Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine
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A glucuronoyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55°C. The esterase displayed broad pH range stability between 4–10 and temperature stability up to 50°C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.
KeywordsGlucuronoyl esterase Nucleophilic serine Active site Lignin–carbohydrate complex Sporotrichum thermophile Pichia pastoris
The authors thank Dr. Peter Biely from Institute of Chemistry, Slovak Academy of Sciences (Bratislava, Slovakia) who supplied the synthetic substrates for assaying GE activity.
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