Functional expression of a thermophilic glucuronyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine.
A glucuronyl 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 alpha-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 degrees C. The esterase displayed broad pH range stability between 4-10 and temperature stability up to 50 degrees 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.
Topakas
Evangelos
E
BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece.
Moukouli
Maria
M
Dimarogona
Maria
M
Vafiadi
Christina
C
Christakopoulos
Paul
P
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005965:Glucuronates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics* / Q000378:metabolism*; D013175:Sporothrix / Q000201:enzymology*; D013696:Temperature
D005656:Fungal Proteins; D005965:Glucuronates; D050505:Mutant Proteins; D012694:Serine; D004950:Esterases