## 3 Results and Discussion
### Partial Purification of Man 58  
Man 58 was partially purified by a combination of ultrafiltration and gel filtration chromatography on Sephacryl S-100. This is the first report on the partial purification and characterization of a \(\beta\)-mannanase from _A. foetidus_. The ultrafiltration procedure showed that the \(\beta\)-mannanase activity was only found in the retentate fraction and displayed an activity level of 6.392 IU/mL. Previous ultrafiltration experiments also indicated that the \(\beta\)-mannanase from _Trichoderma harzianum_ (_T. harzianum_) was only found in the retentate [2]. In this present work, xylanase and pectinase activities were detected in both retentate and ultrafiltrate fractions, with activity levels of 5.577 IU/mL (xylanase) and 5.686 IU/mL (pectinase). For further purification, the retentate was subjected to gel filtration chromatography on Sephacryl S-100. The sample elution resulted in separation of two major peaks of protein. In this case, the \(\beta\)-mannanase activity was only found in the first protein peak (Figure 2). The yield of \(\beta\)-mannanase activity was low for all purification procedures with most of the loss occurring during the different steps of purification. In this case, the partial purification yield was 32%. Fractions containing \(\beta\)-mannanase activity (53-65) were collected and submitted to SDS-PAGE and zymogram analysis (Figure 3). The profiles of these fractions showed several bands staining for \(\beta\)-mannanase activity, migrating at molecular masses varying from 30 to 45 kDa, suggesting multiple forms of \(\beta\)-mannanase (Figure 3). For example, fractions 60-65 showed at least four bands of \(\beta\)-mannanase activity while fraction 58 (designated as Man 58) presented two major bands of \(\beta\)-mannanase activity with molecular masses of approximately 43 and 45 kDa. The multiplicity of forms is commonly described for hemicellulases from fungi and bacteria as result of differential mRNA processing and posttranslational modifications [2,3]. These enzymes may be allozymes, products of different alleles of the same gene, isozymes or same molecules with different postmodifications. The substrate specificity of Man 58 preparation showed that \(\beta\)-mannanase was the major activity (1.2 IU/mL), followed in much lesser extent by activities of xylanase (0.4 IU/mL) and CMCase (0.20 IU/mL). On the other hand, avicalese, FPase and pectinase activities were not detected. Man I from _T. harzianum_ exhibited no detectable activities of CMCase and xylanase [2]. A thermophilic \(\beta\)-mannanase preparation from _Neosartorya fischeri_ was not active against beechwood xylan and barely \(\beta\)-glucan [8].  
Figure 1: Induction profile of mannananase, pectinase and xylanase activities during growth of _Aspergillus foetidus_ (_A. foetidus_) on soybean husk.  
At least three \(\beta\)-mannanase activities were detected in the crude extract of _T. harzianum_[2]. Because of the complexity of mannan structure, the presence of \(\beta\)-mannanases with different specificities is required for a complete hydrolysis of \(\beta\)-mannan, suggesting that a synergistic action of mannan-degrading enzymes of different GH families is required for the degradation of mannan structure [4].