Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan

• Published in: Applied microbiology and biotechnology Vol. 94; no. 6; pp. 1543 - 1552
• Main Authors: Normand, J., Bonnin, E., Delavault, P.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.06.2012; Springer; Springer Nature B.V; Springer Verlag
• Subjects: Acetylation; Amino Acid Sequence; Amino acids; Ascomycetes; Base Sequence; Basidiomycetes; Basidiomycota - chemistry; Basidiomycota - enzymology; Basidiomycota - genetics; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Chemical and Process Engineering; Cloning; Cloning, Molecular; Engineering Sciences; Enzyme Stability; Enzymes; Food engineering; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Expression; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Hydrolases; Irpex lacteus; Life Sciences; Microbial Genetics and Genomics; Microbiology; Molecular Sequence Data; Pectin; Pectins - metabolism; Peptides; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Plasmids; Proteins; Recombinant proteins; Sequence Alignment; Studies; Substrate Specificity; Yeast; Rhamnogalacturonase; Acetylated rhamnogalacturonans; Pichia pastoris; Irpex lacteus
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3705-5

In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5′ and 3′ rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus . The recombinant protein was successfully expressed in active form in Pichia pastoris . SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus . The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme.