3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P77 OPTIMALIZATION OF CuLTIVATION MEDIA
AND hyDROxyLASE ENZyME PRODuCTION
by AureObAsiDiuM PullulANs
STAnISLAVA MATALOVá, JIřInA OMELKOVá and
IVAn ŠIMKOVIČ
Mendel University of Agriculture and Forestry, Zemědělská
1, 602 00 Brno, Czech Republic,
matalovas@volny.cz
Introduction
In many countries, wheat straw is an abundant lignocellulosic
by-product from farming, consisting of cellulose
(35–40 % wt.) and hemicellulose (25–30 % wt.) in close association
with lignin (10–15 % wt.) 1 .
Lignin is a natural, complex, heterogenous, phenylpropanoid
polymer comprising 25–30 % of plant biomass.
Primarily three enzymes, i.e., lignin peroxidase (LiP), manganese
peroxidase (MnP) and laccase 2,3,4 have been held
responsible for lignin degradation.
Laccase (benzenediol: oxygen oxidoreductase, EC
1.10.3.2) belongs to a group of polyphenol oxidases containing
copper atoms in the catalytic centre and usually called
multicopper oxidases.
Laccases catalyze the reduction of oxygen to water
accompanied by the oxidation of a substrate, typically a pdihydroxy
phenol or another phenolic compound. It is difficult
to define laccase by its reducing substrate due to its very
broad substrate range.
Although laccase was also called diphenol oxidase,
monophenols like 2,6-dimethoxyphenol or guaiacol are often
better substrates than diphenols, e.g. catechol or hydroquinone.Syringaldazine[n,n0-bis(3,5-dimethoxy-4-hydroxybenzylidene
hydrazine)] and ABTS are often considered to
be a unique laccase substrates 5 .
Laccase activity has been demonstrated in many fungal
species, but laccase production has never been demonstrated
in lower fungi. There are many records of laccase production
by ascomycetes. Yeasts are a physiologically specific group
of both ascomycetes and basidiomycetes. This basidiomycete
yeast produces a true laccase capable of oxidation of phenols
and aminophenols and unable to oxidize tyrosine 6 . The
production of laccase was not demonstrated in ascomycetous
yeasts.
In this study, the method suitable for production laccase
as lignin-degrading enzymes by Aureobasidium pullulans
was optimalized. Wheat straw was the only carbon source
and only the most essential nutrients were added.
Experimental Microorganism
The fungal strain Aureobasidium pullulans F 8189 used
in the present study was obtained from the culture collection
of the Culture Collection of Yeasts (CCY), SAV, Institute of
Chemistry. The tested culture was maintained at 26 °C on
malt slant agar for 10 days.
s745
C e l l u l o l y t i c S u b s t r a t e
Wheat straw from local sources (0.3–0.5 mm particle
size) was used as natural substrate. This substrates was sterilized
in autoclave at 120 °C for 20 minutes.
O p t i m a l i z a t i o n o f C u l t u r e
C o n d i t i o n s
Growth of A. pullulans was studied in three cultivation
media – glucose medium (GM), glucose medium with wheat
straw (GSM) and basal medium with wheat straw (BSM)
during solid-state (SSF) and submerged (SF) fermentation.
The glucose medium contained in grams per 1,000 g:
KnO 3 , 2; K 2 HPO 4 , 1; MgSO 4 , 0.5; glucose, 50. The basal
medium has the same composition as glucose medium but
without glucose. The pH of the both media was adjusted to 5.6.
These media were then autoclaved for 30 min at 121 °C and
poured into Petri dishes with diameter of 9 cm and in 100 ml
Erlenmayer flasks. Cultivation media with wheat straw also
contained sterilized wheat straw (1 g wheat straw 100 ml –1 of
medium).
Petri dishes and Erlenmayer flasks were inoculated
by three ways – with fungal spores obtained from 4 days
grown cultures on malt agar, with three mycelial discs (each
1 × 1 cm) obtained from 4 days fungal culture grown on glucose
agar and inoculation by loops. Growth of A.pullulans
was observed at 26 and 32 °C.
E n z y m e A s s a y
Laccase (EC 1.10.3.2) activity was determined by the
oxidation of 2,2‘-azino-bis(3-ethylthiazoline-6-sulfonate)
(ABTS) according to Buswell et.al. 7 The reaction mixture for
the standard assays contained 100 µl ABTS solution (1mM),
300 µl sodium acetate buffer pH 5.0 (0.1M) and 600 µl
enzyme extract. Oxidation was followed via the increase in
absorbance at 420 nm (ε 420 = 36,000 M –1 cm –1 ). All enzyme
assays were performed in triplicate.
Protein concentration was determined by Lowry et al.
method 8 with bovine serum albumin as standard.
Results
A comparison of growth pattern of A.pullulans in various
cultivation conditions as medium composition, temperature
or method of inoculation was made and the results are
presented in Table I. As Table I demonstrates, A. pullulans
were grown in different type of solid media. In the course
of liquid cultivations less growth was observed regardless of
temperature. The best growth rate was obtained in GSM or
BSM medium in comparison to GM during solid-state fermentation.
The effect of method of inoculation varied from inoculation
with fungal spores (maximum growth) to inoculation by
loops (minimum growth).
For testing of production of hydroxylase were chosen
microorganism Aureobasidium pullulans by methods
of Azure-B agar and ABTS agar 9 . In an attemp to compare
the production laccase during solid-state and submerged