Evaluation of magnetic polymer micro-beads as carriers of immobilised biocatalysts for selective and stereoselective transformations.

The kinetic, selective and stereoselective properties of enzyme immobilised on magnetic polymer beads with diameters in the range 1 microm was studied with penicillin amidase from E. coli. The enzyme was immobilised on epoxy and glutaraldehyde-activated poly(vinyl alcohol), poly(methylmetacrylate) and poly(vinyl acetate-divinylbenzene) magnetic beads. The amount of covalently bound active protein was dependent on the chemical modification of the matrix and increased at higher ionic strength of the immobilisation buffer. The small size of the magnetic beads, that reduces mass transfer limitations, and the decreased charge density in the electric double layer resulted in lower apparent Km values and higher efficiency for benzylpenicillin hydrolysis, higher stereoselectivity in condensation of R-phenylglycine amide with S- and R-Phe and in hydrolysis of racemic phenylacetyl-Phe and higher selectivity in kinetically controlled synthesis of cephalexin compared to the enzyme immobilised on larger and porous carriers.

Stability of immobilized soybean lipoxygenases: influence of coupling conditions on the ionization state of the active site Fe.

The potential application of lipoxygenase as a versatile biocatalyst in enzyme technology is limited by its poor stability. Two types of soybean lipoxygenases, lipoxygenase-1 and -2 (LOX-1 and LOX-2) were purified by a two step anion exchange chromatography. Four different commercially available supports: CNBr Sepharose 4B, Fractogel((R)) EMD Azlactone, Fractogel((R)) EMD Epoxy, and Eupergit((R)) C were tested for immobilization and stabilization of the purified isoenzymes. Both isoenzymes gave good yields in enzyme activity and good stability after immobilization on CNBr Sepharose 4B and Fractogel((R)) EMD Azlactone. Rapid decay in activity associated with change in the ionization state of Fe, as shown by EPR measurements was observed within the first 5 days after immobilization on epoxy activated supports (Eupergit((R)) C and Fractogel((R)) EMD Epoxy) in high ionic strength buffers. Stabilization of the biocatalyst on these supports was achieved by careful adjustment of the immobilization conditions. When immobilized in phosphate buffer of pH 7.5 and low ionic strength (0.05 M), the half-life time of the immobilized enzyme increased 20 fold. The dependence of the stability of LOX immobilized on epoxy activated supports on the coupling conditions was attributed to a modulation of the ligand environment of the iron in the active site and consequently its reactivity.

pH dependence of penicillin amidase enantioselectivity for charged substrates.

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these pK values, a pH dependence of E is also expected. This was experimentally verified around these pK values. The S'(1)-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phenylacetyl-S,R-PhG. The S(1)-stereospecificity was investigated for the hydrolysis of the enantiomeric S/R-PhG-NH(2). The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5-9) was found to be a result of compensatory effects for binding and catalysis. The ratios k(cat, S)/k(cat,R) and K(m,S)/K(m,R) for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5-8. The dependence was stronger for the S'(1)- than for the S(1)-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S'(1)-subsite than that of the substrate amino group in the S(1)-subsite on the pK of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground-state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.

Temperature effects on S1- and S'1-enantioselectivity of alpha-chymotrypsin.

The temperature dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) has been studied for the alpha-chymotrypsin catalysed hydrolysis of the enantiomeric N-Boc-L/D-TyrOMe, L/D-TyrOMe, Ac-L/D-PhgOMe, L/D-PhgOMe and for the kinetically controlled synthesis of the diastereomeric dipeptides N-Ac-L-Tyr-L/D-ArgNH2 and N-Ac-L-Tyr-L/D-ValNH2. The results show that the S1- and S'1-enantioselectivity can be modulated by the temperature (3-15 fold for the studied substrates in the range 5-45 degrees C). For L/D-PhgOMe a reversal in stereospecificity was found in this temperature interval. For the studied substrates both an increase or decrease of the enantiomeric ratio with increasing temperature was observed. For these processes the following relation for the temperature dependence of E has been derived ln E = ln(kL/kD) = -(delta deltaH# - delta deltaHb)/RT + (delta deltaS# - delta deltaSb)/R where kL and kD are apparent second order rate constants for the reactions with the L- and D-enantiomers, respectively. Delta delta denotes the differences between the thermodynamic parameters for transformation of the enantiomeric substrates. The subscript b applies for the binding of the substrate or the nucleophile and the superscript # for the formation of the transition state of the enzyme acylation or deacylation. For the studied processes either the enthalpy (delta deltaH# - delta deltaHb) or the entropy (delta deltaS# - delta deltaSb) term was found to control the discrimination. Thus, the enantioselectivity decreases or increases with temperature, respectively. The influence of ground-state interactions and transition-state stabilisation on enzyme enantioselectivity has been discussed.

Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530.

A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.

Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri.

The cultivation of the extremely thermophilic archaeon Thermococcus stetteri in a dialysis membrane reactor was paralleled by the production of an extremely heat-stable proteinase(s). By applying preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, an SDS-resistant proteinase was purified 67-fold in one step with a yield of 34%. The purified enzyme, which was composed of a single polypeptide chain with a molecular mass of 68 kDa, showed a broad temperature and pH profile (50 to 100(deg)C; pH 5 to 11). The optimal activity with substantial thermal stability was measured with casein at 85(deg)C and pH 8.5 to 9. Inhibition by phenylmethylsulfonyl fluoride and diisopropylfluorophosphate demonstrated that the enzyme was a serine proteinase. The enzyme displayed a relatively narrow substrate specificity, catalyzing the hydrolysis only of N-protected p-nitroanilides or p-nitrophenyl esters of basic (Arg or Lys) or hydrophobic (Phe or Tyr) l-amino acids. l-Phenylglycine amide was also attacked by the proteinase, but with a lower specificity constant. Within the detection limit, no hydrolysis of d-amino acid derivatives was observed. The catalytic efficiency of the enzyme at 80(deg)C (k(infcat)/K(infm) for benzoyl-Arg-p-nitroanilide, 10(sup4)) is the same order of magnitude when compared with that of functionally similar mesophilic enzymes. The proteinase also acts as a transferase, catalyzing the acyl transfer from protected amino acid ester or amide to amino acid amide. The observed thermostability, SDS resistance, relatively narrow substrate specificity, high stereospecificity, and limited catalytic efficiency probably reflect the tighter packing of the thermostable protein molecule and its limited flexibility. This was supported by fluorescence spectra of the enzyme, mainly due to tryptophan residues, in the temperature range of 30 to 90(deg)C. Structural reorganization was observed at temperatures over 100(deg)C. The results obtained could be of relevance for the better understanding of the structure-function relationship of enzymes from extreme thermophiles and suggest possible biotechnological application of the proteinase for resolution of racemic mixtures.

Enzyme catalyzed biotransformations in aqueous two-phase systems with precipitated substrate and/or product.

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.

Penicillin amidase-catalysed transfer of low specific acyl moiety. Synthesis of 7-benzoxazolonylacetamido desacetoxycephalosporanic acid.

The methyl ester of 2-benzoxazolon-3-yl-acetic acid was used as an acyl donor in the penicillin amidase-catalysed transfer reaction to 7-aminodesacetoxycephalosporanic acid. The synthesis of 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid was carried out as a kinetically controlled reaction. A characteristic feature of this system is that the benzoxazolone derivatives are very low specific substrates for penicillin amidase (the kcat/Km values for their hydrolysis were shown to be 10(5)-fold lower compared to the corresponding values for phenylacetyl derivatives). Nevertheless, penicillin amidase proved to be an effective catalyst for the synthesis of these new cephem derivatives (50% yield for 6 h). The reason is the observed unusually high value for the transferase-hydrolase activity ratio. The determined value for (k3'/k3)app = 120,000 implies that in this case of low specific acyl moiety, penicillin amidase acts more like a transferase than a hydrolase. The maximum yield has been increased up to 70% by lowering the reaction temperature and stepwise feeding of the reaction medium with the acyl component. The results obtained extend the potential of the penicillin amidase as a catalyst for the synthesis of a new group of biologically active cephem derivatives.