A new alpha/beta hydrolase gene from Pelosinus fermentans (Pfe_Lip) was optimized against Escherichia coli codon usage and cloned into the pET26b(+) vector, containing a 6xHis-Tag downstream the polylinker, and transformed into the bacterial cells. The Pfe_Lip is a member of the serine hydrolases containing a modification in the common –GxSxG– motif in the first glycine to have the –AxSxG– motif. This enzyme can be grouped in the I.5 subfamily (Nardini M, 1999). To improve the expression of this enzyme in the soluble fraction, a lactose-containing medium was used for the expression of the enzyme. Western blotting was carried out for revelation of the enzyme with an antibody against the His-Tag and further purification was carried out by means of Immobilized Ion Metal Affinity Chromatography (IMAC) with Nickel-coated columns for the binding of the His-Tagged enzyme. The Pfe_Lip purified was tested in hydrolase activity assay with p-nitrophenylbutyrate (p-NPB) (Herrero Acero E, 2013). Pfe_Lip had the pH optimal at 8 (0.1 M sodium phosphate buffer). Analysis of homology shows high identity with enzymes containing a zinc ion in the structure and a lid covering the active site, containing serine, aspartic acid and histidine. The enzyme was able to hydrolyse polyesters like Ecoflex based on HPLC quantification of the solubilized hydrolysis products.
Characterization of a new alpha/beta hydrolase from Pelosinus fermentans for polyesters hydrolysis
Antonino Biundo;
2014-01-01
Abstract
A new alpha/beta hydrolase gene from Pelosinus fermentans (Pfe_Lip) was optimized against Escherichia coli codon usage and cloned into the pET26b(+) vector, containing a 6xHis-Tag downstream the polylinker, and transformed into the bacterial cells. The Pfe_Lip is a member of the serine hydrolases containing a modification in the common –GxSxG– motif in the first glycine to have the –AxSxG– motif. This enzyme can be grouped in the I.5 subfamily (Nardini M, 1999). To improve the expression of this enzyme in the soluble fraction, a lactose-containing medium was used for the expression of the enzyme. Western blotting was carried out for revelation of the enzyme with an antibody against the His-Tag and further purification was carried out by means of Immobilized Ion Metal Affinity Chromatography (IMAC) with Nickel-coated columns for the binding of the His-Tagged enzyme. The Pfe_Lip purified was tested in hydrolase activity assay with p-nitrophenylbutyrate (p-NPB) (Herrero Acero E, 2013). Pfe_Lip had the pH optimal at 8 (0.1 M sodium phosphate buffer). Analysis of homology shows high identity with enzymes containing a zinc ion in the structure and a lid covering the active site, containing serine, aspartic acid and histidine. The enzyme was able to hydrolyse polyesters like Ecoflex based on HPLC quantification of the solubilized hydrolysis products.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
