Journal of Proteomics & EnzymologyISSN: 2470-1289

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Research Article, J Biocatal Biotransformation Vol: 2 Issue: 2

Cellulases and Xylanases Production by Penicillium Echinulatum in Submerged Cultivation: Statistical Optimization of Process Parameters

Carla Eliana Todero Ritter*, Marli Camassola, Mauricio Moura da Silveira and Aldo José Pinheiro Dillon
Institute of Biotechnology, University of Caxias do Sul, Caxias do Sul, Brazil
Corresponding author : Carla Eliana Todero Ritter
Institute of Biotechnology, University of Caxias do Sul, Caxias do Sul, RS 95070-560, Brazil
Tel/Fax: 555432182100R2681
E-mail: [email protected]
Received: November 08, 2013 Accepted: December 19, 2013 Published: December 25, 2013
Citation: Todero Ritter CE, Camassola1 M, da Silveira MM, Pinheiro Dillon AJ (2013) Cellulases and Xylanases Production by Penicillium Echinulatum in Submerged Cultivation: Statistical Optimization of Process Parameters. J Biocatal Biotransformation 2:2. doi:10.4172/2324-9099.1000111

Abstract

Enhancing Thermal Stability of Immobilized Raw Starch Digesting Amylase (RSDA) Using Different Additives

The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. The production of cellulases and xylanases is difficult in submerged cultures, because the cellulose used as an inducing substrate is insoluble, which causes rheological difficulties, such as homogenization of the medium, aeration and agitation, factors that influence the oxygen transfer.

Keywords: Cellulases; Sorbitol; Penicillium echinulatum; Statistical optimization

Keywords
Cellulases; Sorbitol; Penicillium echinulatum; Statistical optimization
Abbreviations
RSM: Response Surface Methodology; CCRD: Central Composite Rotational Design; FPA: activity paper filter
Introduction
The production of commercially viable lignocellulosic hydrolysates is a great challenge in the creation of second generation ethanol, leaving to the enzymatic cellulase and xylanase complexes the key role in facilitating this process [1]. Low cost cellulases may be obtained from cheap and accessible substrates, thus making the process economically attractive. Moreover, the advantage of enzymatic hydrolysis in relation to other hydrolysis technologies lies in the fact that it does not use costly equipment, such as cooling systems, electricity and gas, in addition to avoiding the equipment’s corrosion [2].
The production of the cellulase and xylanase complexes in submerged cultures is difficult, because the cellulose used as an inducing substrate is insoluble, which causes rheological difficulties, such as homogenization of the medium, aeration and agitation, factors that influence the oxygen transfer [3]. Data indicating the inducing potential of cellulase secretions from insoluble substances, such as Solka Floc®, cotton and paper, as well as information on soluble substances capable of inducing the cellulase complex are available in the literature. In Trichoderma, it has been found that the disaccharide sophorose (2-O-β-D-glucopyranosyl-D-glucose) provides high inducing capacity of the cellulase complex, while other sources, such as starch, glucose and glycerol do not act as inducers of these enzymes [4]. It was also found that different concentrations of caffeine and theophylline can increase the production of β-glucosidase and FPA by Penicillium echinulatum [5].
Trichoderma reesei studies [6] indicated that the polyhydric alcohols sorbitol and glycerol allow fungal growth without causing catabolite repression, but these substrates have not yet been studied with the aim of producing cellulases and xylanases. Pail et al. [7] also observed the growth of Hypocrea jecorina in D-sorbitol, similar to other poly galactitol and D-talitol. Also, sorbitol is secreted by Zymomonas mobilis for osmotic pressure regulation [8], and it is metabolised as a carbon source for the production of cell mass. Mutants of P. echinulatum are potential producers of cellulases through cellulose hydrolysis, because they offer relatively good thermal stability of filter paper activity (FPA) and β-glucosidase enzymes at 50°C [9], high levels of enzymatic production [5] as well as good proportions of FPA and β-glucosidase for the efficient hydrolysis of cellulose, as compared to the cellulases of T. reesei [10].
However, as it occurs in the production of cellulases by T. reesei, high concentrations of cellulose in the culture medium cause problems for the fermentation process, hampering oxygen transfer, medium homogenization and sampling [3,11].
Studies with Trichoderma reesei have shown that the polyols glycerol and sorbitol allow fungal growth without causing catabolite repression [6]. According to these studies, sorbitol can be considered a neutral carbon source for cellulase expression. During the present work, submerged cultures of the fungus 9A02S1 P. echinulatum were used, varying the concentrations of both soluble (sorbitol) and insoluble (cellulose) carbon sources, as well as the addition time of inducer to improve the yield by optimising the growing conditions. To this end, experiments were performed to optimise the process parameters, and the production of cellulases and xylanases by P. echinulatum in submerged culture by performing a central rotational composite design based on Response Surface Methodology (RSM).
Materials and Methods
Microorganism
The mutant P. echinulatum 9A02S1 strain (DSM 18942) was used during this study. This strain was obtained by exposing wild type P. echinulatum strain 2HH to different mutagenic agents [12]. Both strains are stored in the culture collection of the Laboratory of Biomass and Enzyme, Institute of Biotechnology, Caxias do Sul, Rio Grande do Sul, Brazil. The strain was maintained on cellulose agar (agar C) consisting of distilled water containing 1% (v/v) swollen cellulose, 10% (v/v) × 10 MS, 0.1% (w/v) proteose peptone (Oxoid L85) and 2% (w/v) agar. The strain was grown on C-agar slants for up to 7 days at 28°C until conidia were formed, then stored at 4°C for later use.
Culture conditions
The assay was performed in 500 ml Erlenmeyer flasks containing 100 ml of liquid medium, which was inoculated with a suspension containing 1x105 conidia/ml. The production medium was formulated with 0.1% soybean meal, 0.14% KNO3, 5% (v/v) salts solution 20x [13], without (NH4)2SO4, 0.1 ml of Tween 80® and 0.1% (v/v) 4.4% gentamicin solution. Various concentrations of sorbitol were employed as a carbon source, according to experimental design. Cellulose supplementations with concentrations ranging from 0 to 0.0075 g/ml were made; the cellulose was added at 0h (beginning of the process) and after 12h, 36h and 48h from the beginning of the process. The flasks were kept under reciprocal shaking at 180 rpm and at 28 ± 1°C. All assays were done in triplicate.
Enzyme assay
Enzyme activity was assayed on filter paper (FPA), according to Ghose (1987) [14]. The β-glucosidase activity was determined using salicin as substrate, according to Chahal (1985) [15]. Endoglucanase activity was determined according to Ghose (1987) [14] using 2% (w/v) carboxymethylcellulose solution in sodium citrate buffer. The reducing sugars were estimated as glucose equivalent by the dinitrosalicylic acid (DNS) method according to Miller (1959) [16]. Xylanase activity was measured by the method of Bailey et al. (1992) [17] using oat spelt xylan. Reducing sugar was measured by the DNS method using xylose as standard. One international unit (U) of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar from the appropriate substrates per minute under assay conditions.
Experimental design
The selection of cellulose and sorbitol concentrations and the addition time of cellulose followed the experimental planning CCRD (Central Composite Rotational Design), according to Table 1. A full factorial 23 was done, including 6 axial points and 6 replicates at the central point, totalling 20 tests performed in triplicate. The enzymatic activity can be written as a function of the independent variables, as in the model with coded variables (Equation 1).
Table 1: Experimental design of cellulose and sorbitol concentration and time of addition of cellulose to the medium for filter paper activity in submerged cultures on 5th day.
    (1)
Statistical tests
The results were statistically analysed using analysis of variance with the PrismGraphPad program (Graph Pad, San Diego, CA), Tukey post-test for a p<0.05 and software Statistica 10.0 (StatSoft Inc. Tulsa, OK,USA).
Results
The effect on the production of cellulases and xylanases by P. echinulatum 9A02S1 was evaluated in media containing different concentrations of sorbitol at the beginning of cultivation, and combined with the supplementation of cellulose at different times.
It appears from Table 1 that some cellulose may be replaced by sorbitol in submerged culture for cellulases production, as it can be seen in experiments 3 and 7. The importance of sorbitol supplementation for the production of cellulases is clear in experiments 11 and 12. Comparing experiment 12 to experiment 11, it appears that the production doubled without the need of adding cellulose to the medium.
The fitted response surface for the cellulose production was generated using STATISTICA 10.0 (StatSoft Inc. Tulsa, OK, USA) and it is presented in Figure 1. Figure 1 shows the interaction between the concentrations of sorbitol and cellulose; it was found that the concentrations of 0.25% (v/v) sorbitol and 0.75% (v/v) cellulose showed the highest activities.
Figure 1: Response surface of the filter paper activity depending on the concentration of sorbitol and cellulose. The enzymatic activities were obtained on the 5th day of production, using the fungus Penicillium echinulatum 9A02S1 in submerged cultivations.
The data obtained for the activity of FPA on the 5th day of culture also indicate that all parameters were highly significant and may produce a second order polynomial equation with coded variables (Equation 2).The variances for FPA were significant at p<0.05 and 93.5% of the explained variability (Table 2). These results indicate agreement between the experimental values and those predicted by the model.
Table 2: Estimates of the coefficients, an association of values and significance level for Filter Paper Activity on the 5th day of culture.
FPA(IU / mL)(5th day)=1.426+0.303.x1+0.12.x2−0.18.x12−0.11.x22−0.11.x32− 0.09.x1 .x2 − 0.07.x1 .x3     (2)
Regarding the addition time of cellulose (Figure 2), it appears that this variable conjugated with the sorbitol concentrations has significance in the process. Also, the addition of cellulose contributed to increase the FPA activities up to the first 24h of culture, although the supplementation of this inducer did not have positive effect after 24h. This fact can be observed in media formulated with 0.5% sorbitol and supplemented with 0.5% cellulose. Enzymatic titers were obtained for FPA of 1.07, 1.52 and 1.05 IU/ml for cellulose supplemented cultures with 0h, 24h and 48h from culture, respectively. After 24h from the beginning of the process, at the same concentrations and supplementation, the determination on the 6th day had a maximum value of 1.91 IU/ml.
Figure 2: Surface response for filter paper activity according to the concentration of cellulose and addition time in submerged cultures of Penicillium echinulatum the 5th day of cultivation.
Endoglucanase activity was increased when some of the cellulose was replaced by sorbitol, in the range of 0.6% to 0.8%. When 0.75% cellulose was added to a medium containing 0.25% sorbitol, 12 hours from the beginning of cultivation, the endoglucanase activity was 4.19 IU/ml. For the same situation, but in cultures with 0.75% sorbitol, the activity has increased to 6.85 IU/ml (Figure 3).
Figure 3: Response surface endoglucanase activity on 5th day of cultivation depending on the concentration of cellulose and sorbitol.
For the response of endoglucanase activity, all regression coefficients were statistically significant (p < 0.05) (Table 3) and the explained variation of 92% indicate a good agreement between experimental and predicted values.
Table 3: Regression coefficients for response of endoglicanase activity in submerged cultures, on 4th day of cultivation.
For the xylanase activity, on the 5th day it is observed that there is an explained variation of 92% (Figure 4). The model equation which defines the behaviour, in coded variables, is shown in Equation 3-5.
Figure 4: Observed values x estimated values of xylanase on the 5th day of cultivation, with variation explained 92%.
Endoglucanase (IU / mL)( 4thday) = 6.03 +0.85.x1+0.11.x 2-1.18.x12-0.86x22-0.6x+0.16x1.x2-0.73.x1.x1-0.16.x2.x3    (3)
Xylanase( IU mL) = 12.1 +1.77.x1 + 0.463.x3-1.26.x12-1.52.x22-0.61x32-0.76.x1.x2-0.43.x1.x2-0.60x2.x3
β- glucosidase (IU / mL)( 4thday) = 0, 402 + 0.039.x1- 0.04.x2- 0.07.x12- 0.04.x2- 0.04.x2+ 0.03.x1.x2- 0.3.x1.x3-0.09. x2.x3
It was found that the addition time of cellulose in xylanase activity is representative. The highest titers occurred when the addition was performed previously to 36h (Figure 5); a decrease happened after this time. The medium containing 0.25% cellulose and 0.25% sorbitol demonstrated activity of 5.81 and 6.56 U/ml, when cellulose addition occurred in 12h and 36h, respectively. In similar situations, but increasing the cellulose concentration to 0.75%, activity results were 11.51 and 12.06 IU/ml, respectively, indicating that the cellulose acts as an inducing source for xylanase.
Figure 5: Response surface of the xylanase activity according to the cellulose concentration and the addition time on the 5th day of cultivation of Penicillium echinulatum.
According to the data obtained in this study, concentration of up to 0.3% (v/v) sorbitol and above 0.7% (w/v) cellulose contributed to increase the xylanase activity (Figure 6). Also, the medium only containing sorbitol (0.5%) showed maximum enzymatic activity of 4.06 IU/ml.
Figure 6: Response surface of the xylanase activity of Penicillium echinulatum depending on the concentration of cellulose and sorbitol in submerged on 5th day of cultivation.
Discussion
The cost of the enzyme cellulase is a major factor in the economic process of ethanol from biomass [18]. Cellulose, as a carbon source, is an inducer of cellulases and xylanases in T. reesei [6]. However, in high concentrations – a condition necessary to achieve high enzyme levels [19] – problems occur in the oxygen transfer through the cultivation medium, with negative repercussion on growth and enzymes production [3,20]. In addition, the presence of cellulose in the medium can reduce the quantity of free cellulases, as these enzymes tend to become adsorbed to their substrates [21].
Environmental factors, such as temperature, pH, oxygen levels, and concentrations of nutrients and products in the medium can significantly affect microbial growth and product formation. A judicious selection of these parameters can dramatically improve the yield of enzymes.
According to the data obtained in this study, it is possible to partially replace the cellulose of the culture media used for cellulases and xylanases production. In the case of endoglucanase production, it was increased when part of the cellulose was replaced by sorbitol in the range of 0.6% to 0.8%. For production of xylanase, sorbitol concentrations of up to 0.3% (v/v) and cellulose above 0.7% (w/v) contributed to increase the enzymatic titer of this enzyme.
The determination of xylanase activity in media formulated with sorbitol (0.5%) only confirms that this carbon source did not cause catabolite repression in P. echinulatum, as noted earlier for T. reesei. It was not possible to detect any transcript of the major cellulases of T. reesei (cbh1, cbh2, egl1 and egl2) in sorbitol-grown cultures [6].
The beneficial effect of sorbitol-formulated culture media may be related to growth, as this polyol can be converted into fructose by the L-iditol 2-dehydrogenase or by sorbitol dehydrogenase, thus favouring microbial growth [22]. Additionally, as previously observed for T. reesei, a link could occur between D-sorbitol utilization and cellulase formation, since D-sorbitol can be converted to L-sorbose by an NADP-dependent ketose reductase [23], while L-sorbose can induce cellulases in T. reesei [24].
Optimizations in enzyme production using experimental design have been observed by other authors. Wen et al. [25] optimized parameters of pH, temperature and concentration of animal waste to produce cellulases and β-glucosidase, using T. reesei and Aspergillus phoenicis. According to Mekala et al. [26], the optimization studies performed using response surface Box–Behnken design on process variables affecting cellulase production under SSF by T. reesei RUT C 30 on sugarcane bagasse was effective in enhancing the production of enzyme from 5.11 to 24.15 FPA U/gds, a 4.7-fold increase in production.
Conclusion
The results of this study indicate that some cellulose may be replaced by sorbitol in submerged processes for the production of cellulases and xylanases. Also, with this substitution, the rheology of the medium is favored, with consequent changes in oxygen transfer. What is more, the addition time of the inductor is specific for each enzyme and the experimental design allows not only economise time and substrate, but also to check the correlation between the determinants of the process.
The present results also indicate that models of polynomial equation for the enzymatic activities of FPA, xylanase and endoglicanase have the same profile, demonstrating the positive contribution of cellulose and sorbitol concentrations and time of addition, and the negative association between the variables.

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