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Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica

Hashim, Noor Haza Fazlin and Mahadi, Nor Muhammad and Md. Illias, Rosli and Feroz, Shevin Rizal and Abu Bakar, Farah Diba and Abdul Murad, Abdul Munir (2018) Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica. Extremophiles, 22 (4). pp. 607-616. ISSN 1431-0651

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Official URL: http://dx.doi.org/10.1007/s00792-018-1021-z

Abstract

Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s−1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.

Item Type:Article
Uncontrolled Keywords:Dienelactone hydrolase, Esterase
Subjects:T Technology > TP Chemical technology
Divisions:Chemical and Energy Engineering
ID Code:84361
Deposited By: Widya Wahid
Deposited On:28 Dec 2019 09:48
Last Modified:28 Dec 2019 09:48

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