Food Biochemistry and Food Processing

12 Pectic Enzymes in Tomatoes 275

consists primarily of cellulose and matrix glycans
with only about 5% remaining pectin. Extracts are
dialyzed exhaustively against water at 4°C, and
uronic acid (UA) content of dialyzed extracts (ex-
tractable polyuronide) is determined using the
method of Blumenkrantz and Asboe-Hansen (1973),
with galacturonic acid as a standard.

BIOCHEMISTRY OF PECTIC
ENZYMES IN TOMATO

Tomato possesses an elaborate ensemble of polysac-
charide-degrading enzymes that is involved in cell-
wall disassembly during ripening (Giovannoni 2001,
Brummell and Harpster 2001). The enzymes that act
on the pectin network are polygalacturonase (PG),
pectin methylesterase (PME),
-galactosidase (
-
GALase), and putatively, pectate lyase (PL) and exo-
polygalacturonate lyase (PGL).

POLYGALACTURONASE

Polygalacturonases are enzymes that act on the
pectin fraction of cell walls and can be divided into
endo-PGs and exo-PGs. Exo-PG (EC3.2.1.67) re-
moves a single galacturonic acid residue from the
nonreducing end of HGA. Endo-PG [poly(1,4--D-
galacturonide)glycanohydrolase, EC 3.2.1.15] is
responsible for pectin depolymerization by hydro-
lyzing glycosidic bonds in demethylesterified reg-
ions of homogalacturonan (Fig. 12.3).

Endo-PGs are encoded by large multigene fami-

lies with distinct temporal and spatial expression

profiles (Hadfield and Bennett 1998). In ripening

tomato fruit, the mRNA of one gene is accumulated

at high levels, constituting 2% of the total poly-

adenylated RNA (DellaPenna et al. 1986), in a fash-

ion paralleling PG protein accumulation. PG gene

expression during ripening is ethylene responsive,

with very low levels of ethylene (0.15 l/L) being

sufficient to induce PG mRNA accumulation and

PG activity (Sitrit and Bennett 1998). PG mRNA

levels continue to increase as ripening progresses,

and persist at the overripe stage. Immunodetectable

PG protein accumulation follows the same pattern

as mRNA accumulation during ripening and fruit

senescence. This mRNA encodes a predicted poly-

peptide 457 amino acids long that contains a signal

sequence of 24 amino acids targeting it to the cell’s

endomembrane system for further processing and

secretion. The mature protein is produced by cleav-

ing a 47 amino acid amino-terminal prosequence

and a sequence of 13 amino acids from the carboxyl

terminus followed by glycosylation. Two isoforms,

differing only with respect to the degree of glycosy-

lation, are identified: PG2A, with a molecular mass

of 43 kDa, and PG2B, with a molecular mass of 45

kDa (Sheehy et al. 1987, DellaPenna and Bennett

1988, Pogson et al. 1991). Another isoform (PG1)

accumulates during the early stages of tomato fruit

ripening, when PG2 levels are low. PG1 has a mo-

lecular mass of 100 kDa and is composed of one or

Figure 12.3.Action pattern of PG and PME on pectin backbone. Endo-PG hydrolyzes glycosidic bonds in
demethylesterified regions of homogalacturonan. PME catalyzes the removal of methyl ester groups (OMe) from
galacturonic acid (GalUA) residues of pectin.