3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P27 COMPARISON OF PECTATE hyDROLASES
FROM PARSLEy ROOT CELLS
DAnA FLODROVá a,b , EVA STRATILOVá b , SOňA
GARAJOVá b and JIřInA OMELKOVá a
a Brno University of Technology, Faculty of Chemistry, Purkyňova
118, 612 00 Brno,
b Institute of Chemistry, Slovak Academy of Sciences, Dúbravská
cesta 9, 845 38 Bratislava, Slovakia,
flodrova@fch.vutbr.cz
Introduction
Plant pectate hydrolases are in general supposed to be
bound on primary cell wall where they cause the homogalacturonan
degradation. Polygalacturonases can be classified
into two groups depending on action pattern; enzymes randomly
cleaving substrate (polygalacturonases, EC 3.2.1.15)
and enzymes terminally cleaving substrate (exopolygalacturonases,
EC 3.2.1.67) 1 . The biological function, structure as
well as gene-expression of polygalacturonases have been studied
in detail, while the research of exopolygalacturonases
(exoPGs) is still on its beginning. ExoPGs have not yet been
fully characterized in terms of developmental roles but could
clearly have significant involvement in cell expansion processes.
These enzymes are supposed to play a key role in the
turnover of biologically active oligogalacturonates as signalling
molecules affecting plant growth and development.
Plant exoPGs were supposed to prefer only polymeric
substrate and the ability for cleaving substrate with lower
degree of polymerization (DP) was strictly attributed to
enzymes produced by microorganisms. First description of
plant enzyme preferring oligogalacturonates (oligogalactu-
Scheme 1
s630
ronate hydrolase, OGH) appeared in 2005 when an enzyme
from carrot roots was described 2 .
Experimental
P u r i f i c a t i o n o f E x o p e c t a t e
H y d r o l a s e s
In this work, exopectate hydrolases were isolated from
parsley root juice and pulp extracts. The pulp protein mixture
presents a very heterogeneous material what requires
more complicated purification process. Accordingly, two different
purification pathways were developed using different
separation methods involving gel-permeation, affinity chromatography,
chromatofocusing as well as preparative IEF
(Scheme 1.).
C h a r a c t e r i z a t i o n o f E x o p e c t a t e
H y d r o l a s e s
Activities of pectate hydrolases, utilizing substrates with
various DP and different pHs, were determined by Somogyi
assay 3 . The zymogram technique after IEF 4 with a colourless
d-galacturonan DP 10 followed by staining of non cleaved
substrate with ruthenim red was used for determination of
pI. Molecular mass analysis of native and deglycosylated
enzymes was done using SDS-PAGE with silver or Commasie
Blue staining method for band visualization. Presence of
Fig. 1. The ph optima of pectate hydrolases in parsley roots;
enzyme activity on: • – 0,5% sodium pectate, ○ – 1 µmol ml –1
pentagalacturonate, A – enzymes from parsley juice, b – enzymes
from parsley pulp
A
B