1 Divisi�on de Ingenier�ıa Qu�ımica y Bioqu�ımica, Tecnol�ogico de Estudios Superiores de Ecatepec, Av. Tecnol�ogico esq. Av. Carlos Hank
Gonz�alez, Ecatepec, CP 55210, M�exico
2 Universidad Tecnol�ogica de Tec�amac, Km 37.5 carretera M�exico-Pachuca S/N, Sierra Hermosa, Tec�amac, CP 55740, M�exico
(Received 17 September 2012; Accepted in revised form 15 March 2013)
Summary Xylanolytic rich filtrates were obtained by A. niger sp in both submerged and solid-state culture using rice
husk or wheat bran as the only carbon source. Filtrates obtained on rice husk showed the highest activi-
ties (~6500 and 5200 U g�1, respectively). Independent of carbon source, these filtrates were very stable in
an acidic pH range (4–7) and mild temperatures, with high half-life time values (more than 7 h at 50 °C)in the corresponding inactivation kinetic models. Also the effect of different metallic ions and denaturing
substances was verified finding that these enzymes are not metaloproteins, and metals as Hg2+ and Pb2+
caused the greatest loss of xylanolytic activity (not higher than 30%). Xylanases produced by this A. niger
strain showed important features that make them potential candidates for applications on human and
Microbial xylanases catabolise the complete hydrolysisof xylan, which requires the interaction of a numberof specific enzymes with the ability to cleave main andside chains. The major enzymes are b-1,4-endoxylan-ase, which cleaves the internal glycosidic linkages ofthe backbone, resulting in a lower-level polymerisationof the chains, and b-D-xylosidase, which hydrolysesshort xylo-oligosaccharides and xylobiose into xylose(Michelin et al., 2010). This enzyme system can beobtained from several microorganisms, among whichfilamentous fungi are the most attractive because theyrelease high titres of them into the medium, with amore complete and specific function on the substrates(Polizeli et al., 2005).
Xylanases have attracted a great deal of attention inthe last decade, particularly due to their biotechnologi-cal potential in various industrial processes. Amongthe different functions suggested for xylanases arebiodegradation in order to provide a source of meta-bolisable energy, degradation of cell wall componentsin concert with other polysaccharide degrading
enzymes, degradation of xylan during germination ofbarley and digestion of dietary vegetation (Loera &Villase~nor-Ortega, 2006). One of the most importantuses is in the paper and pulp industries, in whichthermo-alkaliphilic enzymes are required for efficientbiobleaching (Beg et al., 2001). Thermostable xylanasewould be advantageous in animal feed production pro-cess too, because this is carried out at 70–95 °C,although for this application, the enzyme also must bestable and highly active at the temperature and pH ofthe digestive tract that is 40 °C and 4.8, respectively.Xylanases, which are most active at low and interme-diate temperatures, are required in the food industryand their process is usually carried out at temperaturesbelow 35 °C (Collins et al., 2005).Although xylanase production by fungi involves
high enzymatic activity, stability of the enzyme inalkaline environments and high temperature conditionsis generally not observed (Techapun et al., 2003). Theoptimum pH for most of the fungal xylanases isaround 5.0 although they are normally stable at pH3.0 to 8.0. Most of the fungi produce xylanases whichtolerate temperatures below 50 °C (Subramaniyan &Prema, 2002).Xylanases can be produced using two culture meth-
ods. Most manufacturers produce these enzymes using*Correspondent: E-mail: [email protected]†Both authors contributed equally to this work.
International Journal of Food Science and Technology 2013, 48, 1798–1807
submerged fermentation (SmF) techniques. In fact,SmF as a producing system accounts nearly for 90%of total xylanase sells worldwide. There is, however, asignificant interest in using solid-state fermentation(SSF) techniques to produce a wide variety ofenzymes, including xylanases from fungal origins(Loera & Villase~nor-Ortega, 2006; Dhiman et al.,2008), besides pectinases (D�ıaz-God�ınez et al., 2001).
Most of the research about xylanases productionhas used SmF, because this system allows the controlof some environmental factors required for the opti-mum growth of microorganisms. However, the use ofSSF for xylanases production has received renewedinterest in recent years, being employed very often(Techapun et al., 2003). This is because the use ofabundantly available and cost-effective agriculturalresidues, such as wheat bran, corn cobs, rice bran, ricehusk, sugar cane bagasse, orange bagasse, soybeanand other similar substrates, that allow achievinghigher xylanase yields (Beg et al., 2001; Heck et al.,2002; Christopher et al., 2005; da Silva et al., 2005;Sanghi et al., 2008; Betini et al., 2009). Depending ofculture methods and conditions, diverse forms of theseenzymes are produced, displaying varying folds, mech-anisms of action, substrate specificities, hydrolyticactivities and physicochemical characteristics. Thismultiplicity may be the result of genetic redundancyand in some cases, differential post-translational pro-cessing have also been reported (Lucena-Neto & Ferre-ira-Filho, 2004; Collins et al., 2005). In some cases,this multiplicity can be observed simply in a SDS-PAGE gel (Brijwani et al., 2010; Kiddinamoorthyet al., 2008).
As culture system can provoke differences in theproduced enzymes, several research groups havefocused their attention on compare enzyme produc-tions and the characteristics of the resultant enzymesobtained with SmF and SSF. For example, MateosDiaz et al. (2006) identified differences in the lipasesproduced on both culture systems with respect to theoptimum operation temperature, specific activity andthermal stability, attributable to some non-proteiccompounds produced in dependence of the culturesystem used. On the other hand, Asthera et al. (2002)find at least two different proteins on SSF comparingto SmF production of feruloyl esterases. In environ-mental biotechnology, this comparison led to knowthat the production of oxygenases by SmF was lowerthan that obtained on SSF (Flores-Flores et al.,2011).
The aim of this study was to identify, from its char-acteristics, the potential of application of xylanolyticfiltrates produced by a wild strain of Aspergillus nigeron rice husk by SmF and SSF, and compare theenzyme titres obtained with those produced on wheatbran with the same strain.
Materials and methods
Microorganism
The wild strain Aspergillus niger sp was used in thisstudy. The strain was isolated from reception area ofraw material of a fruit juice industry. Cultures weremaintained on potato dextrose agar (PDA) at 4 °C byperiodic sub-culturing.Inoculums were prepared on PDA medium, and the
resulting spores were harvested from 72-h-old culturesat 37 °C. The conidia were dispersed in 0.01% Tween80 solution. Spores were counted in a Neubauer chamber.
Production of xylanolytic filtrates
For obtaining the xylanolytic filtrates, a simple mineralmedium was used (in g L�1: K2HPO4, 2.0; KH2PO4,2.0 and (NH4)2SO4, 5.0; dissolved in distilled water),with an initial pH value of 5.0. Rice husk and wheatbran were used as the only carbon source on eachexperiment.For SmF, 10% (w/v) of the corresponding carbon
source was used. Fermentations were carried out in50-ml conic tubes containing 10 mL of mineral med-ium on a rotatory shaker at 200 rpm.For SSF, medium constituents were dissolved in
distilled water and sterilised properly after adjusting thepH at 5.0. Separately, 1 g of the corresponding carbonsource was placed on 50-ml conic tubes and sterilisedfor 15 min at 121 °C. After cooling, both fractions weremixed, obtaining a mixture with moisture of 80%. Afterthe inoculation, experiments were maintained at a con-stant humidity by means of a H2O saturated atmosphere.All the experiments were inoculated using 1 9 109 sporesg of substrate�1 and incubated at 37 °C during 72 h.Xylanases obtained on each case were recovered by
filtration, directly from the conic tube (in the case ofSmF) or after the extraction with acetate buffer pH5.0 and a constant agitation at 4 °C (for SSF). Theobtained filtrates were maintained on refrigerationuntil the corresponding analysis.
Analytical techniques
The xylanolytic activity of each filtrate was assayed bydetermining the liberated reducing end products afterthe reaction of the appropriate amount of filtrate with1 mL of 1% (w/v) xylan in 50 mM acetate buffer, pH5.0, at 40 °C for 1 h. One unit of enzyme activity wasdefined as the amount of enzyme that liberates 1 lmolof xylose per minute under standard assay conditions(Mendicuti Castro et al., 1997).Thermal stabilities of aliquots of the xylanolytic
filtrate were estimated in a temperature range of 30,40, 50 or 60 °C. Residual activity was quantified after
International Journal of Food Science and Technology 2013
Applicability of Aspergillus niger xylanases I. Membrillo Venegas et al. 1799
8, 16 and 24 h of incubation of the aliquots, using thepH of the activity assay.
To monitor pH stabilities, the xylanolytic filtrate wasdiluted in citrate-phosphate buffer, with the proper pHvalue: 3, 4, 5, 6 or 7; and incubated at 37 °C. Residualactivity was estimated after 8, 16 and 24 h of incubation.
For studying the influence of some metallic ions andchemical substances on the xylanolytic activity filtrate,the corresponding 10 mM solutions prepared onacetate buffer at pH 5 were incubated with aliquots ofthe xylanolytic filtrate during two hours at 30 °C, esti-mating the residual xylanolytic activity. All the experi-ments were developed in triplicate, expressing theobtained results as an average � standard deviation.
Productivity was calculated for both culture systemsusing the maximum enzymatic activity obtained oneach experiment and considering the time in which thismaximum activity was achieved.
Kinetics of inactivation
The residual enzyme activities were subjected to math-ematical procedures to find a kinetic model that repre-sents inactivation. Three models were tested: in thefirst one, considered as a first-order type, inactivationdepends only on residual activity (A); it was repre-sented in eqn 1 in the integrated form (eqn 2). Inacti-vation depends on the square of residual activity (A2),represented in eqn 3 in the integrated form (eqn 4), asa second-order type. Finally, a third model was pro-posed, which supposed that enzyme inactivationdepends on both the residual activity and incubationtime (eqn 5), integrated as eqn 6.
� dA
dt¼ kA ð1Þ
lnA0
A
� �¼ ktþ c ð2Þ
� dA
dt¼ kA2 ð3Þ
1
A� 1
A0¼ ktþ c ð4Þ
� dA
dt¼ kAt ð5Þ
lnA0
A
� �¼ k
t2
2
� �þ c ð6Þ
SDS-PAGE
In order to identify the differences in proteinsproduced on each culture system and/or carbon
sources, filtrates were concentrated by salting in with(NH4)2SO4 at 80% of saturation and submitted todenaturing sodium dodecyl sulphate-polyacrylamide(SDS-PAGE) with a 10% gel according to the methodof Laemmli (1970). The resulting gel was stained withCoomassie blue R250.
Results
Production of xylanolytic filtrates
Xylanolytic enzyme production obtained by SmF washigher than obtained on SSF on both substrates used(Fig. 1). On SmF, this production was similar in bothcarbon sources, in which approximately 6500 U g ofxylanases�1 was reached.Xylanolytic production obtained with our Aspergillus
strain showed high xylanolytic activities, which weregenerally higher than those reported for other fungaland bacterial strains. With respect to filtrates obtainedwith wheat bran in SmF, the obtained productionswere about 3.32- and 5.64-fold higher than thosereported for A. nidulans (Taneja et al., 2002) andA. tamari (de Souza et al., 2001). Filtrates were11-fold higher than those obtained with Trichodermalongibrachiatum after an optimisation procedure (Azinet al., 2007) in the same carbon source. They were also13-fold higher than those obtained with Trichodermaharzianum on cellulose (Sater & Said, 2001) and twicehigher than that reported for T. reesei Rut C-30 onlactose (Xiong et al., 2004). In same manner, produc-tions were more than thirty times higher than thoseobtained by Penicillium janthinellum on cassava peeland oat husk hidrolisates (Oliveira et al., 2006). Onthe other hand, productions obtained on SSF withwheat bran were 303.2-, 5.68- and 1.14-fold higherthan those reported for xylanases produced by P. ox-alicum (Muthezhilan et al., 2007), P. janthinellum (Ad-sul et al., 2009) and A. foetidus (Christov et al., 1999),
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Figure 1 Xylanolytic production of Aspergillus sp on rice husk ( )
International Journal of Food Science and Technology 2013
Applicability of Aspergillus niger xylanases I. Membrillo Venegas et al.1800
respectively. The productions obtained were higher thanproductions obtained by T. harzianum F416 on sorghumflour (Fadel, 2001) or sugar cane bagasse (Rezende et al.,2002) and Melanocarpus albomyces IIS-68 on sugarcanebagasse (Jain et al., 1998). Also, our xylanolytic filtratesactivities were comparable in magnitude with thosereported for the mutant strain of P. janthinellum (Adsulet al., 2009), although lower than those reported forBacillus pumilus ASH (Battan et al., 2006), T. aurantiacusIMI 216529 (Kalogeris et al., 1998) and Paecilomycesthemophila J18 (Yang et al., 2006) on wheat straw.
Nonetheless, considering that hemicellulose contentof rice husk is generally lower than that reported forwheat bran (Singh & Chen, 2008), the high xylanolyticactivities obtained with this carbon source must behighlighted. First, these activities were comparable(those from SmF) or even higher than that obtainedon wheat bran (that from SSF). Activities of the fil-trates obtained in SmF with this carbon source were5.11-fold higher than the activity reported for B. pumi-lus (Asha Poorna and Prema, 2006) and very similarto that obtained by a mutant strain of A. niger by anoptimisation procedure (Park et al., 2002). All theseresults suggest a potential of this agriculture residuefor being exploited adequately in several processes,related to xylanases production and/or its applications.
One of the benefits of xylanases productions usingSSF over SmF is the high productivity level than can bereached (D�ıaz-God�ınez et al., 2001). In fact, it has beenreported that SSF is a better system than SmF for pro-ducing enzymes of industrial interest, such as xylanases(Beg et al., 2001), exopectinases (D�ıaz-God�ınez et al.,2001), tannases (Aguilar et al., 2002) and feruloylesterases (Asthera et al., 2002), although at our experi-mental conditions, the highest xylanase productions wereobtained on SmF on both carbon sources (Fig. 1). Never-theless, referring to productivities, filtrates obtained bySSF had threefold higher values than those from SmFon both substrates (Table 1). This behaviour couldbe explained under several points of view. From aphysicochemical approach, it is probably that in theSSF system, a little amount of oxygen is available inthe substrate due to the heterogeneity, which could beprovoking the presence of compacted zones and stressto the cells. In fact, a recent fungal secretome reviewrevealed that the cultivation conditions significantlyaffect the types and concentrations of secreted proteinsby ascomycetes (Bouws et al., 2008), and in a proteomicanalysis of protein extracts of A. oryzae obtained fromSmF and SSF systems developed on wheat bran, itwas found that every culture condition have specificenzymes associated and consequently some of them
Table 1 Comparison of productions and productivities for xylanases produced on either SmF or SSF on several agricultural residues
International Journal of Food Science and Technology 2013
Applicability of Aspergillus niger xylanases I. Membrillo Venegas et al. 1801
are observed on SmF but not in SSF, or vice verse(Oda et al., 2006).
Finally, it is also true that, even when xylanasesactivities obtained on SmF were higher than theobtained on SSF, if we compare the total productivity(in U g�1 day�1) among them, filtrates from SSF weremore than four times higher than those fromSmF (31 588.24 vs. 9247.22 and 35 606.58 vs.9032.66 U g�1 day�1 for wheat straw and rice husk,respectively). In this case, we must note that our pro-ductivities were higher than those for other fungal spe-cies previously reported (Table 1), representing a greatxylanolytic production potential with this strain in twoculture systems. Even when other previous researchesit has been reported a similar behaviour where xylan-ases production was higher on SmF than on SSF, theproductivities for SSF were higher than that fromSmF (Shah & Madamwar, 2005). As far as we know,this is the first work in which both productions andproductivities are considerably higher and comparableto those reported for several bacterial and fungalstrains.
Considering that our strain produces high titres ofxylanase activity, on subsequent works we could pro-pose the scaling of the processes, using a technique
similar to that reported for Varzakas et al. (2008) forthe continuous production of glucoamylases on acounter-current reactor by solid-state fermentation.
Xylanolytic filtrates stability
As we were looking for the potential applications thatmay have the xylanolytic filtrates produced by ourAspergillus strain, we considered that one importantdetermination was the stability of these enzymes underseveral environmental conditions. Considering thatmost of the applications of xylanases involve solid sub-strates and that apparently there is no relationshipbetween diffusional limitation and enzyme inactivation(Varzakas et al., 2006), we only checked the effect ofpH of the medium and the incubation temperature forthe stability of xylanolytic filtrates.Filtrates obtained in all experimental conditions
were very stable in a wide range acid pH from 4 to 7.Nonetheless, when filtrates obtained on rice husk inSmF and on wheat bran in SSF were incubated at pH3.0, they lost about 30% and 40% of the initial activ-ity (Fig. 2a,d, respectively), the half-life time (t1/2) inthese cases were 55.4 and 103.5 h, respectively (seeconstants in Table 2).
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Figure 2 Stability of xylanolytic filtrates incubated at 40 °C con different pH 3 (♦), 4 (■), 5 (▲), 6(X) and 7 (●). (a) Rice husk on SmF,
(b) wheat bran on SmF, (c) rice husk on SSF and (d) wheat bran on SSF. Results are the mean of three measurements, which have a standard
International Journal of Food Science and Technology 2013
Applicability of Aspergillus niger xylanases I. Membrillo Venegas et al.1802
About the stability of our xylanolytic filtrates, wemust note that those obtained by SmF were compara-ble with those produced by other fungal species in thisfermentation system, such as xylanases obtained onsugar cane bagasse by P. janthinellum, that were stablebetween pH 4 and 9 (Curotto et al., 1994); filtratesproduced on wheat bran by A. oryzae NRRL1808,that showed stability at pH range of 5–7 (Christovet al., 1999); and xylanases produced by A. japonicuson wheat bran by SSF, that showed a broad pH activ-ity profile (Facchini et al., 2011).
On the other side, kinetic parameters of these filtratesat different pH incubation values are very interesting incomparison with other xylanases obtained by SSF, whichshowed a much lower stability, that is, Thermoascusaurantiacus xylanases obtained on wheat straw had at1/2 = 1 h in a pH range of 4–5 (Kalogeris et al., 1998);andMelanocarpus albomyces xylanolitic filtrates producedon cane bagasse had a t1/2 lower than 5 h in the pH rangeof 5-9.5 (Jain et al., 1998).
The high stability of our xylanolitic extracts undersome pH values must be considered for their applica-tion, especially if they should be used in food industry,where the main desirable properties for xylanases arehigh stability and optimum activity at an acid pH(Polizeli et al., 2005). Also, in feed applications, theenzyme must be highly active at temperature (approxi-mately 40 °C) and pH (approximately pH 4.8) of thedigestive tract (Collins et al., 2005). So, the high stabil-ity of xylanases produced in this work points out tothe use of these enzymes on feed, as proposed forxylanases of A. japonicus with the same stability char-acteristics (Facchini et al., 2011).
With respect of thermal stability of these filtrates, ourresults suggest that at temperatures upper than 30 °C, thekinetic model that best represents the inactivation is firstorder, depending only on residual activity, meanwhile
at mild conditions (30 °C or lower), inactivationdepends on time too. So, the low t1/2 calculated at thistemperature (data in bolds on Table 2) can beexplained by this double dependency. The filtratesobtained on rice husk by means of SmF were generallymore stable than those produced by SSF; althoughat the highest temperatures, this condition changeddrastically (Fig. 3).In general, the xylanolitic filtrates of our A. niger
strain showed greater thermal stability than those fromother species of the genus. For example, the maximumt1/2 reported for filtrates obtained on SmF was close to128 h (those produced on rice husk and incubated at40 °C) meanwhile the maximum of filtrates producedon SSF was 88.3 h (those produced on wheat branand incubated at 30 °C). These values were higherthan those reported by Techapun et al. (2003) forxylanases of A. oryzae grown on wheat bran at 65 °C(t1/2 = 4 h) and Danusa & Cano (2003) for xylanoliticfiltrates of A. giganteus grown on oat spelts xylan at40 °C and pH 6 (t1/2 = 2 h).Regarding to xylanases produced by SSF with our
A. niger strain, they had remarkable half-life values (48and 83 h for rice husk and wheat bran, respectively at30 °C); at higher temperature (40 °C), the results werepromising (35 and almost 52 h for rice husk and wheatbran, respectively) as compared to those from Pleurotusostreatus xylanases produced by SSF on sugarcanebaggasse (nearly 8 h) at pH 7 and 39 °C (Membrillo,2008), xylanolitic filtrates obtained byM. albomyces grownon cane bagasse (2 h at 70°, pH 10) and T. aurantiacusxylanases produced on wheat straw (2 h at 80 °C, pH 5)(Techapun et al., 2003).On the other hand, some studies have showed that
temperatures of around 50–60 °C favoured the diffu-sivity of fungal endoglucanases and endoxylanases onsolid substrates as soybean (Varzakas et al., 2005). So,
Table 2 Adjustment of deactivation kinetic for xylanolitic activity
Temperature (°C)
Rice husk Wheat bran
Kinetic model k t1/2 (h) R2 Kinetic model k t1/2 (h) R2
International Journal of Food Science and Technology 2013
Applicability of Aspergillus niger xylanases I. Membrillo Venegas et al. 1803
considering the good thermal stability of our A. nigerxylanolitic filtrates, it can be suggested a potential field ofapplication of our enzymes on several processes in whichsolid substrates must be processed at high temperatures.
Effect of metalic salts and denaturing substances onxylanolytic filtrates
The effect of substances as EDTA, ionic salts and dena-turing agents was tested, to identify some important char-acteristics of xylanolytic filtrates. Only those cases inwhich a decrease in xylanolytic activity bigger than 20%from the control was observed were reported (Fig. 4).
EDTA did not cause any effect on filtrates. The ionicsalts tested (10 mM Ag+, Cu2+, Ba2+ and Fe2+) did notcaused any effect on the obtained xylanolytic filtrates.
As happened with the other characteristics, xylano-lytic filtrates obtained with SmF were more stable thanthose from SSF. Divalent ions Mn2+, Hg2+ and Pb2+
had the most drastic effect on filtrates.Among denaturing solutions tested, filtrates
obtained by SmF were also more stable than thoseobtained by SSF. In this case, b-mercaptoethanol andSDS showed the negative effect on those xylanases,
although these lost a maximum of 20% and 40% oftheir original activity (Fig. 4c,d).In general, none of the ionic salts tested increased
xylanases activity of the filtrates. These characteristicsindicated that xylanases of the filtrates are not metallo-proteins. These results differ with those obtained withother Aspergillus species, which have shown somemetal dependency, as the case of an isolated strain ofA. foetidus (Shah & Madamwar, 2005).The negative effect of Mn2+ ion could limit the use
of the filtrates on pulp and paper process (Isil & Nilu-fer, 2005), meanwhile the negative effect caused byHg2+, Pb2+ and b-mercaptoethanol on the filtratescould indicate the presence of xylanases in which acystein group is present on the corresponding catalyticsite, as suggested for exopolygalacturonases producedby A. giganteus (Biscaro Pedrolli & Cano Carmona,2010) and xylanases produced by Thermomyces lanugi-nosus (Triches Damaso et al., 2002).Finally, although certain negative effect of some diva-
lent ions on xylanolytic filtrate was observed, thesefiltrates were highly stable for some denaturing solu-tions, which make them easy to renaturing when aresubmitted to a SDS-PAGE process.
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Figure 3 Stability of xylanolytic filtrates incubated at pH 5.0 on different temperatures 30 (▲), 40 (●), 50 (■) and 60 °C (♦).(a) rice husk on
SmF, (b) wheat bran on SmF, (c) rice husk on SSF and (d) wheat bran on SSF.
International Journal of Food Science and Technology 2013
Applicability of Aspergillus niger xylanases I. Membrillo Venegas et al.1804
In feed production, enzymes are used as additivesfor increasing energy and organic matter digestibility.Our xylanolytic filtrates are not metalloproteins andalso were very stable at the presence of several metallicsalts. These characteristics represent an advantage ifthey were used for feed production, because theenzymes do not require the presence of any specificmetallic ion for its activity, and therefore, all mineralscontained in diets are available for livestock. So, thedigestibility of these products would increase, reducingthe environmental load due to decrease in faeces excre-tion, which is ecologically sound (Wenk, 1998). On theother hand, the high t1/2 values of our xylanolyticfiltrates under mild temperature conditions aresuperior than that reported as desirable for their useas additives in several feed livestock (Beaucheminet al., 2004), which is other advantage of the filtratesproduced by this wild A. niger strain.
Electrophoretic analysis
Electrophoretic patterns obtained show a clear differ-ence in the proteins obtained on each case. In SmF onboth carbon sources, there was a low mass proteinthat was not observed on SSF, which probably confers
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Figure 4 Effect of several metallic salts and denaturing solution of xylanolytic filtrates obtained on SmF( ) or SSF (□) on rice husk (a,c) or
wheat bran (b,d). Results are the mean of three measurements, which have a standard deviation of around 5%.
Figure 5 Electrophoretic pattern of xylanases filtrates. (1) rice husk
on SmF, (2) rice husk on SSF, (3) wheat bran on SmF and (4) wheat
bran on SSF. The proteins observed in all experimental conditions are
shown in the box, meanwhile those that means a difference among
International Journal of Food Science and Technology 2013
Applicability of Aspergillus niger xylanases I. Membrillo Venegas et al. 1805
the stability of the filtrates to ionic metals as Hg2+,Pb2+ and Cu2+. On rice husk, there were a few pro-tein bands, but considering the high xylanolytic activ-ity observed in this carbon source, this is an importantresult because it suggests that the specific activity ofthese enzymes is high, as indicated by the productivityof the filtrates, and points out the potential to use thiscarbon source for producing xylanases (Fig. 5). Thedifferences of electrophoretic patterns could explainthe observed characteristics of each filtrate.
Conclusions
Our A. niger strain produces higher xylanases produc-tions, productivities and half-life time values thanthose reported for other Aspergillus strains when it iscultured on submerged or solid-state culture usingagricultural residues as carbon source. Filtrates fromSmF had higher xylanolytic activities and were gener-ally more stable than those produced by solid-statefermentation. The potential of rice husk for xylanasesproduction was highlighted. Xylanases produced bythis A. niger strain showed some important featuresthat make them potential candidates for applicationson human and livestock food industries.
Acknowledgments
This work was supported by a fortifying programfrom “Consejo Mexiquense de Ciencia y Tecnolog�ıa”(EDOMEX-CO1-78011). JFH thanks to “ConsejoNacional de Ciencia y Tecnolog�ıa” the scholarship.
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