Characterization of extremely thermostable enzymatic breakers (alpha-1,6-galactosidase and beta-1,4-mannanase) from the hyperthermophilic bacterium Thermotoga neapolitana 5068 for hydrolysis of guar gum

An alpha-galactosidase and a beta-mannanase produced by the hyperthermophilic bacterium, Thermotoga neapolitana 5068 (TN5068), separately and together, were evaluated for their ability to hydrolyze guar gum in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil and gas well stimulation. In such applications, premature guar gum hydrolysis at lower temperatures before the fracturing process is completed is undesirable, whereas thermostability and thermoactivity are advantageous. Hyperthermophilic enzymes presumably possess both characteristics. The purified alpha-galactosidase was found to have a temperature optimum of 100-105 degrees C with a half-life of 130 minutes at 90 degrees C and 3 min at 100 degrees C, while the purified beta-mannanase was found to have a temperature optimum of 91 degrees C and a half-life of 13h at this temperature and 35 min at 100 degrees C.These represent the most thermostable versions of these enzymes yet reported. At 25 degrees C, TN5068 culture supernatants, containing the two enzyme activities, reduced viscosity of a 0.7% (wt) guar gum solution by a factor of 1.4 after a 1.5-h incubation period and by a factor of 2.4 after 5 h. This is in contrast to a viscosity reduction of 100-fold after 1.5 h and 375-fold after 5 h for a commercial preparation of these enzymes from Aspergillus niger. In contrast, at 85 degrees C, the TN5068 enzymes reduced viscosity by 30-fold after 1.5 h and 100-fold after 5 h compared to a 2.5-fold reduction after 5 h for the control. The A. niger enzymes were less effective at 85 degrees C (1.6-fold reduction after 1.5 h and a 4.2-fold reduction after 5 h), presumably due to their thermal lability at this temperature. Furthermore, it was determined that the purified beta-mannanase alone can substantially reduce viscosity of guar solutions, while the alpha-galactosidase alone had limited viscosity reduction activity. However, the alpha-galactosidase appeared to minimize residual particulate matter when used in conjunction with the beta-mannanase. This could be the result of extensive hydrolysis of the alpha-1,6 linkages between mannose and galactose units in guar, allowing more extensive hydrolysis of the mannan chain by the beta-mannanase. The use of thermostable enzymatic breakers from hyperthermophiles in hydraulic fracturing could be used to improve well stimulation and oil and gas recovery. (c) 1996 John Wiley & Sons, Inc.     

Beta-mannosidase (EC 3.2.1.25) activity during and following germination of tomato (Lycopersicon esculentum Mill.) seeds. Purification,cloning and characterization

Beta-mannosidase, a high-salt-soluble enzyme, increases in activity in seeds of tomato prior to the completion of germination. This increase occurs in both the lateral and micropylar endosperm and becomes more evident during post-germinative seedling growth. The beta-mannosidase activity profile is similar to that of endo beta-mannanase although it is the first to increase in the lateral endosperm. Tomato seed beta-mannosidase was purified to homogeneity and its cDNA (LeMside1) obtained by 3'-RACE PCR using oligonucleotide sequences based on four peptide sequences obtained from the purified enzyme. The derived amino acid sequence of the tomato beta-mannosidase shows the enzyme is a member of the Glycosyl Hydrolases Family 1 (GHF1) but has a very low sequence identity with that of beta-mannosidases from non-plant sources; no other plant sequence for the enzyme is known. There appears to be only one gene encoding beta-mannosidase in tomato, the sequence of which has been determined (LeMSide2). Its expression occurs first in the micropylar endosperm, and then declines after germination. This is followed by an increase in its expression in the lateral endosperm, which precedes that of the gene for endo beta-mannanase. Expression of the beta-mannosidase gene increases appreciably in the growing seedling embryo. With this report, the cloning of all three of the enzymes involved in galactomannan mobilization (endo beta-mannanase, alpha-galactosidase and beta-mannosidase) in tomato seeds has now been achieved.     

Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068.

Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.     

Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.     

Mannan-degrading enzymes purified from the crop of the brown garden snail Helix aspersa Müller (Gastropoda Pulmonata)

Two mannan-degrading enzymes were purified from the crop of the terrestrial snail Helix aspersa Müller. The crude extracts were taken from dormant (for 4 months) snails. The enzymes were a betaD-mannanase of 37.4 +/- 0.3 kDa (EC 3.2.1.78) and a betaD-mannosidase of 77.8 +/- 1.9 kDa (EC 3.2.1.25). Both enzymes degraded insoluble mannan, releasing either mannose only (beta-mannosidase) or oligosaccharides, possibly mannotriose and mannopentaose (beta-mannanase). The beta-mannanase had a typical endo-activity pattern, while the beta-mannosidase was an exoenzyme. The incubation of both enzymes with mannan increased the catalysis by 83%. The best synergy was found with 75% mannosidase combined with 25% mannanase. The beta-mannanase also hydrolysed beta-linked heteromannans and its affinity for different galactomannans was studied. The Km values, varying from 2.89 +/- 0.47 mg x ml(-1) to 0.26 +/- 0.01 mg x ml(-1), revealed the inhibitory effect of the alphaD-galactosyl residues released. The beta-mannosidase was acidic (optimum pH = 3.3) and heat-sensitive (50% residual activity at 42 degrees C after 5 min of pre-incubation), while the beta-mannanase remained stable until 48.5 degrees C (50% residual activity) and over a pH range of 4.3-7.5. The properties of these mannanolytic enzymes are discussed in this paper compared with those purified in other gastropods and in a bacterium, Enterococcus casseliflavus, a quite similar strain previously isolated from this snail intestine. The occurrence of thermostable enzymes in H. aspersa digestive tract could be a zootechnic parameter of great importance for snail farming. J. Exp. Zool. 290:125-135, 2001.     

Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.     

Influence of detergents and pH on the isolation, purification and molecular properties of alpha-galactosidase-C2 from germinating guar (Cyamopsis tetragonolobus)

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.     

Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus

Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and alpha-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 alpha-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.     

Kinetic model for the co-action of beta-amylase and debranching enzymes in the production of maltose

The kinetics of the hydrolysis of starch with beta-amylase and debranching enzymes was studied. The hydrolysis of the alpha-1, 6-glycoside bonds of the substrate by debranching enzymes does not create any new nonreducing ends, so debranching enzyme promotes the action of beta-amylase not by increasing the concentration of the substrate of beta-amylase but by increasing the linear linkage portion of the substrate. The introduction of an effective chain length function was used to formulate a kinetic model.     

Protein release from galactoglucomannan hydrogels: influence of substitutions and enzymatic hydrolysis by beta-mannanase

O-Acetyl-galactoglucomannan (AcGGM) is the major soft-wood hemicellulose. Structurally modified AcGGM and hydrogels of AcGGM were prepared. The degree of substitution (DS) of AcGGM was modified enzymatically with alpha-galactosidase, and chemically with an acrylate derivative, 2-hydroxyethylmethacrylate (HEMA). The hydrolysis of AcGGM with beta-mannanase was shown to increase with decreasing DS. AcGGM hydrogels were prepared from chemically modified AcGGM with varying DS of HEMA. Bovine serum albumin (BSA) was encapsulated in hydrogels. A spontaneous burst release of BSA was decreased with increased DS of HEMA. The addition of beta-mannanase significantly enhanced the BSA release from hydrogels with a DS of 0.36, reaching a maximum of 95% released BSA after eight hours compared to 60% without enzyme. Thus, both the pendant group composition and the enzyme action are valuable tools in the tailoring of hydrogel release profiles of potential interest for intestine drug delivery.     

Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus.

When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces alpha-galactosidase I which is different from alpha-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for alpha-galactosidase I, 6.3 to 6.5 for alpha-galactosidase II). However, alpha-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas alpha-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. alpha-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (Km values of 20.8, 98.1, and 8.5 mM, respectively) than does alpha-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of alpha-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only alpha-galactosidase that was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose.     

Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum.

Bacteroides ovatus, a gram-negative obligate anaerobe from the human colon, can ferment the branched galactomannan guar gum. Previously, three enzymes involved in guar gum breakdown were characterized. The expression of these enzymes appeared to be regulated; i.e., specific activities were higher in extracts from bacteria grown on guar gum than in extracts from bacteria grown on the monosaccharide constituents of guar gum, mannose and galactose. In the present study, we used two-dimensional gel analysis to determine the total number of B. ovatus proteins enhanced during growth on guar gum. Twelve soluble proteins and 20 membrane proteins were expressed at higher levels in guar gum-grown cells than in galactose-grown cells. An unexpected finding was that the expression of the two galactomannanases was induced by glucose as well as guar gum. Three other proteins, one membrane protein and two soluble proteins, had this same expression pattern. The remainder of the guar gum-associated proteins seen on two-dimensional gels and the guar gum-associated alpha-galactosidase were induced in cells grown on guar gum but not in cells grown on glucose. Two transposon-generated mutants (M-5 and M-7) that could not grow on guar gum were isolated. Both mutants still expressed the galactomannanases and the alpha-galactosidase. They also still expressed all of the guar gum-associated proteins that could be detected in two-dimensional gels of glucose-grown or galactose-grown cells. A second transposon insertion that suppressed the guar gum-negative phenotype of M-5 was isolated and characterized. The characteristics of this suppressor mutant indicated that the original transposon insertion was probably in a regulatory locus.     

Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum.

Bacteroides ovatus, a gram-negative obligate anaerobe from the human colon, can ferment the branched galactomannan guar gum. Previously, three enzymes involved in guar gum breakdown were characterized. The expression of these enzymes appeared to be regulated; i.e., specific activities were higher in extracts from bacteria grown on guar gum than in extracts from bacteria grown on the monosaccharide constituents of guar gum, mannose and galactose. In the present study, we used two-dimensional gel analysis to determine the total number of B. ovatus proteins enhanced during growth on guar gum. Twelve soluble proteins and 20 membrane proteins were expressed at higher levels in guar gum-grown cells than in galactose-grown cells. An unexpected finding was that the expression of the two galactomannanases was induced by glucose as well as guar gum. Three other proteins, one membrane protein and two soluble proteins, had this same expression pattern. The remainder of the guar gum-associated proteins seen on two-dimensional gels and the guar gum-associated alpha-galactosidase were induced in cells grown on guar gum but not in cells grown on glucose. Two transposon-generated mutants (M-5 and M-7) that could not grow on guar gum were isolated. Both mutants still expressed the galactomannanases and the alpha-galactosidase. They also still expressed all of the guar gum-associated proteins that could be detected in two-dimensional gels of glucose-grown or galactose-grown cells. A second transposon insertion that suppressed the guar gum-negative phenotype of M-5 was isolated and characterized. The characteristics of this suppressor mutant indicated that the original transposon insertion was probably in a regulatory locus.     

Formation of a self-assembled phenylboronic acid monolayer and its application toward developing a surface plasmon resonance-based monosaccharide sensor

An assay has been developed to quantitate the amount of mannose 6-phosphate in glycoproteins using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The method was tested on a recombinant lysosomal enzyme, human alpha-galactosidase A, that contains mannose 6-phosphate. The assay includes two steps: hydrolysis of the glycoprotein in 6.75 M trifluoroacetic acid to release mannose 6-phosphate and quantitation of the released mannose 6-phosphate using HPAEC with PAD. There is a linear relationship between the amount of mannose 6-phosphate measured and the amount of alpha-galactosidase hydrolyzed. The assay is also sensitive for as little as 2.5 microg alpha-galactosidase, which contains 117 pmol mannose 6-phosphate. Further, the assay has been shown to have good day-to-day and operator-to-operator consistency. In order to evaluate the assay for glycoprotein in crude extract, the glycoprotein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The amount of mannose 6-phosphate in the electroblots following hydrolysis was determined using HPAEC-PAD. The assay was also linear when measuring mannose 6-phosphate on electroblots. Therefore, this assay has been shown to be specific, sensitive, and reproducible.     

Antagonistic action of low-fluence and high-irradiance modes of response of phytochrome on germination and beta-mannanase activity in Datura ferox seeds

Seed germination is often induced by a pulse of red light perceived by phytochrome and cancelled by a subsequent pulse of far-red light. When the pulse of red light is followed by several hours of darkness, a pulse of far-red light is no longer effective and prolonged far-red is necessary to block germination. The aim was to investigate whether the red light pulse and prolonged far-red light act on the same or different processes during germination of Datura ferox seeds. Forty-five hours after the inductive red light pulse, germination could not be blocked by one pulse or six hourly pulses of far-red light, but was significantly reduced by 6 h of continuous far-red light. The pulse of red light increased embryo growth potential and the activities of beta-mannanase and beta-mannosidase extracted from the micropylar region of the endosperm. Continuous far-red light had no effect on embryo growth potential or beta-mannosidase activity, but severely reduced the activity of beta-mannanase. The effect of far-red light had the features of a high-irradiance response of phytochrome. Both germination and beta-mannanase activity were restored by a pulse of red light given after the end of the continuous far-red treatment. It is concluded that the low-fluence response and the high-irradiance response modes of phytochrome have antagonistic effects on seed germination and that the control of beta-mannanase activity is one process where this antagonism is established.     

Purification and characterization of a cell-associated, soluble mannanase from Bacteroides ovatus.

Bacteroides ovatus, a human colonic anaerobe, utilizes the galactomannan guar gum as a sole source of carbohydrate. Previously, we found that none of the galactomannan-degrading enzymes were extracellular, and we characterized an outer membrane mannanase which hydrolyzes the backbone of guar gum to produce large fragments. We report here the purification and characterization of a second mannanase from B. ovatus. This enzyme is cell-associated and soluble. Using ion-exchange chromatography, gel filtration, and chromatofocusing steps, we have purified the soluble mannanase to apparent homogeneity. The enzyme has a native molecular weight of 190,000 and a monomeric molecular weight of 61,000. It is distinct from the membrane mannanase not only with respect to cellular location but also with respect to stability and isoelectric point (pI of 6.9 for the membrane mannanase and pI of 4.8 for the soluble mannanase). The soluble mannanase, like the membrane mannanase, hydrolyzed guar gum to produce large fragments rather than monosaccharides. However, if galactosyl side chains were removed from the galactomannan fragments by alpha-galactosidase, both the soluble mannanase and the membrane mannanase could degrade guar gum to monosaccharides. Thus either or both of these two enzymes, working together with alpha-galactosidase, appear to be sufficient for the breakdown of guar gum to the level of monosaccharides.     

Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30.

Trichoderma reesei RUT C-30 formed an extracellular alpha-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain alpha-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced alpha-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of alpha-galactosidase formation by galactose. alpha-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 +/- 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several alpha-galactosides (p-nitrophenyl-alpha-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei beta-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50 degrees C resulted in recovery of 70% of activity), and its highest level of activity occurred at 60 degrees C. Its action on galactomannan was increased by the presence of beta-mannanase.     

Purification and properties of alpha-mannosidase from bakers' yeast.

The yeast alpha-mannosidase [EC 3.2.1.24] was purified 1160-fold from the crude extract of the autolysate. The purified preparation was practically free from alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-mannosidase, and beta-N-acetylhexosaminidase activities. After the separation of yeast mannan during the purification procedures the enzyme became unstable but could be stored at 5 degrees C for three weeks with 50% loss of activity. The purified enzyme hydrolyzed both aryl and alkyl mannosides, but hydrolysis of yeast mannan proceeded slowly. Yeast mannan and Zn2+ increased the enzyme catalyzed hydrolysis of p-nitrophenyl mannoside, whereas NaN3, monoiodoacetate and methyl alpha-D-mannoside acted as inhibitors. The molecular weight was estimated to be 450,000 by gel filtration.     

Glycosidases of Ehrlich ascites tumor cells and ascitic fluid--purification and substrate specificity of alpha-N-acetylgalactosaminidase and alpha-galactosidase: comparison with coffee bean alpha-galactosidase

Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.     

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.