Purification and the effect of manganese ions on the activity of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Carboxymethylcellulases (CMCases) fromAspergillus niger andCellulomonas biazotea were purified by a combination of ammonium sulfate precipitation, anion-exchange and gel-filtration chromatography with a 12- and 9-fold increase in the purification factor. The native and subunit molar mass of CMCase fromA. niger were 40 and 25–57 kDa, respectively, while those fromC. biazotea were 23 and 20–30 kDa, respectively. Low concentrations of Mn²⁺ activated the enzymes from both organisms (mixed activation) with apparent activation constants of 0.80 and 0.45 mmol/L of CMCases fromA. niger andC. biazotea, respectively, while at higher CMC concentrations Mn²⁺ inhibited the enzymes (mixed and partial uncompetitive inhibition). The reason for this complex behavior is that more than one Mn²⁺ bind to the same enzyme form with the apparent average inhibition constants of 2.7 and 1.3 mmol/L for CMCases fromA. niger andC. biazotea, respectively.