Purification and chemical characterisation of a cell wall-associated b-galactosidase from mature sweet cherry (Prunus avium L.) fruit

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Abstract

Using four different chromatographic steps, b-galactosidase was purified from the ripe fruit of sweetcherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunusavium b-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of fourdifferent active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and bgalactosidase-activity staining. The active polypeptides were individually excised from the gel andsubjected to SDS-PAGE. Each of the four native enzymes showing b-galactosidase activity was composedof two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjectedto N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry b-galactosidaseshowed a 43% identity with the 44 kDa subunit of persimmon and apple b-galactosidases and the 48 kDasubunit of carambola galactosidase I. The sweet cherry b-galactosidase exhibited a strict specificitytowards p-nitrophenyl b-D-galactopyranoside, a pH optimum of 4.0 and Km and Vmax values of 0.42 mMand 4.12 mmol min1 mg1 of protein respectively with this substrate. The enzyme was also activetowards complex glycans. Taken together the results of this study prompted a role for this class ofenzymes on sweet cherry fruit ripening and softening.

Autore Pugliese

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  • Gerardi C.; Blando F.; Santino A.

Titolo volume/Rivista

Plant physiology & biochemistry

Anno di pubblicazione

2012

ISSN

0254-3591

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