Characterisation of an α-galactosidase with potential relevance to ripening related texture changes

• Published in: Phytochemistry (Oxford) 2006-02, Vol.67 (3), p.242-254
• Main Authors: Soh, Chin-Pin, Ali, Zainon Mohd, Lazan, Hamid
• Format: Article
• Language: English
• Subjects: Ajuga reptans; alpha-galactosidase; alpha-Galactosidase - chemistry; alpha-Galactosidase - isolation & purification; alpha-Galactosidase - physiology; Amino Acid Sequence; amino acid sequences; Biological and medical sciences; Carica - chemistry; Carica - enzymology; Carica - growth & development; Carica papaya; Carica papaya L; Caricaceae; Cations - metabolism; Cell Wall - enzymology; Cell wall modification; cell walls; Characterisation; enzyme activity; firmness; Fructification and ripening; Fruit; Fruit - enzymology; fruit crops; Fundamental and applied biological sciences. Psychology; galactosyltransferases; glycoproteins; Isoenzymes - chemistry; Isoenzymes - isolation & purification; isozymes; Kinetics; Molecular Sequence Data; Monosaccharides - metabolism; Nitrophenylgalactosides - metabolism; papayas; Pectin modification; pectins; Pectins - chemistry; plant biochemistry; Plant physiology and development; plant proteins; ripening; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Texture; Vegetative and sexual reproduction, floral biology, fructification; α-Galactosidase
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2005.09.032

α-Gal 2 is the dominant isoform and its activity and protein level increase during ripening of papaya. Purified α-gal 2 from ripe fruit was isolated and characterised. This isoform was capable to markedly catalyse increased pectin solubility and depolymerisation. α-Galactosidase (EC 3.2.1.22) from ripe papaya ( Carica papaya L.) fruit was fractionated by a combination of ion exchange and gel filtration chromatography into three forms, viz., α-galactosidase 1, 2 and 3. The predominant isoform, α-gal 2, was probably a tetramer with a native molecular mass of about 170 kDa and 52 kDa-sized subunits and an estimated p I of 7.3. The subunit’s N-terminal amino acid sequence shared high identity (97%) with the deduced sequence of a papaya cDNA clone (accession no. AY25329) encoding a putative α-galactosidase PAG2 as well as with an Ajuga reptans L. GGT1 clone (accession no. AY386246) encoding a galactan: galactan galactosyltransferase (66%). During ripening, α-galactosidase activity increased concomitantly with firmness loss and this increase was largely ascribed to α-gal 2. The protein level of α-gal 2 as estimated by immunoblot was low in developing fruits and generally increased with ripening. α-Galactosidase 2 also had the ability to markedly catalyse increased pectin solubility and depolymerisation while the polymers were still structurally attached to the cell walls mimicking, in part, the changes that occur during ripening. The close correlation between texture changes, α-gal 2 activity and protein levels as well as capability to modify intact cell walls suggest that the enzyme might contribute to papaya fruit softening during ripening. The purported mechanism of α-gal 2 action as a softening enzyme was discussed in terms of its functional capacity as a glycanase or perhaps, as a transglycosylase.