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

Wang Y, Vilaplana F, Brumer H, Aspeborg H - Planta (2013)

Bottom Line: However, the galactose-rich and highly branched guar gum was not as efficiently degraded.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Glycoscience, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Centre, 106 91, Stockholm, Sweden.

ABSTRACT
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 Co(2+) and inhibited by Mn(2+). 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.

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Oligosaccharide profiles using HPAEC-PAD after enzymatic hydrolysis of different mannan oligo- and polysaccharides. a Unassigned hydrolysis products, b mannan oligosaccharides present in the blank
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Fig7: Oligosaccharide profiles using HPAEC-PAD after enzymatic hydrolysis of different mannan oligo- and polysaccharides. a Unassigned hydrolysis products, b mannan oligosaccharides present in the blank

Mentions: To further investigate the AtMan5-1 mode of action, the AtMan5-1p hydrolysis products released from various mannan polysaccharides and mannan oligosaccharides were analyzed by HPAEC-PAD (Fig. 7; Table 3). Only the Pichia-produced recombinant protein was included in the study since the yield of soluble AtMan5-1e was relatively low. The major products from carob galactomannan depolymerization were M2, M3 and a galactomanno-oligosaccharide that can be assigned as a GM3. From konjac glucomannan hydrolysis the main identified oligosaccharide was M2, although M3 was also moderately abundant. Two abundant hydrolysis products could not be identified using HPAEC-PAD, although these fragments most probably consist of short glucomanno-oligosaccharides (Albrecht et al. 2011). Guar gum was not efficiently degraded by AtMan5-1p, although peaks for M2 and M3 could be identified. For the most complex mannan polysaccharide spruce galactoglucomannan, the hydrolysis yielded predominantly equal amounts of M3 and M4. Arabinoxylan, hydroxyethylcellulose (HEC) and xyloglucan incubated with AtMan5-1p yielded no hydrolytic products (data not shown). In addition to the polymer degradation product analysis, the hydrolysis of manno-oligosaccharides was investigated. Here, M3, M4 and G2M5 were not cleaved by AtMan5-1p (data not shown), whereas M2 and M3 were identified as the main products of M5 although not in high amounts. The digestion of M6 released M2, M3 and M4 among which M3 was the most abundant product. Comparison of the relative amounts of digested M5 and M6 revealed that the catalytic rate of AtMan5-1 towards M6 was 2.6 times higher than that of M5. The absence of products with a higher degree of polymerization (d.p.) than the examined oligosaccharides indicated that no transglycosylation reaction occurred. However, when in the substrate concentration of M5 was increased in order to favor transglycosylation the larger manno-oligosaccharides M6, M8 and M9 were observed indicating that recombinant AtMan5-1 is able to catalyze transglycosylation reactions in vitro at higher substrate concentrations (Fig. 8a, b). Moreover, transglycosylation at higher substrate concentrations occurs more favorably for mannohexaose (M6) than for mannopentaose (M5), suggesting a size dependence of the substrate on the transglycosylation capability (Fig. 8c, d). Salt addition had no apparent effect on the transglycosylation activity.Fig. 7


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

Wang Y, Vilaplana F, Brumer H, Aspeborg H - Planta (2013)

Oligosaccharide profiles using HPAEC-PAD after enzymatic hydrolysis of different mannan oligo- and polysaccharides. a Unassigned hydrolysis products, b mannan oligosaccharides present in the blank
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3928506&req=5

Fig7: Oligosaccharide profiles using HPAEC-PAD after enzymatic hydrolysis of different mannan oligo- and polysaccharides. a Unassigned hydrolysis products, b mannan oligosaccharides present in the blank
Mentions: To further investigate the AtMan5-1 mode of action, the AtMan5-1p hydrolysis products released from various mannan polysaccharides and mannan oligosaccharides were analyzed by HPAEC-PAD (Fig. 7; Table 3). Only the Pichia-produced recombinant protein was included in the study since the yield of soluble AtMan5-1e was relatively low. The major products from carob galactomannan depolymerization were M2, M3 and a galactomanno-oligosaccharide that can be assigned as a GM3. From konjac glucomannan hydrolysis the main identified oligosaccharide was M2, although M3 was also moderately abundant. Two abundant hydrolysis products could not be identified using HPAEC-PAD, although these fragments most probably consist of short glucomanno-oligosaccharides (Albrecht et al. 2011). Guar gum was not efficiently degraded by AtMan5-1p, although peaks for M2 and M3 could be identified. For the most complex mannan polysaccharide spruce galactoglucomannan, the hydrolysis yielded predominantly equal amounts of M3 and M4. Arabinoxylan, hydroxyethylcellulose (HEC) and xyloglucan incubated with AtMan5-1p yielded no hydrolytic products (data not shown). In addition to the polymer degradation product analysis, the hydrolysis of manno-oligosaccharides was investigated. Here, M3, M4 and G2M5 were not cleaved by AtMan5-1p (data not shown), whereas M2 and M3 were identified as the main products of M5 although not in high amounts. The digestion of M6 released M2, M3 and M4 among which M3 was the most abundant product. Comparison of the relative amounts of digested M5 and M6 revealed that the catalytic rate of AtMan5-1 towards M6 was 2.6 times higher than that of M5. The absence of products with a higher degree of polymerization (d.p.) than the examined oligosaccharides indicated that no transglycosylation reaction occurred. However, when in the substrate concentration of M5 was increased in order to favor transglycosylation the larger manno-oligosaccharides M6, M8 and M9 were observed indicating that recombinant AtMan5-1 is able to catalyze transglycosylation reactions in vitro at higher substrate concentrations (Fig. 8a, b). Moreover, transglycosylation at higher substrate concentrations occurs more favorably for mannohexaose (M6) than for mannopentaose (M5), suggesting a size dependence of the substrate on the transglycosylation capability (Fig. 8c, d). Salt addition had no apparent effect on the transglycosylation activity.Fig. 7

Bottom Line: However, the galactose-rich and highly branched guar gum was not as efficiently degraded.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Glycoscience, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Centre, 106 91, Stockholm, Sweden.

ABSTRACT
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 Co(2+) and inhibited by Mn(2+). 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.

Show MeSH