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.     

The isolation of lytic enzymes from Cytophaga and their application to the rupture of yeast cells

Yeast–lytic enzymes have been isolated on a pilot scale from Cytophaga species by precipitation and the light, enzyme-rich solid phase recovered by liquid-liquid separation. The enzyme complex was immobilized to soluble polymeric carbohydrates and the effectiveness of the free and immobilized enzyme for protein release and cell debris dissolution has been assessed.     

Immobilization of cellulolytic and hemicellulolytic enzymes on inorganic supports

Commercial cellulase preparations from Trichoderma viride and Aspergillus niger were immobilized on porous silica glass and ceramics such as alumina and titania with titanium tetrachloride (TiCl(4)) and on their silanized derivatives with glutaraldehyde (GLUT). The amounts of the immobilized enzymes were in the range 10-50 mg/g carrier (dry) depending on the kind of carrier and immobilization method. Their activities toward carboxymethyl cellulose (CMC), xylan, aryl-beta-glucoside, and aryl-beta-xyloside were 3-53% of those of the native enzymes. The optimum pH of the enzymes shifted to the acidic side in most cases, whereas the optimum temperatures were nearly the same as those of native ones. The activity of immobilized enzyme preparations towards CMC did not change significantly during continuous operation over a periods of 60 days. Finally, xylan was hydrolyzed with the immobilized enzymes, and the sugars formed were investigated.     

Urease immobilization on macroporous silicas

Urease was immobilized on macroporous silicas using gamma-aminopropyl triethoxysilane and glutaraldehyde. The amount of protein on the surface, the structure of pores of the support and the purity of the initial enzyme were varied, the enzymic activity of the immobilized preparations being controlled. After the immobilization of sufficiently large quantities of the enzyme (about 3 mg protein per m2 support) about 35% of the specific activity was retained. The maximum activity per unit weight of the support was observed for silicagels and silochromes with the mean diameter of pores 70-90 nm and the specific surface area about 70 m2/g. The use of purified urease produced highly active preparations of the immobilized enzyme (17,000 U per g dry support). Freeze-drying of the immobilized enzyme in the presence of sorbitol yielded dry preparations retaining their activity.     

Transfer of Sucrose-Fermenting Ability and Nisin Production Phenotype among Lactic Streptococci

Transfer of sucrose fermentation ability, nisin production, and nisin resistance from Streptococcus lactis to S. lactis and Streptococcus lactis subsp. diacetylactis occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Transconjugants were able to act as donors to transfer the Suc-Nis phenotype in subsequent mating. No changes in sensitivity to lytic phage c2 were noted in S. lactis transconjugants. However, temperature-independent restriction of lytic phage 18-16 was noted in transconjugants of S. lactis subsp. diacetylactis 18-16. Adsorption studies with phage-resistant transconjugants showed that resistance was not due to lack of adsorption by the lytic phage. Physical evidence for the presence of introduced plasmid DNA was not found in lysates of transconjugants.     

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.     

Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.     

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.     

Purification of pancreatic kallikrein by the method of affinity chromatography]

Pancreatic Kallikrein was purified by affinity chromatography on BPTI-Sepharose. Immobilized BPTI was prepared via three stages: a) formation of the BPTI--acetylated trypsin complex; b) coupling of the resultant complex with CNBr-activated Sepharose; c) dissociation of the bound complex at pH 2.0 to yield immobilized BPTI and acetylated trypsin. The final product contained 59 X 10(-3) micronmoles insolubilized BPTI/ml Sepharose. Trypsin occurring in commercial Kallikrein preparations was separated by the batch procedure with ovomucoid-Sepharose. This method allows 100-fold purification of commercial Kallikrein.     

Comparison of the efficiency of polygalacturonase and beta-glucosidase enzyme preparations in stabilization of cherry plum wine material

Pektofoetidin and Pectinex, enzyme preparations with the highest polygalacturonase and beta-glucosidase activities, were covalently immobilized on DEAE cellulose and Aminosilochromes 10 and 30. After treatment of cherry plum wine material with the soluble and immobilized enzyme preparations, the content of phenolics increased by 26 and 40%, respectively. The increase was accompanied by a decrease in the protein content (by up to 37%), carbohydrate content (by 17% on the average), and antioxidant activity (5-37%). The most efficient treatment involved Pektofoetidin immobilized on Aminosilochrome 10. It increased the clarity of the wine material and its antioxidant activity by 100 and 10%, respectively.     

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.     

Enzymatic synthesis of beta-lactam antibiotics using penicillin-G acylase in frozen media.

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.     

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Reduction and activity of cytochrome c in the cytochrome c-cytochrome aa3 complex.

An interaction between rat liver glucocorticoid--receptor complex and immobilized ATP was identified. Rat liver cytosol preparations were incubated with [3H]triamcinolone acetonide for 4 h at 4 degrees C and partially purified by precipitation with (NH4)2SO4 before use. The resulting glucocorticoid--receptor complex could be selectively adsorbed on to columns of ATP--Sepharose. The freshly prepared cytosol [3H]triamcinolone acetonide--receptor complex had very little affinity for binding to the ATP--Sepharose column, but acquired this ability on temperature- or salt-activation. The presence of 10 mM-sodium molybdate during this salt- or temperature-dependent activation blocked the binding of the receptor complex to ATP--Sepharose. The interaction is reversible, since it can be disrupted by high-salt conditions. A competitive binding assay, using free nucleotides in samples to be chromatographed, revealed a preferential interaction between ATP and the glucocorticoid--receptor complex. Buffer containing ATP was also used to elute the glucocorticoid--receptor complex from ATP--Sepharose columns successfully. When ATP was added to the preparations containing [3H]triamcinolone acetonide--receptor complexes, the steroid specificity or sedimentation properties of the complex remained unaltered. Our results demonstrate an interaction between rat liver glucocorticoid--receptor complex and immobilized ATP and suggest a role of this nucleotide in receptor function.     

Biochemical studies on the cell wall degradation of Fusarium oxysporum f. sp. lycopersici race 2 by its own lytic enzymes for its biocontrol

An exhaustive cell wall fractionation of Fusarium oxysporum f. sp. lycopersici race 2 ( Fol 2) with alkali in a sequential procedure yields only three polysaccharide fractions: F1_s (alkali and water soluble), F1₁ (alkali soluble and water insoluble) and F4 (alkali-insoluble residue). These fractions amounted respectively to 15, 1.3 and 52% of the cell wall and have been characterized by infra-red spectroscopy and gas liquid chromatography-mass spectrometry (GLC-MS). F1_s is a β-gluco-galacto-mannan, F1₁ is mainly composed of a β-1, 3-glucan and F4 is a β-1,3-glucan-chitin complex. The F1_s is a very complex polysaccharide and its hydrolysis requires the action of different enzymes. The lysis of the cell wall and its three fractions with lytic enzymes from Fol 2 has been studied and a correlation between the lysis of the cell wall and the lysis of these fractions was found. The amount of glucose, galactose and mannose in F1_s and cell wall hydrolysates were quantified by GLC and they indicated the hydrolysis of the gluco-galacto-mannan polysaccharide. In the hydrolysis of F4 and cell walls N -acetylglucosamine was also found and quantified. When chlamydospores of this fungus were treated with Fol 2 lytic enzymes, the sugars liberated were principally mannose and N -acetylglucosamine. These results indicate that Fol 2 produces during its autolysis the necessary enzymes to hydrolyse its own cell walls. This fact suggests that a biological control of Fol 2 with its own lytic enzymes, conveniently immobilized, could be developed.     

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.     

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.     

Preparation of an alcohol-dehydrogenase--NAD(H)--sepharose complex showing no requirement of soluble coenzyme for its activity.

1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3. A molar ratio of 1:1 between the amount of bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.     

Immobilization of β-glucosidase on an inorganic carrier

The enzymatic hydrolysis of cellobiose, an important intermediate of the decomposition of cellulose containing materials, with immobilized β-glucosidase preparations from Geotrichium candidum, Trichoderma lignorum and Aspergillus foetidus was examined At first it was the aim to prepare from differently purified samples with different specific cellobiase activities high active preparations on the basis of the inorganic carrier Silochrom S-80. Characteristics e.g. thermal stability and temperature and pH optimum of immobilized preparations were compared with those of soluble preparations Kinetics of cellobiose hydrolysis by immobilized enzyme preparations were studied.     

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.