Protease Subtilisin Carlsberg Research Articles

ChemInform Abstract: Efficient Asymmetric Synthesis of Lamivudine via Enzymatic Dynamic Kinetic Resolution.

AbstractThe protease subtilisin Carlsberg treated with octyl β‐D‐glucopyranoside and Brij 56 surfactants is used for the novel enzymatic DKR reaction of dithianediol (I) with aldehyde (II) and phenyl acetate.

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.

A Flow Cytometry–Based Screening System for Directed Evolution of Proteases

Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and β-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance.

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.

Immobilized protein films for assessing surface proteolysis kinetics

Enzymatic cleavage of protein substrates at solid surfaces is important in the food and detergent industries, and in biomedical applications. Creation of a reproducible protein substrate to study surface proteolysis is difficult as protein monolayers may not necessarily provide complete coverage of the surface, and protein multilayer systems are often unstable and nonuniform. We present a method to form a reproducible, immobilized, multilayer protein substrate. A 100-nm ovalbumin protein film is spin-cast onto an amine-functionalized silicon wafer and chemically cross-linked using glutaraldehyde to create a multilayer film. This protein film is stable in the presence of non-protease components such as detergents, and can be tailored to include different proteins and their mixtures, and varying degrees of susceptibility to proteolysis. Ellipsometry was used to measure the protein-film thickness as the substrate is cleaved by the protease subtilisin Carlsberg. The decrease in film thickness over time was found to be linear, indicating the depth-homogeneity of the model substrates. Lateral-homogeneity of the substrates was corroborated by atomic force microscopy (AFM) and by the reproducibility of the ellipsometric film thickness measured across different spots on the sample substrates. AFM of the multilayer protein surface before and after exposure to enzyme suggests uniform areal surface cleavage by the protease.

One- and two-step enzymatic synthesis of polymerizable vinyladipoyl mono- and diesters of non-reducing trisaccharides

The enzymatic transesterification of non-reducing trisaccharides raffinose and melezitose with divinyl adipate using two lipases (Novozym 435, Lipozyme TL IM) and the protease subtilisin Carlsberg is described. Polymerizable vinyladipoyl sugar esters were obtained with good to excellent selectivity and high yields. Monoesters prepared with lipase catalysis have also been used as substrates for a second acylation step catalyzed by the protease. Diesters were obtained with high selectivity although the isolated yields were slightly lower than those obtained for the corresponding monoester derivatives. It is important to note that the preferential acylation positions for subtilisin are the same in the free sugar and in the corresponding used monoesters for both trisaccharides. This two-step enzymatic approach allows regioselective control in the incorporation of the sugar inside the polymer structure.

Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-α secretion in Abca1-deficient macrophages

Lipid rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of theta-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kappaB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-alpha (TNF-alpha) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kappaB and TNF-alpha were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-alpha secretion. We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-alpha secretion.

On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg

BackgroundEnzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.ResultsAs model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-β-cyclodextrin (MβCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.ConclusionThese results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

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.

Preliminary X-ray analysis of a human V(H) fragment at 1.8 A resolution.

Small antibody fragments are more useful than full-size antibodies for achieving efficient biodistribution. As a first step towards the design of a clinically desirable antibody fragment, the crystallization of a human V(H) fragment has been achieved. The fragment was derived from the single-chain antibody scFvM12, which recognizes a cancer-specific hypoglycosylated form of mucin. The V(H) fragment was obtained by in-drop digestion of the scFvM12 with a low concentration of the broad-spectrum protease subtilisin Carlsberg. The crystal belongs to the monoclinic space group C2. The crystal diffracted to 1.8 A resolution when analysed at 100 K using a rotating-anode X-ray generator.

Proline versus charge concept for protein stabilization against proteolytic attack.

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.

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.