L.M. Simon

Immobilization and characterization of porcine pancreas lipase

Abstract Porcine pancreas lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) was immobilized with the highest activity (2,187 U g−1 solid) on polyacrylamide beads possessing carboxylic functional groups activated by a water-soluble carbodiimide. The optimum pH for catalytic activity was pH 8.9. The apparent optimum temperature for the immobilized enzyme was about 7°C higher than that for the soluble enzyme. The immobilization stabilized the enzyme against heat and urea treatment. Cross-linking of the immobilized enzyme with glutaraldehyde or 3,5-difluoronitrobenzene improved the thermal stability. Application of the immobilized lipase for olive oil hydrolysis is also presented.

Stability of hydrolytic enzymes in water-organic solvent systems

Abstract The effects of organic solvents on the stabilities of bovine pancreas trypsin, chymotrypsin, carboxypeptidase A and porcine pancreas lipase were studied. Water-miscible solvents (ethanol, acetonitrile, 1,4-dioxane and dimethyl sulfoxide) and water-immiscible solvents (ethyl acetate and toluene) were used in 100 mM phosphate buffer (pH 7.0) or 100 mM Tris/HCl buffer (pH 7.0) in concentrations of 20–80% (v/v). All hydrolytic enzymes studied were inactivated by mixtures containing dimethyl sulfoxide at higher concentrations. Trypsin and carboxypeptidase A resisted solvent mixtures containing acetonitrile, 1,4-dioxane and ethanol. They preserved more than 80% of their starting activities during 20-min incubations. The activities of lipase and chymotrypsin decreased with increasing concentration of water-miscible polar organic solvents, but at higher concentrations (80%) 70–90% of the activity remained. In mixtures with water-immiscible solvents, the decrease in activity of carboxypeptidase A was pronounced. Trypsin and chymotrypsin underwent practically no loss in activity in the presence of toluene or ethyl acetate. In respect of stability, the polar solvent proved to be more favorable for lipase. These results suggest that the conformational stabilities of hydrolytic enzymes are highly dependent on the solvent-protein interactions and the enzyme structure.

Effects of Cd2+, Cu2+, Pb2+ and Zn2+ on activities of some digestive enzymes in carp (Cyprinus carpio L.)

Abstract The effects of haevy metals (Cd2+, Cu2+, Pb2+ and Zn2+) on activities of carp trypsin, α‐chymotrypsin, carboxypeptidase A and lipase were studied. The enzymes were isolated from the gastrointestinal tract and the effects of metal ions were investigated during incubation for 5 min. The presence of Cd2+ did not influence the activities of CPA and trypsin and 10–20 % inhibiton was observed with α‐chymotrypsin and lipase. Cu2+ only slightly influenced the trypsin and lipase activities, whereas the α‐chymotrypsin activity was decreased. All enzyme activities decreased at higher Zn2+ concentrations. The inhibition was most pronounced at α‐chymotrypsin (more than 50%). With the exception of trypsin, Pb2+ inhibited the activities of the investigated enzymes.

Effects of synthetic pyrethroids and methidation on activities of some digestive enzymes in carp (Cyprinus carpio L.).

The effects of pyrethroid pesticides (deltamethrin, permethrin and cypermethrin) and an organophosphate ester (methidation) on the activities of carp trypsin, alpha-chymotrypsin, carboxypeptidase A and lipase were studied. The enzymes were isolated from the gastrointestinal tract and the effects of the pesticides were investigated during incubation for 5 min. The activity of trypsin was influenced only slightly by the presence of deltamethrin and methidation, whereas permethrin and cypermethrin caused significant inhibition. The pyrethroid pesticides at lower concentrations resulted in a slight activation of alpha-chymotrypsin. Methidation inhibited the alpha-chymotrypsin activity by about 20%. These pesticides modified the lipase activity to a lesser extent; the highest inhibition was measured with cypermethrin. The carboxypeptidase A activity was inhibited by both pyrethroid pesticides and methidation. The results suggest that these pesticides might interact with the active conformation of the studied hydrolytic enzymes, resulting in changes in their activities.

Preparation and characterization of immobilized glucose-phosphate isomerase

Abstract Glucose-phosphate isomerase ( d -glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) isolated from rabbit muscle was covalently attached to Sepharose 4B, Akrilex C-100, Akrilex P-100 and Silochrome aldehyde, respectively. The highest immobilized activity (110 units mg −1 solid) was achieved when the enzyme was bound to Akrilex C-100, a polyacrylamide support possessing carboxylic functional groups. The catalytic properties and the stability of this Akrilex-bound enzyme were studied in detail relative to those of the soluble enzyme. For both soluble and immobilized glucose-phosphate isomerase the pH optimum was about pH 8.0. The apparent optimum temperature of the immobilized enzyme was about 50°C, while that for the soluble enzyme lay between 50 and 55°C. The apparent K m for glucose 6-phosphate as substrate was higher (2 × 10 −1 mM) in the case of the immobilized enzyme. Little difference was found between the thermal stabilities of the soluble enzyme (at 45° C t 1 2 = 42 min ; at 50° C t 1 2 = 12 min ) and the immobilized enzyme (at 45° C t 1 2 = 82 min ; at 50° C t 1 2 = 10 min ). As a result of immobilization, the stability against urea was increased.

Preparation, characterization and application of immobilized carboxypeptidase A

Abstract Bovine carboxypeptidase A (peptidyl- l -amino-acid hydrolase, EC 3.4.17.1) was covalently attached to polyacrylamide beads and to silica-based supports, and also adsorbed on polyethylene terephthalate. The highest immobilized activity (125 U g −1 solid) was achieved when a polyacrylamide bead support (Akrilex C) possessing carboxylic functional groups activated by a water-soluble carbodiimide was used. The catalytic properties and stability of Akrilex C-carboxypeptidase A were studied and compared with the corresponding properties of the soluble enzyme. The optimum pH for the catalytic activity of the immobilized carboxypeptidase A was practically identical to that for the soluble enzyme (pH 7.5–8.0). The apparent optimum temperature of the immobilized carboxypeptidase A was about 20°C higher than that of the soluble enzyme. With hippuryl- l -phenylalanine as substrate, K m app for the immobilized enzyme (1.65 mM) was somewhat higher than K m for the soluble enzyme (1.07 mM). The conformational stability of the enzyme was markedly enhanced by the strongly hydrophilic microenvironment. The immobilized carboxypeptidase A was used for the C-terminal amino acid analysis of peptides.

Immobilization of pig muscle 3-phosphoglycerate kinase

3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g−1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent Km values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced.

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ∼pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent Km value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ∼pH 6.0.

N-acetyl-L-arginine ethyl ester synthesis catalysed by bovine trypsin in organic media

The bovine trypsin-catalysed synthesis of N-acetyl-l-arginine ethyl ester from N-acetyl-l-arginine and ethanol was studied in various organic solvents (dimethyl sulfoxide, dioxane, dimethylformamide, acetonitrile, acetone, tetrahydrofuran, chloroform, toluene, carbon tetrachloride, cyclohexane and n-hexane). The highest yield was achieved in acetonitrile after incubation for 6 or 24 h. The optimal conditions for ester synthesis in acetonitrile for 6 h were as follows: 5.0 mM N-acetyl-l-arginine, 10.0 M ethanol, 7.2 mg trypsin, 2.87% water, total volume 10.3 ml, pH 7.0 and 30°C. The hydrolytic activity of trypsin was determined after incubation for 6 days, when 87.7% of the original activity remained, suggesting that acetonitrile caused little inactivation of the enzyme. The synthetic reaction resulted in a maximal 79.3% conversion under optimized conditions after incubation for 48 h.

Coenzyme production using immobilized enzymes. I. Preparation, characterization, and laboratory-scale application of an immobilized NAD+ kinase

Abstract NAD+ kinase (ATP: NAD+ 2-phosphotransferase, EC2.7.1.23) isolated from chicken liver was immobilized on a silica-based support possessing aldehyde functional groups. The highest catalytic activity achieved was 16 U g−1 solid. The optimal pH for the catalytic activity of the immobilized NAD+ kinase was pH 7.1–7.3. The apparent optimum temperature for the immobilized enzyme was about 5°C higher than that of the soluble enzyme. There were no significant differences in the Km app values. The immobilization improved the conformational stability of the enzyme. In preliminary experiments, a 95% conversion of NAD+ to NADP+ was achieved with use of the immobilized NAD+ kinase, which preserved its starting activity practically unchanged up to 36 days.