To isomerize D-galactose for preparing D-tagatose,a novel L-arabinose isomerase gene was cloned from Paenibacillus polymyxa KF-1 and expressed in Escherichia coli BL21(DE3)cells.The recombinant protein PpoLAI was purified by Ni Sepharose 6FF column,and immobilized by chitosan-glutaraldehyde crosslinking method.The results show that the optimal temperature and pH of purified PpoLAI are 50℃and 7.0,respectively,using D-galactose as substrate.The enzyme is significantly activated by Mn2+,and the activity of PpoLAI reaches the highest when Mn2+concentration is 0.8 mmol/L.The Michaelis constant and maximal velocity of PpoLAI with D-galactose as substrate are 161.4 mmol/L and 98.84 μmol/(mg·min),respectively.The optimal immobilization conditions of PpoLAI are achieved with following conditions:mass fraction of chitosan is 3％,volume fraction of glutaraldehyde is 3％,amount of free enzyme addition is 0.9 mg/g,adsorption temperature is 25 ℃,and adsorption time is 4 h.Under these conditions,the immobilization rate reaches 80.12％,and the thermal stability and pH stability of immobilized PpoLAI are significantly improved compared with the free enzyme.
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