Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.     

Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues

A beta-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-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-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 beta-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.     

Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB from Pyrococcus furiosus

The substrate specificity of the beta-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 A resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)(8)-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho-beta-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-beta-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.     

Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C.

Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h.     

Directed evolution of transketolase activity on non-phosphorylated substrates

We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form L-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.     

Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals

The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.     

Pressure-Enhanced Activity and Stability of a Hyperthermophilic Protease from a Deep-Sea Methanogen

We describe the properties of a hyperthermophilic, barophilic protease from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This enzyme is the first protease to be isolated from an organism adapted to a high-pressure-high-temperature environment. The partially purified enzyme has a molecular mass of 29 kDa and a narrow substrate specificity with strong preference for leucine at the P1 site of polypeptide substrates. Enzyme activity increased up to 116(deg)C and was measured up to 130(deg)C, one of the highest temperatures reported for the function of any enzyme. In addition, enzyme activity and thermostability increased with pressure: raising the pressure to 500 atm increased the reaction rate at 125(deg)C 3.4-fold and the thermostability 2.7-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the M. jannaschii protease is not grossly different from that of several mesophilic proteases; however, the active-site structure may be relatively rigid at moderate temperatures. The barophilic and thermophilic behavior of the enzyme is consistent with the barophilic growth of M. jannaschii observed previously (J. F. Miller et al., Appl. Environ. Microbiol. 54:3039-3042, 1988).     

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.     

Identification, purification, and characterization of a thermophilic imidase from pig liver

This study investigates thermophilic imidase activity of the liver. We demonstrate that imidase catalyzes the hydrolysis of imides at a temperature substantially higher than that of its native environment. Then, a thermophilic imidase is purified to homogeneity from pig liver, and its thermoproperties are studied. About 2500-fold of purification and 15% yield of imidase activity are obtained after ammonium sulfate precipitation, octyl, DEAE, chelation, and gel filtration chromatography. While avoiding heat treatment for the protein purification, this study also indicates that only one enzyme is responsible for the imidase activity. This homogenous enzyme prefers to catalyze hydrolysis of imides at above 60 degrees C rather than at the body temperature of a pig. Although stable at below 50 degrees C, imidase quickly loses its activity at above 65 degrees C. Thus, the temperature effect on imidase activity is limited mainly by its thermostability. Substrate specificity of imidase is also temperature dependent. Our results demonstrate that the hydrolysis of physiological substrates is the most temperature dependent and that of hydantoins is the least temperature dependent. When increasing the reaction temperature from 25 to 60 degrees C, specific activities increase 50- and 60-fold for dihydrouracil and dihydrothymine, respectively. The temperature effect on the K(m) and V(max) of imidase is substrate dependent.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-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 degrees 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%) compared with the wild-type CelB (40%). 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%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.     

Altering substrate specificity of phosphatidylcholine-preferring phospholipase C of Bacillus cereus by random mutagenesis of the headgroup binding site

PLC(Bc) is a 28.5 kDa monomeric enzyme that catalyzes the hydrolysis of the phosphodiester bond of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to provide a diacylglycerol and the corresponding phosphorylated headgroup. Because single replacements of Glu4, Tyr56, and Phe66 in the headgroup binding pocket led to changes in substrate specificity [Martin et al. (2000) Biochemistry 39, 3410-3415], a combinatorial library of approximately 6000 maltose binding protein-PLC(Bc) fusion protein mutants containing random permutations of these three residues was generated to identify PLC(Bc) mutants with altered specificity profiles and high catalytic activities. Members of this library were screened for hydrolytic activity toward the water soluble substrates C6PC, C6PE, and C6PS using a novel protocol that was conducted in a 96-well format and featured the in situ cleavage of the fusion protein to release the mutant PLC(Bc)s. Ten mutant enzymes that exhibited significant preferences toward C6PE or C6PS were selected and analyzed by steady-state kinetics to determine their specificity constants, k(cat)/K(M). The C6PS selective clones E4G, E4Q/Y56T/F66Y, and E4K/Y56V exhibited higher specificity constants toward C6PS than wt, whereas Y56T, F66Y, and Y56T/F66Y were C6PE selective and had comparable or higher specificity constants than wt for C6PE. The corresponding wt residues were singly reinserted back into the E4Q/Y56T/F66Y and E4K/Y56V mutants via site-directed mutagenesis, and the E4Q/F66Y mutant thus obtained exhibited a 10-fold higher specificity constant toward C6PS than wt, a value significantly higher than other PLC(Bc) mutants. On the basis of available data, an aromatic residue at position 66 appears important for significant catalytic activity toward all three substrates, especially C6PC and C6PE. The charge of residue 4 also appears to be a determinant of enzyme specificity as a negatively charged residue at this position endows the enzyme with C6PC and C6PE preference, whereas a polar neutral or positively charged residue results in C6PS selectivity. Replacing Tyr56 with Val, Ala, Thr, or Ser greatly reduces activity toward C6PC. Thus, the substrate specificity of PLC(Bc) can be modulated by varying three of the amino acid residues that constitute the headgroup binding pocket, and it is now apparent that this enzyme is not evolutionarily optimized to hydrolyze phospholipids with ethanolamine or serine headgroups.     

Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. III. Utilization of two thermostable beta-glycosidases in a continuous ultrafiltration membrane reactor and galacto-oligosaccharide formation under steady-state conditions.

Hydrolysis of lactose by hyperthermophilic beta-glycosidases from the archaea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) was carried out at 70 degrees C in a continuous stirred-tank reactor (CSTR) coupled to a 10-kDa cross-flow ultrafiltration module to recycle the enzyme. Recirculation rates of > or =1 min(-1), reaction of proteins with reducing sugars, and enzyme adsorption onto the membrane are major "operational" factors of enzyme inactivation in the CSTR. They cause the half-life times of SsbetaGly and CelB to be reduced two- and eight-fold, respectively, the average value for both enzymes now being approximately 5 to 7 days. Using lactose at initial concentrations of 45 and 170 g/L, the CSTR was operated at a constant conversion level of approximately 80% for more than 2 weeks without the occurrence of microbial contamination. The productivities for the SsbetaGly-catalyzed conversion of lactose were determined at different dilution rates and initial substrate concentrations, and exceed by a factor of < or =2 those observed with CelB under otherwise identical conditions. This difference reflects the approximately eight-fold stronger product inhibition of CelB by D-glucose. While the maximum total galacto-oligosaccharide production (90-100 mM) at 170 g/L lactose in the CSTR was not different from that in the batch reactor (CelB) or was greater by approximately 25% (SsbetaGly), continuous and batchwise reactions with both enzymes differed markedly with regard to relative proportions of the individual saccharide components present at 80% substrate conversion. The CSTR yielded an up to four-fold greater ratio of disaccharides to trisaccharides concomitant with a 5- to 30-fold larger relative proportion of beta-D-Galp-(1-->3)-D-Glc in the product mixture. The results show that apart from continuous hydrolysis of lactose at 70 degrees C, a CSTR charged with SsbetaGly or CelB and operated at steady-state conditions could be a useful reaction system for the production of galacto-oligosaccharides in which composition is narrower and more easily programmable, in terms of the individual components contained, as compared to the batchwise reaction.     

Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and approximately 450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substrate-free form at 2.1 A resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A--4-hydroxybenzaldehyde complex structure at 2.1 A resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.     

Effects of temperature on allosteric and catalytic properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.     

Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Isolation and characterization of a 1,4-beta-D-glucan glucohydrolase from the yeast, Torulopsis wickerhamii

1,4-beta-D-Glucan glucohydrolase (exo-1,4-beta-D-glucosidase) (EC 3.2.1.74) was isolated from growth supernatants of Torulopsis wickerhamii and was subjected to hydrodynamic, optical (CD), and kinetic analysis after purification to homogeneity by ammonium sulfate precipitation, size exclusion chromatography, ion exchange chromatography, and isopycnic banding centrifugation in cesium chloride. The last step was found to separate the enzyme from strongly associating, high molecular weight polysaccharide. Enzyme homogeneity was established by isoelectric focusing, sodium dodecyl sulfate-gel electrophoresis, and analytical high performance size exclusion chromatography using dual detection. The native exo-1,4-beta-D-glucosidase was found to be a dimer of 151,000 +/- 21,100 daltons by high performance size exclusion chromatography and 143,600 +/- 1,800 daltons by sedimentation equilibrium. The enzyme has a 12% linked carbohydrate content (mostly mannose) and no essential metal ions. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside was found to be optimal at pH 4.25 and 50 degrees C. The enzyme was found to produce beta-D-glucose from cellodextrins (indicating retention of anomeric configuration during hydrolysis) and demonstrated depolymerization from the non-reducing polymer terminus. The enzyme followed competitive type inhibition with p-nitrophenyl-beta-D-glucopyranoside as substrate and demonstrated high values of Ki for D-glucose and D-cellobiose inhibition (190 and 230 mM, respectively). The exo-1,4-beta-D-glucosidase was found to hydrolyze cellotetraose more rapidly than D-cellobiose and aryl-beta-D-glycosides more rapidly than all other substrates. Low levels of activity were found for the polymeric substrates beta-glucan (yeast cell walls), Avicel, and Walseth cellulose. Although this enzyme demonstrates broad disaccharide substrate specificity, a characteristic common to beta-D-glucosidases from many sources, the ability to hydrolyze higher cellodextrins more rapidly than cellobiose renders this enzyme the first exo-1,4-beta-D-glucosidase purified from yeast.     

Entrapment in E. coli improves the operational stability of recombinant beta-glycosidase CelB from Pyrococcus furiosus and facilitates biocatalyst recovery

beta-Glycosidase CelB from the hyperthermophilic archaeon Pyrococcus furiosus is a versatile biocatalyst that has been used for the hydrolysis and synthesis of beta-d-glycosidic compounds at high temperatures and in non-conventional solvents. In spite of its outstanding thermal stability, CelB is prone to inactivation in the presence of reducing sugars and through recirculation in loop enzyme reactors. Entrapment into E. coli cells was used here to improve the stability of recombinant CelB under conditions promoting strong inactivation. Glutardialdehyde-mediated protein cross-linking or rigidification of the cell membrane by adding magnesium ions was required to prevent release of CelB from within the cell into the bulk solution. In the presence of 1M glucose or when applying recirculation rates of 2.6 min(-1), the entrapped enzyme was around two-fold more stable at 80 degrees C than free CelB. The significance of the stabilisation was attenuated by the decrease in CelB initial activity which was due to cross-linking and glutardialdehyde concentration-dependent. Entrapment facilitated downstream processing of CelB and biocatalyst recovery in repeated batchwise conversions of lactose at elevated temperatures.     

Purification and characterization of an extracellular beta-glucosidase from Monascus purpureus

An extracellular beta-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A 22 central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at 50 degrees and pH 5.5. The beta-glucosidase showed moderate thermostability, was inhibited by HgCl2, K2CrO4, and K2Cr2O7, whereas other reagents including beta- mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-beta-D-glucopyranoside, and maltose indicates that the beta-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-beta-Dcellobiose. beta-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.     

Substrate specificity in glycoside hydrolase family 10. Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of Pseudomonas cellulosa xylanase 10A

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes beta1, 4-linked xylans but exhibits very low activity against aryl-beta-cellobiosides. The family 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-beta-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the -1 and -2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the -2 subsite of the enzyme. Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the -1 subsite. We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-beta-cellobioside and 4-nitrophenyl-beta-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-beta-xylobioside activity but did not significantly reduce 4-nitrophenyl-beta-cellobioside hydrolysis. The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents. These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed.