Effects of halophilic peptide fusion on solubility and stability of D-phenylglycine aminotransferase

Name: Javid, Hossein.

LCSH: Halophilic microorganisms.

MeSH: Alanine Transaminase.

MeSH: Transaminases.

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

Mahidol University. Mahidol University Library and Knowledge Center

Address: NAKHONPATHOM

Email: liwww@mahidol.ac.th

Name: Suthep Wiyakrutta

Role: Thesis Advisors

Name: Vithaya Meevootisom

Role: Thesis Advisors

Name: Chartchai Changsen

Role: Thesis Advisors

Name: Nuttawee Niamsiri

Role: Thesis Advisors

Created: 2014

Modified: 2019-11-19

Issued: 2019-11-19

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: TH J41e 2014

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degree

Descipline: Microbiology

Grantor: Mahidol University

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