title: Hydrophobic Mutagenesis and Semi-rational Engineering of Arginine Deiminase for Markedly Enhanced Stability and Catalytic Efficiency.
abstract: Due to its systemic arginine degradation, arginine deiminase (ADI) has attracted attentions as an anti-tumor drug. Its low activity at physiological conditions among other limitations has necessitated its engineering for improved properties. The present study describes the hydrophobic mutagenesis and semi-rational engineering of ADI from Pseudomonas plecoglossicida (PpADI). Using an improved ADI variant M13 (D38H/A128T/E296K/H404R/I410L) as parent, site saturation mutagenesis at position 162 resulted in an over 20 % increase in protein solubility. Compared with M13 (15.23 U/mg), mutants M13-2 (M13+S245D) and M13-5 (M13+R243L) exhibited enhanced specific activity of 21.19 and 31.20 U/mg at physiological conditions. M13-5 displayed enhanced substrate specificity with a dramatic reduction in its K m value (from 0.52 to 0.16 mM). It is speculated that the improvements in M13-5 could mainly be attributed to the enhanced structural stability due to an R243L substitution. The hydrophobic contribution of Leu 243 was supported by mutant M13-9 (M13+A276W) generated based on the hydrophobic mutagenesis concept. M13-9 showed a specific activity of 18.68 U/mg, as well as remarkable thermal and pH stability. It retained over 90 % activity over pH range from 4.5 to 8.5. At 60 °C, the half-life of M13-9 was enhanced from 4 to 17.5 min in comparison with M13, and its specific activity at 62 °C (93.0 U/mg) was approximately fivefold of that determined at 37 °C. Our results suggest that the increased hydrophobicity around the active regions of PpADI might be crucial in improving its structural stability and ultimately catalytic efficiency.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Biocatalysis, School of Biotechnology, Jiangnan University, 1800 Lihu Rd., 214122, Wuxi, China.
authors.forename: Serwanja
authors.initials: S
authors.lastname: Jamil
authors.forename: Meng-Han
authors.initials: MH
authors.lastname: Liu
authors.forename: Yong-Mei
authors.initials: YM
authors.lastname: Liu
authors.forename: Rui-Zhi
authors.initials: RZ
authors.lastname: Han
authors.forename: Guo-Chao
authors.initials: GC
authors.lastname: Xu
authors.forename: Ye
authors.initials: Y
authors.lastname: Ni
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011549:Pseudomonas / Q000201:enzymology; D012399:Rotation; D016415:Sequence Alignment; D012995:Solubility; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D006867:Hydrolases; C019723:arginine deiminase