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The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes.

Morrill J, Kulcinskaja E, Sulewska AM, Lahtinen S, Stålbrand H, Svensson B, Abou Hachem M - BMC Biochem. (2015)

Bottom Line: Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme.BlMan5_8 is evolved for efficient deconstruction of soluble mannans, which is reflected by an exceptionally low K m and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules.These features highlight fine tuning of catalytic and binding properties to support specialization towards a preferred substrate, which is likely to confer an advantage in the adaptation to competitive ecological niches.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Lund, Sweden.

ABSTRACT

Background: β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8).

Results: BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan and displays the highest catalytic efficiency reported to date for a GH5 mannanase owing to a very high k cat (1828 ± 87 s(-1)) and a low K m (1.58 ± 0.23 g · L(-1)) using locust bean galactomannan as substrate. The novel CBM of BlMan5_8 mediates increased binding to soluble mannan based on affinity electrophoresis. Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs present only in mannanases. Members of this new subfamily lack an aromatic residue mediating binding to insoluble cellulose in canonical CBM10 members consistent with the observed low mannan affinity.

Conclusion: BlMan5_8 is evolved for efficient deconstruction of soluble mannans, which is reflected by an exceptionally low K m and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules. These features highlight fine tuning of catalytic and binding properties to support specialization towards a preferred substrate, which is likely to confer an advantage in the adaptation to competitive ecological niches.

No MeSH data available.


Related in: MedlinePlus

Initial products from INM and LBG. Manno-oligosaccharide products formed after 30 s of a INM and b LBG hydrolysis by BlMan5_8 as measured using HPAEC-PAD analysis. The chromatograms from BlMan5_8-ΔCBM10 (I), BlMan5_8 (II) and the substrate before hydrolysis (III)
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Fig1: Initial products from INM and LBG. Manno-oligosaccharide products formed after 30 s of a INM and b LBG hydrolysis by BlMan5_8 as measured using HPAEC-PAD analysis. The chromatograms from BlMan5_8-ΔCBM10 (I), BlMan5_8 (II) and the substrate before hydrolysis (III)

Mentions: The hydrolysis profiles of LBG and INM by BlMan5_8 and BlMan5_8-ΔCBM10 were analyzed using HPAEC-PAD. Both enzyme variants produced M3, M4 and M5 as the main initial hydrolysis products from both LBG and INM, along with some mannobiose (M2) (Fig. 1). The main products of BlMan5_8 from M5 were M2, M3 and M4. The formation of 10-fold higher M4 compared to mannose (M1) (Fig. 2) is indicative of transglycosylation activity, which was supported by the detection of mannohexaose (M6), mannoheptaose (M7) and mannooctaose (M8) transglycosylation products by mass-spectrometry following incubation with M5 (Fig. 3). All four productive M5 binding modes were observed, albeit with higher frequency (66 %) for binding modes where the larger part of the substrate was bound at the aglycone binding subsites (Fig. 4).Fig. 1


The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes.

Morrill J, Kulcinskaja E, Sulewska AM, Lahtinen S, Stålbrand H, Svensson B, Abou Hachem M - BMC Biochem. (2015)

Initial products from INM and LBG. Manno-oligosaccharide products formed after 30 s of a INM and b LBG hydrolysis by BlMan5_8 as measured using HPAEC-PAD analysis. The chromatograms from BlMan5_8-ΔCBM10 (I), BlMan5_8 (II) and the substrate before hydrolysis (III)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4642672&req=5

Fig1: Initial products from INM and LBG. Manno-oligosaccharide products formed after 30 s of a INM and b LBG hydrolysis by BlMan5_8 as measured using HPAEC-PAD analysis. The chromatograms from BlMan5_8-ΔCBM10 (I), BlMan5_8 (II) and the substrate before hydrolysis (III)
Mentions: The hydrolysis profiles of LBG and INM by BlMan5_8 and BlMan5_8-ΔCBM10 were analyzed using HPAEC-PAD. Both enzyme variants produced M3, M4 and M5 as the main initial hydrolysis products from both LBG and INM, along with some mannobiose (M2) (Fig. 1). The main products of BlMan5_8 from M5 were M2, M3 and M4. The formation of 10-fold higher M4 compared to mannose (M1) (Fig. 2) is indicative of transglycosylation activity, which was supported by the detection of mannohexaose (M6), mannoheptaose (M7) and mannooctaose (M8) transglycosylation products by mass-spectrometry following incubation with M5 (Fig. 3). All four productive M5 binding modes were observed, albeit with higher frequency (66 %) for binding modes where the larger part of the substrate was bound at the aglycone binding subsites (Fig. 4).Fig. 1

Bottom Line: Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme.BlMan5_8 is evolved for efficient deconstruction of soluble mannans, which is reflected by an exceptionally low K m and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules.These features highlight fine tuning of catalytic and binding properties to support specialization towards a preferred substrate, which is likely to confer an advantage in the adaptation to competitive ecological niches.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Lund, Sweden.

ABSTRACT

Background: β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8).

Results: BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan and displays the highest catalytic efficiency reported to date for a GH5 mannanase owing to a very high k cat (1828 ± 87 s(-1)) and a low K m (1.58 ± 0.23 g · L(-1)) using locust bean galactomannan as substrate. The novel CBM of BlMan5_8 mediates increased binding to soluble mannan based on affinity electrophoresis. Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs present only in mannanases. Members of this new subfamily lack an aromatic residue mediating binding to insoluble cellulose in canonical CBM10 members consistent with the observed low mannan affinity.

Conclusion: BlMan5_8 is evolved for efficient deconstruction of soluble mannans, which is reflected by an exceptionally low K m and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules. These features highlight fine tuning of catalytic and binding properties to support specialization towards a preferred substrate, which is likely to confer an advantage in the adaptation to competitive ecological niches.

No MeSH data available.


Related in: MedlinePlus