mohammed saleemuddin | Aligarh Muslim University (original) (raw)

Papers by mohammed saleemuddin

Research paper thumbnail of DNA damage by ribose: inhibition at high ribose concentrations

Indian journal of biochemistry & biophysics, 2010

Nucleobases and DNA react non-enzymatically with sugars, and generate DNA-advanced glycation end ... more Nucleobases and DNA react non-enzymatically with sugars, and generate DNA-advanced glycation end products (DNA-AGEs). Incubation of plasmid pBr322 with ribose for 3-7 days caused the transformation of the supercoiled plasmid to the open circular and linear forms. Removal of sugar after an initial 24 h incubation resulted in marked enhancement in the transformation rate. Enhancement in transformation was also observed when bovine serum albumin (BSA) exposed to ribose for short durations was incubated with plasmid in absence of the sugar. The effect on DNA was attenuated when excess ribose remained in the incubation mixture of ribose and protein. The data suggested that an increase in ribose concentration in the vicinity of DNA could be damaging and the damage exacerbated, if sugar levels fell subsequently. Incubation of herring sperm DNA with ribose resulted in a concentration and time-dependent increase of N2-carboxyethyl-2'-deoxyguanosine (CEdGA,B). The concentration of CEdGA,B...

Research paper thumbnail of Sucrose hydrolysis using invertase immobilized on concanavalin A-sepharose

Enzyme and Microbial Technology, 1985

The stability and ability of yeast invertase (~D-fructofuranoside fructohydrolase, EC 3.2.1.26) b... more The stability and ability of yeast invertase (~D-fructofuranoside fructohydrolase, EC 3.2.1.26) bound and covalently coupled to concanavalin A-Sepharose continuously to hydrolyse sucrose over long periods has been investigated. The immobilized preparation exhibited high resistance to heat and urea induced denaturation. A small column of the immobilized preparation could hydrolyse relatively high concentrations of sucrose almost quantitatively for more than 60 days.

Research paper thumbnail of Alkaline protease from Spilosoma obliqua: potential applications in bio-formulations

Biotechnology and Applied Biochemistry, 2000

Research paper thumbnail of Phospholipid diversity: Correlation with membrane–membrane fusion events

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2005

The transport of various metabolically important substances along the endocytic and secretory pat... more The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called bfusion proteinsQ. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.

Research paper thumbnail of Bioaffinity layering: A novel strategy for the immobilization of large quantities of glycoenzymes

Journal of Biotechnology, 1997

A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insol... more A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described. The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A. Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one ml Sepharose matrix after seven Con A/glucose oxidase incubation cycles. Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (p) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability. The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering. A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cere6isiae.

Research paper thumbnail of Application of reversible immobilization techniques for biosensors

Analytica Chimica Acta, 1998

Biosensors are analytical devices which incorporate a biologically active component, often an enz... more Biosensors are analytical devices which incorporate a biologically active component, often an enzyme covalently attached to an electrode surface, and are normally used for the measurement of a single analyte at a given time. Attempts were therefore made to develop a¯exible biosensor which enables the measurement of different substrates. In this article we describe a new immobilization system where enzymes like glucose oxidase, invertase and peroxidase were bound to chelating sepharose through different metal ions and the lectin concanavalin A in a way which permits elution and reloading of these enzymes in the conditions irrespective of each other. The thermal stability of the glucose oxidase preparation obtained by immobilization on different metal ions was also studied. After the performance of an experiment, the gels can easily be regenerated and reloaded with the same or different metal ions for an ensuing experiment. Thus this technique with selective removal and new immobilization of the enzymes can also be a useful tool in the ®eld of biosensors.

Research paper thumbnail of Immunoaffinity layering of enzymes

Applied Microbiology and Biotechnology, 1999

A general procedure for the high yield immobilization of enzymes with the help of speci®c antienz... more A general procedure for the high yield immobilization of enzymes with the help of speci®c antienzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ionexchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoanity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioanity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a¯ow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoanity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small¯ow-through cell integrated with a cartridge bearing immunoanity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using ā ow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoanity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaf-®nity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Research paper thumbnail of Additional stabilization of stem bromelain coupled to a thermosensitive polymer by uniform orientation and using polyclonal antibodies

Biochemistry (Moscow), 2007

Stem bromelain was covalently coupled to a thermosensitive polymer of N isopropylacrylamide (p(NI... more Stem bromelain was covalently coupled to a thermosensitive polymer of N isopropylacrylamide (p(NIPAm)) either through the amino groups of the enzyme (randomly coupled) or via the lone oligosaccharide chain (uniformly cou pled). The enzyme coupled via the oligosaccharide chain exhibited better access to the substrate casein as compared to the preparation in which the amino groups formed the point of contact between the enzyme and the polymer. Native bromelain exhibited a pH optimum of 8.0 and a broad pH activity profile. The polymer coupled preparations exhibited broader pH activity profiles and shifting of pH optimum to 10.0 at 35°C. At 25°C, the shifting of pH optimum was observed for the ran domly coupled enzyme only. The temperature-activity profiles of bromelain coupled to p(NIPAm) also showed apprecia ble broadening and the preparations retained greater fraction of maximum activity above the temperature optimum. The optimum temperature of the uniformly oriented preparation also rose to 70°C. Inactivation rates of the polymer coupled bromelain were remarkably low at 60°C as compared to the native protease, and binding of antibromelain antibodies improved the resistance to inactivation of the polymer coupled preparations. The cleavage patterns of hemoglobin and IgG by the native bromelain and the polymer coupled preparations were comparable.

Research paper thumbnail of A fluorometric fiber-optic biosensor for dual analysis of glucose and fructose using glucose-fructose-oxidoreductase isolated from Zymomonas mobilis

Journal of Biotechnology, 1994

A fluorometric fiber-optic biosensor was used to determine the content of glucose and fructose ac... more A fluorometric fiber-optic biosensor was used to determine the content of glucose and fructose according to the measurement principle of flow injection analysis (FIA). The enzyme glucose-fructose-oxidoreductase (GFOR) isolated from Zyrnomonas mobilis was confined in a measurement cell behind an ultrafiltration membrane. The GFOR catalyzes the oxidation of glucose to gluconolactone and the reduction of fructose to sorbitol according to the ping-pong mechanism. This unique enzyme contains NADPH tightly confined in the enzyme complex. The change of NADPH fluorescence was detected by a fluorosensor. The stability could be increased remarkably by crosslinking with glutaraldehyde. The detection range for glucose was within a concentration range of 0.055-55.5 mM and that for fructose within 0.278-331 raM. The response time for glucose and fructose was in the range of 40 s and 1 min, respectively. Steady-state was achieved for glucose injection in 2 min and fructose injection in 6.5 min. Sampling time for glucose could be reduced to 37% by including fructose in excess to the sample. This also makes the regenerating step unnecessary. The specificity of this biosensor was tested for different sugars. The results showed that this biosensor system is highly specific, sensitive and suitable for dual analysis of glucose and fructose.

Research paper thumbnail of Entrapment of Nonproteolytic Enzymes in α2-Macroglobulin Using Immobilized Trypsin

Analytical Biochemistry, 1993

Entrapment in human alpha 2-macroglobulin (alpha 2M) of non-proteolytic enzymes was achieved with... more Entrapment in human alpha 2-macroglobulin (alpha 2M) of non-proteolytic enzymes was achieved with the help of trypsin covalently attached to Sepharose matrix. While it was also possible to achieve entrapment by the exposure of the alpha 2M: enzyme mixtures to soluble trypsin, use of the immobilized proteinase resulted in improved entrapment yields and also prevented the coentrapment of trypsin. Both soluble and immobilized trypsin transformed alpha 2M to the electrophoretically fast form but the immobilized trypsin required relatively longer incubation to bring about the transformation. Horseradish peroxidase was entrapped in higher yield in alpha 2M compared to the relatively high-molecular-weight invertase. alpha 2M-entrapped peroxidase and invertase appeared highly accessible to their respective substrates, as evident from their relatively unaltered Km values. alpha 2M-associated invertase, in spite of its large dimensions, failed to crossreact with the rabbit anti-invertase antiserum, indicating its physical entrapment rather than any other form of association.

Research paper thumbnail of Behaviour of oleic acid-depleted bovine alpha-lactalbumin made LEthal to tumor cells (BAMLET)

Molecular bioSystems, May 25, 2016

Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effect... more Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effective in the killing of a variety of tumor cells. While debate on whether the key component of the complexes is the OA or protein continues, studies probing the mechanism of action of the complexes at the tumor cell surface or in the cell interior assume the action of a molecule in the form of an undissociated complex. Recent evidence however suggests that OA complexes of protein are stripped of bound OA on interaction with artificial or natural membranes before entering the cell. Properties of BAMLET stripped of its OA by exposure to erythrocytes (ET-BAMLET) were investigated in the study. ET-BAMLET resembled α-LA in its inability to induce hemolysis of erythrocytes and behaviour in a gel filtration column. Spectroscopy techniques-fluorescence, far- and near UV CD as well as calorimetry and proteolysis however suggest the molecule to be different both from native α-LA and the apo form. Re...

Research paper thumbnail of Fibrin matrices: The versatile therapeutic delivery systems

International Journal of Biological Macromolecules, 2015

Fibrin sealants, that have been employed for over a century by surgeons to stop post surgery blee... more Fibrin sealants, that have been employed for over a century by surgeons to stop post surgery bleeding, are finding novel applications in the controlled delivery of antibiotics and several other therapeutics. Fibrinogen can be easily purified from blood plasma and converted by thrombolysis to fibrin that undergoes spontaneous aggregation to form insoluble clot. During the gelling, fibrin can be formulated into films, clots, threads, microbeads, nanoconstructs and nanoparticles. Whole plasma clots in the form of beads and microparticles can also be prepared by activating endogenous thrombin, for possible drug delivery. Fibrin formulations offer remarkable scope for controlling the porosity as well as in vivo degradability and hence the release of the associated therapeutics. Binding/covalent-linking of therapeutics to the fibrin matrix, crosslinking of the matrix with bifunctional reagents and coentrapment of protease inhibitors have been successful in regulating both in vitro and in vivo release of the therapeutics. The release rates can also be remarkably lowered by preentrapment of therapeutics in insoluble particles like liposomes or by anchoring them to the matrix via molecules that bind them as well as fibrin.

Research paper thumbnail of Vaccine potential of Plasma beads based dual antigen delivery system against experimental murine candidiasis

International Journal of Biological Macromolecules, 2015

The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic p... more The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic proteins (Cp) as antigen and plasma beads (PB) prepared from plasma as sustained delivery system, is described. The immune-prophylactic potential of various PBs-based dual antigen delivery systems, co-entrapping Cp pre-entrapped in PLGA microspheres were tested in the murine model. Induction of cell mediated immunity was measured by assaying DTH and NO production as well as in vitro proliferation of lymphocytes derived from the immunized animals. Expression of surface markers on APCs (CD80, CD86) and T-cells (CD4+, CD8+) was also evaluated. Humoral immune response was studied by measuring circulating anti-Cp antibodies and their subclasses. When the prophylactic efficacy of the vaccines was tested in mice challenged with virulent C. albicans, the PB-based formulation (PB-PLGA-Cp vaccine) was found to be most effective in the generation of desirable immune response, in terms of suppression of fungal load and facilitating the survival of the immunized animals.

Research paper thumbnail of Selective stabilization of a labile mutant form of bovine pancreatic ribonuclease A by antibodies

The region between the amino acids 31-46 was previously identified as being first exposed during ... more The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a ...

Research paper thumbnail of Immobilization and stabilization of horseradish peroxidase isoforms

IUBMB Life, 1996

S~Y-Purified anionic and cationic isoforms of horseradish peroxidase (HRP) immobilized by couplin... more S~Y-Purified anionic and cationic isoforms of horseradish peroxidase (HRP) immobilized by coupling the amino acid side-chain amino groups and/or carbohydrate moieties to Sepharose have been studied for their resistance to denaturation. The isoforms were treated with periodate followed by ethylenediamine to generate additional amino groups in the glycosyl residues. The immobilized preparations were: Preparation I (Sp-aHRP, Sp-cHRP), in which HRP was covalently immobilized via side-chain amino groups exclusively; Preparation II (Sp-NHaHRP, Sp-NHcHRP), in which periodate and ethylenediamine-treated HRP was covalently immobilized via sidechain amino groups and amino groups incorporated into glycosyl residues. HRP isoforms in preparation II lacked about 33-55 % carbohydrate. Both strategies of immobilization induced significant stabilization against denaturation. Inclusion of 2 mM calcium enhanced isoenzyme stability significantly.

Research paper thumbnail of Batch kinetics and modelling of ethanolic fermentation of whey

International Journal of Food Science and Technology, 2005

The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying la... more The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L)1 , the production of ethanol was 0.618 g L)1 whereas at 50 g L)1 it was 3.98 g L)1. However, an increase in lactose concentration to 100 g L)1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L)1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S 0 ¼ 10 and 50 g L)1 but failed in case of S 0 ¼ 100 g L)1 because of strong substrate inhibition.

Research paper thumbnail of Immobilization of the restriction endonuclease EcoRI usefulness of a polyclonal antibody support

Enzyme and Microbial Technology, 1999

The restriction enzyme EcoRI has been immobilized by using an immunoaffinity-based procedure on S... more The restriction enzyme EcoRI has been immobilized by using an immunoaffinity-based procedure on Sepharose 4B. The antibody support, prepared by coupling the ␥-globulin fraction of serum of immunized rabbits to CNBr-activated Sepharose-4B, was highly effective in binding EcoRI from solution although only about half of the bound activity appeared to be expressed by the immobilized preparations. Restriction activity of EcoRI immobilized on the antibody support was indistinguishable from that of soluble enzyme on the linear-DNA and supercoiled plasmids pBR322 and pBHLUC P. Binding to the antibody support markedly enhanced the resistance of EcoRI to heat inactivation, and the preparation, unlike the native enzyme, retained significant activity after exposure to a temperature of 65°C. It was also possible to immobilize EcoRI directly from the cell lysates of Escherichia coli pMB4, an EcoRI overproducing strain. The immobilized preparation did not possess nonspecific nuclease activity that was prominent in the lysates, suggesting specificity in the binding of EcoRI to the antibody support.

Research paper thumbnail of Stabilization of yeast invertase by insoluble immunocomplex formation using unfractionated glycosyl-specific and peptide specific polyclonal antiinvertase antibodies

Biotechnology Techniques, 1995

A strategy for the production of non-inhibitory polyclonal antisera for the immobilization of gly... more A strategy for the production of non-inhibitory polyclonal antisera for the immobilization of glycoenzymes is described. Insoluble complexes of invertase prepared using antiinvertase glycosyl antisera were more resistant to heat and urea induced denaturation than those prepared using antiinvertase antisera from which the glycosyl specific antibodies were removed. Unfractionated antiinvertase antibodies however gave most stable immunocomplexes with invertase

Research paper thumbnail of Bioaffinity Based Oriented Immobilization of Stem Bromelain

Biotechnology Letters, 2006

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas... more Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 degrees C for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K (m) values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V (max) values remained unaffected on immobilization.

Research paper thumbnail of Entrapment of concanavalin A-glycoenzyme complexes in calcium alginate gels

Biotechnology and Bioengineering, 1985

Glucose oxidase, invertase, and amyloglucosidase were entrapped in calcium alginate gels as conca... more Glucose oxidase, invertase, and amyloglucosidase were entrapped in calcium alginate gels as concanavalin A complexes in order to prevent the leaching out of the enzymes from the porous matrix. The free as well as the gel-entrapped concanavalin A-glycoenzyme complexes exhibited a relatively high effectiveness factor, eta, indicating good accessibility to the substrates. Concanavalin A-invertase complex exhibited marked broadening of pH-activity and temperature-activity profiles and was highly resistant to temperature inactivation even after entrapment in the alginate beads. It was possible to entrap considerable quantities of invertase as concanavalin A complex in the beads without a marked decrease in eta. A column containing crosslinked concanavalin A-invertase complex entrapped in alginate beads retained the ability to completely hydrolyze 1M sucrose even after continuous operation for over four months.

Research paper thumbnail of DNA damage by ribose: inhibition at high ribose concentrations

Indian journal of biochemistry & biophysics, 2010

Nucleobases and DNA react non-enzymatically with sugars, and generate DNA-advanced glycation end ... more Nucleobases and DNA react non-enzymatically with sugars, and generate DNA-advanced glycation end products (DNA-AGEs). Incubation of plasmid pBr322 with ribose for 3-7 days caused the transformation of the supercoiled plasmid to the open circular and linear forms. Removal of sugar after an initial 24 h incubation resulted in marked enhancement in the transformation rate. Enhancement in transformation was also observed when bovine serum albumin (BSA) exposed to ribose for short durations was incubated with plasmid in absence of the sugar. The effect on DNA was attenuated when excess ribose remained in the incubation mixture of ribose and protein. The data suggested that an increase in ribose concentration in the vicinity of DNA could be damaging and the damage exacerbated, if sugar levels fell subsequently. Incubation of herring sperm DNA with ribose resulted in a concentration and time-dependent increase of N2-carboxyethyl-2'-deoxyguanosine (CEdGA,B). The concentration of CEdGA,B...

Research paper thumbnail of Sucrose hydrolysis using invertase immobilized on concanavalin A-sepharose

Enzyme and Microbial Technology, 1985

The stability and ability of yeast invertase (~D-fructofuranoside fructohydrolase, EC 3.2.1.26) b... more The stability and ability of yeast invertase (~D-fructofuranoside fructohydrolase, EC 3.2.1.26) bound and covalently coupled to concanavalin A-Sepharose continuously to hydrolyse sucrose over long periods has been investigated. The immobilized preparation exhibited high resistance to heat and urea induced denaturation. A small column of the immobilized preparation could hydrolyse relatively high concentrations of sucrose almost quantitatively for more than 60 days.

Research paper thumbnail of Alkaline protease from Spilosoma obliqua: potential applications in bio-formulations

Biotechnology and Applied Biochemistry, 2000

Research paper thumbnail of Phospholipid diversity: Correlation with membrane–membrane fusion events

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2005

The transport of various metabolically important substances along the endocytic and secretory pat... more The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called bfusion proteinsQ. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.

Research paper thumbnail of Bioaffinity layering: A novel strategy for the immobilization of large quantities of glycoenzymes

Journal of Biotechnology, 1997

A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insol... more A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described. The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A. Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one ml Sepharose matrix after seven Con A/glucose oxidase incubation cycles. Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (p) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability. The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering. A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cere6isiae.

Research paper thumbnail of Application of reversible immobilization techniques for biosensors

Analytica Chimica Acta, 1998

Biosensors are analytical devices which incorporate a biologically active component, often an enz... more Biosensors are analytical devices which incorporate a biologically active component, often an enzyme covalently attached to an electrode surface, and are normally used for the measurement of a single analyte at a given time. Attempts were therefore made to develop a¯exible biosensor which enables the measurement of different substrates. In this article we describe a new immobilization system where enzymes like glucose oxidase, invertase and peroxidase were bound to chelating sepharose through different metal ions and the lectin concanavalin A in a way which permits elution and reloading of these enzymes in the conditions irrespective of each other. The thermal stability of the glucose oxidase preparation obtained by immobilization on different metal ions was also studied. After the performance of an experiment, the gels can easily be regenerated and reloaded with the same or different metal ions for an ensuing experiment. Thus this technique with selective removal and new immobilization of the enzymes can also be a useful tool in the ®eld of biosensors.

Research paper thumbnail of Immunoaffinity layering of enzymes

Applied Microbiology and Biotechnology, 1999

A general procedure for the high yield immobilization of enzymes with the help of speci®c antienz... more A general procedure for the high yield immobilization of enzymes with the help of speci®c antienzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ionexchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoanity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioanity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a¯ow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoanity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small¯ow-through cell integrated with a cartridge bearing immunoanity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using ā ow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoanity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaf-®nity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Research paper thumbnail of Additional stabilization of stem bromelain coupled to a thermosensitive polymer by uniform orientation and using polyclonal antibodies

Biochemistry (Moscow), 2007

Stem bromelain was covalently coupled to a thermosensitive polymer of N isopropylacrylamide (p(NI... more Stem bromelain was covalently coupled to a thermosensitive polymer of N isopropylacrylamide (p(NIPAm)) either through the amino groups of the enzyme (randomly coupled) or via the lone oligosaccharide chain (uniformly cou pled). The enzyme coupled via the oligosaccharide chain exhibited better access to the substrate casein as compared to the preparation in which the amino groups formed the point of contact between the enzyme and the polymer. Native bromelain exhibited a pH optimum of 8.0 and a broad pH activity profile. The polymer coupled preparations exhibited broader pH activity profiles and shifting of pH optimum to 10.0 at 35°C. At 25°C, the shifting of pH optimum was observed for the ran domly coupled enzyme only. The temperature-activity profiles of bromelain coupled to p(NIPAm) also showed apprecia ble broadening and the preparations retained greater fraction of maximum activity above the temperature optimum. The optimum temperature of the uniformly oriented preparation also rose to 70°C. Inactivation rates of the polymer coupled bromelain were remarkably low at 60°C as compared to the native protease, and binding of antibromelain antibodies improved the resistance to inactivation of the polymer coupled preparations. The cleavage patterns of hemoglobin and IgG by the native bromelain and the polymer coupled preparations were comparable.

Research paper thumbnail of A fluorometric fiber-optic biosensor for dual analysis of glucose and fructose using glucose-fructose-oxidoreductase isolated from Zymomonas mobilis

Journal of Biotechnology, 1994

A fluorometric fiber-optic biosensor was used to determine the content of glucose and fructose ac... more A fluorometric fiber-optic biosensor was used to determine the content of glucose and fructose according to the measurement principle of flow injection analysis (FIA). The enzyme glucose-fructose-oxidoreductase (GFOR) isolated from Zyrnomonas mobilis was confined in a measurement cell behind an ultrafiltration membrane. The GFOR catalyzes the oxidation of glucose to gluconolactone and the reduction of fructose to sorbitol according to the ping-pong mechanism. This unique enzyme contains NADPH tightly confined in the enzyme complex. The change of NADPH fluorescence was detected by a fluorosensor. The stability could be increased remarkably by crosslinking with glutaraldehyde. The detection range for glucose was within a concentration range of 0.055-55.5 mM and that for fructose within 0.278-331 raM. The response time for glucose and fructose was in the range of 40 s and 1 min, respectively. Steady-state was achieved for glucose injection in 2 min and fructose injection in 6.5 min. Sampling time for glucose could be reduced to 37% by including fructose in excess to the sample. This also makes the regenerating step unnecessary. The specificity of this biosensor was tested for different sugars. The results showed that this biosensor system is highly specific, sensitive and suitable for dual analysis of glucose and fructose.

Research paper thumbnail of Entrapment of Nonproteolytic Enzymes in α2-Macroglobulin Using Immobilized Trypsin

Analytical Biochemistry, 1993

Entrapment in human alpha 2-macroglobulin (alpha 2M) of non-proteolytic enzymes was achieved with... more Entrapment in human alpha 2-macroglobulin (alpha 2M) of non-proteolytic enzymes was achieved with the help of trypsin covalently attached to Sepharose matrix. While it was also possible to achieve entrapment by the exposure of the alpha 2M: enzyme mixtures to soluble trypsin, use of the immobilized proteinase resulted in improved entrapment yields and also prevented the coentrapment of trypsin. Both soluble and immobilized trypsin transformed alpha 2M to the electrophoretically fast form but the immobilized trypsin required relatively longer incubation to bring about the transformation. Horseradish peroxidase was entrapped in higher yield in alpha 2M compared to the relatively high-molecular-weight invertase. alpha 2M-entrapped peroxidase and invertase appeared highly accessible to their respective substrates, as evident from their relatively unaltered Km values. alpha 2M-associated invertase, in spite of its large dimensions, failed to crossreact with the rabbit anti-invertase antiserum, indicating its physical entrapment rather than any other form of association.

Research paper thumbnail of Behaviour of oleic acid-depleted bovine alpha-lactalbumin made LEthal to tumor cells (BAMLET)

Molecular bioSystems, May 25, 2016

Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effect... more Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effective in the killing of a variety of tumor cells. While debate on whether the key component of the complexes is the OA or protein continues, studies probing the mechanism of action of the complexes at the tumor cell surface or in the cell interior assume the action of a molecule in the form of an undissociated complex. Recent evidence however suggests that OA complexes of protein are stripped of bound OA on interaction with artificial or natural membranes before entering the cell. Properties of BAMLET stripped of its OA by exposure to erythrocytes (ET-BAMLET) were investigated in the study. ET-BAMLET resembled α-LA in its inability to induce hemolysis of erythrocytes and behaviour in a gel filtration column. Spectroscopy techniques-fluorescence, far- and near UV CD as well as calorimetry and proteolysis however suggest the molecule to be different both from native α-LA and the apo form. Re...

Research paper thumbnail of Fibrin matrices: The versatile therapeutic delivery systems

International Journal of Biological Macromolecules, 2015

Fibrin sealants, that have been employed for over a century by surgeons to stop post surgery blee... more Fibrin sealants, that have been employed for over a century by surgeons to stop post surgery bleeding, are finding novel applications in the controlled delivery of antibiotics and several other therapeutics. Fibrinogen can be easily purified from blood plasma and converted by thrombolysis to fibrin that undergoes spontaneous aggregation to form insoluble clot. During the gelling, fibrin can be formulated into films, clots, threads, microbeads, nanoconstructs and nanoparticles. Whole plasma clots in the form of beads and microparticles can also be prepared by activating endogenous thrombin, for possible drug delivery. Fibrin formulations offer remarkable scope for controlling the porosity as well as in vivo degradability and hence the release of the associated therapeutics. Binding/covalent-linking of therapeutics to the fibrin matrix, crosslinking of the matrix with bifunctional reagents and coentrapment of protease inhibitors have been successful in regulating both in vitro and in vivo release of the therapeutics. The release rates can also be remarkably lowered by preentrapment of therapeutics in insoluble particles like liposomes or by anchoring them to the matrix via molecules that bind them as well as fibrin.

Research paper thumbnail of Vaccine potential of Plasma beads based dual antigen delivery system against experimental murine candidiasis

International Journal of Biological Macromolecules, 2015

The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic p... more The development of prophylactic anti-candidal vaccine comprising the Candida albicans cytosolic proteins (Cp) as antigen and plasma beads (PB) prepared from plasma as sustained delivery system, is described. The immune-prophylactic potential of various PBs-based dual antigen delivery systems, co-entrapping Cp pre-entrapped in PLGA microspheres were tested in the murine model. Induction of cell mediated immunity was measured by assaying DTH and NO production as well as in vitro proliferation of lymphocytes derived from the immunized animals. Expression of surface markers on APCs (CD80, CD86) and T-cells (CD4+, CD8+) was also evaluated. Humoral immune response was studied by measuring circulating anti-Cp antibodies and their subclasses. When the prophylactic efficacy of the vaccines was tested in mice challenged with virulent C. albicans, the PB-based formulation (PB-PLGA-Cp vaccine) was found to be most effective in the generation of desirable immune response, in terms of suppression of fungal load and facilitating the survival of the immunized animals.

Research paper thumbnail of Selective stabilization of a labile mutant form of bovine pancreatic ribonuclease A by antibodies

The region between the amino acids 31-46 was previously identified as being first exposed during ... more The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a ...

Research paper thumbnail of Immobilization and stabilization of horseradish peroxidase isoforms

IUBMB Life, 1996

S~Y-Purified anionic and cationic isoforms of horseradish peroxidase (HRP) immobilized by couplin... more S~Y-Purified anionic and cationic isoforms of horseradish peroxidase (HRP) immobilized by coupling the amino acid side-chain amino groups and/or carbohydrate moieties to Sepharose have been studied for their resistance to denaturation. The isoforms were treated with periodate followed by ethylenediamine to generate additional amino groups in the glycosyl residues. The immobilized preparations were: Preparation I (Sp-aHRP, Sp-cHRP), in which HRP was covalently immobilized via side-chain amino groups exclusively; Preparation II (Sp-NHaHRP, Sp-NHcHRP), in which periodate and ethylenediamine-treated HRP was covalently immobilized via sidechain amino groups and amino groups incorporated into glycosyl residues. HRP isoforms in preparation II lacked about 33-55 % carbohydrate. Both strategies of immobilization induced significant stabilization against denaturation. Inclusion of 2 mM calcium enhanced isoenzyme stability significantly.

Research paper thumbnail of Batch kinetics and modelling of ethanolic fermentation of whey

International Journal of Food Science and Technology, 2005

The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying la... more The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L)1 , the production of ethanol was 0.618 g L)1 whereas at 50 g L)1 it was 3.98 g L)1. However, an increase in lactose concentration to 100 g L)1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L)1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S 0 ¼ 10 and 50 g L)1 but failed in case of S 0 ¼ 100 g L)1 because of strong substrate inhibition.

Research paper thumbnail of Immobilization of the restriction endonuclease EcoRI usefulness of a polyclonal antibody support

Enzyme and Microbial Technology, 1999

The restriction enzyme EcoRI has been immobilized by using an immunoaffinity-based procedure on S... more The restriction enzyme EcoRI has been immobilized by using an immunoaffinity-based procedure on Sepharose 4B. The antibody support, prepared by coupling the ␥-globulin fraction of serum of immunized rabbits to CNBr-activated Sepharose-4B, was highly effective in binding EcoRI from solution although only about half of the bound activity appeared to be expressed by the immobilized preparations. Restriction activity of EcoRI immobilized on the antibody support was indistinguishable from that of soluble enzyme on the linear-DNA and supercoiled plasmids pBR322 and pBHLUC P. Binding to the antibody support markedly enhanced the resistance of EcoRI to heat inactivation, and the preparation, unlike the native enzyme, retained significant activity after exposure to a temperature of 65°C. It was also possible to immobilize EcoRI directly from the cell lysates of Escherichia coli pMB4, an EcoRI overproducing strain. The immobilized preparation did not possess nonspecific nuclease activity that was prominent in the lysates, suggesting specificity in the binding of EcoRI to the antibody support.

Research paper thumbnail of Stabilization of yeast invertase by insoluble immunocomplex formation using unfractionated glycosyl-specific and peptide specific polyclonal antiinvertase antibodies

Biotechnology Techniques, 1995

A strategy for the production of non-inhibitory polyclonal antisera for the immobilization of gly... more A strategy for the production of non-inhibitory polyclonal antisera for the immobilization of glycoenzymes is described. Insoluble complexes of invertase prepared using antiinvertase glycosyl antisera were more resistant to heat and urea induced denaturation than those prepared using antiinvertase antisera from which the glycosyl specific antibodies were removed. Unfractionated antiinvertase antibodies however gave most stable immunocomplexes with invertase

Research paper thumbnail of Bioaffinity Based Oriented Immobilization of Stem Bromelain

Biotechnology Letters, 2006

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas... more Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 degrees C for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K (m) values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V (max) values remained unaffected on immobilization.

Research paper thumbnail of Entrapment of concanavalin A-glycoenzyme complexes in calcium alginate gels

Biotechnology and Bioengineering, 1985

Glucose oxidase, invertase, and amyloglucosidase were entrapped in calcium alginate gels as conca... more Glucose oxidase, invertase, and amyloglucosidase were entrapped in calcium alginate gels as concanavalin A complexes in order to prevent the leaching out of the enzymes from the porous matrix. The free as well as the gel-entrapped concanavalin A-glycoenzyme complexes exhibited a relatively high effectiveness factor, eta, indicating good accessibility to the substrates. Concanavalin A-invertase complex exhibited marked broadening of pH-activity and temperature-activity profiles and was highly resistant to temperature inactivation even after entrapment in the alginate beads. It was possible to entrap considerable quantities of invertase as concanavalin A complex in the beads without a marked decrease in eta. A column containing crosslinked concanavalin A-invertase complex entrapped in alginate beads retained the ability to completely hydrolyze 1M sucrose even after continuous operation for over four months.