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

234 Part 2: Biotechnology and Enzymology

O

OH

OH

HO

HO

HO

O O

O

O

HO HO C

OH

OH OH OH

OH

OH

OH O C OH

OH OH

O O C

O

O

OH

OH

O C

O

O

O

O

O

O

HOO C O C

OH

OH

OH

OH

OH

O O

O

C

O HO

HO

HO

H 3 CO

H 3 CO

OCH 3

HO HO HO HO

HO

OH

Ca2+ Ca2+ Ca2+ Ca2+

OH

C

C C

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O O O O O O O

C

C

C

O

O O

HO HO

HO

HO HO

HO HO O

C OH

O

O

O O C OH

O

O

O

O O

O

O

CO

O

CO

O

CO

O

CO

O

O

O

O

C OCH 3

C

O

O

C OCH 3

O

H 3 CO

O

O

(A)

Figure 12.2.(A) Structure of homogalacturonan showing calcium ion associations. (Continued)

constituent of the cell wall (about 10% of pectin) consisting of

an HGA backbone that exhibits a complex and highly conserved

substitution pattern with many diverse sugars, linked together

with more than 20 different linkages. RGII often is found in

the wall as dimers cross-linked via borate-diol esters at the

apiose residues (Ishii et al. 1999). Another, less-abundant

pectic side chain is XGA, which is composed of a galacturonan

backbone substituted atO-3 with xylosyl side units (Willats

et al. 2004). Pectins are covalently interlinked, in part via

diferulic acid, and further cross-linked to cellulose, matrix

glycans, and other cell wall polymers (Thompson and Fry

2000, Zykwinska et al. 2005, Coenen et al. 2007). The relative

abundance and distribution of these polymers in the primary

cell wall is not constant. Variation in structure occurs within

cell types and plant species, even within the cell wall of a

single cell (Knox 2008, Jarvis 2011).

Fractionation of Pectic Substances

Methods for analyzing cell wall polymeric substances are based

on deconstruction of the cell wall by sequentially extracting

its components based on solubility. Tomato pericarp is boiled

in ethanol or extracted with tris-buffered phenol to inactivate

enzymes (Huber 1992), and cell wall polysaccharides are precip-

itated with ethanol. Alcohol-insoluble solids are sequentially ex-

tracted with 50 mMtrans-1,2-diaminocyclohexane-N,N,N′,N′-

tetraacetic acid (CDTA) and 50 mM sodium acetate buffer

(pH=6.5) to obtain the chelator-soluble (ionically bound)

pectin. Next, the pellet is extracted with 50 mM sodium carbon-

ate and 20 mM sodium borohydride, which results in breaking of

ester bonds, to obtain the carbonate-soluble (covalently bound

pectin via ester bonds) pectin. The remaining residue 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

(extractable polyuronide) is determined using the method of

Blumenkrantz and Asboe-Hansen (1973), with GalA as a

standard.

BIOCHEMISTRY OF PECTIC ENZYMES

IN RIPENING TOMATO FRUIT

Ripening is a genetically coordinated process during which sev-

eral biochemical and physiological modifications of the fruit