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Optimization of thermostable and alkaline-tolerant cellulase-free xylanase production from agricultural waste by thermotolerant Streptomyces sp. Ab106, using the central composite experimental design
Biochemical Engineering Journal 12 (2002) 99–105
Optimization of thermostable and alkaline-tolerant cellulase-free xylanase production from agricultural waste by thermotolerant Streptomyces sp. Ab106, using the central composite experimental design Charin Techapun a , Thanakorn Charoenrat a , Masanori Watanabe b , Ken Sasaki b,* , Naiyatat Poosaran a a b
Department of Biotechnology, Faculty of Agro-Industry, Chiangmai University, Chiangmai 50100, Thailand Materials Science and Engineering, Graduate School of Engineering, Hiroshima Kokusai Gakuin University, 6-20-1, Nakano, Akiku, Hiroshima 739-0321, Japan Received 27 December 2001; accepted after revision 18 March 2002
Abstract Cellulase-free xylanase was produced by Streptomyces sp. Ab106 from the agricultural waste cane bagasse. The effect of external factors pH and temperature on the xylanase production was studied and the optimization by using the central composite experimental design was investigated. The effects of pH and temperature were significant for the xylanase production. A second-order quadratic model and a response surface method showed that the optimum condition for xylanase production was 50 ◦ C and pH 7. The maximum yield of xylanase was about 15 IU without cellulase and manannase activities. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cellulase-free xylanase; Central composite design; Enzyme production; Modeling; Optimization; Thermophiles
1. Introduction Toxic wastes from pulp bleaching industries come from chemical substances used in the bleaching processes [1–3]. Therefore, biological bleaching of pulp mainly uses non-toxic hemicellulolytic enzymes [4,5]. Due to the high temperature and alkalinity of bleaching enzymes at alkaline pH 9–10 and at a high temperature (60–90 ◦ C) are needed [3,5]. The commercial enzyme xylanases have not yet met these requirements in Thailand. Newly isolated thermotolerant Streptomyces sp. Ab106 can produce thermostable xylanase free of cellulolytic and manannase activities at high temperature (55 ◦ C) [6]. This Streptomyces produced alkaline-stable xylanase of more than 70% activity that remained at pH 9.0 at a high temperature (60 ◦ C). However, the real suitable cultural condition for xylanase production was not investigated. Srinivasan et al. [7] used the central composite experimental design for optimization of cell growth by using pH and temperature as the main conditions of ectomycorrhizal fungus growth. However, this method has not been used for enzyme production so far. ∗ Corresponding author. Tel.: +81-82-820-2570; fax: +81-82-820-2560. E-mail address: [email protected] (K. Sasaki).
In this study, the Box–Wilson central composite design (CCD), commonly called a “CCD” was used to evaluate the coefficients in a quadratic mathematical model. Also, a response surface method was used to predict the optimum temperature and pH of xylanase production.
2. Materials and methods 2.1. Microorganism and media Streptomyces sp. Ab106 was used, which was isolated from the soil of gold teak forest in Chiangmai University, Thailand, and was selected as an effective thermotolerant cellulase-free xylanase producer [6]. A modified basal medium comprised KH2 PO4 (1.5 g l−1 ), K2 HPO4 (2 g l−1 ), (NH4 )2 SO4 (4.5 g l−1 ), yeast extract (0.075 g l−1 ), peptone (0.075 g l−1 ), Tween 80 (0.075 ml l−1 ) and a trace element solution (2.7 ml l−1 ) that comprised ZnSO4 ·7H2 O (140 mg l−1 ), MnSO4 ·H2 O (160 mg l−1 ), FeSO4 ·7H2 O (500 mg l−1 ) and CoCl2 ·2H2 O (200 mg l−1 ) in distilled water. The pH was adjusted to 3.0–3.5 by 0.1 N HCl. The agricultural waste cane bagasse (10 g l−1 ) was added to the medium [6].
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An inoculum was prepared by use of the basal medium supplemented with 0.1% (w/v) oat splet xylan, pH 7.0 and was incubated at 55 ◦ C in a 1000 ml Erlenmeyer flask (250 ml medium) on a reciprocal shaker (100 rpm) until the mid-log phase (1–2 days). The amount of seed culture in each culture flask was maintained at 5% (v/v) [6]. 2.2. Enzyme activities and cell growth measurement The activities of enzymes cellulase, manannase and xylanase were measured as described in our previous paper [6]. Cell growth in culture broth was estimated by using the indirect method that measures the total releasable soluble protein by using the Bradford reagent. The culture broth (10 ml) was centrifuged at 8000×g for 10 min at 44 ◦ C. Harvested pellets were re-suspended with distilled water (5 ml) and boiled with 15 ml of 1 M NaOH for 20 min and was centrifuged at 8000 × g for 10 min at 4 ◦ C. The protein was estimated by using the Bradford method [8]. One milligram of protein solubilized for each milliliter corresponded to about 7.5 g l−1 of cell growth measured with the soluble substrate oat splet xylan. 2.3. Experimental design Generally, a prior knowledge of the procedure is needed to achieve a statistical model [9,10]. The three steps of this experimental design included doing statistically designed experiments, estimating the coefficients in a mathematical model and predicting the response, and checking the applicability of the model. CCD was used to find the optimum pH and temperature of xylanase production. The CCD contained an imbedded factorial or fractional factorial matrix with center points and “star points” around the center point that allow estimation of the curvature (Fig. 1) [9]. If the distance from the center of the design space to a factorial point is ±1 unit for each factor, the distance from the center of the design space to a star point is ±α, where |α| > 1. The precise value of α depends on certain properties needed for the design and on the number of factors that are used. Similarly, the number of center point runs that the design must contain also depends on certain properties required for the design. A CCD always contains twice as many star points as factors in the design. The star points represent new extreme values (low and high) for each factor in this design (Fig. 1). To maintain rotatability, the value of α depends on the number of experimental runs in the factorial portion of the CCD. If the factorial is a full factorial then α = [2k ]1/4
(1)
In this study k = 2 factors (pH and temperature). Eq. (1) can be written as: α = 1.414
(2)
The pH and temperature were the most important conditions for xylanase production [6]. Table 1 shows maximum and minimum values of the variables pH and temperature,
Fig. 1. General diagram of a CCD for two factors. CCD contains an imbedded factorial matrix with center points added to a group of star points, ±α, where |α| > 1.
chosen for trials in this study. Xylanase is produced at pH 4.5–8.5 [5,11,12]. Temperature of 65 ◦ C is the maximum temperature for the growth of our Streptomyces strain [6]. Table 1 shows the estimations of the CCD for the pH, and the temperature was estimated. 2.4. Enzyme production experiments Cane bagasse (10 g l−1 ) was added to the basal medium (250 ml in 1000 ml Erlenmeyer flasks) and was adjusted to a variety of pH (Table 2). The inoculum (5%, v/v) was used, and was incubated at various temperatures in a reciprocal shaker at 100 rpm as described above. Three sample flasks of each experimental design treatment were removed after 5 days. Then the activities of cellulase, manannase and xylanase were assayed. 2.5. Quadratic model analysis The variables were pH and temperature and the responses were of the xylanase activity. A second-degree quadratic Table 1 Maximum and minimum levels of pH and temperature used in the central composite experimental design Independent variable
X1 , pH X2 , temperature (◦ C)
Level −α
−1
0
+1
+α
3.6 28
4.5 35
6.5 50
8.5 65
9.3 71
C. Techapun et al. / Biochemical Engineering Journal 12 (2002) 99–105 Table 2 The CCD for the two independent variables, factors 1 and 2
101
Table 4 Least squares linear fit of the variable estimates
Treatment
Factor 1
Factor 2
Temperature
pH
1 2 3 4 5 6 7 8 9 10 11
−1 −1 1 1 −1.414 1.414 0 0 0 0 0
−1 1 −1 1 0 0 −1.414 1.414 0 0 0
35 35 65 65 28 71 50 50 50 50 50
4.5 8.5 4.5 8.5 6.5 6.5 3.6 9.6 6.5 6.5 6.5
Variable
Variable estimate
Intercept X1 X2 X12 X22 X 1 X2
13.87 2.94 −1.10 −4.23 −7.05 −1.53
Degree of freedom 1 1 1 1 1 1
Computation (T) 10.52 3.64 −1.36 −4.41 −7.34 −1.34
P >t
0.0149 0.2317 0.0070 0.0070 0.2390
3. Results and discussion 3.1. Estimation of the coefficients in a mathematical model and model design
model [9,10] was established as Eq. (3) by using the method of least squares as follows: Y = b0 + b1 X1 + b2 X2 + b12 X1 X2 +b11 X12 + b22 X22
(3)
where Y is the predicted response (xylanase yield); X1 and X2 the coded forms of the input variables (pH and temperature, respectively); b0 a constant; b1 and b2 the linear coefficients; b12 a cross-product coefficient; b11 and b22 the quadratic coefficients. The relation between the coded forms of the input variable and the actual value of pH and temperature are described as Eq. (4): Xi =
(Ai − A∗0 ) A
(4)
where Xi is a coded value and Ai the actual value of temperature or pH, A∗0 the actual value of the same variable at the center point, A the step change of the variable. Table 2 shows the design matrix of the variables in both coded and uncoded values (e.g., coded pH, +1.0 is equal to pH 8.5).
The CCD was predicted to obtain the possible conditions for xylanase production from Streptomyces sp. Ab106. Eleven treatments were established by using computer simulation with Eq. (4) (Table 2). Streptomyces sp. Ab106 was cultured in all 11 treatments to obtain xylanase for 5 days in the shaker flask. Treatments nos. 8–11 showed a high level of xylanase activity (Fig. 2) with amounts of enzyme activity at 12.3, 14.8, 13.4 and 13.4 IU, respectively. Treatments nos. 9–11 were the same experiments under the same conditions. This result suggests a data deviation and accuracy of our flask experiments, and the Streptomyces sp. Ab106 tends to produce xylanase at a relatively high temperature and in neutral or alkaline conditions. Cellulase and mannanase were not detected in all experiments. 3.2. Model analysis to estimate the coefficients The “Sigma Stat” program (Version 3.0, Jandel Scientific GmbH, Erkrath, Germany) was used to find out the quadratic mathematical model (Eq. (4)), which can be given as
Table 3 Least squares linear regression analysis for the optimum pH and temperature for xylanase production (quadratic model) Source
Sum of squares
The first model Model error 397.6 Residual error 26.0 Total 386.60 Coefficient of correlation (R2 ), 0.939 Coefficient of determination (adjusted R2 ), 0.877 Coefficient of variation, 6.2% Adjusted model Model error 388.3 Residual error 35.3 Total 423.6 Coefficient of correlation (R2 ), 0.917 Coefficient of determination (adjusted R2 ), 0.861 Coefficient of variation, 5.6%
Degree of freedom
Mean squares
F-Value (P < 0.05)
5 5 10
79.52 5.21
15.3 (P = 0.044)
4 6 10
97.07 5.89 42.36
16.5 (P = 0.0022)
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Fig. 2. Xylanase production from each condition simulated by the CCD. Xylanase activities were assayed after 5 days of cultivation.
Eq. (5): Y = 13.9 + 2.94X1 − 1.10X2 − 4.23X12 −7.05X22 − 1.53X1 X2
(5)
where Y is the predicted xylanase yield, and X1 and X2 the coded variables of pH and temperature, respectively. Tables 3 and 4 summarize the analysis of variance for the model and the pH and temperature.
Fig. 3. Xylanase production shown by three-dimensional graphics for a quadratic response surface optimization compared with pH and temperature. The quadratic model used response surface methodology to predict the best point for xylanase production. Average values of xylanase production from three cultures are shown.
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103
Fig. 4. Improvement of xylanase production with thermotolerant Streptomyces sp. Ab106. Ab106 was cultured in a modified medium at various temperatures between 35 and 65 ◦ C at pH 7.0. Xylanase production: 䊉, 35 ◦ C; 䊏, 45 ◦ C; 䉱, 55 ◦ C; 䉬, 60 ◦ C; 䉲, 65 ◦ C. Cell growth: 䊊, 35 ◦ C; 䊐, 45 ◦ C; , 55 ◦ C; 䉫, 60 ◦ C; , 65 ◦ C.
In this study, the computed F-value (15.3) in Table 3 was near the F-value in statistic table [9], indicating that the model was significant at a high confidence level. The probability P-value was also relatively low (P < 0.05), indicating the significance of the model. The coefficient of variation (R 2 = 0.939) indicates a high correlation between the experimentally observed and predicted values and indicates the degree of precision with which
the treatments are compared. The lower value of coefficient of variation (6.2%) was the greatest of the experiments. Table 4 shows the Student’s t-distribution and corresponding values of the variable estimation. The P-values were used to check the significance of each coefficient. The low values of P of less than 0.05 indicate the more significant correlation of coefficients.
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The independent variables temperature and pH (X1 , X12 , had a significant effect on the xylanase production (Table 4). Interactions between temperature and pH (X1 X2 ) had a low significance (P > 0.05). Thus pH and temperature did not interact with each other (e.g., pH did not change when the temperature changed). The quadratic model was reduced to Eq. (6):
albus [5] that produces 12 IU of xylanase in a xylan medium at 30 ◦ C at pH 7.5 after 5 days and Streptomyces cuspidosporous [12] that produces 18 IU in a xylan medium at 37 ◦ C at pH 7.5 after 5 days. The enzyme production in this study with the agricultural waste cane bagasse was comparable with that of the other actinomycetes.
Y = 13.9 + 2.94X1 − 1.10X2 − 4.23X12 − 7.05X22
3.4. Applicability
X22 )
(6)
This modified model indicates a high correlation between the experimentally observed and predicted values as same as the first model (R 2 > 0.9) with the value of coefficient of variation (5.6%) lower than the first model (Table 3). Furthermore, the probability P-value (P = 0.0022) was also relatively lower than the first model (P = 0.044), indicating the more significance of the second model. 3.3. Optimization of pH and temperature in xylanase production To investigate the effects of pH and temperature on xylanase production, the three-dimensional contour plot (Sigma Plot, Version 5.0, Jandel Scientific GmbH, Erkrath, Germany) was used to assess the effects of pH and temperature on the xylanase production (Fig. 3). A maximum xylanase production of 14.3 IU was estimated at pH 7.0 and temperature 50 ◦ C. Streptomyces sp. Ab106 clearly tended to produce xylanase at the high temperature of 50 ◦ C. Many studies on cellulase-free xylanase production have been mainly of Bacillus sp. and Cellulomonas sp. [1,13–15] with several studies of Streptomyces sp. [3,5,12]. Also, most strains producing cellulase-free xylanase by Streptomyces sp. are mesophilic microorganisms, such as Streptomyces sp. QG113 [3] that produces 7.5 IU of xylanase in a wheat bran medium at 37 ◦ C at pH 8.0 after 5 days, Streptomyces
To confirm the applicability of the model, xylanase production by Streptomyces sp. Ab106 was cultured at various temperatures from 35 to 65 ◦ C at a constant pH 7.0. Fig. 4 shows the experimental results of its growth and enzyme productions. Growth was the greatest at the lowest temperature 35 ◦ C and declined with increase in temperature. But medium temperatures 45 and 55 ◦ C induced greatest xylanase production, instead of lower cell mass production (compare results between 35, 45 and 55 ◦ C). As shown in Fig. 5, the quadratic model that uses Eq. (6) seem to fit with actual experiments of xylanase production by Streptomyces sp. Ab106. When the additional experimental data cultured under various temperatures at constant pH 7.0 after 5 days were plotted. At the constant pH 7.0, we assume that xylanase production was dependent on the changes of temperature variable. The quadratic (polynomial) equation of simulated curve and real data curve (28 plotted) in Fig. 5 are calculated as Eqs. (7) and (8), respectively: Y = −61.88 + 3.125T − 0.032T 2 , R 2 = 0.9987, P < 0.0001
(7)
Y = −82.69 + 4.124T − 0.043T 2 , R 2 = 0.9670, P < 0.0021
(8)
Fig. 5. Response linear plot of simulated data and the actual experimental data. Xylanase production at various temperatures at pH 7.0 was tested for the accuracy and precision of the model fit. (—) Xylanase simulated by the model, (—䊏—) xylanase from the experiments after 5 days culture.
C. Techapun et al. / Biochemical Engineering Journal 12 (2002) 99–105
where Y is the xylanase yield (IU) and T the actual value of temperature (◦ C). The t-test statistical method was used to find a correlation or fit of a curve. It was found that both curve have a correlation or fit of a curve in the same direction with a high confidence at 95% interval, t = 0.4322. These results suggest the possibility of applicability of the optimization.
4. Conclusions We conclude that the CCD, regression analysis and response surface method was effective to find the optimum conditions of pH and temperature for xylanase production. The optimum condition was estimated as 50 ◦ C at pH 7.0. This optimization experiment showed that Streptomyces sp. Ab106 produces a higher xylanase activity at these conditions. Cellulase-free xylanase is produced from Streptomyces sp. Ab106 with xylanase activity higher than all other mesophilic and thermophilic actinomycetes reported so far. This microorganism can grow easily at low cost and with simple substrates, such as cane bagasse. References [1] A. Gessesse, G. Mamo, High-level xylanase production by alkaliphilic Bacillus sp. by using solid-state fermentation, Microbiol. Technol. 25 (1999) 68–72. [2] M. Tuncer, A.S. Ball, A. Rob, M.T. Wilson, Optimization of extracellular lignocellulolytic enzyme production by a thermophilic actinomycete Thermomonospora fusca BD25, Enzyme Microbiol. Technol. 25 (1999) 38–47. [3] Q.K. Beg, B. Bharat, K. Mukesh, G.S. Hoondal, Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and
[4]
[5]
[6]
[7]
[8]
[9] [10] [11]
[12]
[13]
[14]
[15]
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its application in biobleaching of eucalyptus kraft pulp, Enzyme Microbiol. Technol. 27 (2000) 459–466. D.V. Gokhale, S.G. Patil, K.B. Bastawde, Potential application of yeast cellulase-free xylanase in agrowaste material treatment to remove hemicellulase fractions, Bioresource Technol. 63 (1998) 187– 191. V.T. Antanopoulos, M. Hernandez, M.E. Arias, E. Mavrakos, A.S. Ball, The use of extracellular enzymes from Streptomyces albus ATCC3005 for the bleaching of eucalyptus kraft pulp, Appl. Microbiol. Biotechnol. 57 (2000) 92–97. C. Techapun, S. Sinsuwongwat, M. Watanabe, N. Poosaran, K. Sasaki, Feasibility of cellulase-free xylanase production in agricultural waste materials by thermotolerant microorganisms, Biotechnol. Lett. 23 (2001) 1685–1698. M. Srinivasan, K. Natarajan, G. Nagarajan, Growth optimization of an ectomycorrhizal fungus with respect to pH and temperature in vitro, using design of experiments, Bioprocess Eng. 22 (2000) 267– 273. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. G.E.P. Box, K.B. Wilson, On the experimental attainment of optimum conditions, J. Roy. Stat. Soc. 13 (1951) 1–45. G.E.P. Box, W.G. Hunter, S.J. Hunter, Statistics for Experiments, Wiley, New York, 1978, 653 pp. U. Kohli, P. Nigam, D. Singh, K. Chaudhary, Thermostable alkalophilic and cellulase-free xylanase production by Thermomyces thalophilus subgroup C, Enzyme Microbiol. Technol. 28 (2001) 606– 610. M.U. Maheswari, T.S. Chandra, Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus, World J. Microbiol. Biotechnol. 16 (2000) 257–263. D.S. Rani, K. Nand, Production of thermostable cellulase-free xylanse by Clostridium absonum CFR-702, Process Biochem. 36 (2000) 355– 362. M.M. Hoq, C. Hempel, W.D. Deckwer, Cellulase-free xylanase by Thermomyces lanuginosus RT9 effect of agitation, aeration, and medium consumption, J. Biotechnol. 37 (1994) 49–58. H. Balakrishman, M.D. Choudhury, M.C. Srinivasan, M.V. Rele, Cellulase-free xylanase production from an alkalophilic Bacillus species, World J. Microbiol. Biotechnol. 8 (1992) 627–631.
Optimization of thermostable and alkaline-tolerant cellulase-free xylanase production from agricultural waste by thermotolerant Streptomyces sp. Ab106, using the central composite experimental design Charin Techapun a , Thanakorn Charoenrat a , Masanori Watanabe b , Ken Sasaki b,* , Naiyatat Poosaran a a b
Department of Biotechnology, Faculty of Agro-Industry, Chiangmai University, Chiangmai 50100, Thailand Materials Science and Engineering, Graduate School of Engineering, Hiroshima Kokusai Gakuin University, 6-20-1, Nakano, Akiku, Hiroshima 739-0321, Japan Received 27 December 2001; accepted after revision 18 March 2002
Abstract Cellulase-free xylanase was produced by Streptomyces sp. Ab106 from the agricultural waste cane bagasse. The effect of external factors pH and temperature on the xylanase production was studied and the optimization by using the central composite experimental design was investigated. The effects of pH and temperature were significant for the xylanase production. A second-order quadratic model and a response surface method showed that the optimum condition for xylanase production was 50 ◦ C and pH 7. The maximum yield of xylanase was about 15 IU without cellulase and manannase activities. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cellulase-free xylanase; Central composite design; Enzyme production; Modeling; Optimization; Thermophiles
1. Introduction Toxic wastes from pulp bleaching industries come from chemical substances used in the bleaching processes [1–3]. Therefore, biological bleaching of pulp mainly uses non-toxic hemicellulolytic enzymes [4,5]. Due to the high temperature and alkalinity of bleaching enzymes at alkaline pH 9–10 and at a high temperature (60–90 ◦ C) are needed [3,5]. The commercial enzyme xylanases have not yet met these requirements in Thailand. Newly isolated thermotolerant Streptomyces sp. Ab106 can produce thermostable xylanase free of cellulolytic and manannase activities at high temperature (55 ◦ C) [6]. This Streptomyces produced alkaline-stable xylanase of more than 70% activity that remained at pH 9.0 at a high temperature (60 ◦ C). However, the real suitable cultural condition for xylanase production was not investigated. Srinivasan et al. [7] used the central composite experimental design for optimization of cell growth by using pH and temperature as the main conditions of ectomycorrhizal fungus growth. However, this method has not been used for enzyme production so far. ∗ Corresponding author. Tel.: +81-82-820-2570; fax: +81-82-820-2560. E-mail address: [email protected] (K. Sasaki).
In this study, the Box–Wilson central composite design (CCD), commonly called a “CCD” was used to evaluate the coefficients in a quadratic mathematical model. Also, a response surface method was used to predict the optimum temperature and pH of xylanase production.
2. Materials and methods 2.1. Microorganism and media Streptomyces sp. Ab106 was used, which was isolated from the soil of gold teak forest in Chiangmai University, Thailand, and was selected as an effective thermotolerant cellulase-free xylanase producer [6]. A modified basal medium comprised KH2 PO4 (1.5 g l−1 ), K2 HPO4 (2 g l−1 ), (NH4 )2 SO4 (4.5 g l−1 ), yeast extract (0.075 g l−1 ), peptone (0.075 g l−1 ), Tween 80 (0.075 ml l−1 ) and a trace element solution (2.7 ml l−1 ) that comprised ZnSO4 ·7H2 O (140 mg l−1 ), MnSO4 ·H2 O (160 mg l−1 ), FeSO4 ·7H2 O (500 mg l−1 ) and CoCl2 ·2H2 O (200 mg l−1 ) in distilled water. The pH was adjusted to 3.0–3.5 by 0.1 N HCl. The agricultural waste cane bagasse (10 g l−1 ) was added to the medium [6].
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An inoculum was prepared by use of the basal medium supplemented with 0.1% (w/v) oat splet xylan, pH 7.0 and was incubated at 55 ◦ C in a 1000 ml Erlenmeyer flask (250 ml medium) on a reciprocal shaker (100 rpm) until the mid-log phase (1–2 days). The amount of seed culture in each culture flask was maintained at 5% (v/v) [6]. 2.2. Enzyme activities and cell growth measurement The activities of enzymes cellulase, manannase and xylanase were measured as described in our previous paper [6]. Cell growth in culture broth was estimated by using the indirect method that measures the total releasable soluble protein by using the Bradford reagent. The culture broth (10 ml) was centrifuged at 8000×g for 10 min at 44 ◦ C. Harvested pellets were re-suspended with distilled water (5 ml) and boiled with 15 ml of 1 M NaOH for 20 min and was centrifuged at 8000 × g for 10 min at 4 ◦ C. The protein was estimated by using the Bradford method [8]. One milligram of protein solubilized for each milliliter corresponded to about 7.5 g l−1 of cell growth measured with the soluble substrate oat splet xylan. 2.3. Experimental design Generally, a prior knowledge of the procedure is needed to achieve a statistical model [9,10]. The three steps of this experimental design included doing statistically designed experiments, estimating the coefficients in a mathematical model and predicting the response, and checking the applicability of the model. CCD was used to find the optimum pH and temperature of xylanase production. The CCD contained an imbedded factorial or fractional factorial matrix with center points and “star points” around the center point that allow estimation of the curvature (Fig. 1) [9]. If the distance from the center of the design space to a factorial point is ±1 unit for each factor, the distance from the center of the design space to a star point is ±α, where |α| > 1. The precise value of α depends on certain properties needed for the design and on the number of factors that are used. Similarly, the number of center point runs that the design must contain also depends on certain properties required for the design. A CCD always contains twice as many star points as factors in the design. The star points represent new extreme values (low and high) for each factor in this design (Fig. 1). To maintain rotatability, the value of α depends on the number of experimental runs in the factorial portion of the CCD. If the factorial is a full factorial then α = [2k ]1/4
(1)
In this study k = 2 factors (pH and temperature). Eq. (1) can be written as: α = 1.414
(2)
The pH and temperature were the most important conditions for xylanase production [6]. Table 1 shows maximum and minimum values of the variables pH and temperature,
Fig. 1. General diagram of a CCD for two factors. CCD contains an imbedded factorial matrix with center points added to a group of star points, ±α, where |α| > 1.
chosen for trials in this study. Xylanase is produced at pH 4.5–8.5 [5,11,12]. Temperature of 65 ◦ C is the maximum temperature for the growth of our Streptomyces strain [6]. Table 1 shows the estimations of the CCD for the pH, and the temperature was estimated. 2.4. Enzyme production experiments Cane bagasse (10 g l−1 ) was added to the basal medium (250 ml in 1000 ml Erlenmeyer flasks) and was adjusted to a variety of pH (Table 2). The inoculum (5%, v/v) was used, and was incubated at various temperatures in a reciprocal shaker at 100 rpm as described above. Three sample flasks of each experimental design treatment were removed after 5 days. Then the activities of cellulase, manannase and xylanase were assayed. 2.5. Quadratic model analysis The variables were pH and temperature and the responses were of the xylanase activity. A second-degree quadratic Table 1 Maximum and minimum levels of pH and temperature used in the central composite experimental design Independent variable
X1 , pH X2 , temperature (◦ C)
Level −α
−1
0
+1
+α
3.6 28
4.5 35
6.5 50
8.5 65
9.3 71
C. Techapun et al. / Biochemical Engineering Journal 12 (2002) 99–105 Table 2 The CCD for the two independent variables, factors 1 and 2
101
Table 4 Least squares linear fit of the variable estimates
Treatment
Factor 1
Factor 2
Temperature
pH
1 2 3 4 5 6 7 8 9 10 11
−1 −1 1 1 −1.414 1.414 0 0 0 0 0
−1 1 −1 1 0 0 −1.414 1.414 0 0 0
35 35 65 65 28 71 50 50 50 50 50
4.5 8.5 4.5 8.5 6.5 6.5 3.6 9.6 6.5 6.5 6.5
Variable
Variable estimate
Intercept X1 X2 X12 X22 X 1 X2
13.87 2.94 −1.10 −4.23 −7.05 −1.53
Degree of freedom 1 1 1 1 1 1
Computation (T) 10.52 3.64 −1.36 −4.41 −7.34 −1.34
P >t
0.0149 0.2317 0.0070 0.0070 0.2390
3. Results and discussion 3.1. Estimation of the coefficients in a mathematical model and model design
model [9,10] was established as Eq. (3) by using the method of least squares as follows: Y = b0 + b1 X1 + b2 X2 + b12 X1 X2 +b11 X12 + b22 X22
(3)
where Y is the predicted response (xylanase yield); X1 and X2 the coded forms of the input variables (pH and temperature, respectively); b0 a constant; b1 and b2 the linear coefficients; b12 a cross-product coefficient; b11 and b22 the quadratic coefficients. The relation between the coded forms of the input variable and the actual value of pH and temperature are described as Eq. (4): Xi =
(Ai − A∗0 ) A
(4)
where Xi is a coded value and Ai the actual value of temperature or pH, A∗0 the actual value of the same variable at the center point, A the step change of the variable. Table 2 shows the design matrix of the variables in both coded and uncoded values (e.g., coded pH, +1.0 is equal to pH 8.5).
The CCD was predicted to obtain the possible conditions for xylanase production from Streptomyces sp. Ab106. Eleven treatments were established by using computer simulation with Eq. (4) (Table 2). Streptomyces sp. Ab106 was cultured in all 11 treatments to obtain xylanase for 5 days in the shaker flask. Treatments nos. 8–11 showed a high level of xylanase activity (Fig. 2) with amounts of enzyme activity at 12.3, 14.8, 13.4 and 13.4 IU, respectively. Treatments nos. 9–11 were the same experiments under the same conditions. This result suggests a data deviation and accuracy of our flask experiments, and the Streptomyces sp. Ab106 tends to produce xylanase at a relatively high temperature and in neutral or alkaline conditions. Cellulase and mannanase were not detected in all experiments. 3.2. Model analysis to estimate the coefficients The “Sigma Stat” program (Version 3.0, Jandel Scientific GmbH, Erkrath, Germany) was used to find out the quadratic mathematical model (Eq. (4)), which can be given as
Table 3 Least squares linear regression analysis for the optimum pH and temperature for xylanase production (quadratic model) Source
Sum of squares
The first model Model error 397.6 Residual error 26.0 Total 386.60 Coefficient of correlation (R2 ), 0.939 Coefficient of determination (adjusted R2 ), 0.877 Coefficient of variation, 6.2% Adjusted model Model error 388.3 Residual error 35.3 Total 423.6 Coefficient of correlation (R2 ), 0.917 Coefficient of determination (adjusted R2 ), 0.861 Coefficient of variation, 5.6%
Degree of freedom
Mean squares
F-Value (P < 0.05)
5 5 10
79.52 5.21
15.3 (P = 0.044)
4 6 10
97.07 5.89 42.36
16.5 (P = 0.0022)
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Fig. 2. Xylanase production from each condition simulated by the CCD. Xylanase activities were assayed after 5 days of cultivation.
Eq. (5): Y = 13.9 + 2.94X1 − 1.10X2 − 4.23X12 −7.05X22 − 1.53X1 X2
(5)
where Y is the predicted xylanase yield, and X1 and X2 the coded variables of pH and temperature, respectively. Tables 3 and 4 summarize the analysis of variance for the model and the pH and temperature.
Fig. 3. Xylanase production shown by three-dimensional graphics for a quadratic response surface optimization compared with pH and temperature. The quadratic model used response surface methodology to predict the best point for xylanase production. Average values of xylanase production from three cultures are shown.
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Fig. 4. Improvement of xylanase production with thermotolerant Streptomyces sp. Ab106. Ab106 was cultured in a modified medium at various temperatures between 35 and 65 ◦ C at pH 7.0. Xylanase production: 䊉, 35 ◦ C; 䊏, 45 ◦ C; 䉱, 55 ◦ C; 䉬, 60 ◦ C; 䉲, 65 ◦ C. Cell growth: 䊊, 35 ◦ C; 䊐, 45 ◦ C; , 55 ◦ C; 䉫, 60 ◦ C; , 65 ◦ C.
In this study, the computed F-value (15.3) in Table 3 was near the F-value in statistic table [9], indicating that the model was significant at a high confidence level. The probability P-value was also relatively low (P < 0.05), indicating the significance of the model. The coefficient of variation (R 2 = 0.939) indicates a high correlation between the experimentally observed and predicted values and indicates the degree of precision with which
the treatments are compared. The lower value of coefficient of variation (6.2%) was the greatest of the experiments. Table 4 shows the Student’s t-distribution and corresponding values of the variable estimation. The P-values were used to check the significance of each coefficient. The low values of P of less than 0.05 indicate the more significant correlation of coefficients.
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The independent variables temperature and pH (X1 , X12 , had a significant effect on the xylanase production (Table 4). Interactions between temperature and pH (X1 X2 ) had a low significance (P > 0.05). Thus pH and temperature did not interact with each other (e.g., pH did not change when the temperature changed). The quadratic model was reduced to Eq. (6):
albus [5] that produces 12 IU of xylanase in a xylan medium at 30 ◦ C at pH 7.5 after 5 days and Streptomyces cuspidosporous [12] that produces 18 IU in a xylan medium at 37 ◦ C at pH 7.5 after 5 days. The enzyme production in this study with the agricultural waste cane bagasse was comparable with that of the other actinomycetes.
Y = 13.9 + 2.94X1 − 1.10X2 − 4.23X12 − 7.05X22
3.4. Applicability
X22 )
(6)
This modified model indicates a high correlation between the experimentally observed and predicted values as same as the first model (R 2 > 0.9) with the value of coefficient of variation (5.6%) lower than the first model (Table 3). Furthermore, the probability P-value (P = 0.0022) was also relatively lower than the first model (P = 0.044), indicating the more significance of the second model. 3.3. Optimization of pH and temperature in xylanase production To investigate the effects of pH and temperature on xylanase production, the three-dimensional contour plot (Sigma Plot, Version 5.0, Jandel Scientific GmbH, Erkrath, Germany) was used to assess the effects of pH and temperature on the xylanase production (Fig. 3). A maximum xylanase production of 14.3 IU was estimated at pH 7.0 and temperature 50 ◦ C. Streptomyces sp. Ab106 clearly tended to produce xylanase at the high temperature of 50 ◦ C. Many studies on cellulase-free xylanase production have been mainly of Bacillus sp. and Cellulomonas sp. [1,13–15] with several studies of Streptomyces sp. [3,5,12]. Also, most strains producing cellulase-free xylanase by Streptomyces sp. are mesophilic microorganisms, such as Streptomyces sp. QG113 [3] that produces 7.5 IU of xylanase in a wheat bran medium at 37 ◦ C at pH 8.0 after 5 days, Streptomyces
To confirm the applicability of the model, xylanase production by Streptomyces sp. Ab106 was cultured at various temperatures from 35 to 65 ◦ C at a constant pH 7.0. Fig. 4 shows the experimental results of its growth and enzyme productions. Growth was the greatest at the lowest temperature 35 ◦ C and declined with increase in temperature. But medium temperatures 45 and 55 ◦ C induced greatest xylanase production, instead of lower cell mass production (compare results between 35, 45 and 55 ◦ C). As shown in Fig. 5, the quadratic model that uses Eq. (6) seem to fit with actual experiments of xylanase production by Streptomyces sp. Ab106. When the additional experimental data cultured under various temperatures at constant pH 7.0 after 5 days were plotted. At the constant pH 7.0, we assume that xylanase production was dependent on the changes of temperature variable. The quadratic (polynomial) equation of simulated curve and real data curve (28 plotted) in Fig. 5 are calculated as Eqs. (7) and (8), respectively: Y = −61.88 + 3.125T − 0.032T 2 , R 2 = 0.9987, P < 0.0001
(7)
Y = −82.69 + 4.124T − 0.043T 2 , R 2 = 0.9670, P < 0.0021
(8)
Fig. 5. Response linear plot of simulated data and the actual experimental data. Xylanase production at various temperatures at pH 7.0 was tested for the accuracy and precision of the model fit. (—) Xylanase simulated by the model, (—䊏—) xylanase from the experiments after 5 days culture.
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where Y is the xylanase yield (IU) and T the actual value of temperature (◦ C). The t-test statistical method was used to find a correlation or fit of a curve. It was found that both curve have a correlation or fit of a curve in the same direction with a high confidence at 95% interval, t = 0.4322. These results suggest the possibility of applicability of the optimization.
4. Conclusions We conclude that the CCD, regression analysis and response surface method was effective to find the optimum conditions of pH and temperature for xylanase production. The optimum condition was estimated as 50 ◦ C at pH 7.0. This optimization experiment showed that Streptomyces sp. Ab106 produces a higher xylanase activity at these conditions. Cellulase-free xylanase is produced from Streptomyces sp. Ab106 with xylanase activity higher than all other mesophilic and thermophilic actinomycetes reported so far. This microorganism can grow easily at low cost and with simple substrates, such as cane bagasse. References [1] A. Gessesse, G. Mamo, High-level xylanase production by alkaliphilic Bacillus sp. by using solid-state fermentation, Microbiol. Technol. 25 (1999) 68–72. [2] M. Tuncer, A.S. Ball, A. Rob, M.T. Wilson, Optimization of extracellular lignocellulolytic enzyme production by a thermophilic actinomycete Thermomonospora fusca BD25, Enzyme Microbiol. Technol. 25 (1999) 38–47. [3] Q.K. Beg, B. Bharat, K. Mukesh, G.S. Hoondal, Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and
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