Enzymes immobilized on magnetic carriers: efficient and selective system for protein modification

In order to obtain an economical, efficient and selective system for glycoprotein modification we prepared reactors with immobilized neuraminidase or (and) galactose oxidase. High storage and operational stability of the enzyme reactors was obtained by their immobilization through the carbohydrate parts of the enzyme molecules to hydrazide-modified supports. Magnetic and non-magnetic forms of bead cellulose and poly(HEMA-co-EDMA) microspheres were used for immobilization. These reactors can be used almost universally for the activation of ligands and for labelling of substances having a carbohydrate moiety.     

Immobilization of lipase from Candida rugosa on Eupergit C supports by covalent attachment

The present study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit^® C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75 °C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t_1/2 values obtained at 75 °C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system.     

Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glucosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9)) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFP and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.     

Rat iso-alpha1-fetoproteins. Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha 1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adsorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glu-cosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha 1-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFT and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.     

Immobilization of glucose oxidase via carbohydrate moiety onto hydrazide derivative of polyacrylate-coated porous glass

Summary The hydrazide derivative of polyacrylate-coated glass was used for the immobilization of glucose oxidase (GO) from Penicillium vitale after periodate oxidation of its carbohydrate moiety. Periodate oxidation of the carbohydrate residues did not influence catalytic activity of the enzyme. Immobilized GO is extremely stable both at 4°C and 25°C for extended period.     

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.     

Role of carbohydrate moiety in carboxypeptidase Y: structural study of mutant enzyme lacking carbohydrate moiety.

To study the roles of the carbohydrate moiety in the function of carboxypeptidase Y, asparagine residues at 13, 87, 168, and 368, the four-consensus N-linked glycosylation sites, were altered to alanine with site-directed mutagenesis. The mutant enzyme of 51 kDa completely lost the carbohydrate moiety which was present in the 61-kDa wild-type enzyme. Structural studies of the mutant enzyme showed that it maintained the native-like structure; hydrolytic activity, and substrate specificity of the mutant enzyme analogous to those of the wild-type enzyme. Susceptibility of the mutant enzyme toward proteolysis and pressure denaturation was reduced by 10-20%. It is concluded that the carbohydrate moiety functions to maintain the structural integrity of the enzyme under stressed.     

Immobilized chymotrypsin by means of Schiff base copper(II) chelate

A new method for cross-linking of protein was proposed in our previous work. The method is based on the spontaneous chelate formation process involving three components, salicylaldehyde moiety, alpha-amino acid residue and copper(II). In this paper versatility of the method as a purpose of immobilization of enzyme was described. Chymotrypsin-salicylaldehyde conjugate was immobilized to the agarose gel attached alpha-amino acid residue in the presence of copper(II) ion The enzyme was not eluted from the gel by washing with a copper free buffer though it was exclusively eluted by a medium containing EDTA. Catalytic activity of the chymotrypsin salicylaldehyde conjugate was not changed upon the immobilization. The method was proposed as a new tool for reversible immobilization of enzyme.     

Effect of periodate oxidation on the structure and properties of glucose oxidase.

In order to elucidate the molecular structure of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and the roles of its carbohydrate moiety, chemical, physiochemical and immunological experiments were performed with enzyme samples before and after periodate oxidation. Hydrodynamic parameters indicated that the native enzyme was a globular protein with values of 1.21 for the frictional ratio and 43 A for the Stokes radius. The enzyme contained about 12% carbohydrate by weight, of which the main component was mannose. The periodate treatment decreased the carbohydrate content to about 40% of its original value. Slight modifications were detected in the absorbance spectrum and the content of arginyl residue. However, no significant alteration was brought about by this treatment in the catalytic parameters, immunological reactivities of the gross structure, not in the secondary and quaternary structures of the protein moity. Thermal denaturation temperature (about 72.5 degrees C) and the enthalpy of denaturation (about 450 kcal/mol) were common to the native and the periodate-oxodozed enzymes. The native was found to be quite resistant to sodium dodecyl sulfate and fairly stable to urea and heating. The periodate-oxidized enzyme was also stable to heat treatment, but it showed a diminished stability when denaturing agents were present. Kinetic analyses of the thermal inactivation processes showed that the entropy of activation was greatly decreased by the denaturing agents, especially in the case of the periodate-oxidized enzyme. It is concluded that the carbohydrate moiety of the enzyme plays a role in increasing the stability of the protein moiety, but does not directly participate in the catalytic activity, the immunological reactivity, or in maintaining the conformation of the enzyme protein.     

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.     

Effects of inhibitors of N-linked oligosaccharide processing on the secretion, stability, and activity of lecithin:cholesterol acyltransferase

The structure and function of the carbohydrate moiety of human lecithin:cholesterol acyltransferase (LCAT) were determined by using several glycosidases in reaction with the isolated plasma protein or by using specific inhibitors of glycoprotein assembly with cultured cells secreting LCAT activity. Analysis of the plasma enzyme indicated that almost all of the large carbohydrate moiety of LCAT (approximately 25% w/w) was N-linked with part of the high-mannose and part of the complex type. This analysis was confirmed with metabolic inhibitors of carbohydrate processing by using CHO cells stably transfected with the human LCAT gene. Inhibitors of the subsequent processing of the N-linked high-mannose chains formed by glucosidase activity were without effect on either the secretion rate or the catalytic activity of LCAT. The inhibition of catalytic activity by glucosidase inhibitors applied to both the phospholipase and the acyltransferase activities of LCAT. The reduction of the LCAT catalytic rate by terminal glycosidase inhibitors was without effect on apparent Km and did not affect enzyme stability. These data indicate an unusual specific role for high-mannose carbohydrates in the catalytic mechanism of LCAT.     

Fungal glucoamylases

The purification and properties of glucoamylase (α-l,4-glucan glucohydrolase, EC 3.2.1.3) from different fungal sources have been compared. The studies on the conformation and activity of the native enzyme at a function of pH, temperature, substrate concentration and the effect of denaturants and on the role of carbohydrate moiety on structure and stability have been reviewed. The chemical modification of the active centre, binding kinetics of the substrate and active site and the mechanism of action have been summarized. They differ in their fine structure as revealed by their near ultra-violet circular dichroism spectra and contain 30–35 % α-helix, 24–36 %Β-structure and the rest aperiodic structure. The activity of the enzyme is very sensitive to the environment around aromatic aminoacid residues. The glucoamylases are glycoprotein in nature, differ in their content and nature of carbohydrate from different sources. The carbohydrate moiety plays an important role in stabilising the native conformation of the enzyme and is not involved in activity and antigenecity. At the active site of the enzyme, two tryptophan and two carboxyl (glutamate or aspartate) groups are present. It is likely that the histidine and tyrosine residues which are present away from the active site are involved in binding of the substrate. There seems to be seven subsites which are involved in binding of the substrate and the catalytic site is situated in between 1 and 2 subsites. In breaking of α-1,4-, α-1,3-, and α-l,6-bonds only one active centre is involved. Studies on the immobilization of either glucoamylase alone or as a part of a multienzyme system have been reviewed briefly     

Effect of partial deglycosylation on catalytic characteristics and stability of an avocado peroxidase

An isoperoxidase (EC 1.11.1.7) purified from avocado ( Persea americana Mill. cv. Topa Topa) leaves, lost 35% of its glycosidic moiety after incubation with glycopeptidase F (EC 3.2.2.18). The partial removal of carbohydrate chains caused a decrease in the K_m for reductor substrates tguaiacol, syringaldazine and indole-3-acetic acid) and favoured the enzyme activation by Ca²⁺. A decrease in stability against temperature was also observed. Our results are consistent with the hypothesis that the carbohydrate moiety located on the surface of the peroxidase molecule acts as a molecular shield.     

Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme

A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

Purification and characterization of the invertase from Schizosaccharomyces pombe. A comparative analysis with the invertase from Saccharomyces cerevisiae.

Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/1% SDS into identical subunits with an apparent molecular mass of 205 kDa. The carbohydrate moiety, accounting for 67% of the total mass, is composed of equimolar amounts of mannose and galactose. There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H. The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae. The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch. cerevisiae. Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar.     

Peroxidase from cultured carrot cells: affinity purification and characteristics

Peroxidase, from cultured carrot cells, has been isolated and purified over Concanavalin-A-Sepharose by lectin affinity chromatography. The enzyme protein registers a gradual enhancement in activity concomitant with the growth of callus. Extracellular (ECP) & intracellular (ICP) peroxidases have been demonstrated in suspension cultures of carrot.     

Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger was purified to homogeneity by hydrophobic interaction and ion-exchange chromatography. Approx. 95% of the carbohydrate moiety was cleaved from the protein by incubation of glucose oxidase with endoglycosidase H and alpha-mannosidase. Cleavage of the carbohydrate moiety effected a 24-30% decrease in the molecular weight and a reduction in the number of isoforms of glucose oxidase. No significant changes were observed in the circular dichroism spectra of the deglycosylated enzyme. Other properties, such as thermal stability, pH and temperature optima of glucose oxidase activity and substrate specificity were not affected. However, removal of the carbohydrate moiety marginally affected the kinetics of glucose oxidation and stability at low pH. From these results it appears that the carbohydrate chain of glucose oxidase does not contribute significantly to the structure, stability and activity of glucose oxidase.     

Thermal Dissection of Lentil Seedling Amine Oxidase Domains by Differential Scanning Calorimetry

The relationships between the structural and energetic domains of lentil seedling amine oxidase (LSAO) were investigated using modifiers that target the active site and the carbohydrate moiety of the enzyme. An irreversible inhibitor, aminoguanidine, specifically modified the active site of the lentil enzyme, whereas sodium metaperiodate cleaves carbohydrate moieties covalently bound to the native enzyme. Differential scanning calorimetry (DSC) measurements were made on the modified LSAOs. Deconvolution of the reversible thermal DSC profiles of the modified enzyme gave three subpeaks (energetic domains), each of which was assigned to one of the three structural domains of the native protein. Our results led us to conclude that deglycosylation of LSAO has no effect on thermal stability, whereas binding of the inhibitor imparts more stability to the enzyme.     

Properties of the deglycosylatedβ-fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524

Summary The carbohydrate moiety of -fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524 was largely removed by exposure of the enzyme to endo--N-acetylglucosaminidase F; the total carbohydrate content of the enzyme was decreased from 53% (w/w) to 15% (w/w). The stability of the deglycosylated enzyme at pH 4 to 7 and 40 to 50°C was decreased and theK _m value for sucrose was increased from 0.65 to 1.43 M. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is concluded that the carbohydrate moiety of -fructofuranosidaseP-2 contributes to the stability of the enzyme as well as its affinity for sucrose.