Mobile and immobile adhesion of staphylococcal strains to hydrophilic and hydrophobic surfaces
Boks, N.P. ; Kaper, H.J. ; Norde, W. ; Mei, H.C. van der; Busscher, H.J. - \ 2009
Journal of Colloid and Interface Science 331 (2009)1. - ISSN 0021-9797 - p. 60 - 64.
bacterial adhesion - biofilm reactors - infections - attachment - deposition - reversibility - substrata - energies - system - flow
Staphylococcus epidermidis adheres to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in similar numbers, but in different modes. Real-time observation of staphylococcal adhesion under a shear rate of 15 s(-1) revealed different adhesion dynamics on both substrata. The number of adsorption and desorption events to achieve a similar number of adhering bacteria was twofold higher on hydrophilic than on hydrophobic DDS-coated glass. Moreover. 22% of all staphylococci on glass slid over the surface prior to adhering on a fixed site ("mobile adhesion mode"), but mobile adhesion was virtually absent (1%) on DDS-coated glass. Sliding preceded desorption on hydrophilic glass in about 20% of all desorption events, while on hydrophobic DDS-coated glass 2% of all staphylococci desorbed straight from their adhesion site. Since acid-base interactions between the staphylococci and a hydrophobic DDS-coating are attractive, it is suggested that these interactions facilitate a closer approach of the bacteria and therewith enhance immobile adhesion at local, high affinity sites. Alternatively, if the local site is low affinity, this may lead to desorption. In the absence of attractive acid-base interactions, as on hydrophilic glass, bacteria can be captured in the minimum of the DLVO-interaction energy curve, but this does not prevent them from sliding under flow at a fixed distance from a substratum surface until immobilization or desorption at or from a local high or low affinity site, respectively.
Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase
Wolterink-van Loo, S. ; Siemerink, M.A.J. ; Perrakis, G. ; Kaper, T. ; Kengen, S.W.M. ; Oost, J. van der - \ 2009
Archaea : an international microbiological journal 2 (2009)4. - ISSN 1472-3646 - p. 233 - 239.
Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 degrees C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40-60 degrees C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures
Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption
Xu, C.P. ; Boks, N.P. ; Vries, J. de; Kaper, H.J. ; Norde, W. ; Busscher, H.J. ; Mei, H.C. van der - \ 2008
Applied and Environmental Microbiology 74 (2008)24. - ISSN 0099-2240 - p. 7522 - 7528.
streptococcus-thermophilus - intermolecular forces - antigen-i/ii - adherence - systems - cells - adsorption - detachment - microscopy - deposition
Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.
Residence time dependent desorption of Staphylococcus epidermidis from hydrophobic and hydrophilic substrata
Boks, N.P. ; Kaper, H.J. ; Norde, W. ; Busscher, H.J. ; Mei, H.C. van der - \ 2008
Colloids and Surfaces. B: Biointerfaces 67 (2008)2. - ISSN 0927-7765 - p. 276 - 278.
plate flow chamber - bacterial adhesion - colloidal particles - surfaces - deposition - forces - model - stiffness - kinetics
Adhesion and desorption are simultaneous events during bacterial adhesion to surfaces. although desorption is far less studied than adhesion. Here, desorption of Staphylococcus epidermidis from substratum surfaces is demonstrated to be residence time dependent. Initial desorption rate coefficients were similar for hydrophilic and hydrophobic dimethylclichlorosilane (DDS)-coated glass, likely because initial desorption is controlled by attractive Lifshitz-Van der Waals interactions, which are comparable on both substratum Surfaces. However, significantly slower decay times of the desorption rate coefficients are found for hydrophilic glass than for hydrophobic DDS-coated glass. This difference is suggested to be due to the acid-base interactions between staphylococci and these surfaces, which are repulsive on hydrophilic glass and attractive on hydrophobic DDS-coated glass. Final desorption rate coefficients are higher on hydrophilic glass than on hydrophobic DDS-coated glass, due to the so called hydrophobic effect, facilitating a closer contact on hydrophobic DDS-coated glass.
Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels
Kaper, T. ; Talik, B. ; Ettema, T.J.G. ; Bos, H. ; Maarel, M.J.E.C. van der; Dijkhuizen, L. - \ 2005
Applied and Environmental Microbiology 71 (2005)9. - ISSN 0099-2240 - p. 5098 - 5106.
archaeon thermococcus-litoralis - alpha-amylase family - large cyclic glucans - potato d-enzyme - cyclodextrin-glycosyltransferase - hyperthermophilic archaeon - escherichia-coli - crystal-structure - disproportionating enzyme - pyrococcus-furiosus
Amylomaltases are 4-¿-glucanotransferases (EC 126.96.36.199) of glycoside hydrolase family 77 that transfer ¿-1,4-linked glucans to another acceptor, which can be the 4-OH group of an ¿-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli, and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95°C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase.
Inhibition of adhesion of yeasts and bacteria by poly(ethylene oxide-)brushes on glass in a parallel plate flow chamber
Roosjen, A.M. ; Kaper, H.J. ; Mei, H.C. van der; Norde, W. ; Busscher, H.J. - \ 2003
Microbiology 149 (2003). - ISSN 1350-0872 - p. 3239 - 3246.
microbial adhesion - voice prostheses - silicone-rubber - surfaces - brushes - contamination - biomaterial - adsorption - chemistry - growth
Poly(ethylene oxide) (PEO)-brushes are generally recognized as protein-repellent surfaces, and although a role in discouraging microbial adhesion has been established for some strains and species, no study exists on the effects of PEO-brushes on a large variety of bacterial and yeast strains. In this paper, a PEO-brush has been covalently attached to glass and silica by reaction in a polymer melt. Subsequently, the presence of a PEO-brush was demonstrated using contact angle measurements, X-ray photoelectron spectroscopy and ellipsometry. For five bacterial (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus salivarius, Escherichia coli and Pseudomonas aeruginosa) and two yeast strains (Candida albicans and Candida tropicalis), adhesion to PEO-brushes was compared with adhesion to bare glass in a parallel plate flow chamber. The initial deposition rates of Sta. epidermidis, Sta. aureus and Str. salivarius to glass were relatively high, between 2400 and 2600 cm-2 s-1, while E. coli and P. aeruginosa deposited much more slowly. The initial deposition rates of the yeasts to glass were 144 and 444 cm-2 s-1 for C. albicans GB 1/2 and C. tropicalis GB 9/9, respectively. Coating of the glass surface with a PEO-brush yielded more than 98 % reduction in bacterial adhesion, although for the more hydrophobic P. aeruginosa a smaller reduction was observed. For both yeast species adhesion suppression was less effective than for the bacteria and here too the more hydrophobic C. tropicalis showed less reduction than the more hydrophilic C. albicans. The PEO-brush had a thickness of 22 nm in water, as inferred from ellipsometry. Assuming that on bare glass the adhered micro-organisms are positioned only a few nanometers away from the surface and that the brush keeps them at a distance of 22 nm, it is calculated that the brush yields a sevenfold attenuation of the Lifshitz–Van der Waals attraction to the surface between the micro-organisms and the surface. Decreased Lifshitz–van der Waals attraction may be responsible for the suppression of the microbial adhesion observed.
Characterization of (polyethylene oxide) brushes on glass surfaces and adhesion of Staphylococcus epidermidis
Kaper, H.J. ; Busscher, H.J. ; Norde, W. - \ 2003
Journal of Biomaterials Science-Polymer Edition 14 (2003)4. - ISSN 0920-5063 - p. 313 - 324.
self-assembled monolayers - protein adsorption - bacterial adhesion - polyethylene oxide - polymer brushes - model - particles
Poly(ethylene oxide) brushes have been covalently bound to glass surfaces and their presence was demonstrated by an increase in water contact angles from fully wettable on glass to advancing contact angles of 54 degrees, with a hysteresis of 32 degrees. In addition, electrophoretic mobilities of glass and brush-coated glass were determined using streaming potential measurements. The dependence of the electrophoretic mobilities on the ionic strength was analyzed in terms of a softlayer model, yielding an electrophoretic softness and fixed charge density of the layer. Brush-coated glass could be distinguished from glass by a 2-3-fold decrease in fixed charge density, while both surfaces were about equally soft. Adhesion of Staphylococcus epidermidis HBH276 to glass in a parallel plate flow chamber was extremely high and after 4 h, 19.0 x 10(6) bacteria were adhering per cm2. In contrast, the organisms did not adhere to brush-coated glass, with numbers below the detection limit, i.e. 0.1 x 10(6) per cm2. These results attest to the great potential of polymer brushes in preventing bacterial adhesion to surfaces.
The activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH
Perugino, G. ; Trincone, A. ; Giordano, A. ; Oost, J. van der; Kaper, T. ; Rossi, M. ; Moracci, M. - \ 2003
Biochemistry 42 (2003). - ISSN 0006-2960 - p. 8484 - 8493.
agrobacterium beta-glucosidase - oligosaccharide synthesis - sulfolobus-solfataricus - glycosyl transfer - glycosidase - intermediate - pyrococcus - mechanism - evolution - protein
We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus so fataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the P-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.
|Engineering of Thermostable Family 1 B-glycosidases for Saccharide Processing
Kaper, T. ; Oost, J. van der; Vos, W.M. de - \ 2002
In: Carbohydrate bioengineering Interdisciplinary Aproaches / Teeri, T.T., Svensson, B., Gilbert, H.J., Feizi, T., Cambridge : Royal Society of Chemistry - p. 135 - 142.
Persistence of tertiary structure in 7.9M guanidinium chloride : the case of endo B-1,3-glucanase from Pyrococcus furiosus
Chiaraluce, R. ; Oost, J. van der; Lebbink, J.H.G. ; Kaper, T. ; Consalvi, V. - \ 2002
Biochemistry 41 (2002). - ISSN 0006-2960 - p. 14624 - 14632.
The Pyrococcus furiosus endo--1,3-glucanase belongs to the subfamily of laminarinase, which can be classified as "all proteins" as confirmed by deconvolution of far-UV CD and FTIR spectra. The persistence of a significant amount of tertiary structure in 7.9 M GdmCl, as indicated by near-UV CD spectroscopy, accompanied by a red-shift of the maximum fluorescence emission wavelength is a peculiar property of this hyperthermophilic endoglucanase. The possibility to observe tertiary structure elements under extremely denaturing conditions is notable and is limited to only a few examples. The unusual resistance toward guanidinium chloride denaturation is paralleled by a notable stability at extremely low pH and at high temperature. The analysis of the protein spectral properties indicates that the secondary structure elements are preserved down to pH 1.0 and up to 90 C at pH 7.4 and pH 3.0. The study of the conditions that determine the persistence of residual structure at high denaturant concentration and the examination of these structures are particularly interesting because these state(s) may be preliminary or coincident with the coalescence of protein aggregates or to the formation of amyloid-like fibrils, and they may serve as seeds of protein folding.
DNA family shuffling of hyperthermostable beta-glycosidases
Kaper, T. ; Brouns, S.J.J. ; Geerling, A.C.M. ; Vos, W.M. de; Oost, J. van der - \ 2002
Biochemical Journal (2002). - ISSN 0264-6021 - p. 461 - 470.
The structural compatibility of two hyperthermostable family 1 glycoside hydrolases, Pyrococcus furiosus CelB and Sulfolobus solfataricus LacS, as well as their kinetic potential were studied by construction of a library of 2048 hybrid b-glycosidases using DNA family shuffling. The hybrids were tested for their thermostability, ability to hydrolyse lactose and sensitivity towards inhibition by glucose. Three screening rounds at 70°C led to the isolation of three high-performance hybrid enzymes (hybrid 11, 18 and 20) that had 1.5–3.5-fold and 3.5–8.6-fold increased lactose hydrolysis rates compared with parental CelB and LacS respectively. The three variants were the result of a single crossover event, which gave rise to hybrids with a LacS N-terminus and a main CelB sequence. Constructed three-dimensional models of the hybrid enzymes revealed that the catalytic (b a)8-barrel was composed of both LacS and CelB elements. In addition, an extra intersubunit hydrogen bond in hybrids 18 and 20 might explain their superior stability over hybrid 11. This study demonstrates that extremely thermostable enzymes with limited homology and different mechanisms of stabilization can be efficiently shuffled to form stable hybrids with improved catalytic features.
Substrate specificity engineering of beta-mannosidase and betaglucosidase from Pyrococcus by exchange of unique active site residues
Kaper, T. ; Heusden, H.H. van; Loo, B. van; Vasella, A. ; Oost, J. van der; Vos, W.M. de - \ 2002
Biochemistry 41 (2002). - ISSN 0006-2960 - p. 4147 - 4155.
A -mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl--D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s-1). The -mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active -glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.
Improved oligosaccharide synthesis by protein engineering of b-glucosidase from hyperthermophilic Pyrococcus furiosus
Hanson, T. ; Kaper, T. ; Oost, J. van der; Vos, W.M. de - \ 2001
Biotechnology and Bioengineering 73 (2001). - ISSN 0006-3592 - p. 203 - 210.
Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type -glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type -mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95°C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45 percent compared with the wild-type CelB (40 percent Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20 percent lactose concentrations compared to the wild-type. At a lactose concentration of 10 percent the oligosaccharide yield for the mutant was 40 percent compared to 18 percent for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10 percent lactose) compared to the wild-type (0.19, 70 percent lactose). At a lactose concentration as low as 10 percent only trisaccharides were synthesized by CelB wild-type. The -mannosidase BmnA from P. furiosus showed both -glucosidase and -galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44&Eth;The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating -glycosidases in oligosaccharide synthesis from lactose
The ß-glucosidase CelB from Pyrococcus furiosus: Production by Escherichia coli, purification, and in vitro evolution
Lebbink, J.H.G. ; Kaper, T. ; Kengen, S.W.M. ; Oost, J. van der; Vos, W.M. de - \ 2001
Methods in Enzymology 330 (2001). - ISSN 0076-6879 - p. 364 - 379.
Characterization of ß-glycosyl hydrolases from Pyrococcus furiosus
Kaper, T. ; Verhees, C.H. ; Lebbink, J.H.G. ; Lieshout, J.F.T. van; Kluskens, L.D. ; Ward, D.E. - \ 2001
Methods in Enzymology 330 (2001). - ISSN 0076-6879 - p. 329 - 346.
The catalytic potency of ß-glucosidase from Pyroccus furiosus in the direct glucosylation reaction
Roode, B.M. de; Meer, T.D. van der; Kaper, T. ; Franssen, M.C.R. ; Padt, A. van der; Oost, J. van der; Boom, R.M. - \ 2001
Enzyme and Microbial Technology 29 (2001). - ISSN 0141-0229 - p. 621 - 624.
Enzymes from extremophiles operate at conditions that are different from their `normal' counterparts, and are therefore a useful extension of the enzyme toolbox. In this paper, the direct glucosylation reaction mediated by a hyperthermophilic -glucosidase from Pyrocuccus furiosus was investigated. Hexanol was successfully coupled to glucose with this enzyme. A preliminary study was conducted to improve the product yield. A maximum product concentration of 12.9 g.l-1 was attainable by increasing the glucose concentration to the maximum solubility of 2000 g.(kg buffer solution)-1 at the reaction temperature. The highest glucose based yield of 2.64 as achieved with a glucose concentration of 900 g.(kg buffer solution)-1 at a reaction temperature of 65°C and a pH of 6.0. Performing the reaction at higher pH and temperature led to lower product concentrations. This was caused by deactivation of the enzyme accompanied by browning of the reaction mixture. A pH of 4.4 did have a negative effect on both the storage and the operational stability of the enzyme.
Engineering of β-glycosidases from hyperthermophilic Archaea
Kaper, T. - \ 2001
Wageningen University. Promotor(en): W.M. de Vos; J. van der Oost. - S.l. : S.n. - ISBN 9789058085047 - 162
glycosidasen - thermofiele micro-organismen - eiwittechnologie - glycosidases - thermophilic microorganisms - protein engineering
Hyperthermophilic Archaea are microorganisms that grow optimally above 80°C. To be able to live at these temperature extremes their cell components display extreme resistance towards thermal degradation. This characteristic is an attractive feature for use of their enzymes in industrial processes. Examples of thermozymes with potential applications in food and pharmaceutical industry are b -Glycosidases, enzymes that specifically hydrolize b -linked glycosidic bonds, which are present in for instance cellulose or the milk sugar lactose. .
Using the b -glucosidase CelB from Pyrococcus furiosus as a model enzyme, molecular determinants of substrate recognition and catalysis in b -glycosidases have been studied by rational design and directed evolution approaches. A 3D model of CelB was established, and its active site was compared to that of a related enzyme with a distinct specificity, the 6-phospho-b -galactosidase LacG of Lactococcus lactis. The substrate specificity of CelB was adjusted by engineering a phosphate-binding site, which resulted in a significant improvement in the hydrolysis of 6-phospho-b-glycosides. In a second study, the active sites of b -glucosidase CelB was compared to that of a b -mannosidase BglB from Pyrococcus horikoshii , and the substrate affinities and activities of the two enzymes could be swapped by exchange of unique residues in their active sites.
The b -glucosidase CelB of P. furiosus was also compared to that of the related b -glycosidase LacS of the hyperthermophile Sulfolobus solfataricus. While the enzymes are very similar regarding catalytic mechanism and substrate specificity, they have not been stabilized to withstand high temperatures in the same way. While CelB is relatively sensitive to detergents, LacS is readily inactivated in the presence of salts. This strongly suggests that CelB is mainly stabilized by hydrophobic interactions, while ion-pair interactions contribute most to the stability of LacS.
In one of the first laboratory evolution studies on proteins from a hyperthermophile, CelB has been optimized for low-temperature catalysis. In several CelB mutants this was accomplished with retention of wild-type stability. Increased activity at low temperatures seemed to result from mutations that increase protein flexibility. In a second directed evolution study, the genes coding for CelB and LacS were shuffled to functional b -glycosidase hybrids. The hybrids of this DNA shuffling experiment were screened for thermostability and hydrolysis of lactose at 70 °C. Several thermostable high-performance mutants were isolated and characterized. The hybrids consisted of an N-terminal LacS stretch, followed by a CelB core. This resulted in a hybrid active site structure, which could explain the altered catalytic properties.
Finally, site-directed CelB mutants from the previous mentioned studies have been tested for their ability to catalyze oligosaccharide synthesis. Indeed, several variants showed increased yields in galacto-oligosaccharide synthesis with lactose as a substrate, compared to wild-type CelB.
The studies described in the thesis are illustrative for the differences in protein engineering by rational design versus by directed evolution. While rational design can give an initial change in activity or substrate specificity, directed evolution is more likely to be successful for fine-tuning of enzyme properties.
|Activity and stability of hyperthermophilic enzymes : a comparative study on two archaeal b-glycosidases
Pouwels, J. ; Moracci, M. ; Cobucci-Ponzano, B. ; Perugino, G. ; Oost, J. van der; Kaper, T. ; Lebbink, J.H.G. ; Vos, W.M. de; Ciaramella, M. ; Rossi, M. - \ 2000
Extremophiles 4 (2000). - ISSN 1431-0651 - p. 157 - 164.
Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus b-glucosidase CelB by directed evolution
Lebbink, J.H.G. ; Kaper, T. ; Bron, P. ; Oost, J. van der; Vos, W.M. de - \ 2000
Biochemistry 39 (2000). - ISSN 0006-2960 - p. 3656 - 3665.
The -glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date. To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli. This library was screened for increased activity on p-nitrophenyl--D-glucopyranoside at room temperature. Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail. Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers. Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme. Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 C on p-nitrophenyl--D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold. In most cases, this was achieved at the expense of -galactosidase activity at 20 C and total catalytic efficiency at 90 C. Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site. The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl--D-glucopyranoside/p-nitrophenyl--D-galactopyranoside hydrolysis. This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing substrates and axial in galactose-containing substrates.
Comparative structural analysis and substrate specificity engineering of the hyperthermostable b-glucosidase CelB from Pyrococcus furiosus
Kaper, T. ; Lebbink, J.H.G. ; Pouwels, J. ; Kopp, J. ; Schulz, G.E. ; Oost, J. van der; Vos, W.M. de - \ 2000
Biochemistry 39 (2000). - ISSN 0006-2960 - p. 4963 - 4970.
The substrate specificity of the -glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level. Following crystallization and X-ray diffraction of this enzyme, a 3.3 Å resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical ()8-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho--glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis. The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG. To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants. Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0). Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol--D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively. In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold). The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.