Immobilization of glycoenzymes through carbohydrate side chains.

Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Isolation and identification of 2'-phosphoadenosine diphosphate, an alkali-degradation product of nicotinamide adenine dinucleotide phosphate.

Cellulose esters of both alkyl and aryl carboxylic acids have been prepared and tested as noncovalent adsorbents for enzymes. Phenoxyacetyl cellulose strongly bound all 10 of the enzymes tested. The bound enzymes, which were not desorbed by 1 m (NH₄)₂SO₄ or moderate (25–50%) concentrations of nonaqueous solvents but which were at least partially desorbed by solutions containing nonionic detergents, usually exhibited nearly complete retention of catalytic activity. Other materials, paper, string, cotton, and glass beads, have been analogously derivatized with similar results. This simple and effective technique warrants consideration for applications in enzyme immobilization.     

Immobilized enzymes: the catalytic properties of lactate dehydrogenase convalently attached to glass beads

Chick LDH (H₄ and M₄) has been covalently attached to aryl and alkyl amine glass using sodium nitrite and glutaraldehyde respectively. These immobilized enzymes remain active for months at 0°C and exhibit Km values similar to those of the soluble enzyme; however, they have pH-rate profiles that are independent of pH and show decreased substrate inhibition. Disaggregation followed by reassociation indicate the enzymes are bound by all four subunits and the resulting activity restored to the native, aryl amine and glutaraldehyde bound enzyme are 33, 25 and 90% respectively. At a pH of 3.2 and 25°, the soluble and aryl amine glass LDH's are rapidly denatured while the glutaraldehyde bound enzyme shows no loss of activity for at least 35 days.     

The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds

Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH₄)₂SO₄ fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (M_r) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a M_r of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - M_r apparent molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Studies on Horseradish Peroxidase Immobilized onto Arylamine and Alkylamine Glass

Peroxidase from horseradish has been immobilized onto zirconia coated arylamine and alkylamine glass through the process of diazotization and glutaraldehyde coupling, respectively. Arylamine glass bound enzyme retained 77% of the initial activity with a conjugation yield of 18 mg g^-1 support, while alkylamine glass bound enzyme retained 38% of the initial activity with a conjugation yield of 16 mg g^-1 support. The immobilized enzyme showed an increase in optimum pH, temperature for maximum activity, energy of activation (Ea), and thermal stability but decrease in time for linearity and Km for H₂O₂. Vmax value of arylamlne conjugated enzyme decreased but Vmax of alkylamine conjugated enzyme was unaltered compared to free enzyme. Both arylamine and alkylamine bound enzyme showed higher stability in cold compared to that of free enzyme. The application of glass bound peroxidase in discrete analysis of serum urate is demonstrated.     

Interaction of carboxypeptidases A and B with nitrotyrosyl and aminotyrosyl derivatives of the carboxypeptidase inhibitor from potatoes

The single tyrosine residue of the carboxypeptidase inhibitor from potatoes, which is in contact with carboxypeptidase A in the enzyme-inhibitor complex as determined by X-ray diffraction. was converted to 3-nitrotyrosine by treatment with tetranitromethane in buffers containing 75% ethanol. The nitroinhibitor bound both bovine carboxypeptidase A and porcine carboxypeptidase B with apparent K_i values indistinguishable from those of the unmodified inhibitor. Spectral titration indicated that the nitrotyrosyl residue of the inhibitor ionized with pK_a of 7.25 either in the presence or absence of carboxypeptidase A; however, this pK_a was shifted to (ca 7.7 in the presence of carboxypeptidase B. Reduction of the 3-nitrotyrosine residue to 3-aminotyrosine slightly increased the strength of binding to both carboxypeptidases. These data suggest that the tyrosine residue of the inhibitor, is in a polar environment in the enzyme-inhibitor complex and that it is not involved in hydrogen bonding.     

Effect of the Pore Size of a Support on Activity and Action Pattern of Immobilized Endopolygalacturonase

Endopolygalacturonase (E.C. 3.2.1.15) was covalently bound to silica supports of different porosity treated with 3-(2′,3′-epoxypropoxy)propyltrimethoxysilane. The activity and action pattern on sodium pectate and tetra(D-galactosiduronic acid) were investigated in batch and continuous flow-reactors. Pore size of the supports affected the loading of the enzyme as well as its action pattern and kinetics. A decrease in randomness of degradation of D-galacturonan, loss of specificity of (3 + 1) splitting of tetra(D-galactosiduronic acid) and decrease in K_m value were found with the supports containing predominantly micropores. The extent of the changes decreased with increasing pore size of the support. The catalytic behaviour of endopolygalacturonase bound on the supports with large pores was quite analogous to that of the free enzymes.     

Specificity of the carboxypeptidase inhibitor from potatoes

Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor. However, several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low affinity (K_i about 10 micromolar) for a carboxypeptidase G₁ from an acinetobacterium was found in this study.     

Separation of proteolytic enzymes originating from Antarctic krill (Euphausia superba) by capillary electrophoresis

Extracts prepared from Antarctic krill (Euphausia superba), mainly consisting of acidic proteolytic enzymes, have been studied with capillary electrophoretic techniques. Approximately 50 repeatable peaks were obtained with capillary zone electrophoresis on an untreated fused-silica capillary using a phosphate buffer containing anionic and cationic fluorosurfactant additives as separation medium. A faster separation was achieved on a polyvinyl alcohol coated capillary. Quantitative variations of individual proteins regarding different krill enzyme batches were noted. In the krill samples trypsin-like serine proteinase, carboxypeptidase A and carboxypeptidase B were tentatively identified.     

Electrochemical properties of hydrogenase on glass carbon electrodes modified with carbon nanotubes

Hydrogenase (H₂ ase) purified from phototropic bacteriumThiocapsa roseopersicina was coassembled with carbon nanotubes (CNTs) on glass carbon electrodes. Both oxidized CNTs and Nafion-CNT composites were used to modify the electrodes. The pure H₂ are formed dot-like domains, while the oxidized CNT-H₂ ase and Nafion-CNT-H₂ ase composites formed wire-like and large closely packed aggregates, respectively. The reductive potentials for the [4Fe-4S]^2+/1+ clusters of H₂ase were at about −500, −650, and −700 mV (vs Ag/AgCl) for the electrodes modified with pure H₂ase, Nafion-SWNT-H₂ase, and Nafion-MWNT-H₂ase composites, respectively. Potential step chronocoulometry measurements indicated a larger charge-transfer diffusion coefficient between the H₂ase and electrodes when the CNTs were co-assembled with H₂ase, suggesting that the CNTs can not only act as a supporting layer to immobilize enzymes, but also act as a highly conductive wire throughout the films.     

Quantitative determination of amino groups on derivatized controlled pore glass: a comparison of methods.

The following methods of determining covalently bound amino groups were tested for self-consistency and convenience on aliquots of the same sample of controlled pore glass which has been derivatized with triethoxyaminopropylsilane (aminopropyl glass): (i) the determination of bound picrate; (ii) the determination of 2-hydroxy-1-naphthaldehyde after Schiff base formation on the aminopropyl glass; (iii) direct nonaqueous titration of the bound amines with HClO₄ in acetic acid and with CF₃SO₃H in acetic acid; and (iv) titration by Hg(NO₃)₂ solution of chloride bound by treatment of the aminopropyl glass with HCl. All methods gave the same results within the precision of measurement. Methods (i) and (iv) appear to be the most convenient.     

Protective effect of the pteroylpolyglutamates and phosphate on the proteolytic inactivation of thymidylate synthetase

Thymidylate synthetase is readily inactivated by trypsin, chymotrypsin, and carboxypeptidase A when incubated in 10–20 mm potassium phosphate buffer (pH 7.0). The loss is activity produced by trypsin and chymotrypsin is accomplished by extensive protein degradation, while inactivation by carboxypeptidase A is accompanied by release of the carboxyl-terminal valine only (Aull et al., 1974, J. Biol. Chem., 249, 1167–1172). In contrast, when the incubations are conducted in 100–200 mm potassium phosphate buffer (pH 7.0), the synthetase is not inactivated by any of the three enzymes and the results of amino acid analysis and sodium dodecyl sulfate disc gel electrophoresis demonstrate that proteolysis is prevented. The resistance of thymidylate synthetase to inactivation was shown not to be due to the inhibition of the proteolytic enzymes by the buffer. The inactivation is not prevented either by pteroylmonoglutamates or by 2′-deoxyuridine 5′-phosphate (dUMP) alone, but the presence of both is partially protective. The pteroylpolyglutamates, however, offer limited protection against carboxypeptidase A and chymotrypsin; in combination with dUMP, proteolytic inactivation of the snythetase by all three enzymes is prevented. Characterization of the properties of carboxypeptidase A-inactivated thymidylate synthetase reveals the following, (i) The binding of deoxynucleotides is unaltered, but the binding of a variety of pteroylpolyglutamate derivatives is reduced or abolished, (ii) Pteroylpolyglutamates are bound provided dUMP or an analog such as 5-fluorodUMP is present, (iii) Ternary complex formation between carboxypeptidase A-inactivated enzyme and (+)5,10-methylenetetrahydropteroyltetraglutamate plus 5-fluorodUMP occurs in the same molar binding ratio (1:2:2) at saturation as with the native enzyme, but differs from the native enzyme ternary complex in that the dissociation constant for 5-fluorodUMP is increased by approximately 10⁵. In addition, there is no evidence for the formation of covalent linkages between the ligands and enzyme, (iv) The treated enzyme cannot catalyze tritium release from [³H₅]dUMP in the presence of either (+)5,10-methylenepteroylmonoglutamate or (+)5,10-methylenetetrahydropteroyltetraglutamate.     

Purification and covalent coupling of calf brain prolidase

We have investigated methods of stabilizing prolidase by chemical modification and covalent coupling to various supports, for use in protein hydrolysis and possible use in enzyme replacement therapy. Purified acetone powder of calf brain prolidase was further purified by gel filtration on Sephadex G-200 and chromatography on DEAE-Sephadex A25. Polyacrylamide gel electrophoresis showed that the number of bands was reduced from 11 to 2. Since yields were low, the purified (NH₄)₂SO₄ fraction was used in all experiments. Thiolation of the enzyme reduced the amount of protein coupled to AH-or CH-Sepharose 4B. Activities were highest when the protein was linked through its carboxyl groups. The coupled enzyme showed much greater thermal stability than its free counterpart. Of the bound preparations, the thiolated was less stable than the untreated. Untreated and thiolated enzymes bound to either matrix showed higher activity at low pH and less at high pH than the free material. Thiolation shifted the pH maximum from 6.8 to 7.5. The free thiolated enzyme and that bound to activated SH-Sepharose 4B showed greater thermal stability and a broader pH range of optimal activity than the bound untreated enzyme. These results show that prolidase can be immobilized by coupling to an insoluble matrix through various types of covalent bonds with retention of activity and increased stability.     

Xylan-Containing supports used to separate xylanase from the cellulases of aspergillus terreus F-413

The separation of xylanase from cellulolytic enzymes of A. terreus F-413 by affinity chromatography on xylan-containing supports was investigated. Xylanase purified over tenfold was obtained after column chromatography on xylan bound to controlled porous glass. The molecular weight of the purified enzyme has been found to be 140 000 daltons, and it is homogeneous in polyacrylamide gel electrophoresis. Purified xylanase also showed residual celluloytic activity (perhaps cross-specificity) with cellulosic substrates.     

Spatial organization of digestion,secretory mechanisms and digestive enzyme properties in Pheropsophus aequinoctialis (Coleoptera: Carabidae)

Aminopeptidase (soluble form M_r 110,000), carboxypeptidase A (soluble form M_r 47,000), maltase (a dimer composed of two identical M_r 60,000 subunits) and trypsin (two charge isomers with M_r 34,000) are found in major amounts in the crop and midgut tissue, whereas amylase (a trimer of three identical M_r 18,000 subunits) and cellobiase (a trimer of three identical M_r 27,000 subunits) occur mainly in the crop and midgut contents. Subcellular fractions of midgut cells were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation. The results suggest that part of the aminopeptidase and carboxypeptidase A activity is bound to microvilli, that major amounts of trypsin and maltase are trapped in the cell glycocalyx and finally that soluble aminopeptidase, amylase and cellobiase occur in intracellular vesicles. The data support the hypothesis that most protein and carbohydrate digestion takes place in the crop under the action of enzymes passed forward from the midgut, after being secreted by exocytosis. Nevertheless, part of the intermediate and final digestion occurs at the surface of the midgut cells. The peculiar features of the digestion of P. aequinoctialis beetles, including their partly fluid peritrophic membranes, are thought to be derived from putative Coleoptera ancestors.     

The pyruvate: Ferredoxin oxidoreductase in heterocysts of the cyanobacteriuim Anabaena cylindrica

Heterocyst preparations have been obtained which actively perform nitrogen fixation (C₂H₂ reduction) and contain the enzymes of glycolysis and some of the tricarboxylic acid cycle. Pyruvate: ferredoxin oxidereductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO₂ and reduced methyl viologen (ferredoxi) from pyruvate, coenzyme A and oxidized methyl viologen (ferredoxin) as well as by the synthesis of pyruvate from CO₂, acetylcoenzyme A and reduced methyl viologen. Pyruvate supports C₂H₂ reduction by isolated heterocysts, however, with lower activity than Na₂S₂O₄ and H₂. α-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle.     

Immobilization of hydrogenase on glass beads

Hydrogenase from $\text{Clostridium pasteurianum}$ was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.     

The role of exopolymers in the attachment of sphingomonas paucimobilis

The importance of exopolymers in the adhesion of Sphingomonas paucimobilis was established by studying the attachment to glass of three mutants with defective gellan production. The attachment assays were performed in either phosphate buffered saline (controls) or in the exopolymeric solutions produced by the mutants. The exopolymer was found to have surface active properties, changing the glass surface from hydrophilic to hydrophobic, making adhesion thermodynamically favourable. Only the cells that had a substantial polymeric layer surrounding their walls were able to significantly colonise glass coated with the exopolymer. It is hypothesised that the exopolymer bound to the glass and the exopolymer present at the surface of the bacteria bound together, overcoming the energy barrier created by the negative charge of both surfaces. It is concluded that the exopolymer from S. paucimobilis has a dual role in the process of adhesion by both coating the surface thereby strengthening adhesion and by enhancing adhesion through the establishment of polymeric bridges.