An extremely alkaline mannanase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS428 hydrolyzes galactomannan producing series of mannooligosaccharides

An alkaline-thermostable mannanase from Streptomyces sp. CS428 was produced, purified, and biochemically characterized. The extracellular mannanase (Mn428) was purified to homogeneity with 12.4 fold, specific activity of 2406.7 U/mg, and final recovery of 37.6 %. The purified β-mannanase was found to be a monomeric protein with a molecular mass of approximately 35 kDa as analyzed by SDS-PAGE and zymography. The first N-terminal amino acid sequences of mannanase enzyme were HIRNGNHQLPTG. The optimal temperature and pH for enzyme were 60 °C and 12.5, respectively. The mannanase activities were significantly affected by the presence of metal ions, modulators, and detergents. K_m and V_max values of Mn428 were 1.01 ± 3.4 mg/mL and 5029 ± 85 µmol/min mg, respectively when different concentrations (0.6–10 mg/mL) of locust bean gum galactomannan were used as substrate. The substrate specificity of enzyme showed its highest specificity towards galactomannan which was further hydrolyzed to produce mannose, mannobiose, mannotriose, and a series of mannooligosaccharides. Mannooligosaccharides can be further converted to ethanol production, thus the purified β-mannanase isolated from Streptomyces sp. CS428 was found to be attractive for biotechnological applications.     

A celluloytic complex from <Emphasis Type="Italic">Clostridium cellulovorans</Emphasis> consisting of mannanase B and endoglucanase E has synergistic effects on galactomannan degradation

In our previous study using a fluorescently labeled cohesin biomarker, we detected and identified a putative cellulosomal mannanase belonging to the glycosyl hydrolase family 26 from Clostridium cellulovorans in xylan-containing cultures. In this study, a mannanase gene, manB from C. cellulovorans, was expressed in Escherichia coli. The optimal pH of a purified enzyme was around pH 7.0 and the optimal temperature was 40°C. The purified mannanase B (ManB) showed high hydrolytic activity toward galactomannan. An assembly of ManB with mini-CbpA, which contains a carbohydrate-binding module that provides proximity to insoluble substrates, increased the activity toward galactomannan [locust bean gum (LBG) and guar gum] 1.7- and 2.0-fold over those without mini-CbpA. We tested the synergistic effects on galactomannan (LBG and guar gum) degradation using cellulosomal mannanase ManB with cellulosomal endoglucanase E, which was predicted to have mannanase activity in C. cellulovorans as a cellulolytic complex. When assembled with the mini-CbpA, the mixture of endoglucanase E (EngE) and ManB at a molar ratio of 1:2 showed the highest synergistic effect (2.4-fold) on LBG. The mixture at a ratio of 1:3 showed the highest synergistic effect (2.8-fold) on guar gum. These synergistic actions indicated that ManB assembled with mini-CbpA hydrolyzed insoluble galactomannan, which in turn promoted soluble galactomannan degradation by EngE.     

A beta-mannanase from Bacillus subtilis B36: purification, properties, sequencing, gene cloning and expression in Escherichia coli

MANB36, a secrete endo-beta-1,4-D-mannanase produced by Bacillus subtilis B36, was purified to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 degrees C. The enzyme activity of MANB36 is remarkably thermostable at 60 degrees C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoethanol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted molecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.     

Crystallization and Some Properties of Mannanase

A mannan-hydrolyzing enzyme produced by a certain strain of Bacillus subtilis was purified from the culture broth and isolated in a crystalline state by being treated with several ion-exchangers. The optimal pH of the enzyme was 6.0. The enzyme was stable in a pH region of 5.0 to 9.5 and at temperatures less than 55°C. The enzyme attacked only β-1,4-mannosidic linkages in the main chain of galactomannan of soybean seed coat, guar gum and coffee bean, and of glucomannan of konjak (Amorphophalus konjac). Investigation of the hydrolysis mode revealed that the enzyme attacked coffee bean galactomannan endowise to form mannobiose, mannotriose and mannotetraose. The action patterns on several mannohomooligomers prepared from a partial hydrolysate of coffee bean galactomannan were also investigated, indicating that the enzyme preferentially attacked the β-1,4-mannosidic linkages that were present apart three to four mannose residues from the non-reducing end of the mannose chain.     

Sequencing and Expression of a β-Mannanase Gene from the Extreme Thermophile Dictyoglomus thermophilum Rt46B.1, and Characteristics of the Recombinant Enzyme

A β-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.1. ManA is a single-domain enzyme related to one group of β-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80°C and 5.0. In the absence of substrate, the enzyme showed no loss of activity at 80°C over 16 h, while at 90°C the enzyme had a half-life of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and mannotriose, confirming that ManA is an endo-acting β-mannanase. Sequence comparisons with related β-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related genes. Received: 7 June 1999 / Accepted: 6 July 1999     

Characterization and large-scale production of recombinant <Emphasis Type="Italic">Streptoverticillium platensis</Emphasis> transglutaminase

Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was ~33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.     

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus.

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.     

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

Analysis of a Clostridium josui cellulase gene cluster containing the man5A gene and characterization of recombinant Man5A

A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.     

Hydrolysis of isolated coffee mannan and coffee extract by mannanases of Sclerotium rolfsii

Different mannanase preparations obtained from the filamentous fungus Sclerotium rolfsii were used for the hydrolysis of coffee mannan, thus reducing significantly the viscosity of coffee extracts. Mannan is the main polysaccharide component of these extracts and is responsible for their high viscosity, which negatively affects the technological processing of instant coffee. Coffee mannan was isolated from green defatted Arabica beans by delignification, acid wash and subsequent alkali extraction with a yield of 12.8%. Additionally, coffee extract polysaccharides were separated by alcohol precipitation and were found to form nearly half of the coffee extract dry weight. These isolated mannans as well as the mannan in the coffee extract were efficiently hydrolysed by the S. rolfsii mannanase, which resulted in significant viscosity reductions. Concurrently, the reducing sugar content increased continuously due to the release of various mannooligosaccharides including mannotetraose, mannotriose, and mannobiose. Both a partially purified, immobilised and a soluble, crude mannanase preparation were successfully employed for the degradation of coffee mannan.     

Biocatalytic characterization of an endo-β-1,4-mannanase produced by <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. strain HY-8

Objectives

To evaluate the biocatalytic characteristics of a new endo-β-1,4-d-mannan-degrading enzyme (ManP) from Paenibacillus sp. strain HY-8, a gut bacterium of the longicorn beetle Moechotypa diphysis.

Results

Purified ManP (32 kDa) with an N-terminal amino acid sequence of APSFAVGADFSYVPG displayed the greatest degree of biocatalytic activity toward locust bean gum (LBG) at 55 °C and pH 7.0. The enzyme degraded LBG, guar gum, ivory nut mannan, and mannooligosaccharides (M₂–M₅), but did not exhibit any hydrolytic activity against structurally unrelated substrates. The biocatalytic activity of ManP against LBG and guar gum was 695 and 450 U mg^?1, respectively. Especially, enzymatic hydrolysis of mannobiose yielded a mixture of mannose (16.6 %) and mannobiose (83.4 %), although the degree of mannobiose degradation by ManP with was relatively limited.

Conclusion

The present results suggest that ManP is an endo-β-1,4-mannanase and is distinct from various other characterized endo-β-1,4-mannanases.

     

Biocatalytic activity of recombinant human β-mannosidase immobilized onto magnetic nanoparticles for bioprocess

Recombinant human β-mannosidase (rhMANB) is an important glycosidase enzyme that degrades mannose-linked glycoproteins and mannan polysaccharides. rhMANB was purified and covalently immobilized onto magnetic nanoparticles. The immobilization of the enzyme was confirmed by Fourier-transform infrared spectroscopy (FTIR) and magnetic nanoparticles linked immunosorbent assay (MagLISA). Antibodies against rhMANB were raised, purified and characterized for MagLISA. The binding of rhMANB onto magnetic nanoparticles was found to be 65%. The V( max ) and K( m ) of immobilized rhMANB was observed 3.0-fold higher and 2.024-fold lower, respectively, as compared to unbound rhMANB. The stability and activity of immobilized enzyme was observed at different pH, temperature, and after storage at 4°C. Metal chelators (oxalic acid, citric acid, and ascorbic acid) did not affect the enzyme activity of immobilized enzyme, whereas ethylenediamine tetraacetic acid reduced the activity. The results obtained from thin-layer chromatography indicate that immobilized rhMANB is more efficient than the unbound form to hydrolyze mannobiose, mannotriose, mannotetraose, mannopentose, galactoglucomannan, and locust bean gum. Magnetic nanoparticles suspended gel-permeation chromatography showed that 29% locust bean gum hydrolyzed efficiently during flow in the column. The immobilization of rhMANB will be a good process for gelling and saccharification of mannan polymers at industrial scale.     

Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production

A locally isolated strain, Bacillus subtilis NM-39, was selected as an active mannan-utilizing bacterium based on high saccharifying activities on coconut residue and locust bean gum galactomannan. The optimal pH and temperature ranges for activity of the crude enzyme were 5.0 to 6.0 and 50 to 60°C, respectively. The organism gave maximum mannanase activity when grown in liquid mineral salts medium containing 1% (w/v) each of coconut residue and soybean flour, as carbon and nitrogen sources, respectively, at pH 7.0 and in aerobic growth for 28 h at 37°C. High saccharifying activity on coconut mannan was also observed.The authors are with the Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are also currently with the Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; T. Yoshida is also with the Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l⁻¹ cellulose and 10 g l⁻¹ xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity by being able to hydrolyze galactomannan (locust bean gum). Adsorption studies revealed that the smaller of the two enzymes was not able to bind to cellulose. Similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller one was lacking the cellulose-binding domain or an essential part of it. The basic xylanase (pI>9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12 of the glycosyl hydrolases.     

Characterization of an outer membrane mannanase from Bacteroides ovatus

Bacteroides ovatus utilizes guar gum, a high-molecular-weight branched galactomannanan, as a sole source of carbohydrate. No extracellular activity was detectable. Approximately 30% of the total cell-associated mannanase activity partitioned with cell membranes. When inner and outer membranes of B. ovatus were separated on sucrose gradients, the mannanase activity was associated mainly with fractions containing outer membranes. Enzyme activity was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or by Triton X-100 at a detergent-to-protein ratio of 1:1. The enzyme was stable for only 4 h at 37 degrees C and for 50 to 60 h at 4 degrees C. Analysis of the products of the CHAPS-solubilized mannanase on Bio-Gel A-5M and Bio-Gel P-10 gel filtration columns indicated that the enzyme breaks guar gum into high-molecular-weight fragments. The CHAPS-solubilized mannanase was partially purified by chromatography on a FPLC Mono Q column. The partially purified mannanase preparation contained three major polypeptides (Mr 94,500, 61,000, and 43,000) and several minor ones. High mannanase activity was seen only when B. ovatus was grown on guar gum. Cross-absorbed antiserum detected two other guar gum-associated outer membrane proteins: a CHAPS-extractable 49,000-dalton polypeptide and a 120,000-dalton polypeptide that was not solubilized by CHAPS. Neither of these polypeptides was detectable in the partially purified mannanase preparation. These results indicate that there are at least two guar gum-associated outer membrane polypeptides other than the mannanase.     

Production and properties of beta-mannanase by free and immobilized cells of Aspergillus oryzae NRRL 3488

Seven fungi were tested for production of mannanases. The highest mannanase activities were produced by Aspergillus oryzae NRRL 3488 after 7 days in static cultures. Mannanases were induced by gum locust bean (1.0%). The highest mannanase activity was produced when a mixture of peptone, urea and ammonium sulphate was used as nitrogen source. Zn2+ or Co2+ favoured enzyme production. The immobilized cells on Ca-alginate and agar were able to produce beta-mannanase for four runs with a slight decrease in the activity. The optimum temperature for enzyme reaction was 50-55 degrees C at pH 6.0. In the absence of substrate the enzyme was thermostable retaining 75% activity for 1 h at 50 degrees C, and 68% activity for 1 h at 60 degrees C.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.     

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.     

Enzymatic hydrolosis of isolated and fibre-bound galactoglucomannans from pine-wood and pine kraft pulp

Fibre-bound and isolated galactoglumanans from pine-wood and pine kraft pulp were hydrolysed with purified mannanases from Trichoderma reesei and Bacillus subtilis. The isolated galactoglucomannans from both wood and pulp could be hydrolysed fairly extensively with both enzymes. In addition to mixed oligomers, the fungal mannase produced mannobiose as the main hydrolysis product whereas the bacterial mannanase produced mannobiose, mannotriose and mannotetraose. Both enzymes hydrolysed the native galactoglucomannan in finely ground pinewood, whereas galactoglucomannan in pine kraft pulp was only hydrolysed by the T. ressei mannanase. Thus, mannanases exhibit different specificities on fibre-bound, modified substrates. In spite of the high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 10% of the theoretical, probably due to poor accessibility of the substrates. Correspondence to: M. Rättö