Amtul Jamil Sami

Production of free and substrate-bound cellulases of cellulomonas flavigena

Abstract Conditions for the production of extracellular carboxymethyl cellulase ( CMCase ) and avicelase activities by a locally isolated Cellulomonas species identified as C. flavigena were optimized. The microbe produced maximal levels of free cellulases after 72 h of fermentation, when cultivated in the presence of 0.2% yeast extract, 0.5% Avicel, and 0.1% Tween 80 at 30°C. Conditions for the elution of substrate-bound cellulases from the residual Avicel were optimized. Higher proportions of the extracellular cellulases were bound to the substrate when higher concentrations of Avicel were used. When 2.0% Avicel was used as a carbon source, maximal levels of extracellular CMCase and avicelase produced were 13.2 and 3 U ml −1 of the culture medium, respectively. Both the cellulases were most active at pH 6.5 and 50°C and were inhibited by β-mercaptoethanol. Calcium chloride activated CMCase but had no effect on avicelase activity. Each of the enzymes lost activity when incubated at 70°C for 30 min. A comparative study of free and substrate-bound CMCase on gradient polyacrylamide gel electrophoresis ( PAGE ) showed that free CMCase activity was composed of at least seven active fractions, while bound activity had four major bands of CMCase activity. Since a considerable level of protease was secreted by C. flavigena, the additional fractions in the free CMCase activity could be a result of proteolysis .

Purification and characterization of two low-molecular weight endoglucanases of Cellulomonas flavigena

Abstract Two isoforms of endoglucanases of Cellulomonas flavigena were purified to homogeneity after a series of purification steps including precipitation with 80% acetone, gel filtration, ion-exchange chromatography, and nondenaturing gradient 5–20% preparative polyacrylamide gel electrophoresis. Each of the endoglucanases, CM-Cellulase 1 and CM-Cellulase 2, appeared as a single band on SDS-PAGE and had an apparent molecular weight of 20,400. Both of the endoglucanases were comparable to the substrate-bound endoglucanases and the recombinant DNA-derived endoglucanases of C. flavigena expressed in Escherichia coli. The two isoforms of endoglucanase, CM-Cellulase 1 and CM-Cellulase 2, were different in their mobilities on native nondenaturing 5–20% gradient PAGE. The purified endoglucanases displayed significantly similar characteristics, including pH optima (6.5 and 7) and temperature optima (50°C). The purified CM-Cellulases were unable to hydrolyze Avicel, xylan, and filter paper and were only active against CMC. The purified proteins had a K m value of 0.78 g l −1 . Heavy metal ions like Ag 2+ and Fe 2+ inactivated the enzymes. NaCl and CaCl 2 had no effect on enzyme activities. β-Mercaptoethanol also inhibited enzyme activity, possibly due to the involvement of sulfhydryl groups .

Formulation of novel chitosan guargum based hydrogels for sustained drug release of paracetamol

The report presents the formulation of hydrogel based on biopolymers chitosan and guar gum after cross-linking for sustained release of a commonly used orally prescribed analgesic Paracetamol. The oral ingestion of Paracetamol is associated with complications of the gastric tract and liver metabolism that can be effectually avoided by using transdermal drug delivery systems. The formulated transdermal patch was characterized for physicochemical properties including swelling, bonding pattern (using FTIR Fourier Transform Infra-Red and Scanning Electron Microscopy SEM) and antimicrobial activity. Biocompatibility and cytotoxicity was examined in vitro using cell culture in HeLa cell lines. After characterizing the novel formulated hydrogel were employed for the preparation of drug encapsulated in alginate beads as a transdermal patch. After formulation of the transdermal patch, the drug release was studied using an avian skin model. The results followed zero order kinetics and Non-Fickian law for diffusion. Paracetamol due to its small molecular mass (151.163g/mol) released in a sustained manner. The released drug successfully retained its biological effects including anti-inflammatory and anti-protease activity, indicating no interaction between the drug and the formulated hydrogel. It was shown that the formulated hydrogels could be safely used as a dermal patch for the sustained drug release of Paracetamol.

Production, purification and characterization of two recombinant DNA-derived N-terminal ovine growth hormone variants: oGH3 and oGH5

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.

Comparison in effect of different metal ions, pH and reducing agent on the protease activity in human hyper mature and mature cataract

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 °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 °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. β mercapotethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates.

Deletion of amino acid residues 33-46 in growth hormone alters the hydrophobicity of the molecule

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 hydrophobicity. 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.