Abstract
A novel esterase gene (designated RmEstB) from the thermophilic fungus Rhizomucor miehei was cloned and functionally expressed in Escherichia coli. Sequence analysis revealed a 960-bp open reading frame encoding a protein of 319 amino acids. The deduced protein sequence contained an HGGG motif, suggesting that the enzyme is a hormone-sensitive lipase (HSL) family esterase. It showed highest identity of 52% with the esterase from Pseudomonas mandelii. The recombinant esterase was purified to homogeneity at 5.1-fold purification with a recovery yield of 85%. The molecular mass of RmEstB was estimated to be 37kDa by SDS-PAGE. RmEstB was most active at pH 7.5 and 50°C. The enzyme was highly stable in the presence of 30% ethanol, methanol, acetone, isopropanol, dimethyl sulfoxide and acetonitrile. RmEstB showed a broad range of substrate specificities toward various p-nitrophenol (pNP) esters (C2–C10) and triglycerides (C2–C6), with the highest specific activities obtained for pNP acetate (255U/mg) and triacetin (1330U/mg), respectively. In addition, RmEstB efficiently catalyzed the hydrolysis of sterically hindered esters of tertiary alcohols. This study presents a novel fungal HSL family esterase with potential for some industrial applications.
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