A thermostable non-xylanolytic alpha-glucuronidase of Thermotoga maritima MSB8

A putative alpha-glucosidase belonging to glycosyl hydrolase family 4 of Thermotoga maritima (TM0752) was expressed in Escherichia coli and it was found that the recombinant protein (Agu4B) was a p-nitrophenyl alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase, not alpha-glucosidase. It did not hydrolyze 4-O-methyl-D-glucuronoxylan or its fragment oligosaccharides. Agu4B was thermostable with an optimum temperature of 80 degrees C. It strictly required Mn(2+) and thiol compounds for its activity. The presence of NAD(+) slightly activated the enzyme. The amino acid sequence of Agu4B showed higher identity with Agu4A (another alpha-glucuronidase of T. maritima, 61%) than with AglA (alpha-glucosidase of T. maritima, 48%).     

Gene cloning and characterization of alpha-glucuronidase of Bacillus stearothermophilus no. 236

The alpha-glucuronidase gene of Bacillus stearothermophilus No. 236 was cloned, sequenced, and expressed in Escherichia coli. The gene, designated aguA, encoded a 691-residue polypeptide with calculated molecular weight of 78,156 and pI of 5.34. The alpha-glucuronidase produced by a recombinant E. coli strain containing the aguA gene was purified to apparent homogeneity and characterized. The molecular weight of the alpha-glucuronidase was 77,000 by SDS-PAGE and 161,000 by gel filtration; the functional form of the alpha-glucuronidase therefore was dimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and 40 degrees C, respectively. The enzyme's half-life at 50 degrees C was 50 min. The values for the kinetic parameters of Km and Vmax were 0.78 mM and 15.3 U/mg for aldotriouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucopyranosyluronic)-D-xylobiose]. The alpha-glucuronidase acted mainly on small substituted xylo-oligomers and did not release methylglucuronic acid from intact xylan. Nevertheless, synergism in the release of xylose from xylan was found when alpha-glucuronidase was added to a mixture of endoxylanase and beta-xylosidase.     

A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase

4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity. A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase. The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.     

极端嗜热菌海栖热袍菌α-葡萄糖醛酸酶的高效表达和重组酶的纯化

α-葡萄糖醛酸酶作为木聚糖降解的限速酶之一,在木聚糖类半纤维素的生物转化中起着重要的作用。海栖热袍菌Thermotoga maritima是一个嗜极端高温的厌氧细菌,其产生的极耐热性酶类具有非常可观的工业应用前景。但热袍菌属Thermotoga的基因在大肠杆菌中的表达一般较困难。研究了T. maritima中的极耐热性α葡萄糖醛酸酶基因在大肠杆菌不同菌株中的表达水平及纯化技术。结果表明,稀有密码子AGA、AGG和AUA限制了该基因在大肠杆菌中的表达,在大肠杆菌BL21-CodonPlus(DE3)RIL可得到高效表达,重组蛋白表达量达20%,比酶活比野生菌株提高5倍;重组蛋白经热处理和金属Ni²⁺的亲和层析提纯后,达到了电泳纯,提纯倍数为5.1倍,收率为55.1%。对重组菌诱导表达条件的研究表明,营养丰富的TB培养基有助于重组菌的生长, 重组菌生长至OD₆₀₀为0.7～0.8时添加IPTG诱导5h后重组蛋白的表达量最高。     

alpha-Glucuronidase and Other Hemicellulase Activities of Fibrobacter succinogenes S85 Grown on Crystalline Cellulose or Ball-Milled Barley Straw

Fibrobacter succinogenes produces an alpha-glucuronidase which cleaves 4-O-methyl-alpha-d-glucuronic acid from birch wood 4-O-methyl-alpha-d-glucuronoxylan. Very low levels of alpha-glucuronidase activity were detected in extracellular enzyme preparations of F. succinogenes on birch wood xylan substrate. The release of 4-O-methyl-alpha-d-glucuronic acid was enhanced when the birch wood xylan substrate was predigested by either a purified Schizophyllum commune xylanase or a cloned F. succinogenes S85 xylanase. These data suggest that the alpha-glucuronidase is unable to cleave 4-O-methyl-alpha-d-glucuronic acid from intact xylan but can act on unique low-molecular-weight glucuronoxylan fragments created by the cloned F. succinogenes xylanase. The cloned xylanase presumably must account for a small proportion of the indigenous xylanase activity of F. succinogenes cultures, since this xylanase source does not support high glucuronidase activity. The alpha-glucuronidase and associated hemicellulolytic enzymes exhibited higher activities in culture fluid from cells grown on ball-milled barley straw than in that of cellulose-grown cells. The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on barley straw was more complex than that of culture filtrates from cells grown on cellulose. These data demonstrate that F. succinogenes produces an alpha-glucuronidase with an exacting substrate specificity which enables extensive cleavage of glucuronic acid residues from xylan as a consequence of synergistic xylanase action.     

Genetic relationship between lysosomal and lamellar body-specific alpha-glucosidases in human lung

In lamellar bodies isolated from adult human lung at least two acid alpha-glucosidases are present: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme. In the present study we measured the activity of this lamellar body-specific alpha-glucosidase and of lysosomal alpha-glucosidase in a patient with an inherited deficiency of lysosomal alpha-glucosidase. The activity of the lamellar body-specific alpha-glucosidase was not affected in the patient, whereas the lysosomal alpha-glucosidase activity was strongly depressed. The results strongly suggest that the lysosomal alpha-glucosidase and the lamellar body-specific alpha-glucosidase are different gene products.     

Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids

The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.     

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1.

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.     

An alpha-glucuronidase of Schizophyllum commune acting on polymeric xylan

The main alpha-glucuronidase (EC 3.2.1.131) of the fungus Schizophyllum commune was purified to homogeneity using standard chromatographic methods; anion exchange, hydrophobic interaction chromatography and gel filtration. The enzyme had a molecular mass of 125 kDa as determined by SDS-polyacrylamide gel electrophoresis and a pI value of 3.6 according to isoelectric focusing. The N-terminal amino acid sequence of the S. commune alpha-glucuronidase did not show any homology with other alpha-glucuronidases. It exhibited maximal activity at pH values from 4.5 to 5.5 and was stable for 24 h between pH 6 and 8 at 40 degrees C. The highest temperature at which the enzyme retained its full activity for 24 h at pH 5.8 was 40 degrees C. The alpha-glucuronidase of S. commune was able to remove almost all 4-O-methylglucuronic acid groups from water-soluble polymeric softwood arabinoglucuronoxylans. The action of the enzyme on birchwood acetyl-glucuronoxylan was limited due to the high amount of acetyl substituents. The degree of hydrolysis of partially soluble deacetylated glucuronoxylan did not exceed 50% of the theoretical maximum. However, together with a xylanase hydrolysing the xylan backbone the action of the alpha-glucuronidase of S. commune on glucuronoxylan was clearly enhanced. It was apparent that the enzyme was able to remove the 4-O-methylglucuronic groups mainly from soluble substrates.     

Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an alpha-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and alpha-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the alpha-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the alpha-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual alpha-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch-degrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.     

Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.     

The Lec23 Chinese hamster ovary mutant is a sensitive host for detecting mutations in alpha-glucosidase I that give rise to congenital disorder of glycosylation IIb (CDG IIb)

Lec23 Chinese hamster ovary cells are defective in alpha-glucosidase I activity, which removes the distal alpha(1,2)-linked glucose residue from Glc(3)Man(9)GlcNAc(2) moieties attached to glycoproteins in the endoplasmic reticulum. Mutations in the human GCS1 gene give rise to the congenital disorder of glycosylation termed CDG IIb. Lec23 mutant cells have been shown to alter lectin binding and to synthesize predominantly oligomannosyl N-glycans on endogenous glycoproteins. A single point mutation (TCC to TTC; Ser to Phe) was identified in Lec23 Gcs1 cDNA and genomic DNA. Serine at the analogous position is highly conserved in all GCS1 gene homologues. A human GCS1 cDNA reverted the Lec23 phenotype, whereas GCS1 cDNA carrying the lec23 mutation (S440F in human) did not. By contrast, GCS1 cDNA with an R486T or F652L CDG IIb mutation gave substantial rescue of the Lec23 phenotype. Nevertheless, in vitro assays of each enzyme gave no detectable alpha-glucosidase I activity. Clearly the R486T and F652L GCS1 mutations are only mildly debilitating in an intact cell, whereas the S440F mutation largely inactivates alpha-glucosidase I both in vitro and in vivo. However, the S440F alpha-glucosidase I may have a small amount of alpha-glucosidase I activity in vivo based on the low levels of complex N-glycans in Lec23. A sensitive test for complex N-glycans showed the presence of polysialic acid on the neural cell adhesion molecule. The Lec23 Chinese hamster ovary mutant represents a sensitive host for detecting a wide range of mutations in human GCS1 that give rise to CDG IIb.     

A zinc finger protein from Candida albicans is involved in sucrose utilization.

A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans.     

Purification and characterization of an extracellular alpha-D-glucuronidase from Phlebia radiata

Alpha-D-glucuronidase was isolated from the culture filtrate of Phlebia radiata grown on wheat bran and purified to homogeneity by chromatographic methods. The final enzymic preparation was purified 65-fold with an activity yield of 58%; it showed a high level of specific activity (over 23,000 nkat/mg protein). The molecular and hydrolytic properties of the purified enzyme were studied. The secreted alpha-glucuronidase had a molecular weight of 110 kDa, as established by gel permeation chromatography (GP HPLC), had a determined pI just below 4.4, and was stable at pH 5.5 for prolonged times. The carbohydrate content in protein molecules was found to be 15%. The activity of alpha-D-glucuronidase peaked at pH 3,8 and 60 degrees C with aldouronic acids preparation as the substrate. The Michaelis-Menten constant (K(m)), the maximum reaction velocity (V(max)), and the activation energy (E(a)) were 0.18 mM, 0.13 microM/min and 5.91 kJ/mol, respectively. The alpha-glucuronidase was active mainly on small substituted xylooligomers. When this enzyme was used with endoxylanase for the degradation of oat xylan, synergistic effects were observed.     

Purification and characterization of an extracellular alpha-glucosidase protein from Trichoderma viride which degrades a phytotoxin associated with sheath blight disease in rice

AIMS: To purify and characterize an extracellular alpha-glucosidase from Trichoderma viride capable of inactivating a host-specific phytotoxin, designated RS toxin, produced by the rice sheath blight pathogen, Rhizoctonia solani Kühn. METHODS AND RESULTS: The host-specific RS toxin was purified from both culture filtrates (culture filtrate toxin, CFTox) and R. solani-inoculated rice sheaths (sheath blight toxin, SBTox). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses of extracellular proteins, purified from a biocontrol fungus T. viride (TvMNT7) grown on SBTox and CFTox separately, were carried out. The antifungal activity of the purified high molecular weight protein (110 kDa) was studied against RS toxin as well as on the sclerotial germination and mycelial growth of R. solani. Enzyme assay and Western blot analysis with the antirabbit TvMNT7 110-kDa protein indicated that the protein was an alpha-glucosidase. The 110-kDa protein was highly specific to RS toxin and its Michaelis-Menten constant value was 0.40 mmol l-1 when p-nitrophenyl alpha-D-glucopyranoside was used as the substrate. The isoelectric point of the protein was 5.2. N-terminal sequencing of the alpha-glucosidase protein showed that its amino acid sequence showed no homology with other known alpha-glucosidases. CONCLUSION: This appears to be the first report of the purification and characterization of an alpha-glucosidase capable of inactivating a host-specific toxin of fungal origin. The alpha-glucosidase is specific to RS toxin and is different from the known alpha-glucosidases. SIGNIFICANCE AND IMPACT OF THE STUDY: As RS toxin could be inactivated by the microbial alpha-glucosidase enzyme, isolation of the gene that codes for the enzyme from T. viride and transfer of the gene to rice plants would lead to enhanced resistance against sheath blight pathogen by inactivation of RS toxin.     

Novel alpha-glucosidase from Aspergillus nidulans with strong transglycosylation activity

Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity.     

The membrane-bound alpha-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylan

The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by alpha-glucuronidases. In this report we describe the primary structures of the alpha-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the alpha-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with alpha-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad alpha-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.