanase activities (Cel I and Cel II) with molecular weight of 55 kDa and 35 kDa.
[Show abstract][Hide abstract] ABSTRACT: Two recombinant DNA-derived variants of ovine growth hormone were produced, purified, characterized and compared with the authentic pituitary derived GH. The variants oGH3 and oGH5 were isolated by differential centrifugation method and were purified after refolding by ion-exchange chromatography and gel filtration. Both the proteins showed single band on SDS-PAGE and had molecular weight and iso-electric point closer to authentic pituitary GH. The variants oGH3 and oGH5 were compared with the authentic pituitary derived GH in radio immuno assays, radio receptor assays and binding with the monoclonal antibodies OA 11 and OA12.
AFRICAN JOURNAL OF BIOTECHNOLOGY 07/2008; 7(12):1859-1864. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the production and characterization of endo-β-1, 4-glucanase from isolated phytopathogenic bacterium Enterobacter cloacae . The bacterium was grown on different carbon sources including carboxymethyl cellulose (CMC) and 2% Avicel, for the production of endo-1, 4-β –D-glucanases enzyme. E. cloacae produced maximum levels of cellulases after 96 h of fermentation. Higher levels of endoglucanases were produced when microbe was grown on CMC. Endo-1, 4- β-D- glucanase had optimum pH and temperature of 5.8 and 40°C. The enzyme was inactivated by calcium chloride and a reducing agent β-mercaptoethanol.
AFRICAN JOURNAL OF BIOTECHNOLOGY 05/2008; 7(9). · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at -20 degrees C. The total protein extract was isolated from 5 samples in each case (mature and hyper mature cataract) and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 degrees C. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. beta mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates.
Journal of Zhejiang University SCIENCE B 09/2007; 8(8):599-603. DOI:10.1631/jzus.2007.B0599 · 1.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Somatotropin, commonly known as growth hormone (GH) is a polypeptide chain containing about 190 amino acid residues, produced by the pituitary gland in mammals and is responsible for a number of anabolic processes. It has two disulphide bridges, with 4 alpha helices arranged in anti-parallel distinctive manner. GH molecule binds with two receptor molecules to exhibit its full biological activity. In this review, the information regarding characterization, structure and function is updated. A number of human growth hormone variants (naturally occurring and produced by recombinant DNA- technology) are visualised, and structure related functions are revealed. 1) The di-sulphide bridges are not essential for the biological activity of the molecule. The two chain variants of GH are able to show full biological activity. 2) The different domains of GH could be related to its functions 3) N-terminus of the molecule is involved in the galactopoietic activity of the molecule. 4) A single amino acid residue at a particular position could determine the magnitude of hormone receptor binding. 5) Role of Trp 86 is critical in packing of the apha helices bundles of the molecule. 6) Hydrophobic cores are essential for the stability of GH molecule 7) Salt bridges and hydrogen bonds are also important for the binding of the molecule with its receptors. 8) GH molecule has two binding sites for receptor molecules, Site I and Site II which are sterically coupled. The placental growth hormone has also been discussed and compared with the pituitary derived GH for its structure and function.
Current Protein and Peptide Science 07/2007; 8(3):283-92. DOI:10.2174/138920307780831820 · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Somatotropin or growth hormone (GH) was isolated from pituitaries of freshly slaughtered animals in slaughter house of Lahore. Proteins were first concentrated with ammonium sulfate in a homogenate of pituitaries in saline buffer pH 9.5 and then passed through an ion-exchange column (pH 9.5), using a linear salt gradient. GH eluted as a shoulder of major protein peak, was further purified on preparative poly-acrylamide gel electrophoresis. Bubaline GH appeared as a single band on SDS-PAGE. Properties of bubaline GH were compared with the authentic bovine GH.
Pakistan journal of zoology 01/2006; 38(4). · 0.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A number of analogues of ovine growth hormone (GH), in which regions of the hormone had been deleted, were produced by site-directed mutagenesis, and characterised by radioimmunoassays and radioreceptor assays. These analogues were based on a previously described variant (oGH1) in which an 8-residue extension replaces the N-terminal alanine of pituitary-derived ovine GH. Three analogues with deletions near the N-terminus were studied, with shorter extensions of 7 or 1-2 residues (oGH14, oGH5) or with the N-terminal sequence Ala-Phe-Pro- of pituitary-derived ovine GH replaced by Thr-Met-Ile-Thr- (oGH11). These modifications had little effect on potency in radioimmunoassays based on a polyclonal antibody and five different monoclonal antibodies (MABs), or in a radioreceptor assay, indicating that the N-terminal sequence was not included in the epitope binding to any of the monoclonal antibodies, or a major epitope binding to the polyclonal antibody, or in receptor binding site 1. A variant in which residues 133-139 were deleted retained full binding to 4 of the 5 MABs, suggesting correct folding, but markedly reduced binding to MAB OA16, suggesting that the epitope for this MAB includes some or all of these residues. This variant also failed to displace about 35% of labelled hormone from the polyclonal antibody studied, suggesting that residues 133-139 may be involved in a major epitope for this antibody. This variant showed slightly lower receptor binding activity than ovine GH. Two other deletion variants - oGH1Delta33-46 (equivalent to the naturally occurring 20K variant of human GH) and oGH1Delta180-191 (lacking the C-terminal 12 residues) showed poor folding efficiency and solubility, and low binding to all MABs except OA15, which has a linear epitope. The results suggest that these variants were incorrectly folded, but interestingly they did retain some activity in the receptor-binding assay (respectively about 5% and 0.5% of the activity of ovine GH itself).
[Show abstract][Hide abstract] ABSTRACT: The expression levels of coding sequences for pituitary growth hormone, introduced into Escherichia coli by genetic manipulation techniques, vary markedly according to the precise sequence introduced. In order to understand the basis of this variation more fully, we have studied the relationship between the level of expression in E. coli of a series of ovine growth hormone variants and the nucleotide sequences coding for their N-terminal regions. Sequence variation resulted from the introduction of deletions, or site-directed mutations, into a plasmid containing the coding sequence for ovine growth hormone preceded by the initiation codon and 25 bases derived from beta-galactosidase or linker regions of plasmid pUC8. The expression levels of the variants varied from less than 0.01% to over 34% of the total cell protein, indicating that changes in the nucleotide sequence close to the initiation codon had a marked effect on expression level. The results of a comparison of closely related sequences in pairs of plasmids giving poor or good expression are consistent with the hypothesis that poor translation of growth hormone mRNAs is caused by the presence of secondary structures close to the initiation codon. Secondary structures are identified that appear to explain the variation in expression levels.