M. Saleemuddin

Alkaline proteases: A review

In recent years there has been a phenomenal increase in the use of alkaline proteases as industrial catalysts. These enzymes offer advantages over the use of conventional chemical catalysts for numerous reasons, for example they exhibit high catalytic activity, a high degree of substrate specificity, can be produced in large amounts and are economically viable. Although many other classes of enzymes are currently in industrial use, the focus of this review is on alkaline proteases from various sources. This is because the organisms producing enzymes capable of catalysing the reactions at the extremes of pH above neutrality, having been considered as oddities thereby receiving little attention until the last decade. A fresh look at some aspects of stabilization of alkaline proteases, their limitations and future strategies is also included.

Concanavalin A: A useful ligand for glycoenzyme immobilization—A review

Concanavalin A is finding increasing applications as a useful ligand in glycoenzyme immobilization. An attempt therefore, has been made to summarize the work available in the area. Glycoenzymes that are recalcitrant to immobilization procedures involving covalent coupling to solid supports can be immobilized in high yields by binding to matrices precoupled with concanavalin A. In addition, glycoenzymes associated with concanavalin A matrices usually exhibit high retention of activity and enhanced stability against various forms of inactivation. Binding of the glycoenzymes on the concanavalin A supports, being noncovalent, can be reversed by incubating the preparation with a high concentration of sugars/glycosides or at acidic pH. The association can be, however, rendered covalent by crosslinking the preparations with bifunctional reagents like glutaraldehyde. Crosslinking may be accompanied by further increase in stability, albeit at the expense of the loss of some enzyme activity. Several laboratory-size reactors containing concanavalin A matrix-bound glycoenzyme have been successfully operated for reasonably long durations with only small losses in catalytic activity. Insoluble glycoenzyme preparation can also be obtained by precipitating them from solution as concanavalin A complexes. Such complexes have small particle dimensions but can be successfully used in column reactors after a subsequent immobilization step. Insoluble concanavalin A-flocculates containing various microorganisms and glycoenzymes that successfully carry out multistep transformations have also been obtained by several investigators.

Alkaline protease from Spilosoma obliqua: potential applications in bio‐formulations

Some properties of the purified alkaline protease from larvae of the insect Spilosoma obliqua (Lepidoptera) and its potential application as an additive in various bio‐formulations are reported. The novel feature of the present study is the use of insect protease. The protease was found to be compatible with some of the commercial detergents tested, and was also effective in cleaving various protein substrates tested, albeit to different extents, implying broader substrate specificity and effectiveness of the protease against a wide variety of stains. This property of the protease can also be exploited by using it as an active component in enzymic debriders in view of its ability to digest various protein substrates. The insect protease appears to be potentially useful as an additive in detergent, stain remover and other bio‐formulations.

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

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 (eta) 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 cerevisiae.

A simple, rapid, and sensitive procedure for the assay of endoproteases using coomassie brilliant blue G-250

Abstract A rapid colorimetric method for the assay of proteolytic enzymes based on the binding of Coomassie brilliant blue G-250 to unhydrolyzed protein substrate is described. Considerable assay time is saved since the method does not require the separation of the hydrolyzed products from the undergraded protein substrate. The procedure is applicable to crude as well as purified preparations of various proteolytic enzymes and compares well with the procedure of M. L. Anson.

Glycoprotein Targeting and Other Applications of Lectins in Biotechnology

Glycoconjugates comprise a variety of structures, include glycoproteins and glycolipids and are found on the surfaces of animal and plant cells, as well as on the surface of microorganisms. Determination of the structure and the distribution of glycoconjugates on cell surfaces are important for the understanding their biological function. Lectins are useful to investigate protein-carbohydrate interactions, because they have specificity for defined carbohydrate structure. They have been implicated in cell-to-cell recognition and signaling, blood group typing, in immune recognition process, and various other biological processes, such as viral, bacterial, mycoplasmal and parasitic infections, fertilization, cancer metastasis, growth and differentiation. Once thought to be confined to plant seeds, lectins are now recognized as ubiquitous in virtually all living systems, ranging from viruses and bacteria to animals. Plant lectins provide a rich source of carbohydrate-recognizing protein reagents for glycobiologists and biotechnologists. Biotechnology offers the therapeutic use of lectin against certain life threatening diseases such as human immunodeficiency virus and cancer. This review presents a comprehensive summary of research efforts that focus on the actual and potential uses and advantages of using lectins to target glycoproteins and also glycoproteins to target lectins.

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.

Oleic Acid May Be the Key Contributor in the BAMLET-Induced Erythrocyte Hemolysis and Tumoricidal Action

A chance discovery of the tumoricidal action of a human milk fraction led to the characterization of the active component as oleic acid complex of the α-lactalbumin, which was given the acronym HAMLET. We report in this study that the oleic acid complex of bovine α-lactalbumin (BAMLET) is hemolytic to human erythrocytes as well as to those derived from some other mammals. Indirect immunofluorescence analysis suggested binding of BAMLET to erythrocytes prior to induction of hemolysis. Free OA was hemolytic albeit at higher concentrations, while sodium oleate caused hemolysis at far lower concentrations. Amiloride and BaCl2 offered protection against BAMLET-induced hemolysis suggesting the involvement of a cation leak channel in the process. BAMLET coupled to CNBr-activated Sepharose was not only hemolytic but also tumoricidal to Jurkat and MCF-7 cells in culture. The Sepharose-linked preparation was however not toxic to non-cancerous peritoneal macrophages and primary adipocytes. The tumoricidal action was studied using the MTT-assay while apoptosis induction measured by the annexin V-propidium iodide assay. Repeated incubation of the immobilized BAMLET with erythrocytes depleted oleic acid and decreased the hemolytic activity of the complex. Incubation of MCF-7 and Jurkat cells with OA, soluble or immobilized BAMLET resulted in increase in the uptake of Lyso Tracker Red and Nile red by the cells. The data presented support the contention that oleic acid plays the key role, both in BAMLET-induced hemolysis and tumoricidal action.

Effects of chemical modification on the stability of invertase before and after immobilization

Invertase from bakers yeast immobilized by coupling the amino acid side-chain amino groups or glycosyl residues to the matrix has been studied for its resistance to heat and urea-induced denaturation, to understand the influence of the nature of immobilization procedure on these processes. The possible role of chemical modifications and/or carbohydrate depletion to which the enzyme was subjected was also examined by investigating the properties of soluble modified enzyme. The immobilized preparations obtained were Sp-INV by direct coupling of invertase to Sepharose, Sp-PEA-INV by coupling of periodate and ethanolamine-treated invertase to Sepharose, Sp-PEDA-INV by coupling of periodate and ethylenediamine-treated invertase to Sepharose, and Sp-PEDA-2-4-6-trinitrobenzene sulfonic acid (TNBS)-INV by coupling of TNBS followed by periodate and ethylenediamine-treated invertase to Sepharose. All of the immobilized preparations exhibited higher stability against heat and urea-induced inactivation as compared to native invertase. Among the procedures employed for immobilization of invertase, the Sp-PEDA-INV preparation exhibited highest yield of immobilization, η value, and thermal and storage stability. The yield of immobilization and stabilization was followed by Sp-PEA-INV, Sp-INV, and Sp-PEDA-TNBS-INV preparations.

Stabilization of pancreatic ribonuclease A by immobilization on Sepharose-linked antibodies that recognize the labile region of the enzyme

The stabilizing potential of the antibodies recognizing the labile region of pancreatic ribonuclease A (RNase) has been investigated. The dodecapeptide SRNLTKDRAKPV corresponding to the labile region 32--43 on RNase was synthesized by the solid-phase method. Antiserum raised against the dodecapeptide-bovine serum albumin conjugate showed good cross-reactivity with the peptide and native RNase. RNase immobilized on Sepharose support precoupled either with the antipeptide immunoglobulin (IgG) or anti-RNase IgG proved to be more resistant to thermal inactivation than the soluble enzyme. Besides, stability against inactivation by trypsin at 55 degrees C was markedly high when enzyme was immobilized on the antipeptide IgG support, as compared to the soluble and other immobilized preparations. These results suggest that matrices bearing antibodies recognizing specific labile regions of enzyme may be useful in selectively improving their stability against specific forms of inactivation.