title: Recombinant expression and characterization of L-asparaginase II from a moderately thermotolerant bacterial isolate.
abstract: A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 °C, was isolated from cow dung; L-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37 °C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 °C with a 33% and 28% of activity retention after 45 and 60 min. The kinetic parameters for the enzyme were calculated from Lineweaver-Burk plot, and K(m) and V(max) were 0.89 mM and 0.18 U/mg, respectively.
authors.affiliation: Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Trivandrum 695019, India. vidyabt@gmail.com
authors.forename: Jalaja
authors.initials: J
authors.lastname: Vidya
authors.forename: Ashok
authors.initials: A
authors.lastname: Pandey
mesh_terms: D000818:Animals; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004755:Enterobacteriaceae / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D004795:Enzyme Stability; D005243:Feces / Q000382:microbiology; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D001215:Asparaginase