Cloning, expression and biochemical characterization of a novel, moderately thermostable GDSL family esterase from Geobacillus thermodenitrificans T2

• Published in: Journal of bioscience and bioengineering 2013-02, Vol.115 (2), p.133-137
• Main Authors: Yang, Zhenxing, Zhang, Yong, Shen, Tiantian, Xie, Yi, Mao, Yumin, Ji, Chaoneng
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Bacillus thermodenitrificans; Biological and medical sciences; Biotechnology; Characterization; Cloning; Cloning, Molecular; DNA; Enzyme Stability; esterases; Esterases - antagonists & inhibitors; Esterases - chemistry; Esterases - genetics; Esterases - isolation & purification; Esterases - metabolism; Fundamental and applied biological sciences. Psychology; GDSL family; genes; Geobacillus; Geobacillus - enzymology; Geobacillus - genetics; Hydrogen-Ion Concentration; Indicators and Reagents - pharmacology; Models, Molecular; Molecular Sequence Data; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Temperature; thermal stability; Thermostable esterase
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2012.08.016

A thermostable GDSL family esterase-encoding gene, EstL5, was directly obtained from the genomic DNA of Geobacillus thermodenitrificans T2. Recombinant hexahistidine-tagged EstL5 was overexpressed, purified, and its biochemical properties were partially characterized. EstL5 was observed to be active within the temperature range of 0–80°C, having maximal activity at 60°C. Unlike most other thermostable enzymes, EstL5 displayed 24% of its highest activity at 0°C. EstL5 exhibited a high level of activity within a pH range of 6.0–11.0, showing the highest activity at pH 8.0. EstL5 also retained 100% of its activity after a 12-h incubation at 55°C. Furthermore, this enzyme was observed to be strongly inhibited by 10% (w/v) SDS and 0.1 mM PMSF.