Effects of halophilic peptide fusion on solubility and stability of D-phenylglycine aminotransferase
Issued Date
2023
Copyright Date
2014
Language
eng
File Type
application/pdf
No. of Pages/File Size
xv, 116 leaves : ill.
Access Rights
restricted access
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (Ph.D. (Microbiology))--Mahidol University, 2014
Suggested Citation
Javid, Hossein, 1980- Effects of halophilic peptide fusion on solubility and stability of D-phenylglycine aminotransferase. Thesis (Ph.D. (Microbiology))--Mahidol University, 2014. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/89527
Title
Effects of halophilic peptide fusion on solubility and stability of D-phenylglycine aminotransferase
Author(s)
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 concentrations. With the aim of increasing protein solubility, 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 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 to the wild-type enzyme (WT-D-PhgAT). In addition, all the fused D-PhgAT displayed a higher enzymatic reaction rate than the WT-D-PhgAT at all concentrations of L-glutamate monosodium salt used. The highest one, 23.82 ± 1.47 mM h-1, was that obtained from having ALAL-D-PhgAT reacted with 1500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT in the presence of NaCl and KCl, 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. In addition a higher thermal stability has been seen in halophilic fused variants as the hydrophobicity (log p) of miscible organic solvents increased
Degree Name
Doctor of Philosophy
Degree Level
Doctoral Degree
Degree Department
Faculty of Science
Degree Discipline
Microbiology
Degree Grantor(s)
Mahidol University