title: Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity.
abstract: The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K(M)) towards β-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer β-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K(M) for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K(M) for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-β-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Adiphol
authors.initials: A
authors.lastname: Dilokpimol
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Gotfredsen
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Baumann
authors.forename: Natsuko
authors.initials: N
authors.lastname: Nakai
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D001233:Aspergillus nidulans / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D006031:Glycosylation; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D009844:Oligosaccharides; D011994:Recombinant Proteins; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase