The modification of PCMB, NEM N AI, NBS resulted in complete and partial loss of the activity of �� D mannanase (EC 3 2 1 78) from Nocardioform actinomycetes NA3 540 respectively. It was shown that sulfhydryl residues, tryptophan residues and tyrosine residues might be involved in the active site of the enzyme. In the presence of ligands binding, the �� max emission fluorescence spectrum of the enzyme changed from 336 nm to 332 nm. It appeared that tryptophan residues were involved in a widely hydrophobic micro environment near the active site of protein molecule. The states of disulfide bonds were analyzed by CD spectroscopy. That decreasing thermostability of the enzyme in the presence of �� mercaptoethanol implied that disulfide bonds were essential for the enzyme stability in the high temperature. In addition, CD spectrum indicated that the secondary structure of the enzyme consisted of 16 6% �� helix, 25 4% �� sheet, 20 5% �� turn and 37 5% random coil.