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Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237

Version 2 2024-06-06, 01:04
Version 1 2022-02-03, 08:25
journal contribution
posted on 2024-06-06, 01:04 authored by Hoang Chinh NguyenHoang Chinh Nguyen, SP Wu, CH Su, TS Hwang
Description of the subject. Alkaline phosphatases (APases) are commonly used as nonradioactive markers for detecting specific proteins or DNA targets in clinical medicine and molecular biology. However, their applications in the biotechnology industry require thermostability and storage stability. Our preliminary study revealed that APase from Thermus sp. NTU-237 (TsAPase) is thermostable and exhibits high activity. Therefore, it is desirable to establish the optimal conditions for its application. Objectives. To characterize APase from thermophile Thermus sp. NTU-237 and to evaluate its potential applications. Method. The APase gene of Thermus sp. NTU-237 was cloned and expressed in Escherichia coli. Subsequently, the effects of buffer, glycerol, sodium dodecyl sulfate (SDS), NaCl, and temperature on enzyme activity were studied to establish the optimal conditions for TsAPase assays. In addition, the potential for application of TsAPase was evaluated using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) active staining method. Results. Recombinant TsAPase was identified as having a dimeric structure and a molecular mass of 109 kDa. Sequence alignment analysis of known thermophilic APases with TsAPase and E. coli APase revealed that catalytic and binding residues were highly conserved. This finding suggested that the catalytic mechanism of TsAPase is the same as that of other APases. TsAPase activity was inhibited by glycerol and SDS but enhanced by NaCl and Tris-HCl buffer. The kinetic parameters Km, kcat, and Vmax were determined to be 81 μM, 6.08 s-1, and 6.76 U.mg-1, respectively. The optimum temperature of TsAPase was 80 °C, and TsAPase was stable at room temperature for more than 10 days. Moreover, TsAPase could catalyze the dephosphorylation of BCIP and could elicit blue color development by the BCIP/NBT reaction kit at room temperature. Conclusions. These results illustrate that TsAPase has potential for application in medical or basic research.

History

Journal

Biotechnology, Agronomy and Society and Environment

Volume

21

Pagination

117-126

Location

Bussels, Belgium

ISSN

1370-6233

eISSN

1780-4507

Language

English

Publication classification

C1.1 Refereed article in a scholarly journal

Issue

2

Publisher

FAC UNIV SCIENCES AGRONOMIQUES GEMBLOUX