Native and modified subtilisin Karlsberg as an effective catalyst of peptide bond formation in organic media

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z- ALA-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P1 and P'1 (Glu, Arg, and Asp) containing unprotected side ionogenic groups.     

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.     

Kinetic studies on the binding of Streptomyces subtilisin inhibitor with subtilisin BPN′

The binding mechanism of Streptomyces subtilisin inhibitor and subtilisin BPN′ was studied kinetically with the stopped-flow method by monitoring the protein fluorescence increase due to complex formation. In the lower concentration range of proteins, the reaction followed the second-order kinetics. The pH dependence of the apparent second-order rate constant, k_on, suggested the involvement of the two ionizable groups of pK_a of 7.8 and 10 in the binding. The activation parameters were calculated from the temperature dependence of the apparent second-order rate constants. The value of the apparent activation energy (E_A = 39.7 kJ · mol^?1, 9.50 kcal · mol^?1) and insensitivity of k_on to the viscosity of the medium suggest that the binding is not a simple diffusion-controlled bimolecular association. Further studies with a much broader range of protein concentrations have revealed that the reaction tends to approach first-order kinetics as the inhibitor concentration increases. The binding reaction is, therefore, reconcilable with a two-step mechanism, in which a fast bimolecular association is followed by a slow unimolecular isomerization step; the dissociation constant of the first step, K_L, is estimated to be 1.2 × 10^?4m and the rate constant of the second step, k₊₂, to be 770 s^?1. It was also found that the increase of tryptophan fluorescence due to the complex formation occurs solely in the rate-determining unimolecular process.     

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.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

Crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) at 2.6 A resolution

The crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) was solved at 2·6 Å resolution. Each subunit of the dimeric inhibitor has a five-stranded antiparallel β-sheet and two short α-helices. The subunit-subunit interface formed by a stack of two β-sheets provided by the two subunits resembles the dimer-dimer interface of concanavalin A. Conformation of the reactive site around the scissible bond Met73-Val74 seems very rigid. Between bovine pancreatic trypsin inhibitor (Kunitz) and the Streptomyces inhibitor, the reactive site conformations are almost identical with each other from the P₂ to P₂′ residues, while between the soybean trypsin inhibitor (Kunitz) and the Streptomyces inhibitor they are similar from the P₂ to P₁′ residues. There are overall similarities in conformation extending from the P₃ to P₂′ residues between the Streptomyces inhibitor and a hypothetical substrate presumed (Robertus et al., 1972b) to be bound to subtilisin BPN′ in a productive binding mode. Apart from the reactive site, there seems to be no structural relationship among the Streptomyces, bovine pancreatic and soybean inhibitors, suggesting their convergent evolution from separate ancestral proteins.     

Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

High-alkaline proteases are of great importance because of their proteolytic activity and stability under high-alkaline condition. We have previously isolated a new protease (AprB) which has potential industrial applications based on its high-alkaline adaptation. However, the molecular and structural basis for alkaline adaptation of this enzyme has not been fully elucidated. In the present study, AprB gene was cloned and expressed in the Bacillus subtilis WB600. This gene codes for a protein of 375 amino acids comprised with a 28-residual signal peptide, a 78-residual pro-peptide, and a 269-residual mature protein. The deduced amino acid sequence has the highest homology of 63.2% with that of the high-alkaline proteases. Recombinant AprB was purified and determined to be monomeric with molecular mass of 26.755 kDa. The NH₂-terminal sequence of the purified AprB was A-Q-S-I-P-W-G-I-E-R. This enzyme exhibited high catalytic efficiencies (K_cat/K_m) towards natural, modified, and synthesis substrates with optimal activity at 60 °C and pH 10. AprB was stable over a wide range of pH 5 to 11 and various surfactants, and could be activated by Mg²⁺, Ca²⁺ and Ba²⁺. The structural properties of AprB, like a higher ratio of R/(R + K), a larger area of hydrophobic surface, increased number of ion pairs formed by Arg residue, and the exposure of Asp active residue on the surface, might be responsible for its alkaline adaptation. In contrast with members of subtilisin family, such as M-protease and subtilisin BPN′, AprB harbored a high content of Glu and Asp residues, and a low content of Arg and Lys residues on the surface. Interestingly, these structural characters were similar with that of psychrophilic proteases, which suggested that these molecular factors were not restricted in the psychrophilic proteases, and therefore were not solely responsible for their cold-adaptation. Our results reveal a novel structural feature of AprB unique to subtilisin family and provide clues for its alkaline adaptation.     

Native and modified subtilisin 72 as a catalyst of peptide synthesis in media with low water content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

Improved catalytic efficiency of a monomeric γ-glutamyl transpeptidase from Bacillus licheniformis in presence of subtilisin

Monomeric 30 kDa γ-glutamyl transpeptidase (GGT₃₀) was purified from culture broth of Bacillus licheniformis ER-15 along with a heterodimeric 67 kDa GGT (GGT₆₇). In presence of subtilisin, GGT₃₀ had improved catalytic efficiency (V_max/K_m) of 59 min^?1, altered pH and temperature optima of pH 11 and 70°C.and had salt-tolerant glutaminase activity. Glutaminase activity was retained even in protease-inhibited condition in presence of 2 mM PMSF. GGT₃₀ and subtilisin complexation was also confirmed by relative electrophoretic mobility and fluorescence quenching experiment.     

The Effects of Organic Solvents on Wild-Type and Mutant Subtilisin-Catalyzed Hydrolyses

The kinetic parameters, k_cat and K_M, for the hydrolysis of N-α-tosyl-L-arginine methyl ester (1, TAME) by the wild-type subtilisins Carlsberg and BPN′ as well as the BPN′ mutants Glyl66Ser, GLyl66Asn, and Met222Phe, were determined in the presence of 5 and 15% (v/v) of a selection of water-soluble organic solvents. The goals were to compare and evaluate the solvent effects with a view to expanding their use in organic synthetic applications of the WT and mutant subtilisins. The results showed that subtilisin BPN′ and its mutants were much less affected by organic solvents than subtilisin Carlsberg. The BPN′ mutant Met222Phe demonstrated the greatest resistance to cosolvent inactivation, making it a particularly attractive mutant for peptide synthesis. Dimethyl sulfoxide, acetone, and branched alcohols were found to be the most benign solvents, whereas dioxane, THF, and N-methyl-2-pyrrolidinone seriously reduced catalytic activities, even at low concentrations. The results parallel the solvent-effect data available for other proteinases, including α-chymotrypsin.     

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 mm phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ($\text{w}\text{v}$) when the concentration of SSI is 1.00 mg/ml (87.0 μm as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.     

Catalytic properties of cross-linked enzyme crystals in organic media

The activity and the enantioselectivity of cross-linked enzyme crystals (CLECs) of subtilisin in the transesterification between N-acetyl-l-phenylalanine ethyl ester and n-propanol have been examined in various organic solvents. The activity of CLECs of subtilisin in decane was 780 times greater than that in triethylamine. CLECs of subtilisin preferred l-enantiomer in the transesterification between N-acetyl-phenylalanine ethyl ester and n-propanol, and the (k_cat/K_M)_L/(k_cat/K_M)_D ratio was 20 000 in cyclohexane.     

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.     

The properties of subtilisin,Novo type,immobilized on porous glass

Studies of the properties of subtilisin, Novo type, immobilized on porous glass with the aid of hexamethylene diisocyanate were carried out. The immobilized proteinase preparation shows optimum activity at a pH value of 10.7 and at a temperature between 60–65°C. It was stable in a wider range of pH and temperature values than the native subtilisin. The K_M values for hemoglobin and BAEE were 9.2 × 10^?5 [M] and 139 × 10^?5 [M], respectively. Under relatively non-aqueous conditions, immobilised subtilisin was able to synthesize phenylacetic acid ethyl ester.     

Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white

Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11 700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100°C for 4 h at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation contants are 7.2·10^?9 M for bovine trypsin adn 5.5·10^?7 M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.     

Activation of Pseudomonas cytochrome oxidase by limited proteolysis with subtilisin

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c2: oxygen oxidoreductase; E.C.1.9.3.2) can be digested with subtilisin under controlled conditions that convert the original parent polypeptide chain (Mr on SDS gels approximately equal to 60,000) to a slightly smaller species (Mr on SDS gels approximately equal to 58,000). Under the conditions used (0.33% subtilisin, w/w, pH 7.4), the product formed from the oxidase was relatively stable to further digestion. Cytochrome oxidase activity was assayed at intervals during proteolysis by following the rate of oxidation of Pseudomonas ferrocytochrome c-551 by the enzyme in the presence of oxygen. The activity increased to a plateau that was more than two times the value for an untreated control. These observations suggest that clipping a small peptide from Pseudomonas cytochrome oxidase either facilitates the rate-limiting electron transfer between the intraprotein heme c and heme d1, enhances the interaction of the enzyme with ferrocytochrome c-551, or both.     

Preparation and characterization of cross-linked enzyme aggregates (CLEA) of subtilisin for controlled release applications

Enzyme stabilization is one of the major challenges in the biocatalytic process optimization. Subtilisin was aggregated using ammonium sulphate and polyethylene glycol with surfactants like triton X-100 and tween 20. The resultant aggregates on cross-linking with glutaraldehyde produced insoluble and catalytically active enzyme. The effect of pH, temperature, kinetic parameter, thermal stability and stability in organic solvents were studied. The cross-linked enzyme aggregates (CLEA) exhibited broad pH optima of 9.0 and higher temperature optima of 70 degrees C. Reusability and surface morphology of the CLEA were also studied. CLEA of subtilisin has good stability in nonpolar organic solvents, such as hexane, and cyclohexane and it has high thermal stability up to 60 degrees C and therefore can be used as a catalyst for the biotransformation of compounds which are not soluble in aqueous medium. The CLEAs were entrapped in the hydrogel composite beads of alginate:guar gum (3:1) which were resistant to low pH conditions in the stomach and thus was found to be useful for the oral drug delivery. This process can be used to deliver the protein and peptide drugs which involve high concentrations at the delivery stage, and which usually degrades in the stomach before reaching the jejunum. Application of these pH-sensitive beads for the controlled release of subtilisin in vitro was studied and found to be a feasible strategy.     

Kinetic analysis of the mechanism for subtilisin in essentially anhydrous organic solvents

Steady-state kinetic analysis has been used to confirm the catalytic mechanism of lyophilized subtilisin suspended in a variety of organic solvents. Specifically, this article demonstrates that partial reactions can occur between subtilisin and ester substrates in organic solvents. Partitioning of common intermediates between competing acceptors at a constant ratio of products has also been described. The decomposition of a common intermediate formed from different substrates at the same rate is also further evidence of an acyl-enzyme mechanism for subtilisin suspended in anhydrous solvents. Partitioning of a common intermediate to give two products at a constant total rate, and saturation kinetics at varying substrate concentrations, complete a kinetic investigation of the enzyme mechanism. All the data generated support the formation of a stable acyl enzyme during the transesterification reaction catalzyed by subtilisin in the solvents used.     

Proteolytic susceptibility of food by-product proteins: An evaluation by means of a quantitative index

A proteolytic susceptibility index was proposed to evaluate the reaction performance of different food by-products with subtilisin. Whey, salmon muscle and feather keratine were hydrolyzed at the same peptide bond concentration at 50 °C and pH 8.0 with different subtilisin concentrations. The logarithmic equation P = 1/b ln(abt+1) was fitted to estimate the kinetic constants a and b. The a/b ratio was proposed as a proteolytic susceptibility index, which was correlated with the chemical structure and conformation of protein sources in increasing order of feather keratin, salmon muscle and whey proteins with corresponding a/b values of 0.1, 8.2 and 14.4 mM²/min, respectively. The methodology proposed in this work can be used to evaluate the proteolytic susceptibility of different protein by-products to different proteases and to evaluate the changes in proteolytic susceptibility after treatment.