Food Biochemistry and Food Processing (2 edition)

BLBS102-c12 BLBS102-Simpson March 21, 2012 13:11 Trim: 276mm X 219mm Printer Name: Yet to Come

236 Part 2: Biotechnology and Enzymology

occur. These include the conversion of starch to sugars, accu-

mulation of pigments, organic acids, and aroma volatiles, as well

as fruit softening. Pectin often consists more than 50% of the

fruit cell wall. During ripening, pectin is extensively modified

by de novo synthesized enzymes that bring about fruit softening

(Brummell 2006, Cantu et al. 2008). The pectin backbone is

de-esterified, allowing calcium associations and gelling to oc-

cur (Blumer et al. 2000). Pectin is solubilized from the wall

complex, followed by a decrease in the average polymer size

(Brummell and Labavitch 1997, Chun and Huber 1997). These

changes are accompanied by loss of galactosyl and arabinosyl

moieties from pectin side chains (Gross 1984). The dissolu-

tion of the pectin-rich middle lamella increases cell separation

and contributes to softening. Cell wall disassembly in ripen-

ing tomato fruit has been extensively studied and a battery of

polysaccharide-degrading enzymes involved in this process has

been biochemically and genetically characterized (for review,

see Brummell and Harpster 2001, Giovannoni 2001, Brummell

2006, Goulao and Oliveira 2008). The most thoroughly stud-

ied enzymes that act on the pectic polysaccharides of fruit

are polygalacturonase (PG), pectin methylesterase (PME),

β-galactosidase (β-GALase), and pectate lyase (PL).

Polygalacturonase

PGs are enzymes that act on the pectin fraction of cell walls

and can be divided into endo-PGs and exo-PGs. Exo-PG

(EC 3.2.1.67) removes a single GalA residue from the nonre-

ducing end of HGA. Endo-PG (poly(1,4-α-d-galacturonide)

glycanohydrolase, EC 3.2.1.15) is responsible for pectin depoly-

merization by hydrolyzing glycosidic bonds in demethylesteri-

fied regions of HGA (Fig. 12.3).

Endo-PGs are encoded by large multigene families 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

polyadenylated RNA, in a fashion paralleling PG protein accu-

mulation (DellaPenna et al. 1986). PG gene expression during

ripening is ethylene responsive, with very low levels of ethylene

(0.15μL/L) being sufficient to induce PG mRNA accumulation

and PG activity (Sitrit and Bennett 1998). PG mRNA levels con-

tinue to increase as ripening progresses and persist at the over-

ripe stage. Immunodetectable PG protein accumulation follows

the same pattern as mRNA accumulation during ripening and

fruit senescence. This mRNA encodes a predicted polypeptide

457 amino acids long containing a signal sequence of 24 amino

acids targeting it to the cell’s endomembrane system for fur-

ther processing and secretion. The mature protein is produced

by cleaving a 47 amino acids 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 glycosylation, are identified: PG2A, with a molec-

ular mass of 43 kDa, and PG2B with a molecular mass of 46 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 molecular mass of 100 kDa and is composed of one or pos-

sibly two PG2A or PG2B subunits and the PGβ-subunit. Recent

work has identified yet another member of the PG1 multipro-

tein complex; a nonspecific lipid transfer protein of a molecular

mass of 8 kDa and no detectable PG activity (Tomassen et al.

2007). When this 8 kDa protein was co-incubated with PG2,

an increase in PG2 activity was observed. Moreover, heating of

the 8 kDa–PG2 mixture resulted in increased heat stability of

the PG2 protein. This report is the first to implicate non-specific

lipid transfer proteins in PG-mediated HGA depolymerization

in tomato.

The PGβ-subunit is an acidic, heavily glycosylated protein.

The precursor protein contains a 30 amino acid signal peptide,

an N-terminus propeptide of 78 amino acids, the mature pro-

tein domain, and a large C-terminus propeptide. The mature

protein presents a repeating motif of 14 amino acids and has a

size of approximately 37–39 kDa. This difference in size results

from different glycosylation patterns or different posttransla-

tional processing at the carboxyl terminus of the protein (Zheng

et al. 1992). The PGβ-subunit protein is encoded by a single

Me

O

Me

O

Me

O

Me

O

Me

O

Me

O

Me

O

Me

O

Me

O

Me

O

-GalUA-GalUA- GalUA-GalUA-GalUA-GalUA- GalUA-GalUA-GalUA-GalUA- GalUA-GalUA-GalUA-

Me

O

Me

O

Me

O

Me

O

-GalUA-GalUA- GalUA-GalUA-

Me

O

Me

O

Me

O

Me

O

Me

O

Me

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GalUA-GalUA-

MeOH

GalUA-GalUA-GalUA-

Me

O

Me

O

Me

O

Me

O

GalUA-GalUA-GalUA-GalUA-

MeOH

PG PG

PME PME

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.