Effects of Halophilic Peptide Fusion on Solubility, Stability, and Catalytic Performance of D-Phenylglycine Aminotransferase
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D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful forenzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low proteinsolubility prevents its application at high substrate concentration. With an aim to increase theprotein 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 ahalophilic 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). Inaddition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgATat 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 thesubstrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT inthe presence of NaCl and KCl, being slightly in favor of KCl, where under the same conditionat 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.
Abstract
D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful forenzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low proteinsolubility prevents its application at high substrate concentration. With an aim to increase theprotein 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 ahalophilic 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). Inaddition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgATat 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 thesubstrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT inthe presence of NaCl and KCl, being slightly in favor of KCl, where under the same conditionat 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.
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