T. Kaper

DNA family shuffling of hyperthermostable beta-glycosidases.

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 beta-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 degrees 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 (betaalpha)(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.

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal β-glycosidases

Abstract Sβgly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of Sβgly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure–function relationships in these enzymes.

The ß-glucosidase CelB from Pyrococcus furiosus: Production by Escherichia coli, purification, and in vitro evolution

Publisher Summary One of the key enzymes of the hyperthermophilic archaeon Pyrococcus furiosus involved in growth on B-linked sugars is the inducible B-glucosidase (CelB). CelB from P. furiosus serves as a very suitable model glycosylhydrolase to study substrate specificity as well as adaptations of stability and activity to extreme temperatures. Stable production in E. coli and an efficient purification protocol allow for simple and rapid preparation of pure wildtype and mutant CelB enzymes. This chapter an presents an overview of the state of the art on this hyperthermostable enzyme, protocols for heterologous production and enzyme purification, and development and application of a directed evolution procedure that has resulted in the isolation and characterization of an active site mutant.

Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

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.

The catalytic potency of β-glucosidase from Pyrococcus furiosus in the direct glucosylation reaction

Abstract 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 β-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% was 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.