Biocomposite of subtilisin Carlsberg with chitosan as an effective biocatalyst for hydrolysis and synthesis of peptides

New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplosite (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocatalytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6:4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min.     

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.     

Reversal of enzymatic hydrolysis: Rate and extent of ester synthesis as catalyzed by chymotrypsin and subtilisin Carlsberg at low water concentrations

Immobilized chymotrypsin catalyzes esterification of N-acetyltyrosine in a medium containing high concentrations of alcohols. The hydrophilic support and inclusion of glycerol protect the enzyme activity and allow catalysis to proceed in the presence of only 10% (v/v) water. The same equilibrium concentration of ester is obtained whether reaction proceeds from ester or from free acid. Hates of ester synthesis and hydrolysis are similar when measured under the same conditions, but are at least one order of magnitude slower than optimal rates of hydrolysis. Subtilisin Carlsberg in the free, unmodified form catalyzes ester synthesis at even lower water concentrations; optimal rates are obtained at 5–15% H₂O. Hydrolytic enzymes can thus be utilized as catalysts of synthesis reactions in nonaqueous solvents where synthesis is thermodynamically favored over hydrolysis; in some cases this may provide economic and/or energetic advantages over conventional techniques.     

Dimethyl sulfoxide-induced high enantioselectivity of subtilisin Carlsberg for hydrolysis of ethyl 2-(4-substituted phenoxy)propionates

The enantioselectivity for subtilisin-catalyzed hydrolysis of ethyl 2-(4-substituted phenoxy)propionates in an aqueous buffer solution was improved by addition of DMSO (54–56% v/v). On the basis of the conformational change of subtilisin Carlsberg observed for FT-IR and CD spectra, the high enantioselectivity for subtilisin-catalyzed hydrolysis of racemic ethyl 2-(4-ethylphenoxy)propionate could be related to a partial decrease of the tertiary structure of the enzyme protein arising from an increase of the ratio of DMSO in the reaction medium. This mechanistic model for the enantiorecognition can also be supported by the discussion based on the value of the Michaelis constant (K _m) obtained for each enantiomer of the substrate.     

Molecular dynamics of subtilisin Carlsberg in aqueous and nonaqueous solutions

Crystal structures have recently appeared for the enzyme subtilisin Carlsberg in anhydrous acetonitrile and in water. To gain a mechanistic understanding of how the solvent environment affects protein structure and dynamics, we have performed molecular dynamics simulations on subtilisin Carlsberg in water and acetonitrile. We describe a 480 ps simulation of subtilisin in acetonitrile solution and a 450 ps simulation of subtilisin in water. Each simulation employed the all-atom AMBER force field. The calculated rms deviations, from their respective x-ray structures, were similar in each simulation, but ～0.5 ? higher in the acetonitrile simulation. Only in the acetonitrile simulation does one helix undergo a reversible partial unwinding, which lasted for about 100 ps. The other secondary structure elements remain intact or undergo modest fluctuations. In the aqueous simulation, the calculated and experimental temperature factors agree very well. In the acetonitrile simulation, however, the calculated temperature factors are much higher than the experimental values. The larger rms deviation and thermal fluctuations noted in the acetonitrile simulation are consistent with the requirement for protein cross-linking in this crystal and a recent two-dimensional NH-exchange nmr study on horse heart cytochrome c in nonaqueous solution. ? 1996 John Wiley & Sons, Inc.     

Cation-binding sites of subtilisin Carlsberg probed with Eu(III) luminescence

Two Ca(2+)-binding sites of subtilisin Carlsberg are studied by monitoring static and time-resolved luminescence of selectively substituted Eu(3+) at each site, and they are found to be characteristically quite different from each other. Compared with the coordination sphere of free Eu(3+), two sites are very similar to each other, so that both have a well-defined binding structure with low coordination symmetry. However, compared with the weak site, the strong site is relatively more polar, more symmetrical, and more easily accessible. Furthermore, despite the absence of water reported in the x-ray crystal structure (, Eur. J. Biochem. 166:673-692), one water molecule is found to exist in the coordination sphere of the strong site in aqueous solution. Thus it is suggested that in solution the Ca(2+) bound in the strong site forms an additional coordination bond to a solvent or substrate molecule.     

Effect of sodium dodecylbenzene sulfonate on subtilisin Carlsberg proteolysis of an immobilized ovalbumin film

Enzymatic degradation of immobilized ovalbumin multilayer films by subtilisin Carlsberg was investigated using in situ ellipsometry. Changes in the substrate cleavage rate in the presence of an anionic surfactant, sodium dodecylbenzene sulfonate (SDBS), were assessed. Exposure of the protein film to SDBS prior to introduction of the enzyme increased the measured proteolysis rate threefold. Surfactant increased the measured film thickness, absorbing into the protein film and causing swelling. Surfactant-induced film swelling was reversible upon aqueous rinsing. Nevertheless, exposure of enzyme to the surfactant-rinsed film increased the proteolysis rate, most likely due to irreversible conformational changes induced in the substrate film by the surfactant. Simultaneous addition of SDBS with enzyme after the initial surfactant exposure did not produce additional protein-removal benefit.     

Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin

The preparations of N alpha-Fmoc-3-nitro-L-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.     

Molecular dynamic study of subtilisin Carlsberg in aqueous and nonaqueous solvents

Using molecular dynamics simulations, we have obtained an important insight into the structural and dynamical changes exerted by a nonaqueous solvent on the serine protease subtilisin Carlsberg. Our findings show that the structural properties of the subtilisin–acetonitrile (MeCN) system were sensitive to the amount of water present at the protein surface. A decrease or lack of water promoted the enzyme–MeCN interaction, which increased structural changes of the enzyme primarily at the surface loops. This effect caused variations on the secondary and tertiary structure of the protein and induced the opening of a pathway for the solvent to the protein core. Also, disturbance of the oxyanion hole was observed due to changes in the orientation in the Asn-155 side chain. The disruption of the oxyanion hole and the changes of the tertiary structure should affect the optimal activity of the enzyme.     

Subtilisin Carlsberg in complex with sodium dodecyl sulfate is an effective catalyst for the solid phase segment coupling of peptides on polyvinyl alcohol cryogel

Subtilisin Carlsberg (SC) was shown to catalyze the solid phase segment coupling of peptides in complex with sodium dodecyl sulfate (SDS) in an organic medium on Aminosilochrom and polyvinyl alcohol (PVA) cryogel activated with glutaraldehyde or divinylsulfone. Diamines of different lengths with a general formula NH₂-(CH₂) _n -NH₂ (n = 2, 4, and 6) were used as spacers between the PVA cryogel and the peptide. A model reaction of enzymatic attachment of the Dnp-Ala-Ala-Leu-OMe tripeptide to the PVA cryogel was carried out by treatment with the SDS-SC complex in a mixture of anhydrous ethanol and DMSO (7 : 3, v/v) using a tenfold excess of the carboxyl component. The molar enzyme-substrate ratio was 1 : 88. The effect of the method of matrix activation, length of a spacer, and reaction time on the coupling efficiency was studied. Hexamethylenediamine was found to be the most effective spacer for the enzymatic coupling on the PVA cryogel activated with glutaraldehyde (the reaction proceeded with the highest yield of 60%). The reaction efficiency was considerably lower in the case of ethylenediamine and tetramethylenediamine (10 and 15%, respectively). The best results were obtained on the PVA cryogel activated by divinylsulfone with hexamethylenediamine as a spacer. A two-step condensation of tripeptides was carried out on this support. The second step of condensation was shown to proceed better (in 85% yield) in comparison with the first step (37% yield).     

The effect of urea and guanidinium chloride on activity of subtilisin Carlsberg

As shown by viscosity and optical rotation dispersion measurements, subtilisin Carlsberg is not denatured in the presence of 10 M urea or 6 M guanidinium chloride. This unusual structural stability made it possible to investigate the effects of these hydrophobic-bond breaking solutes on various aspects of the enzymic interaction with substrates and inhibitors. The binding of the competitive inhibitor N-benzoylarginine was decreased by urea or guanidinium chloride. The nature of this effect was such as to implicate hydrophobic interaction as making a major contribution to the binding. By contrast, Ks for the substrates N-acetyltyrosine ethyl ester, N-benzoylarginine ethyl ester and N-trans-cinnamoylimidazole was apparently unchanged by the presence of urea or guanidinium chloride. The influence of these solutes on kcat for the substrates was rather involved. Tentative hypotheses are put forward to account for the effects seen.     

Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media

Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes.     

Crystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg

The crystal structure of the molecular complex of eglin, a serine proteinase inhibitor from leeches, with subtilisin Carlsberg has been determined at 2.0 A resolution by the molecular replacement method. The complex has been refined by restrained-parameter least-squares. The present crystallographic R factor (Formula: see text) is 0.183. Eglin is a member of the potato inhibitor 1 family, a group of serine proteinase inhibitors lacking disulfide bonds. Eglin shows strong structural homology to CI-2, a related inhibitor from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure of subtilisin novo, despite changes of 84 out of 274 amino acids.     

Supercritical fluids are superior media for catalysis by cross-linked enzyme microcrystals of subtilisin Carlsberg

We report on the performance of cross-linked enzyme microcrystals (CLECs) of subtilisin Carlsberg in supercritical fluids (SC-fluids). The catalytic activity of CLECs in SC-ethane was found to be 2- to 10-fold greater than in hexane under the same conditions, using CLECs dried by propanol washing. Air-dried CLECs and lyophilized powders showed much lower activities, reflecting the same hydration hysteresis effects as in organic solvents. Reaction rates were much lower in SC-CO(2), especially at higher water activity, probably as a result of acid-base effects of carbonic acid on the enzyme.     

Progressive rearrangement of subtilisin Carlsberg into orderly and inflexible conformation with Ca(2+) binding

Fluorescence depolarization and decay kinetic profiles, together with differential scanning calorimetric thermograms and circular dichroism spectra, are measured to understand the respective roles of Ca(2+) ions at the strong (Ca1) and weak binding sites (Ca2) of subtilisin Carlsberg (sC). Thermal denaturation temperature decreases considerably with Ca1 removal, whereas it does slightly with Ca2 removal. The fraction of random coil structure increases significantly with Ca2 removal as well as with Ca1 removal. sC shows three fluorescence decay times of 100, 1100, and 3300 ps. Although the fast and the slow do not change noticeably, the medium one decreases progressively with Ca(2+) removal. sC has two fluorescence anisotropic decay components of 340 and 12,000 ps. The fast one arises from the internal rotation of Tyr, whereas the slow results from the global rotation of sC. Although both become significantly faster with Ca2 removal, only the slow one becomes slightly faster with further Ca1 removal. Overall, sC undergoes progressive rearrangement into disorderly and flexible conformation with Ca(2+) removal, indicating that both Ca1 and Ca2 are indispensable for the stable structure of sC.     

Probing the specificity of the S1 binding site of subtilisin Carlsberg with boronic acids

The binding properties and limitations of the key S1 site of subtilisin Carlsberg have been probed with boronic acid inhibitors bearing structurally varied substituents ranging from small alkyl to large aromatic groups. The data permit structural features favoring, and disfavoring, good S1 binding to be clarified. In addition, applications of electrostatic energy calculations have identified a hitherto unsuspected region of positive potential in the fundamentally hydrophobic S1 pocket, whose interactions with electronegative substituents of inhibitors can make significant binding contributions.     

Interactions of alpha-chymotrypsin and Carlsberg subtilisin with methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates

Methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates bearing different alkylthio groups were synthesized and employed as substrates for alpha-chymotrypsin and Carlsberg subtilisin in an attempt to investigate the properties of the hydrophobic pocket or cleft (S1 subsite) of the enzymes which accommodates the side-chain of the P1 amino acid residue of the substrates. The derivatives with ethylthio, 2-hydroxyethylthio, 2,3-dihydroxypropylthio, 2-aminoethylthio, carboxymethylthio, 2-carboxyethylthio, 1,2-dicarboxyethylthio, and 2-amino-2-carboxyethylthio (cysteinyl-S) groups were hydrolyzed by alpha-chymotrypsin but with kcat/Km values 4.6 to 15 times smaller than that of methyl N alpha-acetyl-L-tryptophanoate, due mainly to larger Km values. The glutathionyl derivative was only weakly bound to the enzyme. Analyses of the kinetic parameters suggested that the S1 pocket of alpha-chymotrypsin is rather more spacious than has been supposed and is able to interact flexibly with substrates so as to orient the scissile bond to the catalytic residues. On the other hand, none of the derivatives were hydrolyzed by Carlsberg subtilisin but they all inhibited the enzyme with Ki values which are generally smaller than the Km values for N alpha-acetyl-L-aromatic (modified aromatic) amino acid methyl esters. The S1 cleft of Carlsberg subtilisin interacts rather strongly with the derivatives but lacks the flexibility necessary for catalysis.