固定化黑曲霉β-葡萄糖苷酶制备染料木素活性苷元的研究

比较了以海藻酸钠为载体,用胶囊法、包埋-交联法、交联-包埋法三种不同方法固定化黑曲霉β-葡萄糖苷酶的效果,并研究了最佳固定化方法的固定化条件和固定化酶的部分性质。结果表明,交联-包埋法即β-葡萄糖苷酶与0.20%戊二醛交联后再用2.0%海藻酸钠包埋的固定化方法中酶结合效率和酶活力回收率最高。海藻酸钠浓度和戊二醛浓度对酶结合效率影响较大,戊二醛浓度和包埋颗粒直径大小对酶活力回收率影响显著。与游离酶相比,制备的固定化酶最适温度、最适pH值和Km值分别由50℃、4.5和2.57μg/mL下降到40℃、4.0和2.02μg/mL。固定化酶具有更强的耐酸性和稳定性。该固定化酶用于大豆异黄酮活性苷元染料木素的合成,重复使用6次后,固定化酶的活力仍保持84.94%,染料木苷转化率为56.04%。     

Immobilization of chitosan-invertase neoglycoconjugate on carboxymethylcellulose-modified chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on a carboxymethylcellulose-coated chitin support via polyelectrolyte complex formation. The yield of immobilized protein was determined to be 72% and the enzyme retained 68% of the initial invertase activity. The optimum temperature for invertase was increased by 5 degrees C and its thermostability was enhanced by about 9 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 12.6-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The prepared biocatalyst retained 98% and 100% of the original catalytic activity after 10 cycles of reuse and 70 h of continuous operational regime in a packed bed reactor, respectively. The immobilized enzyme retained 95% of its activity after 50 days of storage at 37 degrees C.     

Immobilization of a soluble chemically thermostabilized enzyme

Cellobiase was coupled to a dialdehyde dextran by reductive alkylation in the presence of sodium cyanoborohydride. The resulting conjugate, obtained without loss of enzymic activity, presents properties of thermoresistance largely superior to those of native enzyme: the rate of inactivation is reduced compared to that of native enzyme and its optimal temperature of activity is 70-75 degrees C instead of 65 degrees C. Finally the conjugate presents increased longevity when subjected to experiments of operational stability; its hydrolytic activity is maintained at 60 degrees C in a 10% (w/v) cellobiose solution for more than 100 h whereas the native enzyme is inactivated after 45 h. The cellobiase-dextran conjugate was immobilized by covalent coupling on aminated silica by reductive alkylation in the presence of NaBH(3)CN. The characteristics of thermoresistance of this stabilized and immobilized conjugate were studied and compared to those of a preparation of native cellobiase immobilized on a silica support activated with glutaraldehyde. Analysis of the thermoresistance of these two cellobiase preparations clearly shows that immobilization has maintained and even enhanced their properties. In particular, the operational stability, measured at 68 degrees C on 10% (w/v) cellobiose shows an increased longevity of the stabilized and immobilized enzyme for 120 h compared to 60 h for the native immobilized enzyme. Two successive incubations of these cellobiase derivatives show that it is possible to obtain 2.5 times more glucose with the stabilized-immobilized enzyme than with the immobilized preparation. The procedure described above enables us to prepare a thermostabilized immobilized cellobiase.     

Flow kinetics of immobilized beta-glucosidase

The enzyme beta-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 degrees C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 degrees C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.     

Enzymatic synthesis of cello-oligosaccharides by rice BGlu1 {beta}-glucosidase glycosynthase mutants

Rice BGlu1 beta-glucosidase is a glycosyl hydrolase family 1 enzyme that acts as an exoglucanase on beta-(1,4)- and short beta-(1,3)-linked gluco-oligosaccharides. Mutations of BGlu1 beta-glucosidase at glutamate residue 414 of its natural precursor destroyed the enzyme's catalytic activity, but the enzyme could be rescued in the presence of the anionic nucleophiles such as formate and azide, which verifies that this residue is the catalytic nucleophile. The catalytic activities of three candidate mutants, E414G, E414S, and E414A, in the presence of the nucleophiles were compared. The E414G mutant had approximately 25- and 1400-fold higher catalytic efficiency than E414A and E414S, respectively. All three mutants could catalyze the synthesis of mixed length oligosaccharides by transglucosylation, when alpha-glucosyl fluoride was used as donor and pNP-cellobioside as acceptor. The E414G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than that of E414S and E414A, respectively, and gave yields of up to 70-80% insoluble products with a donor-acceptor ratio of 5:1. (13)C-NMR, methylation analysis, and electrospray ionization-mass spectrometry showed that the insoluble products were beta-(1,4)-linked oligomers with a degree of polymerization of 5 to at least 11. The BGlu1 E414G glycosynthase was found to prefer longer chain length oligosaccharides that occupy at least three sugar residue-binding subsites as acceptors for productive transglucosylation. This is the first report of a beta-glucansynthase derived from an exoglycosidase that can produce long-chain cello-oligosaccharides, which likely reflects the extended oligosaccharide-binding site of rice BGlu1 beta-glucosidase.     

Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein.

Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.     

A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars

A fungal strain, BCC2871 (Periconia sp.), was found to produce a thermotolerant beta-glucosidase, BGL I, with high potential for application in biomass conversion. The full-length gene encoding the target enzyme was identified and cloned into Pichia pastoris KM71. Similar to the native enzyme produced by BCC2871, the recombinant beta-glucosidase showed optimal temperature at 70 degrees C and optimal pH of 5 and 6. The enzyme continued to exhibit high activity even after long incubation at high temperature, retaining almost 60% of maximal activity after 1.5h at 70 degrees C. It was also stable under basic conditions, retaining almost 100% of maximal activity after incubation for 2h at pH8. The enzyme has high activity towards cellobiose and other synthetic substrates containing glycosyl groups as well as cellulosic activity toward carboxymethylcellulose. Thermostability of the enzyme was improved remarkably in the presence of cellobiose, glucose, or sucrose. This beta-glucosidase was able to hydrolyze rice straw into simple sugars. The addition of this beta-glucosidase to the rice straw hydrolysis reaction containing a commercial cellulase, Celluclast 1.5L (Novozyme, Denmark) resulted in increase of reducing sugars being released compared to the hydrolysis without the beta-glucosidase. This enzyme is a candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.     

Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe. Purification and characterization of intracellular beta-glucosidase

Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).     

Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus beta-glucosidase: effects of donor concentration, water content, and temperature on activity and selectivity in hexanol.

The transglucosylation reaction catalyzed by wild-type beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-beta-glucoside to hexyl-beta-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The selectivity towards transglucosylation was quantified by the S value defined as follows: S = r(S) x a(W)/r(H) x a(hex) where r(S) and r(H) are the initial rates of transglucosylation and hydrolysis and a(w) and a(hex) are the thermodynamic activities of water and hexanol. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-beta-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95 degrees C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis product. The largest increase in S-value (2.6 fold) was obtained by the F426Y mutant, which resulted in increased hexyl-beta-glucoside yield from 56% to 69%. In addition, the F426Y enzyme had higher selectivity over the wide range of temperatures tested. The activity of CelB wild-type and CelB F426Y increased as a function of water activity (a(w)), and complete activation by the water was obtained in a two-phase system with 20% water phase. In contrast to CelB wild-type, the F426Y mutant had transferase activity as low as a(w) = 0.29. Surprisingly, the S value increased with increasing water activity up to a(w) = 0.92. At still higher water content the S value decreased.     

Solid-phase chemical amination of a lipase from Bacillus thermocatenulatus to improve its stabilization via covalent immobilization on highly activated glyoxyl-agarose

In this paper, the stabilization of a lipase from Bacillus thermocatenulatus (BTL2) by a new strategy is described. First, the lipase is selectively adsorbed on hydrophobic supports. Second, the carboxylic residues of the enzyme are modified with ethylenediamine, generating a new enzyme having 4-fold more amino groups than the native enzyme. The chemical amination did not present a significant effect on the enzyme activity and only reduced the enzyme half-life by a 3-4-fold factor in inactivations promoted by heat or organic solvents. Next, the aminated and purified enzyme is desorbed from the support using 0.2% Triton X-100. Then, the aminated enzyme was immobilized on glyoxyl-agarose by multipoint covalent attachment. The immobilized enzyme retained 65% of the starting activity. Because of the lower p K of the new amino groups in the enzyme surface, the immobilization could be performed at pH 9 (while the native enzyme was only immobilized at pH over 10). In fact, the immobilization rate was higher at this pH value for the aminated enzyme than that of the native enzyme at pH 10. The optimal stabilization protocol was the immobilization of aminated BTL2 at pH 9 and the further incubation for 24 h at 25 degrees C and pH 10. This preparation was 5-fold more stable than the optimal BTL2 immobilized on glyoxyl agarose and around 1200-fold more stable than the enzyme immobilized on CNBr and further aminated. The catalytic properties of BTL2 could be greatly modulated by the immobilization protocol. For example, from (R/S)-2- O-butyryl-2-phenylacetic acid, one preparation of BTL2 could be used to produce the S-isomer, while other preparation produced the R-isomer.     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

Preparation of immobilized alpha-amylase covalently attached to granular polyacrylonitrile

In this report, alpha-Amylase originating from Bacillus subtilis (liquefying type) was immobilized on partially imidoesterized polyacrylonitrile (PAN) by covalent bonding. For the preparation of immobilized alpha-amylase, which has a high activity and high stability to repeated use, the optimum conditions for the preparation reaction were investigated. The optimum conditions for the preparation reaction were quantified on the basis of the enzymatic activity, the preservation of the activity during repeated use in batch process and the protein content on the support. Further-more, enzymatic properties of immobilized alpha-amylase prepared at optimum conditions were compared with the native enzyme. The optimum temperature and reaction time for the imidoes-terification reaction were 30 degrees c and 6 h, respectively, whereas those of the amidinatin reaction were 30-40 degrees C and more than 3 h, respectively; the optimum pH range was 9-10. Immobilized alpha-amylase prepared at the optimum conditions was very stable against the repeated use and had more than 90% of relative to activity of the first use after the tenth procedure. The initial reaction rate of immobilized alpha-amylase was lower than native alpha-amylase, but same amount of reducing sugars were produced after the reaction passed for more than 90 min. The immobilized alpha-amylase was less stabel at the high temperature and the more basic media. However, after long incubation time, immobilized alpha-amylase was more stable than the native enzyme in exposure to heat and a storng base.     

Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

The rate of celluose degradation, limited due to the inhibition by cellobiose, can be increased by the hydrolysis of cellobiose to glucose using immobilized beta-glucosidase. Production of beta-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. A study of the immobilization of beta-glucosidase containing cells of Pichia etchellsii on various solid supports was conducted and immobilization by entrapment in calcium alginate gel beads was found to be the most simple and efficient method. A retention of 96.5% of initial activity after ten sequential batch uses of the immobilized preparation was observed. The pH and temperature optima for free and immobilized cells were the same, i.e., 6.5 (0.05M Maleate buffer) and 50 degrees C, respectively. Even though the temperature optimum was found to be 50 degrees C, the enzyme exhibits a better thermal stability at 45 degrees C. Beads stored at 4 degrees C for six months retain 80% of their activity. Kinetic studies performed on free and immobilized cells shown that glucose is a noncompetitive product inhibitor.The immobilized preparation was found to be limited by pore diffusion but exhibited no film-diffusion resistance during packed bed column indicated by a low dispersion number of 0.1348. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to the cellobiose hydrolysis system. The rate of reaction with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. An effectiveness factor of 0.49 was obtained for a particle diameter of 2.5 mm. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column was found to fall rapidly with increase in conversion rate indicating that the operating conditions of the column would have to be a compromise between high conversion rates and reasonable productivity. A half-life of over seven days was obtained at the operating temperature of 45 degrees C in continuous operation of the packed bed reactor. However, the half-life in the column was found to be greatly affected by temperature, increasing to over seventeen days at a temperature of 40 degrees C and decreasing to less than two days at 50 degrees C.     

Overexpression of celB gene coding for beta-glucosidase from Pyrococcus furiosus using a baculovirus expression vector system in silkworm, Bombyx mori

beta-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing beta-D-glucose residues from the end of various oligosaccharides with the release of beta-D-glucose. CelB gene, encoding the thermostable beta-glucosidase, was amplified from the Pyrococcus furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express beta-glucosidase in silkworm. The recombinant beta-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed beta-glucosidase was obtained at pH 5.0 and about 105 degrees C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant beta-glucosidase were similar to the native counterpart. The expressed beta-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable beta-glucosidase using this system.     

Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports

Dextransucrase from Leuconostoc mesenteroides B-512F was immobilized on epoxy-activated acrylic polymers with different textural properties (Eupergit C and Eupergit C 250L). Prior to immobilization, dextransucrase was treated with dextranase to remove the dextran layer covering the enzyme surface, thus increasing the accessibility of its reactive groups to the epoxide centers of the support. Elimination of 99% of the initial carbohydrate content was determined by the anthrone method. To prevent enzyme inactivation, the immobilization was carried out at pH 5.4, at which the coupling to the support took place through the carboxylic groups of the enzyme. The effects of the amount (mg) of dextransucrase added per gram of support (from 0.2:1 to 30:1), temperature and contact time were studied. Maximum activity recovery of 22% was achieved using Eupergit C 250L. Using this macroporous support, the maximum specific activity (710 U/g biocatalyst) was significantly higher than that obtained with the less porous Eupergit C (226 U/g biocatalyst). The dextransucrase immobilized on Eupergit C 250L showed similar optimal temperature (30 degrees C) and pH (5-6) compared with the native enzyme. In contrast, a notable stabilization effect at 30 degrees C was observed as a consequence of immobilization. After a fast partial inactivation, the dextransucrase immobilized on Eupergit C 250L maintained more than 40% of the initial activity over the following 2 days. The features of this immobilized system are very attractive for its application in batch and fixed-bed bioreactors.     

An enzyme-polymer film prepared with the use of poly(vinyl alcohol) bearing photosensitive aromatic azido groups

Photochemical reaction of poly(vinyl alcohol) bearing aromatic azido groups was applied for immobilization of beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21.) in poly(vinyl alcohol) film. Photo-crosslinking and immobilization reactions proceeded by light irradiation for 25 min in air. The immobilized enzyme showed approx. 40% of its native enzyme activity with an apparent Michaelis constant of 3.9 mM. The Michaelis constant of the native enzyme was 2.3 mM. Some properties of the immobilized and native enzyme are compared.     

Purification and partial characterization of a cellodextrin glucohydrolase (beta-glucosidase) from Trichoderma reesei strain QM 9414

A beta-glucosidase (E.C. 3.2.1.21) was isolated from the culture filtrate of fungus Trichoderma reesei QM 9414 grown in continuous culture with biomass retention. The crude extracellular enzyme preparation was fractionated by a three-step purification procedure [chromatography on Fractogel HW-55 (S) and Bio-Gel A 0.5 plus final preparative isoelectric focusing] to yield three beta-glucosidases with isoelectric points at pH 8.4, 8.0, and 7.4. Only one enzyme (pi 8.4) met the stringent criterion of being homogeneous according to titration curve analysis. This enzyme was then characterized not to be a glycoprotein, although the native protein contained 35% carbohydrate (as glucose). It was found to have an apparent molar mass of 7 x 10(4) g/mol (SDS-PAGE), exhibited its optimum activity towards cellobiose at pH 4.5 and 70 degrees C (30 min test), and lost less than 3% activity at 50 degrees C over a period of 7 h. The K(M) values towards cellobiose and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.5mM and 0.3mM, respectively. The enzyme hydrolyzed cellodextrins (cellotriose to cellooctaose) by sequentially splitting off glucose units from the nonreducing end of the oligomers. The extent of the observed transfer reactions varied with the initial substrate concentration. No enzyme activity towards microcrystalline cellulose or carboxymethylcellulose could be detected. The classification of the enzyme as beta-glucosidase or exo-beta-1,4-glucan glucohydrolase is discussed with respect to the exhibited hydrolytic activities.     

锰过氧化物酶的耦合固定化及其性质研究

分别采用海藻酸钠、明胶和壳聚糖为载体,并以戊二醛为交联剂,通过包埋-交联和吸附-交联两种耦合固定化方法制备固定化锰过氧化物酶。探讨了酶的不同固定化条件和固定化酶的部分性能。与游离酶相比,制备的3种固定化酶最适反应pH分别由7·0降低到5·0、5·0和3·0,最适反应温度分别由35℃升高到75℃、55℃和75℃。3种固定化酶的耐热性都显著提高,其中用壳聚糖制成的固定化酶在pH2·2~11的宽范围内表现出很好的酸碱耐受性。30℃连续测定6~9次酶活力,重复使用的3种固定化酶显示出良好的稳定性。将固定化酶应用在偶氮染料的脱色中,用明胶制成的固定化酶在静置和摇床条件下,以及用海藻酸钠制成的固定化酶在摇床条件下,均表现出与游离酶相近的脱色能力,并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。     

Immobilization of Streptomyces flavochromogenes pullulanase on tannic acid and TEAE--cellulose.

Pullulanase was immobilized successfully by simple, inexpensive methods that may be useful for industrial application of this enzyme. A tannin--pullulanase(TP) complex was obtained by addition of tannic acid to the culture filtrate of thermophilic Streptomyces flavochromogenes. TP could be bound to TEAE--cellulose (TTCP). Immobilization in this manner took place with quantitative retention of activity. The immobilized enzymes were stable for more than six months. The optimum temperatures of the native enzyme and TP were both 50 degrees C; that of TTCP was 45 degrees C. In the presence of 5mM Ca2+, the activity of TTCP was increased approximately twofold and the optimum temperature was raised to 50--60 degrees C. Pullulanase was not significantly eluted from TP or TTCP by NaCl solution (0.1--0.5M).     

Enzymatic Synthesis of Carbohydrate Derivatives Using β-Glycosidase of Sulfolobus Solfataricus

Enzymatic synthesis of different β-D-glycosides was obtained using as biocatalyst immobilized cells, crude homogenate, and homogeneous native and recombinat β-glycosidase activity of the thermophilic archaeon Sulfolobus solfataricus. In particular our investigation was concerned with the selectivity in the glycosylation of hydroxybenzyl alcohols, salicin, 1,2-propanediol, and more complex polyols as well as the use of immobilized cells for the synthesis of hexyl-β-D-glucoside. The aromatic glucosides obtained by enzyme-catalyzed transglucosylation were used for kinetic studies of purified Sulfolobus solfataricus enzyme in the hydrolysis reaction.