To determine the physiological and biochemical characteristics,three Bbacillus strains were isolated from Yunnan springs and were identified by 16 SrDNA sequence analysis as Bacillus amyloliquefacie LCEP-1,Bacillus subtilis DL-3-4-2 and Bacillus subtilis DMJS-2-1 with their GeneBank accession numbers of gb.GQ199589,gb.GQ199593 and gb.GQ199594,respectively.Isoamylases from all three strains could hydrolyze corn amylopectin.The optimal pH of enzymatic reaction respectively was 6.8,5.4 and 7.8;optimum temperature was 60℃,55℃ and 60℃.Under optimized conditions,the highest enzyme activity was 36.5 U/mL,18.89 U/mL and 11.08 U/mL.The enzymes react differently to metal ions.It is proposed that the three isoamylases belong to metal enzymes because some metal and EDTA showed stronger inhibitory effect than others on the enzyme activity.Within the pH range of 4.4~8.0 all three enzymes remain stable;the enzyme activities can maintain high under the condition of weak acidic,neutral to weak alkaline.