Purification, biochemical and secondary structural characterisation of ß-mannanase from Lactobacillus casei HDS-01 and juice clarification potential.

A 36.7 kDa extracellular mannanase was originally purified from a generally regarded as safe (GRAS) lactic acid bacteria (LAB) strain Lactobacillus casei HDS-01. The LC-GC/GC determined that HDS-01 mannanase contained 338 amino acid residues. This protein belonged to glycoside hydrolase (GH) 26 Family and shared high similarity (94%) but low coverage (54%) with mannanase from Bacillus licheniformis (GQ859489.1). Circular dichroism (CD) illustrated that the proportion of α + ß structural elements closely related to enzyme activity and were both negatively influenced by temperature. Fourier-transform infrared (FT-IR) showed the existence of typical protein bonds such as CO, CC, NH, CCH and CC. The CD and FT-IR collectively suggested HDS-01 mannanase a ß-conformation rich (60-70%) protein. The optimal reaction condition of HDS-01 mannanase was 40 °C and pH 5.0. More than 50% activity was maintained after 2 h incubation under the condition of pH value 5.0-7.0 and 30-60 °C. This enzyme was strongly activated by Mn2+ and exhibited a high affinity for locust bean gum (Km = 2.68 ± 0.02 mg mL-1, Vmax = 400.03 ± 1.22 µmol mL-1·min-1). The HDS-01 mannanase clarified fruit juices more efficiently than commercial mannanase. These results promised HDS-01 mannanase a bio-safe addictive used in various especially food grade industrial fields.

Selection of Bacillus subtilis US191 as a mannanase-producing probiotic candidate.

ß-Mannanases are crucial enzymes for the breakdown of mannans. As Mannans are being considered as antinutritional factors in poultry production, the search for mannanase-producing probiotic bacteria is now attracting considerable attention as a strategy to enhance nutrients bioavailability. Five soil born Bacilli (US134, US150, US176, US180, and US191) were selected for their ability to produce extracellular ß-mannanases that were biochemically characterized. The probiotic properties of these strains were determined to assess their potential as animal feed supplements. Bacillus subtilis US191 was shown to be sensitive to all antibiotics tested, to inhibit growth of the bacterial pathogens tested, and to produce a thermostable ß-mannanase. It exhibited a notable acid and bovine bile tolerance and high ability to form biofilm. These features may favor its adherence to the intestinal epithelial cells allowing its survival and persistence in the digestive tract. Furthermore, our study revealed that US191 was among the strains showing the highest ability to digest wheat dry matter in vitro when compared to the commercial feed additive Rovabio® Max. Altogether, our findings suggest that the ß-mannanase producer B.subtilis US191 is a promising probiotic candidate for the feed industry.

Thermo and alkali stable ß-mannanase: Characterization and application for removal of food (mannans based) stain.

The present work aims to characterize thermo and alkali stable ß-mannanase (ManSS11) from newly isolated Klebsiella pneumoniae SS11 and its food stain (mannan based) removal efficiency. The enzyme, ManSS11 was stable over a wide range of pH and temperature. The highest activity of ManSS11 was observed at pH 9.0 and temperature, 70 °C, with t1/2 of 135.91 min at the same temperature while, >70% of its initial activity was retained at pH 7.0-10.6. It was purified to 5.50-fold homogeneity with a final recovery of 9.6% and a specificity of 7573.57 U/mg protein. Purified ManSS11 was visible as a single protein band with a molecular weight of ~45 kDa. The kinetic parameters of Km, Vmax and kcat were 1.66 mg/mL, 833.33 µmolmL-1 min-1 and 1190.47 s-1 respectively. The compatibility of ß-mannanase with different detergents together with wash performance test confirmed its potential usability in laundry sector. Wash performance analysis confirmed that the enzyme displayed great efficiency in the removal of stains caused by mannan containing foods.

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology.

Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf-enzyme products and their validation with 15 commercial microbial-enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude-leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast-derived enzymes are stable in crude-leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude-extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker-free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site-specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf-production platform offers a novel low-cost approach by the elimination of fermentation, purification, concentration, formulation and cold-chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.

Cloning and expression of a ß-mannanase gene from Bacillus sp. MK-2 and its directed evolution by random mutagenesis.

A ß-mannanase gene was cloned from Bacillus sp. MK-2 and expressed in Bacillus subtilis WB800. The ORF of the ß-mannanase gene was 1104 bp in length, encoding 367 aa. The deduced amino acid sequence shared high sequence identity with the ß-mannanase from Bacillus subtilis, and belongs to glycosyl hydrolase family 26. The purified recombinant enzyme had a specific activity of 2802 U/mg and displayed optimum activity at pH 6.0 and 55 °C. To obtain an enzyme with high specific activity and facilitate its industrial applications, molecular engineering of Bman26 was undertaken using random mutagenesis in Bacillus subtilis WB800. Three positive mutants with substantially improved specific activities were selected and studied. The best performing mutant was K291E, for which the single amino acid substitution led to a 3.5-fold increase in kcat/Km. Mutants Q112R and L211I also exhibited an apparently increased kcat/Km towards konjac glucomannan, approximately 200% and 80% improvement, respectively. Structural-functional analysis indicated that a slight conformational change could dramatically affect certain enzyme characteristics. In addition, three amino acid sites (Gly88-Leu212-Lys288) in Bman26 were found to have close relationships with the enzyme's thermal stability. These new findings will help promote the development of industrially useful ß-mannanase, with both good thermal stability and high specific activity.

Polysaccharide-Degrading Enzymes From Marine Gastropods.

Seaweed polysaccharides have been widely used as viscosifier, gelling agents, and stabilizer in the various application fields, e.g., food, pharmaceutical, nutraceutical, and chemical industries. Applications of seaweed polysaccharides are further expanding to versatile directions, e.g., biofuels, bioactive compounds, and functional materials for medical and basic researches. Production of functional oligo- and monosaccharides by the use of specific enzymes is also expected to improve the value of seaweed polysaccharides. The enzymes that depolymerize seaweed polysaccharides are distributed largely among seaweed-associating organisms like marine invertebrates and bacteria. Among them, herbivorous marine gastropods such as abalone and sea hare are the most prominent producers of polysaccharide-degrading enzymes. To date, various kinds of polysaccharide-degrading enzymes have been isolated from the digestive fluid and hepatopancreas of these animals. Among them, alginate lyase, ß-1,3-glucanase, mannanase, and cellulase are the major constituents of their digestive fluid. In this chapter, the authors describe the general methods for the preparation and activity assay of the gastropod polysaccharide-degrading enzymes and provide basic knowledge for their primary structures.

Purification of ß-mannanase derived from Bacillus subtilis ATCC 11774 using ionic liquid as adjuvant in aqueous two-phase system.

The partitioning of ß-mannanase derived from Bacillus subtilis ATCC 11774 in aqueous two-phase system (ATPS) was studied. The ATPS containing different molecular weight of polyethylene glycol (PEG) and types of salt were employed in this study. The PEG/salt composition for the partitioning of ß-mannanase was optimized using response surface methodology. The study demonstrated that ATPS consists of 25% (w/w) of PEG 6000 and 12.52% (w/w) of potassium citrate is the optimum composition for the purification of ß-mannanase with a purification fold (PF) of 2.28 and partition coefficient (K) of 1.14. The study on influences of pH and crude loading showed that ATPS with pH 8.0 and 1.5% (w/w) of crude loading gave highest PF of 3.1. To enhance the partitioning of ß-mannanase, four ionic liquids namely 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4), 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4), 1-butyl-3-methylimidazolium bromide ([Bmim]Br), 1-ethyl-3-methylimidazolium bromide ([Emim]Br) was added into the system as an adjuvant. The highest recovery yield (89.65%) was obtained with addition of 3% (w/w) of [Bmim]BF4. The SDS-PAGE analysis revealed that the ß-mannanase was successfully recovered in the top phase of ATPS with the molecular size of 36.7kDa. Therefore, ATPS demonstrated a simple and efficient approach for recovery and purification of ß-mannanase from fermentation broth in one single-step strategy.

Purification, characterization, and overexpression of an endo-1,4-ß-mannanase from thermotolerant Bacillus sp. SWU60.

Endo-ß-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the ß-1,4-linkage in the mannan backbone and release short ß-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60 °C, respectively. The enzyme was stable up to 60 °C for 1 h and at pH 5-9 at 4 °C for 16 h. Its enzyme activity was inhibited by Hg2+. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.

A Recombinant Highly Thermostable ß-Mannanase (ReTMan26) from Thermophilic Bacillus subtilis (TBS2) Expressed in Pichia pastoris and Its pH and Temperature Stability.

A gene encoding a highly thermostable ß-mannanase from a thermophilic Bacillus subtilis (TBS2) was successfully expressed in Pichia pastoris. The maximum activity of the recombinant thermostable ß-mannanase (ReTMan26) was 5435 U/mL, which was obtained by high-density, fed-batch cultivation after 168-h induction with methanol in a 50-L bioreactor. The protein yield reached 3.29 mg/mL, and the protein had a molecular weight of ~42 kDa. After fermentation, ReTMan26 was purified using a 10-kDa cut-off membrane and Sephadex G-75 column. The pH and temperature optima of purified ReTMan26 were pH 6.0 and 60 °C, respectively, and the enzyme was stable at pH 2.0-8.0 and was active at 20-100 °C. HPLC analysis of the products of locust bean gum hydrolysis showed that the mannan-oligosaccharide content was 62.5%. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min, which was higher than any other ß-mannanase reported up to now, suggesting its potential for industrial applications.

Engineering a family 27 carbohydrate-binding module into an Aspergillus usamii ß-mannanase to perfect its enzymatic properties.

A family 27 carbohydrate-binding module of a Thermotoga maritima ß-mannanase (TmCBM27) was chosen from the carbohydrate-active enzyme database by computer-aided design, possessing the lowest binding free energy with mannopentaose. To improve the enzymatic properties of a glycoside hydrolase family 5 ß-mannanase from Aspergillus usamii (AuMan5A), two fusion ß-mannanases, AuMan5A-F-M and AuMan5A-R-M, were designed by fusing a TmCBM27 into its C-terminus linked with a flexible peptide F (GGGGS)3 and rigid peptide R (EAAAK)3. Two fusion enzyme genes, Auman5A-F-m and Auman5A-R-m, were constructed as designed theoretically by overlapping PCR. Then, Auman5A and two fusion genes were expressed in Pichia pastoris GS115. Three recombinant ß-mannanases, reAuMan5A, reAuMan5A-F-M and reAuMan5A-R-M, were purified to homogeneity with specific activities of 230.6, 153.3 and 241.7 U/mg. The temperature optimum of reAuMan5A-R-M was 70°C, identical with that of reAuMan5A, while its thermostability and melting temperature (Tm) reached 68°C and 74.9°C, being 8.0°C and 8.4°C higher than those of the latter, respectively. Additionally, the Km values of reAuMan5A-R-M, towards locust bean gum, konjac gum and guar gum, significantly decreased to 0.9, 1.9 and 2.5 mg/mL from 1.7, 3.8 and 4.2 mg/mL of reAuMan5A, while its kcat/Km (catalytic efficiency) values increased to 287.8, 163.7 and 84.4 mL/mg⋅s from 171.2, 97.6 and 56.0 mL/mg⋅s of the latter, respectively. These results verified that the fusion of a TmCBM27 into the C-terminus of AuMan5A mediated by (EAAAK)3 linker contributed to its improved thermostability and catalytic efficiency.

Purification and Characterization of a Thermostable ß-Mannanase from Bacillus subtilis BE-91: Potential Application in Inflammatory Diseases.

ß-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the ß-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a "two-step" method comprising ultrafiltration and gel chromatography. The purified ß-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5-7.0. The ß-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 µmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, ß-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the ß-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.

A thermostable GH26 endo-ß-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation.

The endomannanase gene em26a from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 26, was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative endomannanase, dubbed MtMan26A, was purified to homogeneity (60 kDa) and subsequently characterized. The optimum pH and temperature for the enzymatic activity of MtMan26A were 6.0 and 60 °C, respectively. MtMan26A showed high specific activity against konjac glucomannan and carob galactomannan, while it also exhibited high thermal stability with a half-life of 14.4 h at 60 °C. Thermostability is of great importance, especially in industrial processes where harsh conditions are employed. With the aim of better understanding its structure-function relationships, a homology model of MtMan26A was constructed, based on the crystallographic structure of a close homologue. Finally, the addition of MtMan26A as a supplement to the commercial enzyme mixture Celluclast® 1.5 L and Novozyme® 188 resulted in enhanced enzymatic hydrolysis of pretreated beechwood sawdust, improving the release of total reducing sugars and glucose by 13 and 12 %, respectively.

An extremely alkaline mannanase from Streptomyces 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 ß-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. Km and Vmax 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 ß-mannanase isolated from Streptomyces sp. CS428 was found to be attractive for biotechnological applications.

Biochemical characterization of an acidophilic ß-mannanase from Gloeophyllum trabeum CBS900.73 with significant transglycosylation activity and feed digesting ability.

Acidophilic ß-mannanases have been attracting much attention due to their excellent activity under extreme acidic conditions and significant industrial applications. In this study, a ß-mannanase gene of glycoside hydrolase family 5, man5A, was cloned from Gloeophyllum trabeum CBS900.73, and successfully expressed in Pichia pastoris. Purified recombinant Man5A was acidophilic with a pH optimum of 2.5 and exhibited great pH adaptability and stability (>80% activity over pH 2.0-6.0 and pH 2.0-10.0, respectively). It had a high specific activity (1356 U/mg) against locust bean gum, was able to degrade galactomannan and glucomannan in a classical four-site binding mode, and catalyzed the transglycosylation of mannotetrose to mannooligosaccharides with higher degree of polymerization. Besides, it had great resistance to pepsin and trypsin and digested corn-soybean meal based diet in a comparable way with a commercial ß-mannanase under the simulated gastrointestinal conditions of pigs. This acidophilic ß-mannanase represents a valuable candidate for wide use in various industries, especially in the feed.

Low-temperature-active and salt-tolerant ß-mannanase from a newly isolated Enterobacter sp. strain N18.

A low-temperature-active and salt-tolerant ß-mannanase produced by a novel mannanase-producer, Enterobacter sp. strain N18, was isolated, purified and then evaluated for its potential application as a gel-breaker in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil field. The enzyme could lower the viscosity of guar gum solution by more than 95% within 10 min. The purified ß-mannanase with molecular mass of 90 kDa displayed high activity in a broad range of pH and temperature: more than 70% of activity was retained in the pH range of 3.0-8.0 with the optimal pH 7.5, about 50% activity at 20°C with the optimal temperature 50°C. Furthermore, the enzyme retained >70% activity in the presence of 0.5-4.0 M NaCl. These properties implied that the enzyme from strain N18 had potential for serving as a gel-breaker for low temperature oil wells and other industrial fields, where chemical gel breakers were inactive due to low temperature.

A novel surfactant-, NaCl-, and protease-tolerant ß-mannanase from Bacillus sp. HJ14.

A glycoside hydrolase family 5 ß-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % ß-mannanase activity at 3.0-30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0-30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5-2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.

A ß-mannanase from Paenibacillus sp.: Optimization of production and its possible prebiotic potential.

A thermotolerant bacterium Paenibacillus thiaminolyticus with an ability to produce extracellular ß-mannanase was isolated from a soil sample. Bacterium produced 45 U/mL ß-mannanase at 50 °C. The culture conditions for high-level production of ß-mannanase were optimized. Optimized MS medium [wheat bran 2% (w/v), ammonium sulfate 0.3% (w/v), yeast extract, and peptone (0.025% each) pH 6.5] was inoculated with 2% of 16 H old culture.  The culture was incubated at 50 °C for 48 H resulting in 24-folds higher ß-mannanase production (1,100 ± 50 U/mL). Optimum pH and temperature for enzyme activity of the crude enzyme was 6.0 and 60 °C, respectively. The enzyme demonstrated 65% relative enzyme activity at 37 °C. The hydrolytic activity of the crude enzymatic preparation was assessed on various agro residues. Thin-layer chromatographic analysis showed that the enzyme activity to saccharify heteromannans resulted in production of a mixture of manno-oligosaccharides (MOS) and enzyme exhibited classic endo-activity. To evaluate the possible prebiotic potential of the MOS thus obtained, initial screening for their ability to support the growth of probiotics was carried out by the pure culture method. Bifidobacterium and Lactobacillus sp. responded positively to the addition of enzymatically derived oligosaccharides and their numbers increased significantly.

Degradation of konjac glucomannan by Thermobifida fusca thermostable ß-mannanase from yeast transformant.

Native konjac glucomannan was used as the substrate for thermophilic actinomycetes, Thermobifida fusca BCRC19214, to produce ß-mannanase. The ß-mannanase was purified and five internal amino acid sequences were determined by LC-MS/MS. These sequences had high homology with the ß-mannanase from T. fusca YX. The tfm gene which encoded the ß-mannanase was cloned, sequenced and heterologous expressed in Yarrowia lipolytica P01 g expression system. Recombinant heterologous expression resulted in extracellular ß-mannanase production at levels as high as 3.16 U/ml in the culture broth within 48 h cultivation. The recombinant ß-mannanase from Y. lipolytica transformant had superior thermal property. The optimal temperature of the recombinant ß-mannanase from Y. lipolytica transformant (pYLSC1-tfm) was 80°C. When native konjac glucomannan was incubated with the recombinant ß-mannanase from Y. lipolytica transformant (pYLSC1-tfm) at 50°C, there was a fast decrease of viscosity happen during the initial phase of reaction. This viscosity reduction was accompanied by an increase of reducing sugars. The surface of konjac glucomannan film became smooth. After 24h of treatment, the DPw of native konjac glucomannan decreased from 6,435,139 to 3089.

Understanding ß-mannanase from Streptomyces sp. CS147 and its potential application in lignocellulose based biorefining.

Hydrolytic enzymes such as cellulase and hemicellulase have been attracted in lignocellulose based biorefinery. Especially, mannanase has been a growing interest in industrial applications due to its importance in the bioconversion. In this study, an extracellular endo-ß-1,4-D-mannanase was produced by Streptomyces sp. CS147 (Mn147) and purified 8.5-fold with a 43.4% yield using Sephadex G-50 column. The characterization of Mn147 was performed, and the results were as follows: molecular weight of ∼25 kDa with an optimum temperature of 50°C and pH of 11.0. The effect of metal ions and various reagents on Mn147 was strongly activated by Ca(+2) but inhibited by Mg(+2) , Fe(+2) , hydrogen peroxide, EDTA and EGTA. Km and Vmax values of Mn147 were 0.13 mg/mL and 294 µmol/min mg, respectively, when different concentrations (3.1 to 50 mg/mL) of locust bean gum galactomannan were used as substrate. In enzymatic hydrolysis of heterogeneous substrate (spent coffee grounds), Mn147 shows a similar conversion compared to commercial enzymes. In addition, lignocellulosic biomass can be hydrolyzed to oligosaccharides (reducing sugars), which can be further utilized for the production of biomaterials. These results showed that Mn147 is attractive in quest of potential bioindustrial applications.

Molecular and biochemical characterizations of a new low-temperature active mannanase.

A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment's performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35-40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K m of 4.2 mg/ml and a k cat of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain.