Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

An inverting ß-1,2-mannosidase belonging to glycoside hydrolase family 130 from Dyadobacter fermentans.

The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of ß-D-mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer_3176 from Dyadobacter fermentans showed no synthetic activity using α-D-mannose 1-phosphate but it released α-D-mannose from ß-1,2-mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer_3176 is a ß-1,2-mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of ß-1,2-mannotriose. The two residues are not conserved among GH130 phosphorylases and are predicted to assist the nucleophilic attack of a water molecule in the hydrolysis of the ß-D-mannosidic bond.

The mannobiose-forming exo-mannanase involved in a new mannan catabolic pathway in Bacteroides fragilis.

We have proposed a new mannan catabolic pathway in Bacteroides fragilis NCTC 9343 that involves a putative mannanase ManA in glycoside hydrolase family 26 (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772). If this hypothesis is correct, ManA has to generate mannobiose from mannans as the major end product. In this study, the BF0771 gene from the B. fragilis genome was cloned and expressed in Escherichia coli cells. The expressed protein was found to produce mannobiose exclusively from mannans and initially from manno-oligosaccharides. Production of 4-O-ß-D-glucopyranosyl-D-mannose or 4-O-ß-D-mannopyranosyl-D-glucose from mannans was not detectable. The results indicate that this enzyme is a novel mannobiose-forming exo-mannanase, consistent with the new microbial mannan catabolic pathway we proposed.

A novel thermophilic endo-ß-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region.

The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular ß-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0-10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent ß-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.

Performance of a new thermostable mannanase in breaking guar-based fracturing fluids at high temperatures with little premature degradation.

A new thermostable ß-1,4-mannanase (DtManB) cloned from Dictyoglomus thermophilum CGMCC 7283 showed the maximum activity towards hydroxypropyl guar gum at 80 °C, with a half-life of 46 h. DtManB exhibited good compatibility with various additives of fracturing fluid, retaining more than 50 % activity in all the cases tested. More importantly, premature degradation could be alleviated significantly when using DtManB as breaker, because at 27 and 50 °C it displayed merely 3.7 and 18.5 % activities compared to those at 80 °C. In a static test, 0.48 mg DtManB could break 200 mL borax cross-linked fracturing fluid dramatically at 80 °C, and merely 18 mPa s of the viscosity was detected even after the broken fluid was cooled down and only 161.4 mg L(-1) of the residue was left after the enzymatic reaction. All these positive features demonstrate the great potential of this mannanase as a new enzyme breaker for application in enhanced recovery of petroleum oil.

Cloning and biochemical characterization of an endo-1,4-ß-mannanase from the coffee berry borer Hypothenemus hampei.

BACKGROUND: The study of coffee polysaccharides-degrading enzymes from the coffee berry borer Hypothenemus hampei, has become an important alternative in the identification for enzymatic inhibitors that can be used as an alternative control of this dangerous insect. We report the cloning, expression and biochemical characterization of a mannanase gene that was identified in the midgut of the coffee berry borer and is responsible for the degradation of the most abundant polysaccharide in the coffee bean. METHODS: The amino acid sequence of HhMan was analyzed by multiple sequence alignment comparisons with BLAST (Basic Local Alignment Search Tool) and CLUSTALW. A Pichia pastoris expression system was used to express the recombinant form of the enzyme. The mannanase activity was quantified by the 3,5-dinitrosalicylic (DNS) and the hydrolitic properties were detected by TLC. RESULTS: An endo-1,4-ß-mannanase from the digestive tract of the insect Hypothenemus hampei was cloned and expressed as a recombinant protein in the Pichia pastoris system. This enzyme is 56% identical to the sequence of an endo-ß-mannanase from Bacillus circulans that belongs to the glycosyl hydrolase 5 (GH5) family. The purified recombinant protein (rHhMan) exhibited a single band (35.5 kDa) by SDS-PAGE, and its activity was confirmed by zymography. rHhMan displays optimal activity levels at pH 5.5 and 30°C and can hydrolyze galactomannans of varying mannose:galactose ratios, suggesting that the enzymatic activity is independent of the presence of side chains such as galactose residues. The enzyme cannot hydrolyze manno-oligosaccharides such as mannobiose and mannotriose; however, it can degrade mannotetraose, likely through a transglycosylation reaction. The K(m) and k(cat) values of this enzyme on guar gum were 2.074 mg ml(-1) and 50.87 s(-1), respectively, which is similar to other mannanases. CONCLUSION: This work is the first study of an endo-1,4-ß-mannanase from an insect using this expression system. Due to this enzyme's importance in the digestive processes of the coffee berry borer, this study may enable the design of inhibitors against endo-1,4-ß-mannanase to decrease the economic losses stemming from this insect.

Studies on extraction of mannanase enzyme by Aspergillus terreus SUK-1 from fermented palm kernel cake.

Microbial mannanases have become biotechnologically important in industry but their application is limited due to high production cost. In presents study, the extraction of mannanase from fermented Palm Kernel Cake (PKC) in the Solid State Fermentation (SSF) was optimized. Local isolate of Aspergillus terreus SUK-1 was grown on PKC in (SSF) using column bioreactor. The optimum condition were achieved after two washes of fermented PKC by adding of 10% glycerol (v/v) soaked for 10 h at the room temperature with solvent to ratio, 1:5 (w/v).

Characterization of mannanase from a novel mannanase-producing bacterium.

Locust bean gum (LBG) was employed to screen mannanase-producing bacteria. The bacterium with highest mannanase ability was identified as Paenibacillus cookii. It revealed highest activity (6.67 U/mL) when cultivated in 0.1% LBG with 1.5% soytone and 0.5% tryptone after 4 days incubation at 27 °C. Its mannanase was purified to electrophoretical homogeneity after DEAE-Sepharose and Sephacryl S-100 separation. The purified mannanase, with an N-terminus of GLFGINAY, had pH and temperature optimum at 5.0 and 50 °C, respectively, and was stable at pH 5.0-7.0, ≤ 50 °C. It was strongly activated by ß-mercaptoethanol, dithiothreitol, cysteine, and glutathione, but inhibited by Hg(2+), Cu(2+), Zn(2+), Fe(3+), PMSF, iodoacetic acid, and EDTA. According to substrate specificity study, the purified mannanase had high specificity to LBG and konjac.

Cloning, high-level expression, purification, and properties of a novel endo-beta-1,4-mannanase from Bacillus subtilis G1 in Pichia pastoris.

A novel gene coding for an endo-beta-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The beta-mannanase showed an identity of 90.2-92.9% (< or =95%) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified beta- mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDITOF mass spectrometry. The recombinant beta-mannanase had an optimum temperature of 45 degrees C and optimum pH of 6.5. The enzyme was stable at temperatures up to 50 degrees C (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions (Hg2+, Pb2+, and Co2+) substantially inhibited the recombinant beta-mannanase. The chemical additives including detergents (Triton X- 100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the beta-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

A Novel endo-1,4-ß-mannanase from Bispora antennata with good adaptation and stability over a broad pH range.

An endo-ß-1,4-mannanase encoding gene, man5, was cloned from Bispora antennata CBS 126.38, which was isolated from a beech stump. The cDNA of man5 consists of 1,299 base pairs and encodes a 432-amino-acid protein with a theoretical molecular mass of 46.6 kDa. Deduced MAN5 exhibited the highest amino acid sequence identity of 58% to a ß-mannanase of glycoside hydrolase family 5 from Aspergillus aculeatus. Recombinant MAN5 was expressed in Pichia pastoris and purified to electrophoretic homogeneity. The specific activity of the final preparation towards locust bean gum was 289 U mg(-1). MAN5 showed optimal activity at pH 6.0 and 70 °C and had good adaptation and stability over a broad range of pH values. The enzyme showed more than 60% of peak activity at pH 3.0-8.0 and retained more than 80% of activity after incubation at 37 °C for 1 h in both acid and alkaline conditions (pH 4.0-11.0). The K (m) and V (max) values were 1.33 mg ml(-1) and 444 µmol min(-1) mg(-1) and 1.17 mg ml(-1) and 196 µmol min(-1) mg(-1) for locust bean gum and konjac flour, respectively. Of all tested metal ions and chemical reagents, Co(2+), Ni(2+), and ß-mercaptoethanol enhanced the enzyme activity at 1 mM, whereas other chemicals had no effect on or partially inhibited the enzyme activity. MAN5 was highly resistant to acidic and neutral proteases (trypsin, α-chymotrypsin, collagenase, subtilisin A, and proteinase K). By virtue of the favorable properties of MAN5, it is possible to apply this enzyme in the paper and food industries.

A highly active endo-ß-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida.

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50°C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, ß-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-ß-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

Recombinant production and characterisation of two related GH5 endo-ß-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity.

The glycoside hydrolase family 5 (GH5) endo-ß-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K(M)) towards ß-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer ß-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K(M) for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K(M) for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-ß-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.

cDNA cloning and bacterial expression of an endo-ß-1,4-mannanase, AkMan, from Aplysia kurodai.

Previously we isolated an endo-ß-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1392bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96Da. Comparison of the primary structure of AkMan with other ß-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 ß-1,4-mannanases indicated that AkMan together with other molluscan ß-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21(DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of the catalytic domain of a hyperthermostable endo-1,4-beta-D-mannanase from Thermotoga petrophila RKU-1.

Endo-1,4-beta-D-mannanases play key roles in seed germination and fruit ripening and have recently received much attention owing to their potential applications in the food, detergent and kraft pulp industries. In order to delineate their structural determinants for specificity and stability, X-ray crystallographic investigations combined with detailed functional studies are being performed. In this work, crystals of the catalytic domain of a hyperthermostable endo-1,4-beta-D-mannanase from Thermotoga petrophila RKU-1 were obtained from three different conditions, resulting in two crystalline forms. Crystals from conditions with phosphate or citrate salts as precipitant (CryP) belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=58.76, b=87.99, c=97.34 A, while a crystal from a condition with ethanol as precipitant (CryE) belonged to space group I2(1)2(1)2(1), with unit-cell parameters a=91.03, b=89.97, c=97.89 A. CryP and CryE diffracted to resolutions of 1.40 and 1.45 A, respectively.

An endo-beta-1,4-mannanase, AkMan, from the common sea hare Aplysia kurodai.

A mannan-degrading enzyme was isolated from the digestive fluid of the common sea hare Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatography. The purified enzyme, named AkMan in the present paper, showed a single band with an approximate molecular mass of 40,000 Da on SDS-PAGE and preferably degraded a linear beta-1,4-mannan from green algae Codium fragile producing tri- and disaccharides. The optimal temperature of AkMan was 55 degrees C at pH 7.0 and temperature that caused 50% inactivation of AkMan during a 20-min incubation was 52 degrees C. AkMan retained high activity at pH 4.0-7.5 and was not inactivated in such acidic pH range by the incubation at 40 degrees C for 20 min. AkMan could degrade glucomannan from konjak root and galactomannan (tara gum and guar gum) as well as the linear beta-1,4-mannan, while not carboxymethyl cellulose, agarose, dextran and xylan. These results indicate that AkMan is a typical endo-beta-1,4-mannanase (EC 3.2.1.78) splitting internal beta-1,4-mannosyl linkages of mannan. The N-terminal and internal amino-acid sequences of AkMan shared approximately 55% amino-acid identity to the corresponding sequences of an abalone beta-1,4-mannanase, HdMan, which belongs to glycosyl hydrolase family 5 (GHF5). Thus, AkMan was also regarded as a member of GHF5 beta-1,4-mannanases.

Comparative analyses of two thermophilic enzymes exhibiting both beta-1,4 mannosidic and beta-1,4 glucosidic cleavage activities from Caldanaerobius polysaccharolyticus.

The hydrolysis of polysaccharides containing mannan requires endo-1,4-beta-mannanase and 1,4-beta-mannosidase activities. In the current report, the biochemical properties of two endo-beta-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-beta-mannanase and endo-1,4-beta-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-beta-mannanase activity and little endo-1,4-beta-glucanase activity; however, this enzyme also exhibited 1,4-beta-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of beta-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

Purification and biochemical characterisation of endoplasmic reticulum alpha1,2-mannosidase from Sporothrix schenckiil.

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, alpha1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one alpha1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound alpha-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-alpha1,2-mannosidase antibodies. The enzyme hydrolysed Man(9)GlcNAc(2) into Man(8)GlcNAc(2) isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This alpha1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised alpha1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi alpha1,2-mannosidases and therefore, the processing of N-glycans by alpha1,2-mannosidases is similar to that present in lower eukaryotes.

Purification and biochemica characterisation of endoplasmic reticulum á 1-2-mannosidase from Sporothrix schenckiil

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, á1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one á1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound á-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-á1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This á1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised á1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi á1,2-mannosidases and therefore, the processing of N-glycans by á1,2-mannosidases is similar to that present in lower eukaryotes.

Purification and characterization of a low molecular weight of beta-mannanase from Penicillium occitanis Pol6.

The highest beta-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified beta-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 degrees C and pH 4.0. It was stable for 30 min at 70 degrees C and has a broad pH stability (2.0-12.0). ManIII showed K (m), V (max), and K (cat) values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s(-1) with locust bean gum as substrate, respectively. It was inhibited by mannose with a K (I) of 0.610(-3) mg/ml. ManIII was activated by CuSO4 and CaCl2 (2.5 mM). However, in presence of 2.5 mM Co2+, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.