Expression and purification of an ArsM-elastin-like polypeptide fusion and its enzymatic properties

Autor:	Chungui Zhao, Changdong Ke, Hui Xiong, Xiaolan Zhao, Guangya Zhang, Christopher Rensing, Zhigang Zhang, Suping Yang
Rok vydání:	2019
Předmět:	S-Adenosylmethionine Recombinant Fusion Proteins medicine.disease_cause Methylation Applied Microbiology and Biotechnology Arsenic 03 medical and health sciences chemistry.chemical_compound Escherichia coli medicine 030304 developmental biology Arsenite chemistry.chemical_classification 0303 health sciences biology 030306 microbiology Temperature Methyltransferases General Medicine Fusion protein Enzyme assay Trimethylarsine Elastin Biodegradation Environmental Enzyme Biochemistry chemistry biology.protein Specific activity Genetic Engineering Peptides Biotechnology
Zdroj:	Applied Microbiology and Biotechnology. 103:2809-2820
ISSN:	1432-0614 0175-7598
DOI:	10.1007/s00253-019-09638-w
Popis:	Enzymes could act as a useful tool for environmental bioremediation. Arsenic (As) biomethylation, which can convert highly toxic arsenite [As(III)] into low-toxic volatile trimethylarsine, is considered to be an effective strategy for As removal from contaminated environments. As(III) S-adenosylmethyltransferase (ArsM) is a key enzyme for As methylation; its properties and preparation are crucial for its wide application. Currently, ArsM is usually purified as a His-tag fusion protein restricting widespread use due to high costs. In this study, to greatly reduce the cost and simplify the ArsM preparation process, an Elastin-like polypeptide (ELP) tag was introduced to construct an engineered Escherichia coli (ArsM-ELP). Consequently, a cost-effective and simple non-chromatographic purification approach could be used for ArsM purification. The enzymatic properties of ArsM-ELP were systematically investigated. The results showed that the As methylation rate of purified ArsM-ELP (> 35.49%) was higher than that of E. coli (ArsM-ELP) (> 10.39%) when exposed to 25 μmol/L and 100 μmol/L As(III), respectively. The purified ArsM-ELP was obtained after three round inverse transition cycling treatment in 2.0 mol/L NaCl at 32 °C for 10 min with the yield reaching more than 9.6% of the total protein. The optimal reaction temperature, pH, and time of ArsM-ELP were 30 °C, 7.5 and 30 min, respectively. The enzyme activity was maintained at over 50% at 45 °C for 12 h. The enzyme specific activity was 438.8 ± 2.1 U/μmol. ArsM-ELP had high selectivity for As(III). 2-Mercaptoethanol could promote enzyme activity, whereas SDS, EDTA, Fe2+, and Cu2+ inhibited enzyme activity, and Mg2+, Zn2+, Ca2+, and K+ had no significant effects on it.
Databáze:	OpenAIRE
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