Production of xylanase by Fusarium oxysporum using agro- industrial waste

This study aimed to optimize the production of xylanase, under submerged conditions using Fusarium oxysporum. Firstly, it was carried out the selection of the best source of carbon from agricultural waste, such as soybean husk, cassava peel, pineapple crown, corn straw, rice husk, bacaba, barley bagasse and, corncob. They were milled and sieved with a maximum granulation of 1 mm. The cultures were carried out by 132 hours 30 °C and 180 rpm. Among the agroindustrial residues, corn straw was the one that stood out with a greater enzymatic activity of 12.6 U/mL. In the second step, it was used Plackett-Burman design to screen the nutrients sources important to xylanase production. Thus, independent variables significant were urea and MgSO4. These variables selected by Plackett-Burman were then used in a Central Composite Rotational Design present activity of 26.6 U/mL and the predicted was 34.5 U/mL. So, the xylanase production by F. oxysporum can be optimize using corn straw, a low-cost waste found in large quantities. Keyword: xylanase, agroindustrial waste, enzymatic activity.


INTRODUCTION
Plant cell walls are composed of polysaccharides, lignins, proteins that together form a complex structure composed of different arrangements and interconnections forming a polysaccharide block, these arrangements and the interconnections of structural polysaccharides inside the cell walls are very restricted to enzyme access, explaining why these glycan compounds are recalcitrant to biological degradation [1].
Cellulose is the most challenging material to degrade and deconstruct into carbohydrate monomers. Hemicellulose is the second-largest component of the plant wall after cellulose. It comprises a group of heterogeneous polysaccharides, representing 20-35% of the total dry weight of the biomass, consisting essentially of a large fraction of xylan with a linear β-(1-4)-D-xylose skeleton [2,3]. The enzymatic hydrolysis of these lignocellulosic materials by cellulolytic and hemicellulolytic enzymes is the most promising approach to obtain high yields of products vital for economic success. For its complete degradation, hemicellulose mainly requires the enzymes endo-1,4-β-xylanase (EC 3.2.1.8) and 1,4-β-xylosidase (EC 3.2.1.37), where the first enzyme acts in the chain β(1,4) D-xylopyranose bond homopolymer for the production of xylooligomers and the second enzyme acts on these xylooligomers to release xylose. Importantly, xylanases (EC 3.2.1.8) belong to the glycoside hydrolases (GH) group (EC 3.2.1.-), based on their amino acid sequences (CAZy database) and are part of the GH families 5,7,8,9,10,11,12,16,26,30,43,44, 51 and 62 [3]. In addition to their importance in the cycling of nutrients in nature, xylanases are widely used commercially in processes of bioconversion of lignocellulosic material or agroindustrial residues, biobleaching in the paper industry, improvement of bread texture and volume, clarification of juice and wine, improvement of the nutritional value of animal feedstock, extraction of vegetable oil, coffee and starch [4].
Fusarium oxysporum is a saprophytic soil fungus and can infect and cause disease in a plant host, due to the fungus penetrating the vascular system and degrading its cell walls. Another feature of this fungus is that it can convert cellulose and hemicellulose into ethanol, from the enzymes it produces. In addition, F. oxysporum can produce alkaline xylanase, widely used in the paper and biofuel industries, as well as acidic xylanase applied in juice clarification and production of xylo-oligosaccharides that can be used in pharmaceutical products [5,6].
The production of xylanases is normally carried out by solid state fermentation (FES) and submerged fermentation (FSm) processes. FSm represents more than 90% of industrial enzyme production due to the ease of control during the aerobic fermentation process, ensuring greater availability of nutrients, oxygen supply, and less fermentation time when compared to other techniques [3,7]. However, the high cost of producing these enzymes makes their application difficult because the purified xylan substrate is extremely expensive, and an alternative to reduce the cost is the use of low-cost agricultural residues that are normally incorporated into the soil after harvesting, or are destined for animal feed, which can be used as carbon and nitrogen sources in the formulation of fermentation media [8]. Statistical analysis of the medium formulation helps to minimize the number of unused components at the end of fermentation, evaluating the effect of various components on enzyme production [3]. This study aimed to evaluate the production of xylanase by F. oxysporum in different residual agro-industrial biomass (soybean husk, cassava husk, pineapple crown, corn straw, rice husk, bacaba, barley bagasse, and corn cob) and estimate the number of enzymes produced from the best chemical composition of the culture medium under submerged conditions.

Microorganism
The strain of F. oxysporum isolated from decaying pequi fruits [9], was maintained in malt extract agar medium, at 4 o C, by Castellani method [10], at the Laboratory of Food Biotechnology and Purification of Bioproducts, University Campus of Gurupi, Federal University of Tocantins/TO. The stock culture was maintained on Vogel salts agar medium [11], containing wheat bran. The peals were performed periodically, in inclined tubes and petri dishes, kept at 30 °C for 7 days and later used or stored at 4 °C.
The conidia of the Fusarium oxysporum strain, stored in the LABAP-UFT, which were subcultured in slant tubes, were suspended at a concentration of 10 6 spores/mL in sterile saline (NaCl) 0.85%. One millilitre of this suspension was used to inoculate the liquid culture media.

Selection of carbon source for Xylanase production
Submerged cultures were carried out in 125 mL Erlenmeyers containing 20 mL of supplementary basal medium proposed by Mandels and Weber (1969) [12], composed of: pineapple crown, corn straw, rice husk, bacaba, barley bagasse, and corn cob. These were previously ground in knife mills and sieved with a maximum granulation of 1 mm. The cultures were carried out in three repetitions and incubated for 132 hours, 30 °C, and 180 rpm. After the incubation period, the fermented broth was separated from the biomass by filtration on filter paper with the aid of a Buchner funnel, kitassate and vacuum pump and then the fermented broth was used for activity analysis enzymatic.

Xylanolytic activity
The xylanolytic activity followed the Miller method (1959) [13], where it was determined from a 1% (w/v) xylan solution in McIlvaine 5.5 buffer. 0.4 mL of the solution was added to the test tubes. The tubes were kept for 5 min in a water bath at 50 °C. Test tubes containing 0.2 mL of 3,5-dinitrosalicylic acid (DNS) solution were prepared. 0.4 mL of the appropriately diluted enzymatic extract was added, this way the reaction was started, and 0.2 mL samples were removed at time intervals of 0 min and 5 min, adding immediately to the DNS solution. After completion of the reaction, the DNS was heated to 100 °C for 5 min. Waited to cool down and 2.0 mL of distilled water was added, and then the determination of reducing sugars was carried out, using xylose as a standard. Readings were taken in duplicates and read at an absorbance to 540 nm. Where a unit is defined as the amount of enzyme releasing 1 µmol of xylose per milliliter per minute of reaction. Enzyme activity is calculated with the following equation:

Placktt & Burman
The Plackett & Burman experimental design was used with 12 trials and three central points, with 15 runs, at three coded levels (-1; 0; +1) to select the significant variables. Multivariate experiments using appropriate statistical models will explain their effect on the fermentation process [14]. The independent variables tested were: KH2PO4 concentration; (NH4)2SO4; Urea; MgSO4.7H2O; CaCl2; meat peptone; yeast extract and Tween-80, presented in Table 2, which shows the coded variables and actual values.
The experiment was carried out in random order and the answers were used to adjust the linear and/or quadratic model and consequently the response surface. All results were evaluated at a significance level of 5%.

Composite central rotational design
Central Composite Rotational Design (CCRD), for two factors with 5 repetitions at the central point and axial points, was used in this investigation with a significance level of 5%. The variables used were Urea and MgSO4.7H20 and each in five coded levels (-1.41; -1; 0; +1; + 1.41), as shown in Table 3 [15].

Analysis of results
The results of the experimental planning were analyzed using STATISTICA software version 10, whose values were submitted to analysis of variance (ANAVA). Results were expressed as means of 3 independent experiments followed by standard deviation. The Scott-Knott test was applied to the averages, with a 5% significance level using the ASSISTAT computer system. The results were presented in the form of graphs and tables.

Selection of the best carbon source for xylanase production
F. oxysporum was cultivated on different carbon sources under submerged conditions to verify the production of xylanase. The carbon sources used were soybean tegument, cassava husk, pineapple crown, corn straw, rice straw, bacaba, barley bagasse and corn cob ( Table 1). The highest xylanase production (p<0.05) was obtained in the medium containing corn straw (12.57 U/mL), with a yield equal to 1306.94 U.g -1 and productivity equal to 2.0 U.g -1 .h -1 . Similar results were described by Pritsch et al. (2020) [16] using the Penicillium crustosum where it obtained a production of 25.45 U/mL and 19.35 U/mL in stationary and agitated culture conditions, respectively, showing that this carbon source is great for enzyme production. This fact can be explained by the fact that corn straw is a residue composed of a high concentration of hemicellulose (58%) and cellulose (41%) inducing the production of xylanase [17]. In addition to these substrates described above and used in the work, other agro-industrial residues, such as wheat bran [3] and sugarcane bagasse [18] are also potential sources for the xylanase production. In the present study, corn cob was not able to induce expressive results for production xylanase, however, corn straw showed potential for such purpose. Several factors are involved in the enzymatic production process, such as the composition of the medium, carbon source, pH value, temperature, presence of inducers or additives, aeration and growth time, these variables are considered of essential importance for the development of bioprocesses, because the increase in productivity is directly related to the optimization of the environment and the conditions of the entire production process [19,20]. Agroindustrial residues stand out as ideal substrates due to their organic richness, lower cost and being widely available, in addition to having a lower environmental impact when compared to pure substrates [21].
Thus, the choice of substrate is of great importance to obtain a high production of the xylanase. Therefore, corn straw was selected for the next step, due to its great ability to induce high amounts of the enzyme, being easy to obtain and having a low cost.

Plackett & burman
The next stage of the work consisted of conducting screening to determine the best chemical composition of the culture medium for xylanase production to investigate the influence of KH2PO4, (NH4)2SO4, urea, MgSO4.7H2O, CaCl2, meat peptone; yeast extract and Tween 80 in the production of the xylanase by F. oxysporum. The enzymatic activity values of such design are shows in Table 2. The significant independent variables (p<0.05) in inducing the production of the xylanase, under submerged conditions, 30 °C at 180 rpm are presented in the Pareto chart ( Figure 1).

Table 2 -Codified variables and real values of the Plackett & Burman design for the composition of the culture medium for xylanase production.
Independent variables encoded outside parentheses and real independent variables inside parentheses. Nota: Cthe central point.
As can be seen, the variables that were significant were urea and MgSO4.7H2O, where the first one had a positive effect, that is, the increase in the amount of this nitrogen source can increase the enzyme's productivity. On the other hand, the second had a negative effect, that is, the smaller the amount of salt used in the medium, the better for enzymatic activity. Thus, these variables were selected to be evaluated in the subsequent step, and the other variables that were not significant in the process were kept according to the basal medium proposed by Mandel and Weber (1969) [12]. According to the results, it is possible to see that the concentrations of nutrients in a medium significantly interfere in the production of xylanase. Each microorganism has its own nutritional requirement, being able to use macronutrients such as carbon, hydrogen, oxygen, phosphorus, potassium, nitrogen, sulfur, calcium and magnesium and micronutrients or trace elements such as iron, manganese, cobalt, copper and zinc, secreting a variety of different metabolites in their environment [3,22]. In the present study, several macronutrients had their concentrations studied, but that of micronutrients remained fixed since the amount of these trace elements was already proposed by Mandel and Weber (1969) [12] as being essential in secondary metabolism, for enzyme production to occur, as they are enzyme activators [3]. Some of these salts also stand out for being important cofactors for enzymes, which in turn can stimulate enzyme production, stabilizing their molecular structure or inhibiting them. As an example, there is potassium phosphate, responsible for the buffering of the medium, and MgSO4, which in the present work inhibited the production of the xylanase enzyme.
Although Tween 80 has not shown a significant effect, it is of great importance for enzyme production, as it facilitates access of the enzyme to the substrate, can increase thermal stability and positive interactions between substrates and enzymes, which justifies its use [23].

Composite central rotational desigh (CCRD)
The last step of the present study consisted of the CCRD experimental design, which is an important tool to determine the level of constituents in the medium and their interaction. Based on the results of Plackett & Burman, the variables urea and MgSO4 were used to assess their effects on the production of xylanase. The enzymatic activity values can be seen in Table 3.
The results presented in Table 3 showed that the concentrations of each factor it is noted that the highest enzymatic activity was obtained in assay 2 (26.6 U/mL), with high a yield (Yp/s) and productivity (Pp), in which the amount of MgSO4 used in the culture medium is greater than the amount of urea. Furthermore, it can be seen that in the central points and in tests, where the amount of urea was greater than the amount of MgSO4, it was observed lower values of enzymatic activity. In order to estimate the effects of each compound, and confirm the better influence of urea and MgSO4, already seen through the absolute values, the data in Table 3 were analyzed using the Statistica software, whose effects are shown in Table 4. Note that the factor corresponding to the linear effect of the urea variable had a negative effect, while the factor corresponding to the quadratic effect of the MgSO4 variable had a positive effect. Such effects were significant, at the 5% level, in the induction of enzyme production, under submerged conditions, 30 °C at 180 rpm. The regression model obtained to predict the enzymatic activity of xylanase as a function of urea (X1) and MgSO4 (X2) are represented by Equation 4, which predicts that the maximum activity according to the proposed CCRD is 34,523 U/mL. The reparametrized model contains only the statistically significant terms, with a confidence level of 95%, and represents the mathematical equation for optimal production of the enzyme From the results of the analysis of variance (ANAVA), the calculated F value for xylanase was 44.820, being greater than and the tabulated F value (2:10 = 4.103) (p<0.05), in the range of 95% reliable. Thus, the model can be considered statistically significant, according to the F test. In addition, the model presented a good coefficient of determination (R² = 0.802), demonstrating that the closer to 1, the greater the agreement between the experimental results and the theoretical values predicted by the equation. Only 34% of the observed results were not fitted to the model (R² = 0.659). To illustrate the effects of independent variables on xylanase production, a response surface graph (Figure 2) was made, generated from the quadratic mathematical model, which presented an optimal point at the lowest urea concentrations and highest concentrations of magnesium sulfate. Interesting reports on the interaction of magnesium sulfate and/or urea with the production of xylanase, seen in the present study, are also shown in other works. Cui and Zhao (2012) [24] carried out an optimization of the xylanase production from Penicillium sp.WX-Z and found that the enzyme production could be increased to 44 U/mL, when it increases the amount of MgSO4, in addition, the authors stated that Mg 2+ may have has a positive effect on the stabilization of the ribosome and cell membranes. When Mg 2+ is combined with Fe 2+ it plays an important role in xylanase production by Streptomyces sp. and when combined with Zn 2+ and Fe 2+ it can stimulate xylanase production by Streptomyces thermodiasticus.

CONCLUSION
The results obtained in this work demonstrated that corn straw, as a carbon source, is effective for the production of xylanase. In addition, it is a low-cost and sustainable source, being of paramount importance to minimize the cost of the enzyme and make the hydrolysis step cheaper, which is the most costly step in the biofuel production process. The Plackett-Burman experimental design helped to select the variables that significantly influenced the production of the enzyme, selecting urea and magnesium sulfate as the components with a significant effect on the production of xylanase. CCRD planning contributed to adjusting the culture medium requirements for the production of xylanase. Furthermore, the absolute activity obtained was equal to 26.57 U/mL and the maximum activity predicted by the CCRD was equal to 34.52 U/mL. The present work is of great importance, as it evaluated corn straw as being excellent for the production of xylanase from F. oxysporum, under submerged conditions, together with the concentrations of urea and magnesium sulfate, which is unprecedented.