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.

Reduction of acrylamide level through blanching with treatment by an extremely thermostable l-asparaginase during French fries processing

“…2). While the sequence displayed very low identity with those non-archaeon strains, such as 19 % with E. chrysanthemi 3937 (Kotzia and Labrou 2007), 18 % with Bacillus subtilis W168 (Onishi et al 2011), and 14 % with Rhizobium etli (Moreno-Enriquez et al 2012). In previous crystallographic studies on P. horikoshii (Yao et al 2005), eight residues, including Thr11, Tyr21, Ser52, Thr53, Thr83, Asp84, Lys154, and Lys274, were found to be involved in the active site and to play a pivotal role in substrate recognition and catalysis.…”

Optimization, purification and characterization of recombinant L-asparaginase II in Escherichia coli

“…Relative activity was expressed as a percentage of control (100% rASPG activity was 203.67 U/mg). respectively (Onishi et al 2011). The K m , K cat and K cat /K m value of rASPG from Erw.…”

“…The specific activity was very different: The activity of purified recombinant Lasparaginase II from E. coli K-12 express in E. coli BLR(DE3) was 190 U/mg (Khushoo et al, 2004), recombinant L-asparaginase II from Erw. chrysanthemi 3937 express in E. coli BL21(DE3) pLysS was 118.7 U/mg (Kotzia and Labrou, 2007), L-asparaginase II from B. subtilis express in E. coli JM109 (DE3) was 45.5 U/mg (Onishi et al, 2011) L-asparaginase from Rhizomucor miehei express in E. coli was 1,985 U/mg (Huang et al, 2014) and activity of purified L-asparaginase from B. licheniformis was 697.09 U/mg (Mahajan et al, 2014).…”

Characterization of a Recombinant Glutaminase-Free l-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis

“…7). LAsparagine was hydrolyzed by the enzyme with a Michaelis and Menten constant (K m ) of L-asparagine compared to L-asparaginase from Erwinia aroideae [31,32], Corynebacterium glutamicum [33], Cylindrocarpon obtusisporum [34], Mycobacterium phlei [35], Thermus thermophilus [36], Tetrahymena pyriformis [37], and B. subtilis [38]. The K m values of Lasparaginases from different microbial sources are shown in Table 2.…”