Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties

Abstract

Expression of the ansZ gene encoding a putative l-asparaginase II (BsAII) from Bacillus subtilis in Escherichia coli was examined. No expression was detected in E. coli transformed with a plasmid containing the full-length ansZ gene. Three N-terminal truncated enzymes (BsAIIT18M, BsAIIS40M, and BsAIID49M) were prepared based on comparison with the N-terminal sequences of other type II l-asparaginases. BsAIIT18M became easily inactivated during DEAE-Toyopearl column chromatography. The purified N-terminal-truncated enzymes BsAIIS40M and BsAIID49M had tetrameric subunit structures and V max values of 45.5 and 45.8 U/mg towards l-asparagine, respectively. Their K m values were 2.06 and 7.02 mM, respectively. The enzymes differed from asparaginase II from E. coli and Erwinia carotovora in substrate specificity and affinity for l-asparagine. BsAIIS40M and BsAIID49M retained over 80% of their original activities in the presence of 15% NaCl, and thus may find application in the food industry for products in which NaCl is used. This study also revealed that BsAII is rather different from the type I enzyme (BsAI) from B. subtilis in substrate specificity and salt-tolerance.