title: Enhanced catalysis of L-asparaginase from Bacillus licheniformis by a rational redesign.
abstract: L-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel L-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7IU/mg, which is higher than native (rBliA) (407.65IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with Km value of 0.42mM and Vmax of 2778.9μmolmin(-1).
authors.affiliation: BRD School of Biosciences, Sardar Patel Maidan, Sardar Patel University, Vadtal Road, Satellite Campus, P.O. Box 39, Vallabh Vidyanagar 388 120, Gujarat, India.
authors.forename: Ankit P
authors.initials: AP
authors.lastname: Sudhir
authors.affiliation: BRD School of Biosciences, Sardar Patel Maidan, Sardar Patel University, Vadtal Road, Satellite Campus, P.O. Box 39, Vallabh Vidyanagar 388 120, Gujarat, India.
authors.forename: Viplove V
authors.initials: VV
authors.lastname: Agarwaal
authors.affiliation: BRD School of Biosciences, Sardar Patel Maidan, Sardar Patel University, Vadtal Road, Satellite Campus, P.O. Box 39, Vallabh Vidyanagar 388 120, Gujarat, India.
authors.forename: Bhaumik R
authors.initials: BR
authors.lastname: Dave
authors.affiliation: P.D Patel Institute of Applied Sciences, Charotar University of Science and Technology (CHARUSAT), Changa, Anand, Gujarat, India.
authors.forename: Darshan H
authors.initials: DH
authors.lastname: Patel
authors.affiliation: BRD School of Biosciences, Sardar Patel Maidan, Sardar Patel University, Vadtal Road, Satellite Campus, P.O. Box 39, Vallabh Vidyanagar 388 120, Gujarat, India. Electronic address: asp.fus@gmail.com.
authors.forename: R B
authors.initials: RB
authors.lastname: Subramanian
mesh_terms: D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000069976:Bacillus licheniformis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D019020:Directed Molecular Evolution; D015195:Drug Design; D006207:Half-Life; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001215:Asparaginase
keywords: B. licheniformis; Enhanced catalysis; Site directed mutagenesis; l-asparaginase