Introduction
Hemicelluloses are polymeric carbohydrates containing polysaccharides such as xylan, arabinoxylan, xyloglucan, glucose, and mannose glycans. After cellulose, hemicelluloses are the second most abundant fraction in nature (
Selinheimo et al., 2006). Xylan is a complex polysaccharide consisting of a backbone of β-1,4-d-xylopyranose residues with arabinofuranose, glucuronic acid side chains (
Wong et al., 1988;
Coughlan and Hazlewood, 1993), and acetyl groups. Xylans are widely distributed in nature, and occur in bacteria, fungi, snails, marine algae, and terrestrial plant tissue. They constitute the major portion of plant cell wall hemicelluloses, accounting for approximately 15%-30% and 7%-12% of the total dry biomass in angiosperms and gymnosperms, respectively.
Xylanase (EC3.2.1.8) is an enzyme that hydrolyzes xylan, and is widely used in animal feed digestion (
Bastawde, 1992), food industries (
Silversides et al., 2006), and brewing science. For example, xylanases can break down the arabinoxylans present in feed ingredients, thereby reducing the viscosity of the raw material for animal feed. Similar to other hemicellulases, xylanases break down hemicelluloses present in wheat flour, helping in the redistribution of water and leaving the dough softer and easier to knead (
Polizeli et al., 2005). Recently, many xylanases have been isolated and characterized from fungi and bacteria and used for application in the abovementioned fields. Feed conversion ratio and pulp bleaching had been improved by xylanase; however, the low enzyme activity of xylanase has become the main problem in the industrialized application of this enzyme.
The objective of the present study was to apply Plackett-Burman and response surface methodology to optimize the fermentation parameters such as medium composition, initial pH, percentage inoculum, substrate concentration, and cultivation time to improve xylanase activity. The Box-Wilson central composite design was employed to evaluate the correctness in a quadratic mathematical model, to provide the basis for industrial production and application of xylanase.
Materials and methods
Strain, media, and cultivation
The xylanase-producing strain Xw2 (The Xw2 strain was isolated from 50 soil samples by hydrolysis circle and transparent circle.The branching pattern was generated by neighbor-joining method, the homology of xylanase producting strain Xw2 and Trichoderma harzianum (HQ596967.1) up to 94%,which indicates that Xw2 belongs to Trichoderma)was maintained in the laboratory of Fujian Normal University. The medium used for xylanase production was composed of 1% wheat bran, 0.5% yeast extract, 0.05% MgSO4·7H2O, 0.1% K2HPO4·3H2O, 0.5% NaCl, and 0.5% (NH4)2SO4. The microorganisms were cultured in 50 mL of medium in 250-mL Erlenmeyer flasks. After inoculation, the flasks were incubated on a rotary shaker at 28°C with shaking at 150 rpm.
Enzyme assays
Xylan solution was added to sodium dihydrogen phosphate-citrate buffer solution (pH 5.0) and incubated for 5 min (
Uysal et al., 2007). The assay mixture contained 0.5 mL 1g/mL substrate and 0.5 mL 10mg/g enzyme solution suitably diluted in buffer. This mixture was incubated at 50°C for 10 min and the reaction was stopped by addition of 1.5 mL 1g/mL dinitrosalicylic acid (DNS) reagent followed by incubating the reaction mixture in boiling water for 15 min. One unit (U) of xylanase activity was expressed as 1 µmol of reducing sugar (xylose equivalent) released in 1 min (
Ding et al., 2004).
Plackett-Burman experimental design
The Plackett-Burman experimental design was used to screen the important variables that influence xylanase production (
Li et al., 2007). According to the Plackett-Burman experimental design, a total of 12 trials were required, there are 8 factor of viable and 3 factor of error analysis items (Table 1).
Central composite design
Response surface methodology (RSM) was used to optimize the fermentation parameters for enhancing xylanase production (
Li et al., 2007). RSM has four steps: procedures to move into the optimum region, behavior of the response in the optimum region, estimation of the optimal condition, and verification (
Tanyildizi et al., 2005). We used the single-factor method to determine the center of the significant factors (compound nitrogen source, inoculation, and fermentation time).
Box-Behnken design
A response surface is a graph of a response variable as a function of variable factors. The typical meta-model used in a simulation application is a regression model. With the aid of RSM, a meta-model can be used to determine the optimal values for a set of factors. The results of the Plackett-Burman and central composite designs were used to determine three levels for each meaningful factor, which were then evaluated according to the Box-Behnken experimental design. The experimental results were then analyzed by SAS version 9.0, and a multivariate quadratic regression equation was obtained. The regression equation was used to map the response surface stereo analysis diagram to ascertain the best level and to determine the optimum culture conditions.
Statistics
Data are presented as means±SE. The significance of differences in mean values within and between multiple groups was evaluated using a one-way ANOVA, followed by a Tukey’s multiple range test. Student’s t-test was used to evaluate statistical significance of differences between two groups. P<0.05 was considered statistically significant.
Results
Optimization of the Xw2 strain fermentation conditions by the Plackett-Burman experimental design
Twelve experimental runs with different combinations of 11 factors were carried out (Table 2). The P values of compound nitrogen source concentration, amount of inoculum, and fermentation time (Pr>F) were<0.1, suggesting that these three were the main factors affecting xylanase production, and their response values are very significant. Treatment run 2 showed a high level of xylanase production based on enzyme activity at 327.28 IU/m. The response values of the concentration of compound nitrogen source, percentage of inoculum, and fermentation time were found to be significant at (Pr>F) at 7.02, 4.11, and 1.43 × 10-2, respectively. An R2 value of>0.9 was considered to denote a very strong correlation, and the R2 value of the regression model was 0.9480. Under the condition of the fit of the equation, other factors can be neglected.
Optimization of fermentation using the Box-Behnken design
Compound nitrogen source.
Various concentrations of yeast extract+ (NH4)2SO4 (1:1) (0.5%, 0.55%, 0.6%, 0.65%, 0.7%, and 0.75%) were used as the nitrogen source in the fermentation medium. The effect of different composite nitrogen concentrations on xylanase production by strain Xw2 was detected as shown in Fig. 1(A). Maximum activity of 303.32 IU/mL was achieved when 0.6% yeast extract+ (NH4)2SO4 (1:1) was used in the medium.
Amount of inoculum.
The effect of various percentages of inoculum (5%, 6%, 7%, 8%, 9%, and 10%) on xylanase production from strain Xw2 was detected as shown in Fig. 1(B). While inoculum percentages of 5, 6, and 7% had little effect on enzyme production, 5% inoculum resulted in higher enzyme activity.
Fermentation time.
The inoculum was added to the fermentation broth and incubated for 39 h. The enzyme activity of fermentation was measured every 3 h. The effect of fermentation time on xylanase production by strain Xw2 was detected as shown in Fig. 2. The results showed that enzyme activity peaked at 54 h, and started to decline thereafter.
Regression models of response
Knowledge obtained from a study was necessary for achieving a more realistic model in this method. Table 3 shows the maximum and minimum levels of variables chosen for trials in the central composite design. Fifteen experimental runs with unique combinations of the three factors were carried out in the case of RSM based on the Box-Wilson design, which was used for the optimization of cultivation conditions for the xylanase production (Table 4). Treatment runs 13-15 showed a high level of xylanase production based on the enzyme activity at 362.83, 358.83, and 351.15 U/mL, respectively. The maximum xylanase production was achieved in run 13, and the minimum xylanase production (286.20 U/mL) was observed in run 11.
By applying multiple regression analysis on the experimental data, the following second order polynomial equation was found to explain the xylanase production regardless of the significance of coefficients:
Where Y is the predicted response (xylanase production) and X1, X2, and X3 are the coded values denoting composite nitrogen, percentage of inoculum, and fermentation time, respectively.
The F test was used to detect statistical significance, and the analysis of variance (ANOVA) for the response surface quadratic model is shown in Table 5. It is evident that the model was highly significant, as suggested by the model F value and a low probability value (P model>F = 0.0156). Usually, the higher the CV (coefficient of variation) value, the lower would be the reliability of the experiment. A lower CV value (3.322%) indicated better precision and reliability of the experiments. At the same time, the R value (0.8248) indicated close agreement between the experimental results and the theoretical values predicted by the model equation. Therefore, the quadratic model was selected in this optimization study.
Student’s t distribution and the corresponding P value, along with the parameter estimate, are presented in Table 6. The P values were used to check the significance of each of the coefficients, which in turn are necessary to understand the pattern of mutual interactions between the best variables. The parameter estimates and the corresponding P values indicated that, among the independent variables, X1, X2, and X3 significantly affected xylanase production in strain Xw2.
Analyze and determine of optimum condition for enzyme production
The three-dimensional (3D) response surface plots described by the regression model were drawn to illustrate the effects of the independent variables and the interactive effects of each independent variable on the response variables. The shape of the corresponding contour plots indicates whether or not the reciprocal interactions between the independent variables are significant. Figure 3 depicts the 3D plot and its corresponding contour plot, showing the effects of complex nitrogen source concentration, percentage inoculum, and fermentation time on xylanase production. Figure 3(A) shows that the percentage of nitrogen and percentage of inoculum affected xylanase production, while cultivation time was fixed at its middle level (540 h). When the percentage of inoculum was 5%, the xylanase production significantly increased with increasing in the percentage of nitrogen and fermentation time, as shown in Fig. 3(B). At 0.6% composite nitrogen, xylanase production significantly increased with increasing in the percentage of inoculum beyond the initial value, as shown in Fig. 3(C). From the drawings and software analysis, regression equation was found to attain a stable point, which is the maximum value of the principal factors (X1= 0.12661, X2= -0.17733, and X3= -0.0756). According to the formula: X= intermediate coding -step × maximal corresponding to the encoded value. The formula had the following critical values: X1, 0.594%; X2, 5.177%; and X3, 54.23 h, the predicted xylanase activity for these conditions was 358.86 IU/mL.
Validation of the model
The statistical optimal values of the variables were obtained when moving along the major and minor axis of the contour, and the response at the center point yielded maximum xylanase production. These observations were also verified from canonical analysis of the response surface. The canonical analysis revealed a minimum region for the model. The stationary point with maximum xylanase activity had the following critical values: complex nitrogen source, 0.6%; inoculum, 5.0%; and fermentation time, 54.23 h. The predicted xylanase activity for these conditions was 358.86 IU/mL.
Discussion
The Plackett-Burman experimental design is a multifactorial two-level experimental design which is used to filter the main factors affecting the experiment (
Francis et al., 2003). In this study, we used RSM based on the Box-Wilson design to optimize the cultivation conditions for xylanase production. The relationship between the factors and the response can be fitted by a quadratic regression equation, so as to determine the best level and cultivating conditions for optimum xylanase production.
Xylanases have a wide range of potential biotechnological applications such as in feed and food industries (
Subramaniyan and Ptema, 2002). The present study intended to apply the Plackett-Burman design and RSM to optimize the fermentation parameters for the xylanase-producing strain Xw2. The effects of the following eight variables on the production of xylanase were determined using the Plackett-Burman design: corn cob+ wheat bran (1:1), yeast extract+ (NH
4)
2SO
4 (1:1), MnSO
4, K
2HPO
4, initial pH, inoculum, liquid volume, and fermentation time. Three factors, namely, compound nitrogen source, inoculation, and fermentation time, were found to be significant by central composite design. Finally, a medium containing corn cob+ 1.5% wheat bran (1:1), yeast extract+ 0.6% (NH
4)
2SO
4 (1:1), 0.04% MnSO
4, 0.04% K
2HPO
4, at an initial pH of 6.0; 5.0% inoculum; and incubation conditions of 28°C, 150 rpm, and 54.23 h were determined as the optimal fermentation conditions for strain Xw2. Rezende et al. (2002) reported the enzyme activity of
trichoderma viride strain up to 288 IU/mL after optimization of fermentation medium. the enzyme activity up to 288 U/mL. Yue et al. (2011) reported the enzyme activity of a xylanase producing bacteria (
Pestalotiopsis) up to 49.3 IU/mL. Under these conditions, the strain exhibited an obvious improvement in the xylanase production at 358.86 IU/mL compared with that achieved by the fermentation of the other xylanase strains.
In fact, the yield of xylanase production is very low,such as
Penicillium purpurogenum (10.4 U/mL),
Penicillium herquei (3.5 U/mL). So, the substrate was mainly restricted to purified high-cost xylan, limiting the production of xylanases in the industry scale.Thus, the optimization of the fermentation conditions for strain Xw2 provides the basis for the industrial production and application of xylanase. High activity of Xw2 strain is beneficial to lower cost, increase production, so as to obtain greater economic benefits. Nevertheless, xylanase has some limitations, for example, its weak thermal stability, which poses a major obstacle in production (
Panbangred et al., 1983). In the future, studies should implement genetic engineering technology, such as site-directed mutagenesis and molecular cloning, to improve the thermal stability of xylanase, and consequently improve its ease of preparation and scope of application.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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