Classification And Identification Of Two Amylase-producing Strains And A Pectinase-producing Strain

Based on physiological and biochemical characteristics, 16S rDNA sequence homology, (G+C) mol% content and cellular fatty analysis , two new isolates XJU-3 and XJU-4 were accurately identified as Bacillus flexus , XJU-8 was identified as Bacillus claussi. And the paper also discussed the enzyme characteristics of alkaline amylase of the two new strains XJU-3 and XJU-4, and the enzyme characteristics of alkaline pectinase of the new strain XJU-8. The data demonstrated the following conclusions:1,XJU-3 and XJU-4 were Gram-positive, spore-forming, aerobic, motile rod alkaliphilic bacterium. They can grow at a broad range of pH (4. 0～12.5) in Luria broth medium. Their NaCl tolerance were up to 15% and major cellular fatty acids were anteiso-C15:0 and iso-C15: 0. The genomic DNA (G+C) content of our isolates were found to be 39.13 mol % and 38.35 mol % . Owing to the primary component of cellular fatty acid, the 16S rDNA sequences, the hydrolysis of urea, the utilization of D-Xylose, D-Lactose and D-Raffinose, and the excellent pH tolerance, which were different from those of the reference strain B. flexus DSM1320T and the description of the Manuals of Bergey, the strains XJU-3 and XJU-4 were classified as new member of B. flexus and were also denominated (Bacillus flexus XJU-3 , Bacillus flexus XJU-4 and Bacillus claussi XJU-8), registered (EF157300 and EF157301) and conserved (CCTCC AB 207167, CCTCC AB 207168).2,The amylase activity was estimated on the basis of the reduction in blue colour intensity resulting from enzymatic hydrolysis of starch,the activity of crude amylases produced by the two Bacillus flexus was determined. The data showed that XJU-3 amylase was most active at pH10.0 and better stability at alkaline condition (pH8.0～13.0), XJU-4 amylase was most active at pH11.0 and better stability at alkaline condition (pH8.5～13.0)3,The isolate XJU-8 was a Gram-positive, spore-forming, aerobic, motile rod. It can grow at a broad range of pH values (5.0 ¹3.0) in LB medium; Optimum conditions for growth were 37℃and pH10.5. Its NaCl tolerance was high up to 15%. It was obviously catalase-positive and oxidase-negative. Comparative 16S rRNA gene sequence analysis showed that strain XJU-8 was most closely related to B. claussi with 99% similarity. Its genomic DNA G+C content was 43.67 mol %, which falls among the range of the B. claussi. The strain XJU-8 was confirmed as B.claussi by comparing its physiological, biochemical and genetical characteristics with those of the B. claussi GMBAE 42 . Whereas, owing to its excellent pH tolerance and several phenotypic characteristics that were different from the description of the reference strain, the strain was further classified as a new member of the species B. claussi.4,According to DNS methods, the activity of crude pectinase produced by Bacillus claussi XJU-8 was determined. The data showed that the pectinase was most active at pH10.0 and better stability at alkaline condition (pH8.5～12.0), The enzyme activity was strongly enhanced with the presence of Ca²⁺. These properties indicated that pectinase of our isolate may be suitable for paper making, coffee and tea fermentations, textile industries and other industrial applications.