J. Turková

Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12.

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.

Affinity chromatography on hydroxyalkyl methacrylate gels. I. Preparation of immobilized chymotrypsin and its use in the isolation of proteolytic inhibitors.

Abstract Heterogeneous suspension copolymerization of hydrophilic methacrylate monomers with dimethacrylates was used to obtain neutral hydrophilic gels with a defined specific surface and pore distribution. Such macroporous carriers can be activated with bromocyanogen for binding biologically active compounds and applied in affinity chromatography. Chymotrypsin was used to investigate the dependence of the amount of a covalently bound enzyme and of its esterase and proteolytic activity on the specific surface and pore size of the carrier. In a similar manner to chymotrypsin-Sepharose, there was a shift of the pH optimum of esterase activity toward the alkaline region compared with the pH optimum of free chymotrypsin, while the pH optimum of the proteolytic activity of free and immobilized chymotrypsin remained unchanged. Affinity chromatography with covalently bound chymotrypsin was used to isolate a chymotryptic inhibitor from potatoes, and a trypsin inhibitor from lung. If the trypsin inhibitor from lung was bound to a hydroxyalkyl methacrylate carrier, the affinity chromatography of a commercial chymotrypsin sample raised its activity by one order of magnitude. The new hydrophilic carriers are characterized by an excellent mechanical stability which gives them outstanding flow properties.

Isolation and characterization of alkaline proteinase of Aspergillus flavus

Abstract 1. 1. A simple method was developed to isolate an alkaline proteinase from a crude enzymatic product prepared from the culture medium of Aspergillus flavus consisting of (io) adsorption of the crude product on CM-Sephadex at pH 4.5 (ii) desorption of proteinase by 0.5 M sodium acetate at pH 7, and (iii) chromatogrpahy on DEAE-Sephadex in phosphate buffers at pH 6.0. This purification results in a 120-fold increase of proteolytic activity. 2. 2. The proteinase obtained is homogeneous when subjected to disc electrophoresis and sedimentation analysis in the ultracentrifuge. The molecular weight was determined, and the following values were obtained: (i) 19 500 to 22 000 by gel filtration, (ii) 19 000 ± 1000 by light-scattering technique, and (iii) 17 800 when the molecular weight was calculated from the amino acid composition: Lys 11 , His 3−4 , Arg 2 , Asp 21 , Thr 11 , Ser 20 , Glu 12−13 , Pro 4−5 , Gly 20 , Ala 23 , Val 15 , He 9−10 , Leu 9 , Tyr 5 , Phe 3 , Trp 2 , amide-NH 3 , 17. The enzyme does not contain either disulphide bonds or cysteine; the N-terminal end-group residue is glycine; the C-terminal amino acid is alanine. The enzyme contains 1 mole of a sugar component not yet identified. Treatment of the proteinase with diisopropylphosphorofluoridate provided evidence of one active serine residue. 3. 3. Similarities existing between analytical data on alkaline proteinase of Aspergillus flavus and other extracellular enzymes from Aspergilli are discussed.

Pepsin immobilized by covalent fixation to hydroxyalkyl methacrylate gels: Preparation and characterization

Insoluble active derivatives of pepsin (EC 3.4.23.1) were prepared by covalent binding of this enzyme to hydroxyalkyl methacrylate gels modified with 1,6-diaminohexane or epsilon-aminocaproic acid in an acid medium by means of water-soluble carbodiimide. The amount of attached enzyme, its proteolytic activity, pH activity curves of the preparations obtained and the time and pH dependence of their stability were determined.

Affinity chromatography on hydroxyalkyl methacrylate gels III. Adsorption of chymotrypsin to poly(hydroxyalkyl methacrylates) with covalently bound benzyloxycarbonyl-glycyl-d-phenylalanine and -d-leucine as function of pH and ionic strength

Chymotrypsin is specifically adsorbed at low ionic strength and alkaline pH to hydroxyalkyl methacrylate gels with N-benzyloxycarbonylglycl-D-phenylalanine or N-benzyloxycarbonylglycyl-D-leucine attached through 1,6-hexanediamine. Chymotrypsin is not adsorbed either to the unmodified gel (Spheron) or to the gel with attached, 1,6-hexanediamine (NH2-Spheron). The adsorption of chymotrypsin to Z-Gly-D-Phe-NH2-Spheron was investigated as a function of pH and ionic strength. Trypsin is not adsorbed to this gel. Chymotrypsin isolated from a crude pancreatic extract by affinity chromatography on Z-Gly-D-Phe-NH2-Spheron had the same activity as the enzyme isolated on a column of Spheron, to which the naturally-occurring trypsin inhibitor had been coupled.

SH-proteinase from bean Phaseolus vulgaris var. Perlicka

An SH-proteinase (EC 3.4.22.-) has been isolated from beans of the species Phaseolus vulgaris var. Perlicka. The enzyme is homogeneous when subjected to disc electrophoresis, electrofocusing and sedimentation analysis. The molecular weight was determined as 26,000-28,000 by gel filtration, 30,850 +/- 1500 by sedimentation analysis and 26,930-27,410 by calculation from the amino acid composition (Lys20-21, His3, Arg9, Asp21-22, Thr13, Ser18, Pro12-13, Glu23-24, Gly30, Ala16, Cys/29, Val19, Met1, Ile10, Leu13, Tyr14, Phe6, Trp3). The N-terminal amino acid of the proteinase is isoleucine. The effect of concentration, time of hydrolysis, pH, temperature, cations, anions, urea and guanidine - HCl on the proteolytic activity of the SH-proteinase was studied.

Alkaline proteinases of the genus aspergillus

Abstract 1. 1.|Extracellular alkaline proteinases from the molds Aspergillus flavus , Aspergillus oryzae , Aspergillus sojae and Aspergillus sulphureus , isolated and characterized by individual authors in four different laboratories, have been compared using uniform methods under identical conditions in one of our laboratories. 2. 2.|The enzymes studied were compared by gel filtration, disc electrophoresis, amino acid analysis, determination of the pH optima of their proteolytic activity, estimation of their specificity by cleavage of the B-chain of oxidized insulin, and by peptide maps of the tryptic, chymotryptic, and peptic digests of the inactivated proteinases. 3. 3.|The results of this comparison led to the following conclusion: Alkaline proteinases from A. flavus and A. oryzae are probably indentical (or very closely related), alkaline proteinase from A. sojae is closely related but not identical, alkaline proteinase from A. sulphureus is homologous yet less related. 4. 4.|The combination of methods used in this study requires only small quantities of enzymes. It is therefore suitable for preliminary comparison of proteinases and for studies on the degree of their homology.

Reactive carriers of immobilized compounds

Sphericanl macroporous reactive carriers capable of forming covalent bonds with amino acids and proteins were prepared by the suspension copolymerization of 2-hydroxyethyl methacrylate, ethylene dimethacrylate and p-nitrophenyl esters of methacrylic acid and methacryloyl derivatives of glycine, beta-alanine and epsilon-aminocaproic acid. The effect of the spacer length, pH and the type of the buffer used, concentration of reactive groups in the copolymer, concentration of the ligand and the participation of the hydrolytic and aminolytic reaction of p-nitrophenyl functional groups in the attachment of glycine, D,L-phenylalanine and serumalbumin was studied. Macroporous copolymers containing reactive functional groups can be used as active enzyme carriers, if their activity is not blocked by the presence of p-nitrophenol split off in the attachment reaction.

Isolation of aminopeptidase from Aspergillus flavus

A mixture of aminopeptidase and neutral protease from the Aspergillus flavus mold obtained by chromatography on DEAE-Sephadex was fractionated by chromatography on the hydroxyalkyl methacrylate gel with chemically bonded 1,6 hexamethylene diamine and D-leucine. Aminopeptidase thus obtained was electrophoretically homogeneous. Conditions for chromatography were worked out allowing a one stage isolation of a highly active aminopeptidase sample directly from the alcoholic precipitate of the culture medium of the Aspergillus flavus mold.