Abstract
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.
Highlights
Galactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties
Two mannanases from C. japonicus, CjMan5A and CjMan26A, were selected and used to hydrolyze three native mannan substrates: KGMN, LBGN, and spruce GGM (SpGGM). KGMN and LBGN serve as model substrates as they share important structural and chemical features with SpGGM, and are well investigated and understood
The chemically acetylated substrates with medium (KGMA, LBGA) and high degree of substitution by acetylation (DSac) (KGMB, LBGB) were included in the study to investigate the role of acetylation on the mannanase action
Summary
Galactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. 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. Hemicelluloses can be hydrolyzed into fermentable sugars, but may serve as oligomeric and polymeric starting materials for the manufacture of high-value products such as films, coatings, gels, food additives, prebiotics, and biodegradable components in composite materials [4, 6,7,8,9]. Enzymes with high specificities can be important in degrading and modifying hemicelluloses for different purposes
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