Purification And Chemical Modification Of The Thermostable Inulinase From Bacillus Smithii

In this paper,thermostable inulinases from two newly-isolated thermophilic strain of Bacillus smithii in our lab were purified and characterized.The key amino acid residues participating in the active site of the thermostable inulinase were also investigated by the method of chemical modifications and the catalytic mechanism and heat-resistant mechanism was proposed.With respect to the purification of inulinase,the fermental liquid of Bacillus smithii T4 was centrifugated at 4℃and the supernatant was collected as crude enzyme,and then the purified inulinase was achieved through ammonium sulphate fractional precipitation,dialysis desalination,Sepharose Q-FF,DEAE-Sepharose CL-6B and Superdex 75 chromatography. The purified inulinase was identified as a single band by SDS-PAGE and the subunit molecular weight was about 45.5KD.The specific activity was more than 881.6 IU/mg.The characterization of inulinase from Bacillus smithii T4 showed that the optimum temperature for enzyme activity is 60℃,and the half life of the inulinase is 8h and 5h at 60℃and 70℃,respectively.The optimum pH for this inulinase is 5.0 and stable at pH range of 4.5-7.The lower Km(2.73mM) of the inulinase for inulin indicated that the inulinase from Bacillus smithii T4 has better substrate specificity and no invertase activity.When compared with the inulinase from Bacillus smithii T7,it was found that inulinase from T7 has higher catalytic efficiency and heat-resistance than the inulinase from Bacillus smithii T4.The specific activity of inulinase from Bacillus srnithii T7 is up to 1283.9 IU/mg.The amino acid residues participating in the active site of the thermostable inulinase from Bacillus smithii T7 were investigated by chemical modifications.The results showed that serine,tyrosine,lysine and arginine residues of inulinase did not take part in the catalysis of enzyme.There was also non-involvement of disulfide bond in catalysis.The chemical modification of inulinase by DEPC and carbodiimide showed that one histidine residue might play a key role in substrate binding and catalysis,and one carboxylate residue located in the active site may act as a nucleophile base in the cleavage of substrate.The Tsou's plot analysis indicated that the inactivation of the enzyme by N-bromosuccinimide(NBS) is dependent upon the modification of two essential tryptophan residues.Moreover,tryptophan residues could play an important role in the thermal stabilization of the active site conformation at higher temperatures as a result of hydrophobic environment.