The mannan endo-1,4-β-mannosidase of A.niger BK01 and the hypothetical protein (An05 g01320) of A.niger CBS513.18 are highly similar with 99% amino acid sequence identity, and 98% DNA sequence similarity. Their amino acid sequences also match the N-terminal sequence of native A. niger mannan endo-1,4-β-mannosidase that was reported in 1998 . We have thoroughly searched the databases and could not find any report on cloning, expression, and characterization of an A. niger mannan endo-1,4-β-mannosidase. Thus, our work is the first report on cloning, expression and characterization of this enzyme. In addition, this work also helps verify the function of the hypothetical protein (An05 g01320) of A.niger CBS513.18 that has been reported to have strong similarity to mannanase (man1) from A. aculeatus. β-Mannanases from A. niger BK01 , A. niger CBS513.18 and A. niger published by Ademark et al  appear to be closely related and have similar properties, which are superior than those from A. niger NRRL 337 . Mannan endo-1,4-β-mannosidases from other Aspergillus species e.g., those from A. aculeatus, A. fumigatus or A. sulphureus, have previously been cloned and expressed in various hosts [26–31]. A. niger β-mannanase has unique properties that are different from other Aspergillus mannanases. Amino acid sequence of A. niger BK01 mannan endo-1,4-β-mannosidases including putative signal peptide can be found in additional file 1.
Mannan endo-1,4-β-mannosidases or β-mannanases have been classified into two family of glycoside hydrolases, i.e., family 5 and 26, based on their sequence similarity http://www.cazy.org. The A. niger mannan endo-1,4-β-mannosidase belongs to glycoside hydrolase family 5 (GH5). Analysis of the primary sequence of fungal mannan endo-1,4-β-mannosidases belonging to this family revealed amino acid sequence similarities ranging from 43 to 93%. Some fungal β-mannanases have been shown to contain a cellulose-binding domain (CBD) at either the C-terminus or N-terminus of the enzyme. For example, CBD of T. reesei β -mannanase is located at its C-terminus (amino acid position 373-410) preceded by a serine-, threonine-, and proline-rich region , whereas CBD of A. fumigatus β-mannanase is located at its N-terminus (amino acid position 9-44) as shown in Fig. 1. A role of this domain in the hydrolysis of mannan/cellulose complex substrates has been suggested . However, β-mannanases from different fungi including A. niger do not contain a CBD. The protruding N-terminus of Bispora sp. as shown in the sequence alignment in Fig. 1 is not similar to a CBD, and its role in catalysis still remains to be explored.
The mature A. niger mannan endo-1,4-β-mannosidase gene without its signal peptide was expressed in P. pastoris X33 with the C-terminus of the recombinant enzyme fused to the 6xHis tag for affinity purification by immobilized metal affinity chromatography (IMAC). The native signal peptide was replaced with that of the Saccharomyces cerevisiae α-factor, allowing secretion of the active enzyme into the extracellular medium with an expression yield of approximately 243 mg L-1. Since cultivation experiments were done in shake flasks, one can expect that growth of P. pastoris in a fermentor under controlled and optimised conditions will result in considerably higher protein yields . In comparison, the expression level of Trichoderma reesei mannan endo-1,4-β-mannosidase in S. cerevisiae was roughly 150 mg L-1  and that of Bispora sp. MEY-1  and B. subtilis mannan endo-1,4-β-mannosidase in P. pastoris GS115  was 1800 mg L-1 and 150 mg L-1, respectively. Moreover, the enzyme activity during fermentation reached 670 U mL-1, which is significantly higher than those reported for most mannan endo-1,4-β-mannosidases [26, 34].
Amino acid sequence analysis by the NetNGlyc 1.0 sever program indicated two putative N-glycosylation sites in the amino acid sequence of mannan endo-1,4-β-mannosidase i.e., 195NSS197, and 252NFT254 (according to numbering in Fig. 1). The deglycosylation analysis, showed in Fig. 3, confirms the prediction that the enzyme is glycosylated. Purified mannan endo-1,4-β-mannosidase treated with Endoglycosidase H was calculated to be 43 kDa by SDS-PAGE. This result indicated that the A. niger mannan endo-1,4-β-mannosidase expressed in P. pastoris was properly glycosylated, as the enzyme was as active as the native enzyme.
Recombinant A. niger BK01 mannan endo-1,4-β-mannosidase efficiently hydrolysed galactomannans, glucomannans and β-1,4-mannans from different sources. Although determination of kinetic parameters enabled us to demonstrate that A. niger GH5 mannan endo-1,4-β-mannosidase was highly active towards structurally different mannans, this enzyme displayed highest specificity towards unsubstituted carob glucomannan with a Km and kcat of 0.6 mg.mL-1 and 215 s-1, respectively. Based on these kinetic parameters, mannanase activity of this enzyme appears not to be hampered by the presence of side chains, i.e. galactosyl residues. The enzyme can hydrolyse galactomannans as well as glucomannans and unsubstituted β-1,4-mannan. The action of the enzyme on these substrates as well as on Azo-carob galactomannan, a standard substrate for the assessment of mannan endo-1,4-β-mannosidase activity, indicates its function as a true endo-β-1,4-mannanase. This activity was confirmed by TLC analysis of reaction products obtained through hydrolysis of locust bean gum, which gave mannobiose as major product and no detectable mannose, indicating that the enzyme has no β-mannosidase activity. In addition to mannan, A. niger mannanase was capable of degrading birchwood xylan (9.1% relative activity). Its lacks of activity for cellulose is valuable for applications in bleaching pulp and paper .
In general, the properties of the recombinant mannan endo-1,4-β-mannosidase from A. niger are very similar to those of the enzyme from its natural source with only some minor differences. The recombinant A. niger mannan endo-1,4-β-mannosidase furthermore shows properties typical of thermostable enzymes, which is in accordance with the wild-type enzyme. The optimal temperature of its activity is 80°C, similar to that of the native enzyme . Optimal temperatures of different fungal β-mannanases have previously been reported; that of the β-mannanase from Trichoderma reesei C-30 was found at 75°C , whereas β-mannanase produced from A. niger and A. flavus showed their optimum at 65 and 60°C, respectively . Two forms of mannan endo-1,4-β-mannosidase from the thermotolerant fungus A. fumigatus IMI 385708 showed highest activity at 60°C (pH 4.5) . These rather high optima appear to be a common but valuable characteristic of fungal β-mannanases. At such high temperatures (above 60-65°C), enzymatic digestion may not only increase the rate of hydrolysis but also reduce microbial contamination of the material being processed .
Both native and recombinant A. niger mannan endo-1,4-β-mannosidase appear to be among the most thermostable fungal β-mannanases reported to date. The recombinant A. niger mannan endo-1,4-β-mannosidase retained >98% activity after 4 h at 70°C, while mannan endo-1,4-β-mannosidase from Talaromyces emersonii and Aspergillus niger NRRL 337 retained 53% and 20% activity after 1 h at 65°C, respectively . The half-life of thermostable acidic mannan endo-1,4-β-mannosidase from Sclerotium (Athelia) rolfsii at 70°C and pH 4.5 was 1.5 h , while that of recombinant A. niger mannan endo-1,4-β-mannosidase at 70°C was 56 h. Bispora sp. MEY-1 mannan endo-1,4-β-mannosidase, which was expressed in P. pastoris, only retained more than 50% of its initial activity after an incubation at 70°C for 20 min .
The strong increase of the enzyme activity in the present of EDTA (1 mM) suggests that metal ions are not present in the active site and are not required for activity. At the same concentration, the effect of EDTA on A. sulphureus β-mannanase activity was only moderate . EDTA might protect the enzyme against the detrimental effect of metal ions present in the enzyme preparation, which inhibit the A. niger β-mannanase to a certain extent while not affecting the A. sulphureus enzyme significantly. For example, Mn2+ strongly inhibited the A. niger β -mannanase but did not effect the latter enzyme. Other ions including Na+, Zn2+, Mg2+, Ca2+, Fe2+ exerted an adverse effect on the activity of A. niger β-mannanase but did not affect or slightly increased the A. sulphureus β-mannanase activity . The β-mannanase activity inhibition in the presence of PMSF (1 mM) was 24.8%, suggesting the role of a serine in the catalytic action of A. niger mannan endo-1,4-β-mannosidase.