title: Cloning, expression, and characterization of L-asparaginase from a newly isolated Bacillus subtilis B11-06.
abstract: This study focused on the cloning, overexpression, and characterization of the gene encoding L-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11-06. The recombinant enzyme showed high thermostability and low affinity to L-glutamine. The ansZ gene, encoding a putative L-asparaginase II, was amplified by PCR and expressed in B. subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of B. subtilis B11-06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0-9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 °C, respectively. The Km for L-asparagine was 0.43 mM, and the Vmax was 77.51 μM/min. Results of this study also revealed the potential industrial application of this enzyme in reducing acrylamide formation during the potato frying process.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Educationand Lab of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University , Wuxi, Jiangsu Province 214122, China.
authors.forename: Mingmei
authors.initials: M
authors.lastname: Jia
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.forename: Beibei
authors.initials: B
authors.lastname: He
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
mesh_terms: D020106:Acrylamide / Q000032:analysis / Q000378:metabolism; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005506:Food Contamination / Q000517:prevention & control; D005511:Food Handling; D015870:Gene Expression; D005973:Glutamine / Q000378:metabolism; D006358:Hot Temperature / Q000009:adverse effects; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D018517:Plant Roots / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011198:Solanum tuberosum / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D005973:Glutamine; D020106:Acrylamide; D001215:Asparaginase