8/22/2019 Study of hydrolases in lentinus
1/6
Study on Hydrolases Activities Produced byLentinula
edodes During the Degradation of Eucalyptus Residues
EZEQUIEL M. SILVA and ADRIANE M. F. MILAGRES
Faculdade de Engenharia Qumica de Lorena Depto. de
BiotecnologiaCx. Postal 116 - Lorena So Paulo - Brazil
[email protected]
Lentinula edodes (Berkeley) Pegler, known as shiitake, has an
enzymatic complex capable
of decaying lignocellulosic compounds and is currently the
second most cultivated mushroom
in the world. Eucalyptus residues (leaves, branches and barks)
originating from a cellulosic
pulp mill were used in this study for production of spawn. The
eucalyptus residues weresupplemented with rice, wheat and soy brans
at different concentrations (5, 10, 15 and 20%)
in order to evaluate the influence of nutrient supplementation
on mycelium growth and
enzymatic activities. Strains CCB-514, FEB-14 and SJC were
selected for their much greater
capacity for fungal biomass growth and hydrolytic activities.The
three cereal brans testedproduced better results when their
concentration value was 20%. FEB-14 was the strain that
best grew when eucalyptus waste was supplemented with 20% of soy
bran. However, CCB-
514 provided greater production of hydrolytic enzyme with almost
all the cereal brans tested.
INTRODUCTION
The production of shiitake (L. edodes) has to expanding all over
the world in the past fewdecades (ROYSE, SANCHEZ-VAZQUEZ, 2001;
ROYSE et al., 2002; BUSWELL et al.,1995; KES, LIU, 2000; CHIU et
al., 2000). L. edodes produces hydrolytic and oxidativeenzymes
responsible by degradation of the lignocellulosic compounds. The
activity of theseenzymes depends on the substrate composition and
the environmental conditions (MORAISet al., 2000). This
bioconversion process helps to prevent environmental impact caused
byforest and agricultural wastes, reduce pollution and generates
products of commercial andindustrial interest. The aim of the
process may be the production of mushroom or itsmetabolic products,
such as organic acids, flavour and aroma compounds or
enzymes(HATVANI, MCS, 2001; KES, LIU, 2000). The use of sawdust,
usually of hardwood,supplemented with cereal brans has contributed
to expanding production and consumption of
L. edodes in several countries Americans and Europeans (OHGA,
ROYSE, 2001; MORAISet al., 2000; CAMPBELL, RACJAN, 1999). Residues
as wheat straw, malt-containing by-
product of the brewing process and many other agricultural
wastes has been studied to thesepurpose (HATIVANI, MCS, 2001;
ZERVAKIS et al., 2001; MATA, SAVOIE, 1998). Thephase of substrate
colonization with the fungal biomass after inoculation and prior of
theprimordia formation is of direct economic importance, since the
media that are non-thoroughly impregnated with the mycelium are
available to fungal and bacterial infectionsresulting in reduced
yields (ZERVAKIS et al., 2001). Supplements such as wheat and
rice
brans may be added to the lignocellulosic substrates, which
serve as nutrients to provide anoptimum growth medium (ROYSE at
al., 2002; OHGA, ROYSE, 2001). Although studies ofenzymatic
activities generates informations about the development of fungus
inlignocellulosic substrates is necessary to integrate the data of
enzymology with myceliumgrowth in solid substrates. In this study,
three strains ofL. edodes were cultivated in
8/22/2019 Study of hydrolases in lentinus
2/6
eucalyptus residues. The eucalyptus residues were supplemented
with rice, wheat and soybrans at different concentrations to
evaluate the influence of nutrient supplementation onmycelium
growth and enzymes productions.
MATERIALS AND METHODS
OrganismsThree strains ofL. edodes identified as SJC, FEB-14 and
CCB-514 were grown and
maintained on potato-dextrose-agar (PDA) for 15 days at 27 2 C
and stored at 4 C.
FermentationsThe organisms were grown in eucalyptus residues
(leaves, branches and bark)
supplemented with 5, 10, 15 e 20% rice, wheat and soy brans.
These eucalyptus residueswere milled hammer-type mill (< 0.5 mm)
and submerged in distilled water for 24 hours.The excess of water
was drained and the moisture determined (65-70%). The cereal
brans,riches in organic nitrogen, were added in the proportions
above mentioned. Polypropylene
bags were packed with approximately 600 g of mixture and an
assay tube was introduced toform inoculation channel. These bags
were sealed using a PVC collar and waterproof paper.The substrates
were sterilized at 121 C for 2 hours, twice obeying an interval of
24 hours.The cultures media prepared in triplicate, after cooled,
were inoculated in sterile conditionsintroducing five discs (=8mm)
of agar carrying mycelium into the channel formed by assaytube. The
bags were sealed again and incubated at 27 2 C for thirty days in
the dark untilthe substrate was fully colonized with mycelium.
Enzymatic extractionAround of 1/3 superior part each fermented
culture medium was removed and
homogenized. The extraction was realized with 100 ml of sodium
acetate buffer (50 mM;pH=5.0) on 20 g dry mass of each culture
medium. For an hour at 100 rpm and roomtemperature, this mixture
was homogenized in shaker. The enzymatic extracts were thenfiltered
twice through a Whatman n1 filter paper under vacuum. The volumes
of enzymaticextract were stored at 4 C for subsequent enzymatic
activities determinations.
Enzymatic activities assaysCarboxymethyl cellulases
(endo-glucanases), xylanases were assayed by release of
reducing sugars measured with the DNS (3,5-dinitrosalicylic
acid) reagent (MILLER, 1959),using carboxymethyl cellulose (TANAKA
et al., 1981) and xylan (BAILEY et al., 1992)respectively.
-glucosidases and -xylosidases were assayed by release
ofp-nitrophenol from
p-nitrophenyl--D-glucopyranoside and
p-nitrophenyl--D-xylopyranoside respectively(TAN et al., 1987). All
the enzymatic activities were expressed as IU kg -1 defined as
theamount of enzyme producing 1 mol of product min-1 kg 1 of
substrate extracted.
Determination of fungal biomass (FB)The determination of FB was
realized by extraction and quantification of ergosterol
contained in the phospholipid bilayer of cell membranes of
fungi. The amount of FB isdetermined using an ergosterol-to-fungal
biomassa conversion factor from cells growing inliquid culture. The
extraction of ergosterol is realized by saponification of the
phospholipid
bilayer with NaOH micro-wave-assisted and subsequent extraction
with methanol/pentane(MONTGOMERY et al., 2000).
