Serine Protease Subtilisin Carlsberg Research Articles

Pentynyl dextran as a support matrix for immobilization of serine protease subtilisin Carlsberg and its use for transesterification of N-acetyl-l-phenylalanine ethyl ester in organic media

In this study, serine protease (subtilisin Carlsberg) was immobilized on pentynyl dextran (PyD, O-alkynyl ether of dextran, 1) and used for the transesterification of N-acetyl-L-phenylalanine ethyl ester (2) with different aliphatic (1-propanol, 1-butanol, 1-pentanol, 1-hexanol) and aromatic (benzyl alcohol, 2-phenyl ethanol, 4-phenyl-1-butanol) alcohols in tetrahydrofuran (THF). The effect of carbon chain length in aliphatic and aromatic alcohols on initial and average transesterification rate, transesterification activity of immobilized enzyme and yield of the reaction under selected reaction conditions was investigated. The transesterification reactivity of the enzyme and yield of the reaction increased as the chain length of the alcohols decreased. Furthermore, almost no change in yield was observed when the immobilized enzyme was repeatedly used for selected alcohols over six cycles. Intrinsic fluorescence analysis showed that the catalytic activity of the immobilized enzyme in THF was maintained due to retention of the tertiary structure of the enzyme after immobilization on PyD (1).

Zooming In on Metagenomics: Molecular Microdiversity of Subtilisin Carlsberg in Soil

Evolution has led to the development of a gigantic repertoire of microbial genes that can be exploited for industrial purposes. Due to microevolutionary processes, this gene pool is constantly varied and adapted to the prevalent environmental and physiological conditions. It though remains unclear to what extent gene variants coexist in natural habitats and to what extent they vanish due to competition. Here, we tapped the pool of gene variants of the serine protease Subtilisin Carlsberg present in soil habitats, demonstrating a high degree of (micro) diversity on a genetic level, as well as on a functional level. A set of 51 mature enzyme variants each carrying two to eight amino acid changes were recovered. While some mutations were only present in single variants, other changes appear to be rather conserved even across different habitats. The observed spectrum of biochemical properties makes persistent gene variants a potent source for biotechnologically relevant enzymes, expanding the toolbox of metagenomic approaches.

Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme’s initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg

The potential of enzyme catalysis as a tool for organic synthesis is nowadays indisputable, as is the fact that organic solvents affect an enzyme's activity, selectivity and stability. Moreover, it was recently realized that an enzyme's initial activity is substantially decreased after prolonged exposure to organic media, an effect that further hampers their potential as catalysts for organic synthesis. Regrettably, the mechanistic reasons for these effects are still debatable. In the present study we have made an attempt to explain the reasons behind the partial loss of enzyme activity on prolonged exposure to organic solvents. Fluorescence spectroscopic studies of the serine protease subtilisin Carlsberg chemically modified with polyethylene glycol (PEG-SC) and inhibited with a dansyl fluorophore, and dissolved in two organic solvents (acetonitrile and 1,4-dioxane) indicate that when the enzyme is initially introduced into these solvents, the active site environment is similar to that in water; however prolonged exposure to the organic medium causes this environment to resemble that of the solvent in which the enzyme is dissolved. Furthermore, kinetic studies show a reduction on both V max and K M as a result of prolonged exposure to the solvents. One interpretation of these results is that during this prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation. Extrapolating this to an enzymatic reaction we argue that substrates bind in a less catalytically favorable conformation after the enzyme has been exposed to organic media for several hours.

Enantioselective transesterification catalysis by nanosized serine protease subtilisin Carlsberg particles in tetrahydrofuran

Enzyme catalysis in organic solvents is a powerful tool for stereo-selective synthesis but the enantioselectivity is still hard to predict. To overcome this obstacle, we employed a nanoparticulate formulation of subtilisin Carlsberg (SC) and designed a series of 14 structurally related racemic alcohols. They were employed in the model transesterification reaction with vinyl butyrate and the enantioselectivities were determined. In general, short alcohol side chains led to low enantioselectivties, while larger and bulky side chains caused better discrimination of the enantiomers by the enzyme. With several bulky substrates high enantioselectivities with E>100 were obtained. Computational modeling highlighted that key to high enantioselectivity is the discrimination of the R and S substrates by the sole hydrophobic binding pocket based on their size and bulkiness. While bulky S enantiomer side chains could be accommodated within the binding pocket, bulky R enantiomer side chains could not. However, when also the S enantiomer side chain becomes too large and does not fit into the binding pocket anymore, enantioselectivity accordingly drops.

Crystallographic Analysis of Counterion Effects on Subtilisin Enzymatic Action in Acetonitrile

When enzymes are in low dielectric nonaqueous media, it would be expected that their charged groups would be more closely associated with counterions. There is evidence that these counterions may then affect enzymatic activity. Published crystal structures of proteins in organic solvents do not show increased numbers of associated counterions, and this might reflect the difficulty of distinguishing cations like Na(+) from water molecules. In this paper, the placement of several Cs(+) and Cl(-) ions in crystals of the serine protease subtilisin Carlsberg is presented. Ions are more readily identified crystallographically through their anomalous diffraction using softer X-rays. The protein conformation is very similar to that of the enzyme without CsCl in acetonitrile, both for the previously reported ( 1SCB ) and our own newly determined model. No fewer than 11 defined sites for Cs(+) cations and 8 Cl(-) anions are identified around the protein molecule, although most of these have partial occupancy and may represent nonspecific binding sites. Two Cs(+) and two Cl(-) ions are close to the mouth of the active site cleft, where they may affect catalysis. In fact, cross-linked CsCl-treated subtilisin crystals transferred to acetonitrile show catalytic activity several fold higher than the reference crystals containing Na(+). Presoaking with another large cation, choline, also increases the enzyme activity. The active site appears only minimally sterically perturbed by the ion presence around it, so alternative activation mechanisms can be suggested: an electrostatic redistribution and/or a larger hydration sphere that enhances the protein domain.

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.

On the role of protein structural dynamics in the catalytic activity and thermostability of serine protease subtilisin Carlsberg

The effect of structural dynamics on enzyme activity and thermostability has thus far only been investigated in detail for the serine protease alpha-chymotrypsin (for a recent review see Solá et al., Cell Mol Life Sci 2007, 64(16): 2133-2152). Herein, we extend this type of study to a structurally unrelated serine protease, specifically, subtilisin Carlsberg. The protease was incrementally glycosylated with chemically activated lactose to obtain various subtilisin glycoconjugates which were biophysically characterized. Near UV-CD spectroscopy revealed that the tertiary structure was unaffected by the glycosylation procedure. H/D exchange FT-IR spectroscopy was performed to assess the changes in structural dynamics of the enzyme. It was found that increasing the level of glycosylation caused a linearly dependent reduction in structural dynamics. This led to an increase in thermostability and a decrease in the catalytic turnover rate for both, the enzyme acylation and deacylation steps. These results highlight the possibility that a structural dynamics-activity relationship might be a phenomenon generally found in serine proteases.

Hydrogen/deuterium exchange study of subtilisin Carlsberg during prolonged exposure to organic solvents.

It has been previously reported that prolonged exposure of an enzyme to organic solvents leads to substantial decrease of activity. This effect was found to be unrelated to the catalysts' structure or their possible aggregation in organic solvents, and up to the present day the cause for activity loss remains unclear. In the present work, the structural dynamics of the serine protease subtilisin Carlsberg (SC) have been investigated during prolonged exposure to two organic solvents by following hydrogen/deuterium (H/D) exchange of mobile protons. The enzyme, after lyophilization, was incubated in organic solvents at controlled deuteriated water activity for different times and the H/D exchange was allowed to take place. The amount of deuterium exchanged was evaluated by (2)H NMR, which in turn gave us a picture of the changing dynamics of our model enzyme during incubation and under different experimental conditions. Our results show that the flexibility of SC decreases during prolonged storage in 1,4-dioxane (Diox) and acetonitrile (ACN) as indicated by the observed 3- to 10-fold decrease in the apparent rate constants of exchange (k) of fast exchangeable protons (FEP) and slow exchangeable protons (SEP) in the protein. Our study also shows that SC is more flexible in ACN than in Diox (k 3-20 times higher in ACN for the FEP and SEP), suggesting that enzyme dynamics are affected by solvent physicochemical properties. Additionally, the enzyme dynamics are also affected by the method of preparation: decreased flexibility (k decreases 3- to 10-fold for FEP and SEP) is observed when the enzyme is chemically modified with poly ethylene glycol (PEGylated) or colyophilized with crown ethers. A possible relationship between activity, enantioselectivity (E), and structural dynamics is discussed, demonstrating that direct correlations, as have been attempted in the past, are hampered by the multi-variable nature and complexity of the system.

Efficient subtilisin immobilization in chitosan, and peptide synthesis using chitosan–subtilisin biocatalytic films

The immobilization of serine protease subtilisin Carlsberg on/in chitosan, a hydrophilic biopolymer was investigated in this study. Using different techniques, biocatalysts in the form of gels, fibers and films were prepared. Subtilisin loading and biocatalytic properties of obtained samples were studied. The optimal method of enzyme incorporation was pre-mixing of chitosan acetate and enzyme solutions, then air-drying and treatment with glutaraldehyde. It was found that this form of biocomposite has the highest loading capacity (up to 20 mg protein g−1 support) and good mechanical properties. The dependence of subtilisin loading on the starting concentration of the enzyme during immobilization shows the maximum loading at [E] = 15 mg mL−1. The dependence of subtilisin loading and activity on the concentration of the glutaraldehyde was explored. The biocatalytic films were active catalysts for peptide bond formation (with 25–92% product yield) in nonaqueous media, demonstrating high activity and stability under these conditions. They are also active in aqueous media, bringing about the reverse reaction, and can therefore be used for either peptide bond formation or cleavage.

High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals.

Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process. In this study we extended the choice of immobilisation matrix from a simple inorganic salt, K(2)SO(4), to other compounds, both inorganic and zwitterionic, that act as solid-state buffers for biocatalysis in organic media. The catalytic activity of serine proteases subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT) were significantly increased when coated onto the surface of solid-state buffers, as measured in acetonitrile/1wt% H(2)O. SC-PCMC with both organic and inorganic buffer carriers (Na-AMPSO, Na(2)CO(3), and NaHCO(3)) showed a 3-fold greater activity than that observed when using the unbuffered system (PCMC-SC/K(2)SO(4)). In comparison with freeze-dried preparations, this represents an approximately 3,000-fold increase in catalytic activity. Importantly, there is no improvement in catalytic activity upon external addition of any of the solid-state buffers to the reaction mixture. When acting in a solid-state buffer capacity, good buffering capacity was observed with SC-PCMC (3 wt% protein loading) prepared from a 1:1 mixture of AMPSO and AMPSO-Na. Alternatively, increasing the amount of solid-state buffer in the system allows improvement of the buffering. This can be achieved either by decreasing the protein loading of the SC/Na-AMPSO-PCMC or by addition of further external solid-state buffer to the reaction mixture. The catalytic activity of lipase-PCMC prepared from solid-state buffers was found less responsive to immobilisation.

Structure and Energetics of Protein–Protein Interactions: The Role of Conformational Heterogeneity in OMTKY3 Binding to Serine Proteases

Proteins with flexible binding surfaces can interact with numerous binding partners. However, this promiscuity is more difficult to understand in “rigid-body” proteins, whose binding results in little, or no, change in the position of backbone atoms. The binding of Kazal inhibitors to serine proteases is considered a classic case of rigid-body binding, although they bind to a wide range of proteases. We have studied the thermodynamics of binding of the Kazal serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease subtilisin Carlsberg using isothermal titration calorimetry and have determined the crystal structure of the complex at very high resolution (1.1 Å). Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding. These effects emphasize that small, “rigid-body” proteins are still dynamic structures, and these dynamics make contributions to both the enthalpy and entropy of binding interactions.

Subtilisin-catalyzed synthesis of amino acid and peptide esters. Application in a two-step enzymatic ligation strategy.

We describe an efficient enzymatic approach to the synthesis of amino acid and peptide esters. The serine protease subtilisin Carlsberg (EC 3.4.21.62) was found to efficiently catalyze the specific formation of C(alpha)-carboxyl 3-hydroxypropyl or 4-hydroxybutyl esters of certain Boc-amino acids and peptides in high-content 1,3-propanediol or 1,4-butanediol solution, with substrate specificity parallel to that of the normal hydrolytic reaction. This approach can be coupled with kinetic-control reverse proteolysis in a two-step enzymatic peptide ligation scheme. [reaction: see text]

Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers.

Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.

Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content.

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.

Activation of serine protease subtilisin Carlsberg in organic solvents: combined effect of methyl-β-cyclodextrin and water

Methyl β-cyclodextrin (MβCD) increased the activity and enantioselectivity of lyophilized subtilisin suspended in dry THF and acetonitrile in two transesterification model reactions. These beneficial improvements were diminished by the addition of water, in contrast to the observation that water activates subtilisin lyophilized from buffer alone. For example, the initial rate for the S enantiomer in the transesterification of vinylbutyrate with (±)-1-phenylethanol (sec-phenethylalcohol) decreased ca. 4-fold and the enantioselectivity from 59 to 40 when 0.1% (v/v) of water was added to THF.

A serine alkaline protease from the fungus Conidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

A serine alkaline protease from the fungus Conidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

ChemInform Abstract: Probing the Specificity of the Serine Proteases Subtilisin Carlsberg and α‐Chymotrypsin with Enantiomeric 1‐Acetamido Boronic Acids. An Unexpected Reversal of the Normal “L”‐Stereoselectivity Preference.

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Probing enzyme stereospecificity. Inhibition of α-chymotrypsin and subtilisin Carlsberg by chiral amine- and aminoalcohol-derivatives

Various enantiomeric amine and aminoalcohol amide and α-ketoamide derivatives have been evaluated as competitive inhibitors of the representative serine proteases α-chymotrypsin (CT) and subtilisin Carlsberg (SC). Each compound studied was an effective competitive inhibitor of both enzymes. However, only for the best inhibitor, N-pyruvoyl-1-(1-naphthyl)ethylamine (K1 27 μM for the S-enantiomer with CT), was noteworthy enantiomeric discrimination manifest, with the S-enantiomer being a significantly more powerful inhibitor of CT and SC than its R-counterpart by factors of 12.6- and 73-fold, respectively. The enzyme-inhibitor interactions responsible for this strong binding and enantiomeric discrimination were revealed by molecular modelling analyses.

Probing the Specificity of the Serine Proteases Subtilisin Carlsberg and α-Chymotrypsin with Enantiomeric 1-Acetamido Boronic Acids. An Unexpected Reversal of the Normal “l”-Stereoselectivity Preference

Enantiomeric 1-acetamido boronic acids, which are N-acetyl transition state analog inhibitor analogs of l- and d-forms of the amino acids alanine, phenylalanine, p-fluorophenylalanine, p-chlorophenylalanine, and 1-naphthylalanine, have been evaluated as inhibitors of the serine proteases subtilisin Carlsberg (SC) and α-chymotrypsin (CT). All of the boronic acids are powerful competitive inhibitors of both enzymes, with, as expected, the l-enantiomers being generally more potent than the d-enantiomers. However, a dramatic reversal of the normal stereoselectivity preference was observed in the inhibition of CT by [1-acetamido-2-(1-naphthyl)ethyl]boronic acid, with the d-enantiomer becoming a 25-fold more potent inhibitor than the l-enantiomer. Furthermore, the KI of 127 nM for CT inhibition by this d-enantiomer is the lowest of any of the boronic acids evaluated. Molecular modeling analyses of the possible binding modes of the inhibitors suggest that the stereoselectivity reversal is due to S1-pocket orient...