Artigo 2017
Artigo 2017
Artigo 2017
DOI 10.1007/s12649-017-9994-x
ORIGINAL PAPER
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Vol.:(0123456789)
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[9], SSF has many advantages when compared with Microorganism and Inoculum
chemical reactions, such as low water requirements,
low toxic residue generation and simplicity of opera- The fungus P. roqueforti ATCC 10110 (Lot: 041140074,
tion. Fungi cultivation, as the microbiological agent of INCQS: 40074) was donated by the Fundação Oswaldo
transformation in SSF, is justified by their mild growth Cruz (FIOCRUZ, Rio de Janeiro, RJ, Brazil). The inoculum
condition requirements [10]. Among the various fungi was prepared in culture medium with agar–agar and potato
species, those considered safe are classified as GRAS dextrose agar, in Erlenmeyer flasks (250 mL) and incu-
(Generally Recognised as Safe) by the Food and Drugs bated at 27.5 °C for 7 days in a bacteriological incubator
Administration (FDA). Penicillium roqueforti is termed (SOLAB/Piracicaba, Brasil). The spores were suspended in
a GRAS fungi because it is a non-pathogenic microor- aqueous 0.1% Tween 80 and counted in a Neubauer cham-
ganism and is commonly associated with cheese produc- ber using a binocular microscope (Medilux MDL 150 BAI/
tion [11]. BPI) adjusted to 40x magnification.
Another benefit of bioconversions in SSF is the sub-
stantial quantity and variety of enzymes produced by the SSF
microorganism. Among the fungal enzymes, xylanases
(endo-1,4-β-xylanase, EC 3.2.1.8) are responsible for 10 g of rice husk substrate were autoclaved
the hydrolysis of xylan—one of the main components (121 °C/1 atm/15 min) in Erlenmeyer flasks of 125 mL;
of hemicellulose [12]—through the depolymerisation after cooling, the sterile substrate was inoculated with
of the main chain, releasing d-xylose [13]. Xylanases 107 spores/g substrate and moistened with sterile distilled
have diverse industrial applications, such as cellulose water until determining the value of water activity ( aw) was
pulp bleaching, juice clarification, baking and many oth- standardised as 0.984. The incubation was carried out in a
ers [14, 15]. Two other enzymes of industrial value, as bacteriological incubator (SOLAB/Piracicaba, Brasil). A
equally produced by fungi, are endoglucanases (E.C. Doehlert-type experimental design (six different experi-
3.2.1.4) that are responsible for cleaving inner cellu- ments and triplicates of the central point) was conducted to
lose bonds and exoglucanases (E.C. 3.2.1.91) that act in evaluate two independent variables including the incuba-
reducing and non-reducing regions of cellulose. From tion time (t, which varied in three levels, from 48 to 96 h)
a commercial perspective, complex enzymatic extracts and incubation temperature (T, which varied in five levels,
(with diverse enzymatic activities acting synergistically) from 24 to 40 °C). The evaluated responses were the activi-
are more interesting than purified enzymes [16] because, ties of xylanase (U/g) and endoglucanase (U/g). The result-
for example, purification steps often increase the final ing data were analysed [17, 18] with statistical software
product cost. (STATISTICA™ v. 10.0, StatSoft), to perform a quadratic
In this context, the objective of this work was to inves- model fitting and to obtain the response surfaces.
tigate the cultivation of P. roqueforti ATCC 10110 by
SSF, using rice husk as the substrate, to obtain a multi- Multienzymatic Crude Extract
enzymatic crude extract containing xylanases, and endo-
glucanases. Also, some fundamental aspects of the xyla- Sodium citrate buffer (50 mM, pH 4.8) was added to the
nases and endoglucanases obtained, were characterised. fermented substrate (rice husk + fungi cells) at 5:1 vol-
ume (mL): weight (g) ratio and the mixture agitated
(200 rpm/35 °C/20 min) in a shaker-type incubator
(SOLAB). The biomass was then pressed and finally cen-
Materials and Methods trifuged (1250 g/10 min). The resulting crude solution was
analysed for xylanase and endoglucanase. However, no cel-
Fermentation Substrate lulase activity was determined, so the methods applied for
this enzyme were not discussed further in this study.
Tio Mário, a local rice producer (Barreiras, Bahia, Bra-
zil) kindly provided samples of rice husk (O. sativa L.) Determination of the Enzymatic Activities
After the hygiene and size reduction, the samples were
dried in an oven (TECNAL/São Paulo, Brazil) at 70 °C The enzymatic activity of xylanase was determined, as
for 24 h, and milled in a Willey-type knife mill (LABOR/ described by Santos et al. [19], using beechwood xylan
Piracicaba, Brazil) to a granulometry of approximately as a substrate in sodium citrate buffer (50 mM, pH 4.8).
2 mm. The substrate was stored in closed polyethylene Endoglucanase activity was assessed based on the method
containers, until the necessary analysis. reported by Santos et al. [20], using 2 g/L carboxymethyl-
cellulose solution (BIOTEC) as the substrate in sodium
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citrate buffer (50 mM, pH 4.8). The reducing sugars were Xylanase Stability under Freezing Conditions
determined by the 3,5-dinitrosalicylic acid (DNS) method
[21] and absorbance measured using a spectrophotometer For the xylanase produced under the optimised conditions,
(BEL Photonics MV M51). the stability, expressed as residual activity (%, U/Uo), in cit-
rate buffer (50 mM, pH 4.8) under freezing (−20 °C), was
also evaluated (triplicate) over 45 days. The initial xylanase
Determination of Optimal pH and Temperature activity (Uo) was 1.262 U/g.
of the Xylanases and Endoglucanases
The activities of xylanase and endoglucanases were deter- Results and Discussion
mined (triplicate) under the fixed condition of pH = 4.8
(50 mM sodium citrate buffer), at various temperatures Production of a Multienzymatic Crude Extract
(from 5 to 85 °C) and, under the fixed condition of 35 °C,
using buffers (50 mM) of various pH including gly- The culture obtained under SSF by P. roqueforti ATCC
cine–HCl at pH 2, sodium citrate at pH 3–5, sodium phos- 10110 on rice husk meal, was conducted in accordance
phate at pH 6–8 and glycine-NaOH at pH 12. The results with the matrix presented in Table 1, for the factors of
were expressed as residual activity (%, U/Uo). The refer- temperature (T, °C) and incubation time (t, h), as well as
ence activities (Uo) were determined as 0.961 U/g for the the responses of xylanase (U/g) and endoglucanases (U/g)
xylanases and 2.200 U/g for the endoglucanases, at the activities.
optimum temperature investigation, and 1.323 U/g for the For the xylanase activity, the analysis of the effects of
xylanases and 2.290 U/g for the endoglucanases at the opti- each factor on the responses (data not presented) indicated,
mum pH investigation. at the 85% confidence level that three terms were statisti-
cally significant (p < 0.15) to the quadratic model including
Activation/Inactivation of Xylanases the linear terms of temperature and time and the quadratic
and Endoglucanases term of time. After the removal of non-significant terms,
analysis of variance (ANOVA) was performed (Table 2)
H4+
The effects of Cu2+, Co2+, Mg2+, Na+, Al3+, K+ and N and the model obtained (Eq. 1) presented a good fit to the
at 1.0 mol, EDTA (ethylenediamine tetraacetic acid) at data (R2 and Radj2 > 0.88). Biological/biochemical systems,
1% v/v, H2O2 (hydrogen peroxide, oxidising agent) at 5% due to their variable characteristics, allow the selection of
v/v, and the solvents acetone, ethanol, isopropanol, dichlo- less rigorous confidence levels (up to 85%) to maintain a
romethane and formaldehyde at 10% v/v, on the enzymatic greater number of significant terms in the model.
activities of xylanase and endoglucanases were evaluated Xylanase activity (U∕g) = 1.04 + 0.08(T) + 0.017(t) − 0.023(t2 )
(triplicate). In all instances, the activities were determined (1)
initially (Uo) and after incubation in the presence of the Equation 1 also allowed estimation of the maxi-
compound evaluated (U). The results were expressed as mum theoretical xylanase activity at 32 °C for about 82 h
residual activity (%, U/Uo) and the analyses were performed
in triplicate. Incubations were performed in sodium citrate
buffer (50 mM, pH 4.8) for both the xylanases and endo- Table 1 Doehlert matrix with coded values (real values are pre-
glucanases. The xylanase Uo values were 1.344 U/g for the sented in parenthesis) for the factors: temperature (T, °C) and time
solvents and 1.262 U/g for the other compounds, whereas (t, h) and the responses: xylanases (XYL, U/g) and endoglucanases
(CMC, U/g) activities obtained from the cultivation of Penicillium
the endoglucanase Uo was 2.40 U/g for all compounds. roqueforti in 10 g of rice husk and aw = 0.984
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endoglucanase activities. In this instance, the best condition greater than 20%, reaching 46% at 85 °C. Khandeparkar and
could be selected as the central point condition or any other Bhosle [27], when evaluating a xylanase from Enterobacter
combination under the conditions applied in the experi- sp. MTCC 5112, observed optimal activities under more
ments (Table 1). Pericin et al. [26], also using P. roqueforti drastic conditions (glycine-NaOH buffer, pH 9.0 at 100 °C)
but on a markedly different substrate (pumpkin oil cake), than those observed in this work. Knob and Carmona [28]
produced substantially more endoglucanases, with a maxi- and Terrasan et al. [29] observed a more similar optimum
mum productivity of almost 0.5 U/g.h. pH (6.0), with xylanases from Penicillium sclerotiorum and
Penicillium janczewskii, respectively. Also, similar temper-
Optimum pH and Temperature of Xylanases atures were defined by Querido et al. [30], for a Penicillium
and Endoglucanases expansum xylanase (40 °C), and Saha and Ghosh [31], for
a Penicillium citrinum xym2 xylanase (45 °C). In contrast,
Figure 2a, b present the various pH and temperature pro- the xylanase produced by Myceliophthora thermophila, had
files of xylanase activity. For pH, it was possible to observe an optimum pH of 12 [32].
(Fig. 2a) residual activities greater than 100% between Figure 2c, d illustrate the effect of pH and temperature
pH 5.0 and 8.0, suggesting different levels of enzymatic for the endoglucanases activities contained in the multien-
activation, possibly by charge equilibrium. Even at pH 3, zymatic extract by SSF of P. roqueforti ATCC 10110 on
the residual activity obtained was around 80% but at the rice husk. In Fig. 2c, it is observed that the endoglucanases
extreme pHs analysed (2.0 and 12.0), there was a consider- were inhibited at all pH values tested, concluding that for
able loss of activity. Among the various temperatures eval- the endoglucanases produced by P. roqueforti ATCC 10110
uated, the xylanases presented (Fig. 2b) the highest residual the optimum activity occurs at pH 4.8. However, the loss of
activity (100%) at the reference condition of 35 °C and activity was not greater than 15% at any pH tested, reveal-
demonstrated a reduction of 10% at 45 °C. For the other ing a good pH range of enzyme activity. Silva et al. [33]
temperatures investigated, the reduction in activity was determined the action of endoglucanases was optimal at pH
Fig. 2 Residual activities (%, U/Uo) of a and b xylanase and c and (at pH = 4.8). The values of Uo (pH 4.8/35 °C) employed in a and c
d endoglucanase produced (from Penicillium roqueforti ATCC 10110 were: 0.961 and 2.20 U/g respectively, and for b and d were 1.323
in rice husk) at different values of a pH (at 35 °C) and b temperature U/g and 2.20 U/g, respectively
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5.5 and for endoglucanases from Streptomyces sp., Azzed- ~97%), whereas, the presence of Na+ was inhibitory (resid-
dine et al. [34] determined an optimum at pH 6. For the ual activity <90%). Khandeparkar and Bhosle [27] also
temperature (Fig. 2d), the endoglucanases presented the determined, an enzymatic activation (202%) with Cu2+ and
highest residual activity (100%) at the reference condi- Co2+ ions, which are frequently identified as enzymatic
tion (50 °C), whereas at 5 and 25 °C, the largest reduction co-factors, for an Enterobacter sp. MTCC 5112 xylanase.
in activity (~22%) was observed. At the other tempera- Terrasan et al. [29] determined an increase in activity in
tures tested, the reduction in activity was not greater than the presence of NH4+ (~131%), for xylanases from P. janc-
11%. Similar results were found by Lin et al. [35], with zewskii but an inhibition was observed with Co2+ (~84%),
Jonesia quinghaiensis, and by Das et al. [36], with Penicil- while Mg2+ presented a minor influence (~104%). Guan
lium notatum NCIM NO-923, and Huang et al. [37], with et al. [40] determined an increase in activity of around 52%
Arthrobacter sp. HPG166. All these studies also demon- with Mg2+, for xylanase from Cladosporium oxysporum.
strate low endoglucanases activities at temperatures lower Among the solvents analysed, acetone and ethanol
than 40 °C. resulted in activation of the xylanases from P. roqueforti
ATCC 10110 (Table 3). Polar solvents, such as these, are
Activation/Inactivation of Xylanases capable of interfering with the three-dimensional configu-
and Endoglucanases ration of proteins (being good precipitating agents at higher
concentrations [41]) but do not suggest that the enzymatic
The activity of enzymes can be increased or decreased activity is preserved under long incubation periods. The
by certain compounds in solution (such as metal ions and presence of formaldehyde had no strong influence on xyla-
solvents) by interfering with their three-dimensional struc- nase, while isopropanol resulted in inhibition (Table 3).
ture or blocking the active site [38, 39]. Therefore, in this The activities of xylanases by Bacillus have been reported
study, some compounds were analysed for their influence, practically unchanged in the presence of 5% (v/v) etha-
during incubation of the multienzymatic crude extract, on nol [42], activated by 50% (v/v) acetone and inhibited by
the residual activities of the xylanases and endoglucanases 50% (v/v) formaldehyde [43]. The use of EDTA, resulted
(Table 3). Considering the xylanase activities, activation in an inhibitory effect on the xylanase from P. roqueforti
(residual activity > 100%) by salts was observed in the ATCC 10110 (Table 3). Similar results were observed by
descending order C o2+ > Cu2+ > NH4+ > Al3− > K+. The Boonchuay et al. [38] and Sorgatto et al. [44], in which the
2+
presence of Mg had a minor effect (residual activity xylanases from Streptomyces thermovulgaris TISTR1948
and Aspergillus terreus, respectively, were reduced by up to
40% of their initial activity in the presence of EDTA. Con-
Table 3 Residual activities (triplicate) for xylanases and endoglu-
canases (from Penicillium roqueforti) incubated with different addi- versely, H2O2 showed a minor activation (Table 3), prob-
tives ably due to its oxidative character but this phenomenon, as
well as that observed with ethanol and acetone, is unlikely
Additives Xylanases (%, U/Uo) Endoglucan-
anses (%, U/ to occur during relatively longer incubations. Sanghvi et al.
Uo) [45] determined a residual activity of 15% in the pres-
ence of only 5% (v/v) acetone, for a Bacillus sp. BHO502
Cu2+ 154.28 ± 2.21 136.31 ± 3.54
xylanase.
Co2+ 211.71 ± 3.15 244.84 ± 1.16
Regarding the residual endoglucanase activities
NH4+ 127.26 ± 3.42 92.66 ± 4.39
(Table 3), activations were observed with Cu2+ and Co2+,
Mg2+ 97.10 ± 3.99 ND
whereas NH4+, K+ and Pb+ caused inhibition. Na+ did
Na+ 88.90 ± 1.50 102.70 ± 2.92
not result in any significant influence. Pol et al. [46] also
Al3+ 107.72 ± 0.41 NT
determined Co2+ as an activator and K+, Pb+ and Cu2+ as
Pb+ NT 93.43 ± 1.34
inhibitors of Penicillium pinophilum MS 20 endoglucanase
K+ 114.23 ± 1.10 74.12 ± 1.34
activity. In accordance with our results, Sadhu et al. [47]
EDTA 91.07 ± 1.10 79.14 ± 8.11
also observed an inhibition by EDTA. Similarly, Azzed-
H2O2 106.76 ± 3.32 NT
dine et al. [34] found C o2+, Cu2+ and NH4+, as activators
Ethanol 167.27 ± 0.39 180.34 ± 0.67
of Streptomyces sp. B-PNG23 endoglucanase action. In the
Acetone 176.56 ± 3.74 176.09 ± 2.41
current endoglucanase study of P. roqueforti ATCC 10110,
Dichloromethane 102.04 ± 3.98 NT
all the solvents tested had a positive effect on the endoglu-
Ethyl Ether 90.26 ± 1.04 NT
canase activity, increasing up to 80% the catalytic activ-
Isopropanol 82.10 ± 3.06 110.82 ± 1.77
ity of the enzyme. In contrast, Silva et al. [33] determined
Formaldehyde 98.64 ± 4.92 169.91 ± 3.72
isopropanol as an activator of endoglucanase activity,
ND not detected, NT not tested whereas ethanol and acetone did not affect or improved the
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