Immobilization of Escherichia coli cells containing aspartase activity with kappa-carrageenan. Enzymic properties and application for L-aspartic acid production.

Whole cells of Escherichia coli having high aspartase (L-asparate ammonialyase, EC 4.3.1.1) activity were immobilized by entrapping into a kappa-carrageenan gel. The obtained immobilized cells were treated with glutaraldehyde or with glutaraldehyde and hexamethylenediamine. The enzymic properties of three immobilized cell preparations were investigated, and compared with those of the soluble aspartate. The optimum pH of the aspartase reaction was 9.0 for the three immobilized cell preparations and 9.5 for the soluble enzyme. The optimum temperature for three immobilized cell preparations was 5--10 degrees C higher than that for the soluble enzyme. The apparent Km values of immobilized cell preparations were about five times higher than that of the soluble enzyme. The heat stability of intact cells was increased by immobilization. The operational stability of the immobilized cell columns was higher at pH 8.5 than at optimum pH of the aspartase reaction. From the column effluents, L-aspartic acid was obtained in a good yield.     

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.     

Various properties of galactose oxidase from Fusarium graminearum IMV-F-1060 immobilized on aminoorganosilochromes

Galactose oxidase preparations are obtained from Fusarium graminearum IMV-F-N 1060 immobilized on aminoorganosilochromes activated by cyanuron chloride and 2.4-toluylene diizocyanate. The immobilized preparations were studied for their selective action on different carbohydrate substrates and for the pH-medium dependence of the obtained preparation activity. Potassium ferricyanide is established to have an activating effect on the immobilized enzyme. It is shown that the immobilized galactose oxidase preparations may be used for the analysis of galactose and lactose.     

Expression of secreted His-tagged S-adenosylmethionine synthetase in the methylotrophic yeast Pichia pastoris and its characterization, one-step purification, and immobilization

S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.     

Evaluation of the performance of immobilized penicillin G acylase using active-site titration

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.     

Biocomplex of subtilisin Carlsberg with chitosan as an effective biocatalyst for hydrolysis and synthesis of peptides

New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplex (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocata-lytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6 : 4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min.     

Biocomposite of subtilisin Carlsberg with chitosan as an effective biocatalyst for hydrolysis and synthesis of peptides

New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplosite (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocatalytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6:4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min.     

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.     

Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.     

Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus

First, the enzyme immobilized on cyanide bromide agarose beads (CNBr) (that did not involve all enzyme subunits in the immobilization) has been crosslinked with aldehyde-dextran. This preparation did not any longer release enzyme subunits and become fully stable at pH 4 and 25 °C.Then, the stabilities of many different enzyme preparations (enzyme immobilized on CNBr, that derivative further crosslinked with aldehyde-dextran, enzyme immobilized on highly activated amino-epoxy supports, GDH immobilized on supports having a few animo groups and many epoxy groups, GDH immobilized on glyoxyl-agarose beads at pH 7, and that preparation further incubated at pH 10, and finally the enzyme immobilized on this support directly at pH 10) were compared at pH 4 and high temperatures, conditions where both dissociation and distortion play a relevant role in the enzyme inactivation. The most stable preparation was that prepared at pH 7 and incubated at pH 10, followed by GDH immobilized on amino and epoxy supports and the third one was the enzyme immobilized on glyoxyl-agarose at pH 10.The incubation of all enzyme preparations in saturated guanidine solutions produced the full inactivation of all enzyme preparations. When not all enzyme subunits were immobilized, activity was not recovered at all. Among the other derivatives, only glyoxyl preparations (the most inert supports and those where a more intense multipoint covalent attachment were expected) gave significant reactivation when re-incubated in aqueous medium. After optimization of the reactivation conditions, the enzyme immobilized at pH 7 and later incubated at pH 10 recovered 100% of the enzyme activity.     

Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. full hydrolysis of lactose in milk

The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.     

Production of palatinose using<Emphasis Type="Italic"> Serratia plymuthica</Emphasis> cells immobilized in chitosan

In recent decades, the production of palatinose has aroused great interest since this structural isomer of sucrose has interesting potential. We describe a simple and effective method of immobilizing Serratia plymuthica cells in chitosan. The sucrose isomerase activity of immobilized preparations was enhanced many times by activation with fresh nutrient medium and subsequent drying. The preparations obtained were physically very stable with high enzyme activity and excellent operational stability. The effect of temperature, pH and substrate concentration on enzyme activity of the immobilized cells was investigated. Using immobilized cells, a complete conversion of sucrose (40% solution) into palatinose was achieved in 4 h in a "batch"-type enzyme reactor. The use of free or immobilized cells had no effect on the composition of the solution, in particular the sugar content. The palatinose content was 80% and that of trehalulose 7%.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Sol–gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents

Lipase PS from Burkholderia cepacia (formerly Pseudomonas cepacia) was successfully immobilized in sol–gels under low methanol conditions using lyophilization in order to dry the gel. The enzyme was also cross-linked with glutaraldehyde to CLEAs without any additives. These immobilized enzyme preparations were employed for the highly enantioselective acylations of 1-phenylethanol (1), 1-(2-furyl)ethanol (2) and N-acylated 1-amino-2-phenylethanol (3) with vinyl acetate in organic solvents. Enzymatic hydrolysis of the obtained ester product was observed as a side reaction of the acylation of 3 in the presence of lipase PS powder. Hydrolysis was suppressed when the immobilized preparations of lipase PS were used.     

Evidence for alpha-ketoglutarate dehydrogenase monomer activity with the aid of enzyme immobilization

Immobilization of pigeon breast muscle alpha-ketoglutarate dehydrogenase on CNBr-activated Sepharose 4B was carried out. Conditions for dissociation of the dimeric enzyme bound to the carrier via a single subunit were determined. Immobilized enzyme monomers with a specific activity higher than that of the dimer were obtained. The immobilized subunits are capable of reassociating with the soluble ones; this is accompanied by the restoration of the initial amount of the matrix-bound protein and the reconstitution of the activity typical of the immobilized enzyme original preparations.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

Stabilization of soluble and immobilized horse liver alcohol dehydrogenase by adenosine 5'-monophosphate

Horse liver alcohol dehydrogenase, which catalyzes oxidoreductions for a broad spectrum of substrates of organic chemical interest, was immobilized on CNBr-activated Sepharose and on decylamine-substituted agarose. The specific activities of the immobilized enzyme preparations were compared with the free enzyme, and the apparent K(m) values of the preparations were determined for a selection of substrates. At pH 9 and 60 degrees C, soluble liver alcohol dehydrogenase was rapidly inactivated, while the enzyme immobilized on CNBr-activated Sepharose was more stable. Adenosine monophosphate (AMP), adenosine diphosphate, and adenosine diphosphoribose protected the free and immobilized alcohol dehydrogenase against heat inactivation. On storage under a variety of conditions, AMP effectively stabilized free horse liver alcohol dehydrogenase and the immobilized preparations.     

Comparison of the thermolability and hydrophobic properties of high- and low-molecular-weight forms of rabbit liver arginyl-tRNA synthetase

Summary Two preparations with arginyl-tRNA synthetase activity have been obtained from rabbit liver post-microsomal fraction: a) a high-molecular-weight containing the multienzyme aminoacyl-tRNA synthetase complex and b) a low-molecular-weight preparation containing free enzymes. Thermal inactivation of arginyl-tRNA synthetase in both preparations has been compared in a solution which was successively supplemented with tRNA, reduced glutathione, L-ascorbic acid, ZnCl₂ and Triton × 100. Moreover, hydrophobic properties of both enzyme preparations have been compared. It was found that the complexed arginyl-tRNA synthetase is more stable than the free enzyme. A role of hydrophobic interactions in the maintenance of the complexed enzyme stability is suggested.Abbreviations DFP Diisopropylfluorophosphate - GSH Glutathione (reduced) - PMSF Phenylmethylsulfonyl Fluoride - Ap₄A Diadenosine 5, 5-P₁, P₄-tetraphosphate - Preparation I high-molecular-weight arginyl-tRNA synthetase preparation - Preparation II low-molecular-weight arginyl-tRNA synthetase preparation     

Activity and stability of glucoamylase preparations in different methods of immobilization

Catalytic activity and stability of glucoamylases immobilized by different methods (adsorption, covalent binding) are studied comparatively. The highest stability is shown to be obtained under covalent binding. The binding efficiency and immobilized glucoamylase properties depend on the nature of insoluble carrier and a purification degree of the enzyme preparations. The choice of the cross-linking agent promoting a binding between the enzyme and the carrier is very significant. The activity and stability of immobilized glucoamylases obtained when using different cross-linking agents rise in such a sequence: 2,4-toluylenediisocyanate, cyanurochloride, glutaric dialdehyde, gossypol. Catalytic properties and stability are determined for soluble and immobilized glucoamylase forms from different sources.