Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins

Abstract

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Simultaneous purification and immobilization of bitter gourd (Momordica charantia) peroxidases on bioaffinity support

“…12 -14 Ease of immobilization, lack of chemical modification and usually accompanying enhancement in stability are some of the advantages offered by the bioaffinity-based procedures, which also offer the additional benefit of orienting the enzyme favorably on the support. 15, 16 These supports have been used for the high yield and stable immobilization of glycoenzymes/enzymes. Bioaffinity-based procedures can be employed for the immobilization of enzymes directly from the crude homogenate or partially purified enzyme preparation.…”

“…Several earlier reports described that the immobilization of glycoenzymes on Con A support resulted in the stabilization of enzymes against various forms of denaturation. 12,16 Protease resistance is an additional attribute of the bioaffinity-based immobilization of BGP. It indicated that Con A-Sephadex-bound BGP preparation has great potential in the treatment of organic pollutants present in industrial effluents.…”

Application of Immobilized Pointed Gourd (Trichosanthes dioica) Peroxidase-Concanavalin A Complex on Calcium Alginate Pectin Gel in Decolorization of Synthetic Dyes Using Batch Processes and Continuous Two Reactor System

“…This process can immobilize enzyme directly from crude homogenate overcoming the high cost of purification which employs commercially available enzyme/expensive supports [12]. Besides conferring ease to immobilize proteins, other advantages of such protocols include lack of chemical modification, proper orientation of enzyme on the support, high yield and enhanced stability of glycoenzymes/enzymes [13].…”

“…Lectins are proteins which recognize and interact with exposed carbohydrate moieties of glycoproteins and glycoenzymes. These proteins are useful in characterizing glycoproteins and certain glycoenzymes have been immobilized on concanavalin A affinity matrices or as Con A-glycoenzyme complexes [13,17].…”

A Study on the Comparative Stability of Insoluble Complexes of Glucose Oxidase Obtained with Concanavalin A and Specific Polyclonal Antibodies

“…Bioaffinity-based procedures, due to their reversibility, lack of chemical modification and their usually accompanying stability enhancement are emerging as powerful strategies for the immobilization of several enzymes (Saleemuddin & Husain 1991;Saleemuddin 1999;Mislovicova et al 2000;McMahon & Mulcahy 2002). The strong affinity of polyclonal/monoclonal antibodies for specific enzymes and those of lectins for glycoenzymes bearing appropriate oligosaccharides have been generally employed for this purpose (Shami et al 1991;Husain & Saleemuddin 1986, 1989.…”