title: Effects of halophilic peptide fusion on solubility, stability, and catalytic performance of D-phenylglycine aminotransferase.
abstract: D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful for enzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low protein solubility prevents its application at high substrate concentration. With an aim to increase the protein solubility, the N-terminus of D-PhgAT was genetically fused with short peptides (A1 α- helix, A2 α-helix, and ALAL, which is a hybrid of A1 and A2) from a ferredoxin enzyme of a halophilic archaeon, Halobacterium salinarum. The fused enzymes A1-D-PhgAT, A2-D-PhgAT, and ALAL-D-PhgAT displayed a reduced pI and increased in solubility by 6.1-, 5.3-, and 8.1- fold in TEMP (pH 7.6) storage, respectively, and 5-, 4.5-, and 5.9-fold in CAPSO (pH 9.5) reaction buffers, respectively, compared with the wild-type enzyme (WT-D-PhgAT). In addition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgAT at all concentrations of L-glutamate monosodium salt used. The highest rate, 23.82 ± 1.47 mM/h, was that obtained from having ALAL-D-PhgAT reacted with 1,500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT in the presence of NaCl and KCl, being slightly in favor of KCl, where under the same condition at 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.
authors: Department of Microbiology, Islamic Azad University, Jahrom, 355/74135, Iran, Faculty of Science, Energy and Environment, King Mongkut's University of Technology North Bangkok (Rayong Campus), Bankhai, Rayong 21120, Thailand.
authors: Hossein
authors: H
authors: Javid
authors: Juntratip
authors: J
authors: Jomrit
authors: Aiya
authors: A
authors: Chantarasiri
authors: Duangnate
authors: D
authors: Isarangkul
authors: Vithaya
authors: V
authors: Meevootisom
authors: Suthep
authors: S
authors: Wiyakrutta
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D015870:Gene Expression; D014644:Genetic Variation; D019617:Halobacterium salinarum / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; C108039:D-phenylglycine aminotransferase; D000637:Transaminases