3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
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Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology<br />
P77 OPTIMALIZATION OF CuLTIVATION MEDIA<br />
AND hyDROxyLASE ENZyME PRODuCTION<br />
by AureObAsiDiuM PullulANs<br />
STAnISLAVA MATALOVá, JIřInA OMELKOVá and<br />
IVAn ŠIMKOVIČ<br />
Mendel University of Agriculture and Forestry, Zemědělská<br />
1, 602 00 Brno, Czech Republic,<br />
matalovas@volny.cz<br />
Introduction<br />
In many countries, wheat straw is an abundant lignocellulosic<br />
by-product from farming, consisting of cellulose<br />
(35–40 % wt.) and hemicellulose (25–30 % wt.) in close association<br />
with lignin (10–15 % wt.) 1 .<br />
Lignin is a natural, complex, heterogenous, phenylpropanoid<br />
polymer comprising 25–30 % of plant biomass.<br />
Primarily three enzymes, i.e., lignin peroxidase (LiP), manganese<br />
peroxidase (MnP) and laccase 2,3,4 have been held<br />
responsible for lignin degradation.<br />
Laccase (benzenediol: oxygen oxidoreductase, EC<br />
1.10.<strong>3.</strong>2) belongs to a group of polyphenol oxidases containing<br />
copper atoms in the catalytic centre and usually called<br />
multicopper oxidases.<br />
Laccases catalyze the reduction of oxygen to water<br />
accompanied by the oxidation of a substrate, typically a pdihydroxy<br />
phenol or another phenolic compound. It is difficult<br />
to define laccase by its reducing substrate due to its very<br />
broad substrate range.<br />
Although laccase was also called diphenol oxidase,<br />
monophenols like 2,6-dimethoxyphenol or guaiacol are often<br />
better substrates than diphenols, e.g. catechol or hydroquinone.Syringaldazine[n,n0-bis(3,5-dimethoxy-4-hydroxybenzylidene<br />
hydrazine)] and ABTS are often considered to<br />
be a unique laccase substrates 5 .<br />
Laccase activity has been demonstrated in many fungal<br />
species, but laccase production has never been demonstrated<br />
in lower fungi. There are many records of laccase production<br />
by ascomycetes. Yeasts are a physiologically specific group<br />
of both ascomycetes and basidiomycetes. This basidiomycete<br />
yeast produces a true laccase capable of oxidation of phenols<br />
and aminophenols and unable to oxidize tyrosine 6 . The<br />
production of laccase was not demonstrated in ascomycetous<br />
yeasts.<br />
In this study, the method suitable for production laccase<br />
as lignin-degrading enzymes by Aureobasidium pullulans<br />
was optimalized. Wheat straw was the only carbon source<br />
and only the most essential nutrients were added.<br />
Experimental Microorganism<br />
The fungal strain Aureobasidium pullulans F 8189 used<br />
in the present study was obtained from the culture collection<br />
of the Culture Collection of Yeasts (CCY), SAV, Institute of<br />
Chemistry. The tested culture was maintained at 26 °C on<br />
malt slant agar for 10 days.<br />
s745<br />
C e l l u l o l y t i c S u b s t r a t e<br />
Wheat straw from local sources (0.3–0.5 mm particle<br />
size) was used as natural substrate. This substrates was sterilized<br />
in autoclave at 120 °C for 20 minutes.<br />
O p t i m a l i z a t i o n o f C u l t u r e<br />
C o n d i t i o n s<br />
Growth of A. pullulans was studied in three cultivation<br />
media – glucose medium (GM), glucose medium with wheat<br />
straw (GSM) and basal medium with wheat straw (BSM)<br />
during solid-state (SSF) and submerged (SF) fermentation.<br />
The glucose medium contained in grams per 1,000 g:<br />
KnO 3 , 2; K 2 HPO 4 , 1; MgSO 4 , 0.5; glucose, 50. The basal<br />
medium has the same composition as glucose medium but<br />
without glucose. The pH of the both media was adjusted to 5.6.<br />
These media were then autoclaved for 30 min at 121 °C and<br />
poured into Petri dishes with diameter of 9 cm and in 100 ml<br />
Erlenmayer flasks. Cultivation media with wheat straw also<br />
contained sterilized wheat straw (1 g wheat straw 100 ml –1 of<br />
medium).<br />
Petri dishes and Erlenmayer flasks were inoculated<br />
by three ways – with fungal spores obtained from 4 days<br />
grown cultures on malt agar, with three mycelial discs (each<br />
1 × 1 cm) obtained from 4 days fungal culture grown on glucose<br />
agar and inoculation by loops. Growth of A.pullulans<br />
was observed at 26 and 32 °C.<br />
E n z y m e A s s a y<br />
Laccase (EC 1.10.<strong>3.</strong>2) activity was determined by the<br />
oxidation of 2,2‘-azino-bis(3-ethylthiazoline-6-sulfonate)<br />
(ABTS) according to Buswell et.al. 7 The reaction mixture for<br />
the standard assays contained 100 µl ABTS solution (1mM),<br />
300 µl sodium acetate buffer pH 5.0 (0.1M) and 600 µl<br />
enzyme extract. Oxidation was followed via the increase in<br />
absorbance at 420 nm (ε 420 = 36,000 M –1 cm –1 ). All enzyme<br />
assays were performed in triplicate.<br />
Protein concentration was determined by Lowry et al.<br />
method 8 with bovine serum albumin as standard.<br />
Results<br />
A comparison of growth pattern of A.pullulans in various<br />
cultivation conditions as medium composition, temperature<br />
or method of inoculation was made and the results are<br />
presented in Table I. As Table I demonstrates, A. pullulans<br />
were grown in different type of solid media. In the course<br />
of liquid cultivations less growth was observed regardless of<br />
temperature. The best growth rate was obtained in GSM or<br />
BSM medium in comparison to GM during solid-state fermentation.<br />
The effect of method of inoculation varied from inoculation<br />
with fungal spores (maximum growth) to inoculation by<br />
loops (minimum growth).<br />
For testing of production of hydroxylase were chosen<br />
microorganism Aureobasidium pullulans by methods<br />
of Azure-B agar and ABTS agar 9 . In an attemp to compare<br />
the production laccase during solid-state and submerged