IDENTIFICATION OF SOME ENZYMES OF THE PECTINOLYTIC SYSTEM OF Aspergillus flavus C' E' GmezSnchez, M'

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IDENTIFICATION OF SOME ENZYMES OF THE
PECTINOLYTIC SYSTEM OF Aspergillus flavusC. E.
Gómez-Sánchez, M. E. Alvarez-Arriaga, A.
Martinez-Trujillo, B. Trejo-Aguilar and G.
Aguilar-OsorioFungal Physiology Group,
Department of Food Science and Biotechnology,
Conjunto E, Faculty of Chemistry, National
University of Mexico, UNAM, C. P. 04510, Mexico
D. F., Mexico. e-mail gao_at_servidor.unam.mx
INTRODUCTION Aspergillus flavus is a
phytopathogenic fungus able to grow in diferents
crops such as corn, cotton and peanuts
contaminating them with aflatoxins. Virulence of
A. flavus strains is related whit its ability to
produce pectinases and is known that some
toxigenic strains produce three
polygalacturonases and a pectinesterase. One of
this polygalacturonase, designed P2c, has shown
to be very important as a virulence factor (1).
Because of pectin is a complex polysaccharide it
is hard to think that A. flavus does not produce
a complex enzymatic system for degrading pectin
that is a key substrate for invasion of plant
tissue. It seems a paradox that A. niger, being a
saprophytic fungus, produce as much as twenty
seven diferents enzymes releated to pectin
degradation in comparation to a phytopathogenic
organism like A. flavus.The aim of this work is
to characterize tha pectinolytic system of A.
flavus growing on pectin at diferents conditions
of pH and temperature. The effect of the carbon
source was also evaluated.
Exo-PG Exo-pectinolytic activity and PL Pectin
lyase activity a) These values were taken at 72 h
of cultivation time
METHODS For this work we use two strains of A.
flavus, toxigenic (CECT 2687) and non-toxigenic
(NRRL 6541) strain. A. flavus was grown on pectin
at 28C and 37C and at initial pH (pHi) of 3.5,
4.5 and 6. Also A. flavus was grown on glucose,
galacturonic acid, polygalacturonic acid, xilose,
galactose, rhamnose and arabinose at 37 C and
pHi 6. Cellular growth was determined as dry
wheight. Pectinolytic activities was determined
as exo-pectinolítyc activity by measuring
reducing groups, endo-pectinolytic activity by
measuring the relative change in viscosity of 1
pectin solution and pectin lyase activity by
messuring the increase in absorbance at 325 nm
using the molar coefficient 5500 M-1cm-1 for the
unsaturated product.
A. flavus CECT 2687 pHi 6.0
RESULTS AND DISCUSSION Aspergillus flavus
NRRL-6541(non-toxigenic strain) and CECT 2687
(toxigenic strain) were grown at different
temperatures and initial pH (pHi) values. Growth
at 28 and 37C was quite similar at pHi of 3.5
and 4.5, only at pHi of 6.0. Cell growth was
better at 37C for both strains. The growth of
toxigenic strain, in any combination of pHi and
temperature, were higher than that of the
non-toxigenic strain. We evaluated exo and
endo-pectinase activities as well as pectin lyase
activitie. For both strains exo-pectinolytic
(Exo-PG) activity was favored at 28C and pHi of
6.0. However, at 37C exo-PG was slightly higher
at pHi 4.5 instead of 6.0. On the other hand,
we found pectin lyase activity, which is best
produced at 28C and pHi value of 6.0 for the
non-toxigenic strain while the toxigenic was
unable to produced significant amounts of this
kind of activity (Figure 1). Very low pectin
lyase activity (0.07 mU/ mg D.W.) was found only
at pHi 6.0 when the strain was grown on pectin.
At 37C both strain produced very limited amount
of the enzyme. The growth on glucose and xylose
was lower than that attained on pectin. On
galacturonic acid, cell growth was very poor,
mainly due to a serious limitation on conidia
germination. Cell growth was improved when
pre-grown mycelia were used. In transfer
experiment using non-induced pre-grown mycelia,
pectin lyase was produced only on galacturonic
acid, polygalacturonic acid and pectin.
Figure1. Efect of the pHi on the pectin lyase
activity produced at 28C by NRRL 6541 and CECT
2687 strains.
Surprisingly no endo-pectinolityc activity was
detected in any of the tested conditions or
strain. This activity has been reported by other
authors for toxigenic A. flavus strains (1 and
2). However, in our conditions this activity was
not found. It is possible that the strain
variability could be very large and that the
virulence given by the presence of one or other
enzyme could not be directly related with the
ability of a specific strain to produce
aflatoxins
REFERENCE 1. Shieh, M., Brown, L., et al. (1997).
Molecular genetic evidence for the involment of a
specific polygalacturonase, P2C, in the invasion
and spread of Aspergillus flavus in cotton bolls.
Applied and Environmental Microbiology. 63
3548-3552. 2. Whitehead, M., Shieh, M., et al.
(1995). Isolation and charaterization of
polygalacturonase genes (pecA and pecB) from
Aspergillus flavus. Applied and Environmental
Microbiology. 61 3316-3322.