Spruce Galactoglucomannan Research Articles

Wood hemicelluloses as effective wall materials for spray-dried microcapsulation of polyunsaturated fatty acid-rich oils

The most commonly-used and effective wall materials (WMs) for spray-dried microencapsulation of bioactive compounds are either costly, or derived from unsustainable sources, which lead to an increasing demand for alternatives derived from sustainable and natural sources, with low calories and low cost. Wood hemicelluloses obtained from by-products of forest industries appear to be attractive alternatives as they have been reported to have good emulsifying properties, low viscosity at high concentrations, high heat stability and low heat transfer. Here, we investigated the applicability of spruce galactoglucomannans (GGM) and birch glucuronoxylans (GX), to encapsulate flaxseed oil (FO, polyunsaturated fatty acid-rich plant based oil) by spray drying; and the results were compared to those of the highly effective WM, gum Arabic (GA). It was found that depending on solid ratios of WM:FO (1:1, 3:1 and 5:1), encapsulation efficiency of GGM was 88–96%, and GX was 63–98%. At the same encapsulation ratio, both GGM and GX had higher encapsulation efficiency than GA (49–92%) due to their ability to produce feed emulsions with a smaller oil droplet size and higher physical stability. In addition, the presence of phenolic residues in GGM and GX powders enabled them to have a greater ability to protect oil from oxidation during spray drying than GA. Physiochemical properties of encapsulated powders including thermal properties, morphology, molecular structure, particle size and water adsorption intake are also investigated. The study has explored a new value-added proposition for wood hemicelluloses which can be used as effective WMs in the production of microcapsules of polyunsaturated fatty acid-rich oils for healthy and functional products in food, pharmaceutical and cosmetic industries.

Sensory profile of hemicellulose-rich wood extracts in yogurt models

Wood is an abundant and sustainable source of emerging food ingredients, namely hemicelluloses that fulfil a number of requirements for functional hydrocolloids. Hemicelluloses, especially spruce galactoglucomannans (GGM) and birch glucuronoxylans (GX), have potential to be used as stabilizers in various foods such as yogurts, beverages, dressings, and desserts. However, in addition to good technological functionality, safety, and low price, the applicability and market potential of new hydrocolloids is determined by their sensory properties. The present study reports, for the first time, the sensory profile of spruce GGM and birch GX in food. Sensory profiles from generic descriptive analysis of GGM- and GX-rich extracts, processed by spray drying or ethanol precipitation, were compared in three types of model food systems: water solutions, yogurt with solutions, and yogurt with emulsions stabilized by GGM or GX. Gum Arabic was included for comparison with a commercial ingredient known to have a mild flavor. The results showed that GGM and GX have a woody flavor, which can be reduced by ethanol precipitation and, in yogurt, masked by other food ingredients.Graphic abstract

Spruce Galactoglucomannan-Stabilized Emulsions Enhance Bioaccessibility of Bioactive Compounds

The increasing public awareness of health and sustainability has prompted the development of functional foods rich in health-promoting ingredients. Processing technologies and sustainable multifunctional ingredients are needed for structuring these formulations. Spruce galactoglucomannan (GGM), the main hemicelluloses in softwood cell walls, are an abundantly available, emerging sustainable food hydrocolloid that have the ability to efficiently emulsify and stabilize oil-in-water emulsions. In this study, we illustrate how this lignocellulosic stabilizer affects the digestion of polyunsaturated fatty acids (PUFAs) in vitro. A 100% decrease in the initial TAG content was observed during the in vitro digestion, suggesting that complete hydrolysis of the TAGs was achieved by the digestive enzymes. Besides, no release of mono-, di-, and oligosaccharides or phenolic compounds from GGM was detected. Our results demonstrate that the GGM-stabilized emulsion could potentially deliver lipophilic bioactive ingredients and enhance their bioaccessibility. In addition, this bio-stabilizer itself would remain stable in the upper gastrointestinal track and serve as a prebiotic for gut microbiota. We anticipate GGM to complement or even replace many of the conventional carriers of bioactive components in future health care products and functional foods.

Altered Thermal and Mechanical Properties of Spruce Galactoglucomannan Films Modified with an Etherification Reaction.

Native hemicellulose lacks many of the properties that make fossil fuel-based polymers excellent for use in today's industrial products and processes. The mechanical and thermal properties of the hemicellulose can, however, be modified, and its processability increased. We functionalized galactoglucomannan to lower its glass transition temperature (Tg) and thereby increase its processability. The functionalization was achieved through an etherification reaction with butyl glycidyl ether used at three molar ratios. Films were produced, and their mechanical and thermal properties were evaluated. Thermogravimetric analysis showed that increased substitution increased the degradation temperature and decreased the water content in the sample, implying increased hydrophobicity upon modification. Dynamic mechanical analysis indicated that butyl glycidyl ether functionalization alters the thermal properties of the modified films both in the absolute values of Tg and in the strength of the films. The etherification reaction resulted in a more ductile material than the unmodified galactoglucomannan (GGM).

Time-dependent self-association of spruce galactoglucomannans depends on pH and mechanical shearing

The demand for naturally derived, functional and cost-effective raw materials for various food applications is escalating. Spruce wood is a sustainable and abundant, but underutilized source of novel hydrocolloids—galactoglucomannans (GGM). Pressurized-hot water extracted GGM with an intermediate molar mass are hypothesized to form colloidal solutions. To design superior quality products from GGM, an understanding of their colloidal stability and their potential effect in multiphasic systems is required. The present study addresses the functionality of GGM by characterizing their properties in a bi-phasic system, and for the first time, their time-dependent colloidal stability at different extrinsic conditions— pH, ionic strength and after the application of high-intensity mechanical shearing. Amongst the conditions studied, the colloidal stability of aqueous GGM solution was highly pH dependent. The results showed that an intermediate molar mass polysaccharide like GGM formed inter-/intra molecular assemblies, which grew over time, depending on the composition and processing of the aqueous medium. The molecular dispersion of GGM and their dynamic behavior was also compared to solutions of known food hydrocolloids—gum Arabic and hydroxypropylmethyl cellulose. The observed solution properties explain the hydrocolloid functionality of GGM and contribute to design of colloidal polysaccharide systems in food application.

Spruce galactoglucomannan-stabilized emulsions as essential fatty acid delivery systems for functionalized drinkable yogurt and oat-based beverage

The food industry has a considerable demand for functional foods, such as emulsions as delivery system for omega-6 and omega-3 fatty acids. Such delivery systems must be stabilized, ideally with a compound that fulfills the criteria for both functionality and sustainability. Spruce galactoglucomannans (GGM) are novel, wood-derived, natural, value-added, versatile, multi-purpose emulsifiers that can physically stabilize oil-in-water emulsions while simultaneously protecting the oil phase against oxidation. In this study, we present for the first time the use of GGM-stabilized emulsions as complex multicomponent delivery systems for omega-6 and omega-3 fatty acids, i.e., (1) cod liver oil in drinkable yogurt and (2) oat oil in a gluten-free vegan beverage. The emulsions and the resulting functionalized beverages were characterized in terms of their physical stabilities. In addition, functionalized, drinkable yogurt was characterized in terms of the oxidative stability of the oil. Results highlighted that oil droplets stabilized with GGM were stable during storage, against thermal treatment, upon addition into the beverages, and GGM protected cod liver oil against oxidation in drinkable yogurt. The results reported here highlight the vast potential for the use of wood lignocellulose-derived, multi-functional hydrocolloids in modern foods.

Boosting of enzymatic softwood saccharification by fungal GH5 and GH26 endomannanases

BackgroundSoftwood is a promising feedstock for lignocellulosic biorefineries, but as it contains galactoglucomannan efficient mannan-degrading enzymes are required to unlock its full potential.ResultsBoosting of the saccharification of pretreated softwood (Canadian lodgepole pine) was investigated for 10 fungal endo-β(1→4)-mannanases (endomannanases) from GH5 and GH26, including 6 novel GH26 enzymes. The endomannanases from Trichoderma reesei (TresMan5A) and Podospora anserina (PansMan26) were investigated with and without their carbohydrate-binding module (CBM). The pH optimum and initial rates of enzyme catalysed hydrolysis were determined on pure β-mannans, including acetylated and deacetylated spruce galactoglucomannan. Melting temperature (Tm) and stability of the endomannanases during prolonged incubations were also assessed. The highest initial rates on the pure mannans were attained by GH26 endomannanases. Acetylation tended to decrease the enzymatic rates to different extents depending on the enzyme. Despite exhibiting low rates on the pure mannan substrates, TresMan5A with CBM1 catalysed highest release among the endomannanases of both mannose and glucose during softwood saccharification. The presence of the CBM1 as well as the catalytic capability of the TresMan5A core module itself seemed to allow fast and more profound degradation of portions of the mannan that led to better cellulose degradation. In contrast, the presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification.ConclusionsThis study identified TresMan5A as the best endomannanase for increasing cellulase catalysed glucose release from softwood. Except for the superior performance of TresMan5A, the fungal GH5 and GH26 endomannanases generally performed on par on the lignocellulosic matrix. The work also illustrated the importance of using genuine lignocellulosic substrates rather than simple model substrates when selecting enzymes for industrial biomass applications.

Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation

BackgroundGalactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. GGM can be hydrolyzed into fermentable sugars, or used as a polymer in films, gels, and food additives. Endo-β-mannanases, which can be found in the glycoside hydrolase families 5 and 26, specifically cleave the mannan backbone of GGM into shorter oligosaccharides. Information on the activity and specificity of different mannanases on complex and acetylated substrates is still lacking. The aim of this work was to evaluate and compare the modes of action of two mannanases from Cellvibrio japonicus (CjMan5A and CjMan26A) on a variety of mannan substrates, naturally and chemically acetylated to varying degrees, including naturally acetylated spruce GGM. Both enzymes were evaluated in terms of cleavage patterns and their ability to accommodate acetyl substitutions.ResultsCjMan5A and CjMan26A demonstrated different substrate preferences on mannan substrates with distinct backbone and decoration structures. CjMan5A action resulted in higher amounts of mannotriose and mannotetraose than that of CjMan26A, which mainly generated mannose and mannobiose as end products. Mass spectrometric analysis of products from the enzymatic hydrolysis of spruce GGM revealed that an acetylated hexotriose was the shortest acetylated oligosaccharide produced by CjMan5A, whereas CjMan26A generated acetylated hexobiose as well as diacetylated oligosaccharides. A low degree of native acetylation did not significantly inhibit the enzymatic action. However, a high degree of chemical acetylation resulted in decreased hydrolyzability of mannan substrates, where reduced substrate solubility seemed to reduce enzyme activity.ConclusionsOur findings demonstrate that the two mannanases from C. japonicus have different cleavage patterns on linear and decorated mannan polysaccharides, including the abundant and industrially important resource spruce GGM. CjMan26A released higher amounts of fermentable sugars suitable for biofuel production, while CjMan5A, producing higher amounts of oligosaccharides, could be a good candidate for the production of oligomeric platform chemicals and food additives. Furthermore, chemical acetylation of mannan polymers was found to be a potential strategy for limiting the biodegradation of mannan-containing materials.

Functionality of spruce galactoglucomannans in oil-in-water emulsions

For a sustainable food chain, the demand for plant-based, functional, and cost-effective food hydrocolloids is on a high-rise. Hemicelluloses from the renewable lignocellulosic biomass are available in abundance from side-streams of the forestry industry to fulfill this demand. Their effective valorization requires a safe, economic extraction method that can be up-scaled to an industrial scale and, simultaneously, understanding of their functionality to develop applications. In this study, an aqueous-based extraction method, pressurized hot water extraction (PHWE) of spruce saw meal was used to obtain galactoglucomannans (GGM), “spruce gum”. Ethanol precipitation was performed to remove non-polysaccharide extractives such as free phenolic compounds, and the emulsion component ratio-dependent interfacial saturation capacity of the remaining purified fraction was studied to understand its functionality. GGM resulted in good to excellent emulsification and stabilization of oil-in-water emulsions and exhibited adsorption at the oil droplet interface, which depended on the amount of oil and droplet size of emulsions. The adsorbed GGM content was determined by gas chromatography after acid methanolysis, and their macromolecular characteristics were studied by size-exclusion chromatography. At GGM to oil ratios 2, 1, and 0.4, stable emulsions with predicted several months of shelf life at room temperature were achieved. The results indicated mechanisms affecting the physical stabilization and breakdown of emulsions containing spruce gum, a novel sustainable hydrocolloid.

Insights on the distribution of substitutions in spruce galactoglucomannan and its derivatives using integrated chemo-enzymatic deconstruction, chromatography and mass spectrometry

Accurate determination of the distribution of substitutions in the primary molecular structure of heteropolysaccharides and their derivatives is a prerequisite for their increasing application in the pharmaceutical and biomedical fields, which is unfortunately hindered due to the lack of effective analytical techniques. Acetylated galactoglucomannan (GGM) is an abundant plant polysaccharide as the main hemicellulose in softwoods, and therefore constitutes an important renewable resource from lignocellulosic biomass for the development of bioactive and functional materials. Here we present a methodology for profiling the intramolecular structure of spruce GGM and its chemical derivatives (cationic, anionic, and benzoylated) by combining chemo-enzymatic hydrolysis, liquid chromatography, and mass spectrometry. Fast identification and qualitative mass profiling of GGM and its derivatives was conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) and electrospray ionization mass spectrometry (ESI-MS). Tandem mass fragmentation analysis and its hyphenation with hydrophilic interaction liquid chromatography (HILIC-ESI-MS/MS) provide further insights on the substitution placement of the GGM oligosaccharides and its derivatives. This method will be useful in understanding the structure-function relationships of native GGM and their derivatives, and therefore facilitate their potential application.

Phenolic residues in spruce galactoglucomannans improve stabilization of oil-in-water emulsions

HypothesisAmphiphilic character of surfactants drives them at the interface of dispersed systems, such as emulsions. Hemicellulose-rich wood extracts contain assemblies (lignin-carbohydrate complexes, LCC) with natural amphiphilicity, which is expected to depend on their chemical composition resulting from the isolation method. Lignin-derived phenolic residues associated with hemicelluloses are hypothesized to contribute to emulsions’ interfacial properties and stability. ExperimentsWe investigated the role of phenolic residues in spruce hemicellulose extracts in the stabilization of oil-in-water emulsions by physical and chemical approach. Distribution and changes occurring in the phenolic residues at the droplet interface and in the continuous phase were studied during an accelerated storage test. Meanwhile, the physical stability and lipid oxidation in emulsions were monitored. FindingsNaturally associated lignin residues in GGM act as vehicles for anchoring these hemicelluloses into the oil droplet interface and further enable superior stabilization of emulsions. By adjusting the isolation method of GGM regarding their phenolic profile, their functionalities, especially interfacial behavior, can be altered. Retaining the native interactions of GGM and phenolic residues is suggested for efficient physical stabilization and extended protection against lipid oxidation. The results can be widely applied as guidelines in tailoring natural or synthetic amphiphilic compounds for interfacial stabilization.

Fibrillar assembly of bacterial cellulose in the presence of wood-based hemicelluloses

Composite materials mimicking the plant cell wall structure were made by culturing cellulose-producing bacteria together with secondary-wall hemicelluloses from wood. The effects of spruce galactoglucomannan (GGM) and beech xylan on the nanoscale morphology of bacterial cellulose were studied in the original, hydrated state with small-angle X-ray scattering (SAXS). The SAXS intensities were fitted with a model covering multiple levels of the hierarchical structure. Additional information on the structure of dried samples was obtained using scanning and transmission electron microscopy and infra-red spectroscopy. Both hemicelluloses induced a partial conversion of the cellulose crystal structure from Iα to Iβ and a reduction of the cross-sectional dimensions of the cellulose microfibrils, thereby affecting also their packing into bundles. The differences were more pronounced in samples with xylan instead of GGM, and they became more significant with higher hemicellulose concentrations.

Softwood-based sponge gels

Crosslinking-aided gelation was utilized to prepare hydrogels from softwood polysaccharides, with spruce galactoglucomannans (GGM)—a group of largely unexploited hemicelluloses—as the main component, aiming at conversion into sponge-like aerogels. Cellulose nanofibrils were used for the formation of a reinforcing network, which was further crosslinked together with a GGM matrix by ammonium zirconium carbonate, an inorganic salt that is regarded as safe for use in food packaging. The hydrogels were freeze-dried into stiff, low-density aerogels with 98 % of their volume composed of air-filled pores. When immersed in water, the aerogels absorbed water up to 37 times their initial weight, demonstrating elasticity and repeatable and reversible sponge capacity. The developed concept reassembles the wood polysaccharides in a new way, creating interesting possibilities for utilizing the abundant “green gold,” GGM. The obtained biobased materials could find application potential, for example, in the field of food packaging and could contribute in the reduction of the usage of petroleum-based plastics in the future.

Spruce galactoglucomannans inhibit lipid oxidation in rapeseed oil-in-water emulsions

Oil-in-water (O/W) emulsions are functional and highly significant systems in industrial settings. Their large interfacial area makes them prone to deterioration, including the oxidation of polyunsaturated lipids. Spruce galactoglucomannans (GGM), which are wood biomacromolecules that are abundantly available from emerging forestry biorefineries, were characterized as novel, bio-based, sustainable stabilizers against lipid oxidation in rapeseed O/W emulsions. The formation of hydroperoxides, volatile oxidation products, and polymerized lipids were followed in emulsions during an accelerated storage test at 40 °C for two weeks. The free and bound phenolic residues associated with plant polysaccharides were quantified to explain the differences in the oxidation pathways of emulsions stabilized with GGM and their carboxymethyl derivatives (CMGGM) in comparison to the “golden standard” food stabilizer, gum arabic (GA), and a promising alternative, corn fiber gum (CFG). GGM and CMGGM efficiently inhibited and altered the pathways of lipid oxidation of rapeseed oil. GGM showed an exceptional capacity to inhibit lipid oxidation and act as a multifunctional stabilizer, enhancing both the physical and oxidative sility of emulsions.

Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana

Plant mannanases are enzymes that carry out fundamentally important functions in cell wall metabolism during plant growth and development by digesting manno-polysaccharides. In this work, the Arabidopsis mannanase 5-2 (AtMan5-2) from a previously uncharacterized subclade of glycoside hydrolase family 5 subfamily 7 (GH5_7) has been heterologously produced in Pichia pastoris. Purified recombinant AtMan5-2 is a glycosylated protein with an apparent molecular mass of 50kDa, a pH optimum of 5.5–6.0 and a temperature optimum of 25°C. The enzyme exhibits high substrate affinity and catalytic efficiency on mannan substrates with main chains containing both glucose and mannose units such as konjac glucomannan and spruce galactoglucomannan. Product analysis of manno-oligosaccharide hydrolysis shows that AtMan5-2 requires at least six substrate-binding subsites. No transglycosylation activity for the recombinant enzyme was detected in the present study. Our results demonstrate diversification of catalytic function among members in the Arabidopsis GH5_7 subfamily.

Spruce galactoglucomannans in rapeseed oil-in-water emulsions: Efficient stabilization performance and structural partitioning

Food emulsions can be stabilized by various components, and their cost is important for the food industry that is always looking for cheaper alternatives. Consumer demand is leading food companies toward a clean label policy, and natural ingredients are the way to go. Modern forestry biorefineries offer an attractive option; novel plant-based hydrocolloids, such as spruce galactoglucomannans (GGM), are available in large quantities. Here we test their functionality and compare it with that of known food stabilizers. Rapeseed oil-in-water (O/W) emulsions stabilized by GGM and their carboxymethyl derivatives (CMGGM) were characterized in detail in terms of droplet size distribution, surface charge, and polysaccharide composition in the aqueous phase and on the droplet surface. The emulsifying and stabilizing capacities of GGM and CMGGM were compared to those of known stabilizers, namely, corn fiber gum (CFG) and gum arabic (GA), the latter of which is the gold standard in food emulsions. During high-pressure homogenization, GGM and CMGGM enabled the formation of emulsions with an average droplet size (D[3,2]) of about 400 nm and stabilized the droplets against creaming more effectively than GA (D[3,2] = 1.27 μm). The GGM and the CMGGM exhibited natural heterogeneity in their molecular composition and were distributed between the aqueous phase and the droplet interface of emulsions according to their molar mass and the CMGGM pattern of carboxymethyl substitution. This study clearly shows that wood-derived natural emulsifiers, GGM and CMGGM, meet the needs of the food industry.

On the formation of lignin polysaccharide networks in Norway spruce

In this study we were mirroring suggested in vivo phenomena of lignin–hemicellulose complex formation in vitro, by cross-linking Norway spruce (Picea abies) galactoglucomannans, xylans and lignin moieties to high molecular weight complexes by laccase treatment. We were able to observe the oxidation and cross-linking of non-condensed guaiacyl-type phenolic moieties attached to both of the hemicelluloses by 31P NMR and size-exclusion chromatography. We suggest that hemicelluloses–lignin complexes form covalently linked structural units during the early stages of lignification via radical enzymatic cross-linking catalyzed by laccase. This work shows that the hemicellulose molecules in wood are covalently linked to two or more lignin units thereby making them suited for forming network structures.

Wood cell wall mimicking for composite films of spruce nanofibrillated cellulose with spruce galactoglucomannan and arabinoglucuronoxylan

Two hemicelluloses (HCs), galactoglucomannan (GGM) and arabinoglucuronoxylan (AGX), and nanofibrillated cellulose (NFC) were isolated from spruce wood and used for the preparation of composite films containing high amounts of cellulose, i.e. 85 and 80 wt% of NFC, respectively. The films were prepared in two ways: (i) by the pre-sorption of HCs on NFC and (ii) by the mixing of components in the usual way. Pre-sorption was applied in an attempt to mimic the carbohydrate biosynthesis pattern during wood cell wall development, where HCs were deposited on the cellulose fibrils prior to lignification taking place. It was assumed that pre-sorption would result in a better film-forming as well as stronger and denser composite films. The mechanical, thermal, structural, moisture sorption and oxygen barrier characteristics of such composite films were tested in order to examine whether the performance of composite films prepared by pre-sorption was better, when compared to the performance of composite films prepared by mixing. The performance of composite films was also tested with respect to the HCs used. All the films showed quite similar barrier and mechanical properties. In general, stiff, strong and quite ductile films were produced. The moisture sorption of the films was comparably low. The oxygen barrier properties of the films were in the range of commercially used poly ethylene vinyl alcohol films. However, the pre-sorption procedure for the preparation of composite films resulted in no additional improvement in the performance of the films compared to the corresponding composite films that had been prepared using the mixing process. Almost certainly, the applied mixing process led to an optimal mixing of components for the film performance achieved. The GGM contributed to a somewhat better film performance than the AGX did. Indications were observed for stronger interactions between the GGM and NFC than that for the AGX and NFC.

Long-Term Physical Stability of Plasticized Hemicellulose Films

Oat spelt arabinoxylan (OsAX) and spruce galactoglucomannan (GGM) are hemicelluloses that can be extracted in large quantities from side-streams of the agriculture and forest industries. They both form self-standing films, making them potential future packaging materials. This systematic study focuses on the effect of long-term storage on the physical stability of hemicellulose-based films. OsAX and GGM films were plasticized with 40% (w/w of the polysaccharide) of glycerol, sorbitol, or their blends, and their stability was followed for four months. Ageing especially affected the glycerol-containing films, in which the tensile strength and Young’s modulus increased and elongation at break decreased. Although the mechanical properties were altered, storage did not affect crystallinity of the films. Oxygen gas permeability (OP) and water vapor permeability (WVP) properties were monitored in OsAX films. Interestingly WVP decreased during storage; more than a 40% decrease was seen when plasticizer blends contained 50% or more glycerol. In contrast, there were no drastic changes in the OP during storage; all the OPs obtained were between 3.7 and 8.9 [cm3 µm/ (m2 d kPa)].

Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana

Each plant genome contains a repertoire of β-mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of Arabidopsis thaliana β-mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 °C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co2+ and inhibited by Mn2+. The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min−1 mg−1 mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.