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Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp. OU01
Bioresource Technology 98 (2007) 1548–1553
Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp. OU01 Yuying Sun a, Baoqin Han a, Wanshun Liu a
a,* ,
Jiquan Zhang b, Xingshuang Gao
a
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, PR China b Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, PR China Received 1 January 2006; accepted 13 June 2006 Available online 22 August 2006
Abstract The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacterium sp. OU01 in shake-flask. A significant influence of (NH4)2SO4, MgSO4 Æ 7H2O and initial pH on chitosanase production was noted with Plackett–Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0 g/L (NH4)2SO4, 1.3 g/L MgSO4 and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6 U/mL to 118 U/mL. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Chitosanase; Induction; Colloidal chitosan; Statistical optimization; Plackett–Burman; Response surface methodology
1. Introduction Chitooligosaccharides, prepared by hydrolyzing chitosan with chitosanase enzyme, have various potential applications in the fields of pharmaceutical (Tokoro et al., 1989), agricultural (Hadwiger et al., 1984), and food (Tsai et al., 2000) industry. In addition, their applications in physiological studies have been impressed (Suzuki et al., 1986, 1992). Most of the chitosanases reported so far have been inducible. In Aspergillus sp. CJ22-326 (Chen et al., 2005), Fusarium solani f. sp. Phaseoli (Shimosaka et al., 1993) and Bacillus cereus S1 (Masahiro et al., 2000), chitosanases were inducible in medium containing colloidal chitosan as carbon source. The molecular mass of colloidal chitosan varies from batch to batch, and, thus, preparation of colloidal chitosan is time consuming. Furthermore, sterilization makes colloidal chitosan caramel. These factors limit the large-scale production of chitosanase.
*
Corresponding author. Tel./fax: +86 532 82032105. E-mail address: [email protected] (W. Liu).
0960-8524/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.07.020
Optimization of fermentation has long been used in enhancing the yield of many bioprocesses. Conventionally, fermentation process was optimized by implementing the variation of one component at a time. Whereas at present, this approach has been replaced with statistical designs for screening of independent variables in generating the response and one of the statistical designs is Plackett–Burman (Plackett and Burman, 1946). This step is generally followed by a process optimization tool, e.g. the steepest ascent method, response surface methodology (RSM) (Simunek et al., 2004; Banik et al., in press; Sharma and Satyanarayana, 2006), etc. However, no investigations have yet been carried out on the statistical optimization of medium composition and culture conditions for chitosanase production. In the present study, an effort was done to maximize the chitosanase production by Microbacterium sp. OU01 in shake-flask culture. The optimization steps were performed as follows: (1) manipulating the variation of one component at a time in order to screen the optimal carbon and nitrogen source; (2) elucidating medium composition and culture conditions that affect enzyme production significantly by
Y. Sun et al. / Bioresource Technology 98 (2007) 1548–1553
Plackett–Burman, (3) accessing the optimal region of these significant variables using the steepest ascent method; (4) optimizing these significant variables by response surface methodology (RSM). 2. Methods 2.1. Materials Chitosan and chitin were purchased from Sigma– Aldrich Co. Colloidal chitosan was prepared according to the methods described by Roberts (1992), and preparation of partially deacetylated chitosan (62–100% DDA) was performed following the methods of Yabuki et al. (1988). A dichroism method (Domard, 1987) was applied in determining the acetyl content of the chitosan samples. All the other reagents were of the highest grade available commercially in China. 2.2. Microorganism and growth medium The bacterial strain was isolated in the laboratory from the soil samples collected from China and maintained on seed culture slants with 1.0% colloidal chitosan at 4 °C and also stored as glycerol stocks at 80 °C. The strain was identified as Microbacterium sp. OU01 (99.5% homology) based on 16S rDNA sequence analysis (16S rDNA sequence was deposited in the GenBank with accession number DQ118082). Seed culture medium consisted (g/L) peptone 5.0, glucose 3.0, MgSO4 Æ 7H2O 0.5, K2HPO4 Æ 3H2O 0.7, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0; and the main culture medium consisted (g/L) chitosan 10.0, NH4NO3 20.0, glucose 0.5, MgSO4 Æ 7H2O 1.0, K2HPO4 Æ 3H2O 0.7, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0. The pH of main culture medium was adjusted to 6.0 with 2 mol/L HCl before sterilization. Seed culture medium was incubated at 30 °C, 150 r/min for 20 h in a rotary shaking incubator and main culture medium was maintained at the same conditions for 96 h. After sterilization at 121 °C for 20 min, main culture medium was inoculated with 4% inoculum (V/V). Shakeflasks (500 mL) containing 80 mL of the medium were used in both seed and main cultures. All experiments were triplicated and results presented were the mean of three values. The standard deviation was within 5%. 2.3. Evaluation of carbon and nitrogen sources for the production of chitosanase In order to optimize the medium composition: a total of twelve different carbon sources, including D-Mannose, colloidal chitin, starch, chitin, Galactose, powder chitosan, sodium carboxymethyl cellulose, N-acetylglucosamine, D-Fructose, carboxymethyl chitosan, glucosamine–hydrochloride, and colloidal chitosan and four kinds of organic nitrogen sources including corn steep liquor, soybean meal, beef extract, and peptone; and two kinds of inorganic
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nitrogen salts including NH4Cl and (NH4)2SO4 were individually evaluated for their performances in chitosanase production. The experimental media and culture conditions were same as described above. For comparison of different carbon and nitrogen sources, the above different carbon (10.0 g/L) and nitrogen (20.0 g/L) sources took the place of chitosan and NH4NO3, respectively. 2.4. Analytical methods Cell growth was monitored by measuring the absorbance at 600 nm after appropriate dilution with distilled water. The supernatant obtained by centrifugation of the culture medium at 10,000g for 10 min (4 °C) was used in chitosanase assay. 2.5. Chitosanase assay Chitosan with 95% DDA was used as the substrate in the chitosanase assay. Chitosanase activity was determined by quantitative estimation of the reducing sugars produced from chitosan. The reaction mixture contained 0.9 mL of 1% soluble chitosan (pH 5.8 sodium acetate buffer), 0.1 mL of diluted enzyme solution and 1 mL of 0.2 mol/L sodium acetate buffer (pH 5.8). The reaction tubes were incubated at 50 °C for 15 min. The reducing sugars formed in the supernatant were estimated spectrophotometrically by using the modified dinitrosalicyclic acid (DNS) method (Miller, 1959), with glucosamine–hydrochloride as the calibration standard. One unit of chitosanase was defined as the amount of enzyme that could liberate l lmol of reducing sugar as GlcN per min under the conditions described above. 2.6. Experimental design data analysis 2.6.1. Optimization of medium composition and culture conditions using Plackett–Burman design The carbon and nitrogen sources, which had been screened earlier, were added to the main culture medium for medium optimization. For the selection of these factors (Table 1), ‘Design ExpertÒ 7.0’ Stat-Ease, Inc., Minneapolis, USA, was used to generate and analyze the experimental design of Plackett–Burman. The Plackett–Burman method allows evaluation of N 1 variables by N number of experiments (N must be a multiple of four). In addition to the variables of real interest, the Plackett–Burman design considers insignificant dummy variables, whose number should be one-third of all variables. The dummy variables, which were not assigned any values, introduce some redundancy required by the statistical procedure. Incorporation of the dummy variables into an experiment allowed an estimation of the variance (experimental error) of an effect (Desai et al., 2006). According to the above depiction, seven variables (X13, X14, X15, X16, X17, X18, X19) were designated as dummy variables. Each parameter
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Y. Sun et al. / Bioresource Technology 98 (2007) 1548–1553
Table 1 Range of different variables studied in the Plackett–Burman design Variables
Names
X1 (g/L) X2 (g/L) X3 (g/L) X4 (g/L) X5 (g/L) X6 (g/L) X7 (g/L) X8 (g/L) X9 (°C) X10 (mL/500 mL) X11 X12 (h) X13 X14 X15 X16 X17 X18 X19
Yeast extracts (NH4)2SO4 MgSO4 Æ 7H2O K2HPO4 Æ 3H2O KH2PO4 NaCl Glucose Power chitosan Incubation temperature Fermentation holding Initial pH Incubation time Dummy 1 Dummy 2 Dummy3 Dummy 4 Dummy5 Dummy 6 Dummy 7
Level 1
+1
3.0 20.0 1.0 0.7 0.3 5.0 0.5 10.0 30 80 2.1 96
4.0 25.0 1.5 1.4 0.7 10.0 1.0 12.5 32 100 2.6 120
was tested at two levels, high (+1) and low (1). Concentration ranges for the variables were decided by extensive literature survey (Table 1). The experiments were performed in 500 mL shake-flasks at 150 r/min following the instruction of design matrix (data not shown). Response was measured in terms of chitosanase activity. The effect of variables on enzyme production was calculated by ‘Design ExpertÒ 7.0’. 2.6.2. Steepest ascent method Variables that significantly influenced chitosanase production were optimized with respect to enzyme activity by applying a single steepest ascent experiment (Montgomeryd, 1991). 2.6.3. Response surface methodology (RSM) Once the ranges of relevant variables were selected, RSM was used to determine the optimum concentration of these variables for the elevation of chitosanase production. A central composite design (CCD) was used to optimize the concentration of the variables and an experimental design of 20 experiments was formulated using the statistical software package ‘Design ExpertÒ 7.0’. Response obtained was the measurement of chitosanase activity. The statistical software package Design ExpertÒ 7.0 (Stat-Ease, Inc., Minneapolis, MN) was used to analyze the experimental design. 3. Results and discussion 3.1. Shake-flask culture using medium reported in literature Based on the literature reports of chitosanase production, the shake-flask cultivation was performed by using main culture medium and the culture conditions described
and then the chitosanase activity was observed as 3.6 U/ mL. As glucose and yeast extract added to promote cell growth during the first period, the optimization of carbon and nitrogen source was not taken into consideration. 3.2. Effect of different carbon and nitrogen sources After extensive screening, the highest chitosanase production was obtained when (NH4)2SO4 was used as the nitrogen source (data not shown) and a high degree of enzyme activity was observed while colloidal chitosan was taken as carbon source (data not shown). Therefore, it seemed that chitosanase produced by Microbacterium sp. OU01 was inducible and the best inducer appeared to be colloidal chitosan. The effect of DDA of chitosan was also investigated (data not shown) and it revealed that 85–100% DDA of chitosan had the highest chitosanase activity. The mass of colloidal chitosan varied from batch to batch, and thus, preparation of colloidal chitosan was time consuming. Furthermore, sterilization made colloidal chitosan caramel. Thus, the pH of the medium was adjusted with 2 mol/L HCl and colloidal chitosan was observed in the medium after sterilization at 121 °C for 20 min (data not shown). An initial pH of 2.0 made the colloidal chitosan content optimal for obtaining highest chitosanase activity in the medium after sterilization than any other pH. 3.3. Plackett–Burman design Plackett–Burman design was used to analyze the effect of 19 variables (including 7 dummy) on chitosanase production by Microbacterium sp. OU01. Variability in three factors ((NH4)2SO4, MgSO4 Æ 7H2O and initial pH) significantly affected enzyme production (Table 2). The F-value Table 2 Identifying significant variables for chitosanase production by Microbacterium sp. OU01 using Plackett–Burman design Source
Sum of squares
DF
Mean square
F-value
Prob > F
Model Yeast extracts (NH4)2SO4 MgSO4 Æ 7H2O K2HPO4 Æ 3H2O K2HPO4 NaCl Glucose Power chitosan Incubation temperature Fermentation holding Initial pH Incubation time Residual Cor total
123.96 1.85 17.77 51.30 5.25 0.64 0.82 8.18 10.30 0.29
12 1 1 1 1 1 1 1 1 1
10.33 1.85 17.77 51.30 5.25 0.64 0.82 8.18 10.30 0.29
4.38 0.79 7.53 21.75 2.23 0.27 0.35 3.47 4.37 0.12
0.0296a 0.4047 0.0287a 0.0023a 0.1792 0.6173 0.5739 0.1049 0.0750 0.7381
6.53
1
6.53
2.77
0.1400
18.34 2.70 16.51 140.48
1 1 7 19
18.34 2.70 2.36
7.77 1.15
0.0270a 0.3201
a
Indicates model terms are significant.
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of 4.38 implied that the model was significant. ‘Prob > F’ of further substantiated model terms were significant (Table 2). An adequate precision of 6.587 indicates an adequate signal as it measures the signal-to-noise ratio. A ratio >4 was desirable. This model could be used to navigate the design space. According to the Plackett–Burman design, the optimum medium composition and culture conditions were as follows: medium was formulated with (g/l) chitosan powder 10.0, glucose 1.0, K2HPO4 Æ 3H2O 1.4, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0 (g/L); the main culture medium was inoculated with 4% inoculum (V/V) and incubated for 96 h at 30 °C, 150 rpm in a rotary shaking incubator. The main culture was grown in 500 mL Erlenmeyer flasks containing 100 mL medium. 3.4. The method of steepest ascent Despite the fact that Plackett–Burman proved to be a valuable tool for screening variables that significantly affected the chitosanase production, it was unable to predict the optimum levels of the variables. Based on Table 3, the path of the steepest ascent was determined to find the proper direction of changing variables by decreasing the concentration of (NH4)2SO4, MgSO4 Æ 7H2O and initial pH to improve chitosanase production. It was found that the yield plateau was reached at the second step. Then, these variables were chosen for further optimization. 3.5. Response surface methodology
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Table 5 Experimental design and results of central composite design Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Level X1
X2
X3
1 1 1 1 1 1 1 1 1.682 1.682 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 1.682 1.682 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 0 0 1.682 1.682 0 0 0 0 0 0
Chitosanase activity/UmL1 94.8 86.54 91.02 85.63 91.89 82.43 86.32 75.21 85.96 95.62 88.75 95.21 75.63 89.12 116.32 113 117.36 113.21 114.25 115.24
analysis of variance (ANOVA) and following quadratic regression equations were obtained in terms of chitosanase production Chitosanase activity ¼ 114:90 þ 2:81X 1 þ 2:08X 2 þ 4:17X 3 8:53X 21 1:01X 1 X 2 0:87X 1 X 3 8:11X 22 þ 0:15X 2 X 3 11:51X 23 ;
Based on the Placket–Burman design and the method of the steepest ascent, three variables ((NH4)2SO4, MgSO4 Æ 7H2O, Initial pH), which significantly influenced the chitosanase production, were used to determine the optimum levels of these parameters. A total of 20 experiments with different combinations of (NH4)2SO4 (X1), MgSO4 Æ 7H2O (X2) and initial pH (X3) were performed (Tables 4 and 5). The results were analyzed by standard
where X1 is the coded value of (NH4)2SO4 concentration, X2 the coded value of MgSO4 concentration, X3 the coded value of initial pH.
Table 3 Experimental design of steepest ascent and corresponding response Experiment no.
(NH4)2SO4 (g/L)
MgSO4 Æ 7H2O (g/L)
Initial pH
Chitosanase activity/UmL1
0 0 + 1D 0 + 2D 0 + 3D
20.0 19.0 18.0 17.0
1.5 1.2 0.9 0.6
2.1 2.0 1.9 1.8
75.23 86.54 75.92 35.36
Table 4 Levels of the variables tested in central composite design Variables
Level 1.682
1
0
1
1.682
DXi
(NH4)2SO4 MgSO4 Æ 7H2O Initial pH
15.6 0.5 1.83
17.0 0.8 1.90
19.0 1.2 2.00
21.0 1.6 2.10
22.4 1.9 2.17
0.2 0.4 0.10
Fig. 1. Response surface plot and its contour plot of chitosanase production by Microbacterium sp. OU01 showing the interaction between (NH4)2SO4 concentration and MgSO4 concentration at X3 = 0.
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Y. Sun et al. / Bioresource Technology 98 (2007) 1548–1553
Fig. 2. Response surface plot and its contour plot of chitosanase production by Microbacterium sp. OU01 showing the interaction between (NH4)2SO4 concentration and initial pH at X2 = 0.
The regression equation obtained after ANOVA indicating that the R2 value of 0.9951 (a value of R2 > 0.75 indicated the aptness of the model) was in reasonable agreement with the adjusted R2 of 0.9907. This ensured a satisfactory adjustment of the quadratic model to the experimental data. The predicted sum of squares (PRESS), which measured how a particular model fitted each point in design, was 48.98. Values of ‘Prob > F’ less than 0.05 indicated that model terms were significant. Six model terms (X1, X2, X3, X 21 , X 22 , and X 23 ) were most significant. An adequate precision of 41.539 indicated an adequate signal as it measured the signal-to-noise ratio. A ratio >4 was desir-
Fig. 3. Response surface plot and its contour plot of chitosanase production by Microbacterium sp. OU01 showing the interaction between MgSO4 concentration and initial pH at X1 = 0.
able. This model could be used to navigate the design space. Figs. 1–3 show the response surface plots and their contour plots of chitosanase production. Evidently chitosanase yield varied significantly upon changing the initial pH, (NH4)2SO4 or MgSO4 concentration. It indicated that the molecular mass of chitosan also significantly induced chitosanase production. The optimum values of each variable was identified based on the hump in the three dimensional plot, or from the central point of the corresponding
Fig. 4. Profile of cell growth and chitosanase production in shake-flask experiments: Main culture medium consisted (g/L) chitosan powder 10, (NH4)2SO4 19.0, glucose 1, MgSO4 Æ 7H2O 1.3, K2HPO4 Æ 3H2O 1.4, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0 (g/L). The pH was adjusted to 2.0 before main culture medium was sterilized. After sterilization at 121 °C for 20 min, main culture medium was inoculated with 4% inoculum (V/V). The main culture was carried out in 500 mL Erlenmeyer flasks containing 100 mL of medium. The flasks were kept at 30 °C under shaking conditions (150 r/min) for 144 h. Samples were withdrawn at regular intervals and analyzed for chitosanase activity.
Y. Sun et al. / Bioresource Technology 98 (2007) 1548–1553
contour plot. The results predicted by the model equation from RSM showed that a combination of adjusting the (NH4)2SO4 concentration to 19.0 g/L (X1 = 0.149), increasing the MgSO4 concentration to 1.3 g/L (X2 = 0.12), and adjusting initial pH to 2.0 (X3 = 0.176) would favor maximum chitosanase production, giving 115.6 U/mL. In order to confirm the optimization results, growth kinetics of the culture were studied using predicted medium composition and culture conditions (Fig. 4). The cell grew rapidly and reached stationary phase just after 60 h. A maximum rate of chitosanase production was observed during the exponential and stationary phase, where it attains a peak of 118.0 U/mL at 96 h, which was almost equal to the actual predicted value. By means of optimizing medium composition and culture conditions, the production of chitosanase was enhanced from 3.6 U to 118 U/ mL. In addition, in this study, the concentration of mineral nitrogen source was very high (19 g/L). Apparently it was probably the characteristic of the strain. It was noteworthy that the level of chitosanlytic activity of Microbacterium sp. OU01 (118 U/mL) was high by contrast to the cloned Streptomyces lividans TK24 (35–40 U/ mL) (Fink et al., 1991), Bacillus sp. MET 1299 (50 U/ mL) (Kim et al., 2004), Bacillus sp. KCTC 0377BP (100 U/mL) (Choi et al., 2004), and other bacteria (not higher than 3 U/mL). Therefore, Microbacterium sp. OU01 sounded good potential in the production of chitosanase. 4. Conclusions From the results it could be concluded that the enzyme production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions. The chitosanase obtained from Microbacterium sp. OU01 could be a promising biocatalyst for the production of chitooligosaccharides. Primarily, colloidal chitosan appeared to be the best inducer and carbon source for chitosanase production. However, work still needs to be done in order to understand the induction mechanism of chitosanase production and practical application of Microbacterium sp. OU01. Acknowledgements This research was supported by the National High Technology Research and Development Program of China (863 Program) in the Tenth five-year Plan (No. 2001BA708B0407). The helpful work of Mr. Bing Liu and Mrs. Xiaojuan Wei is also appreciated. References Banik, R.M., Santhiagu, A., Upadhyay, S.N., in press. Optimization of nutrients for gellan gum production by Sphingomonas Paucimobilis ATCC-31461 in molasses based medium using response surface methodology. Bioresource Technol., doi:10.1016/j.biortech.2006.03.012.
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Chen, X., Xia, W., Yu, X., 2005. Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326. Food Res. Int. 38, 315–322. Choi, Y.J., Kim, E.J., Piao, Z., Yun, Y.C., Shin, Y.C., 2004. Purification and characterization of chitosanase from Bacillus sp. Strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Appl. Environ. Microbiol. 70, 4522–4531. Desai, K.M., Akolkar, S.K., Badhe, Y.P., Tambe, S.S., Lele, S.S., 2006. Optimization of fermentation media for exopolysaccharide production from Lactobacillus plantarum using artificial intelligence-based techniques. Process Biochem. 41, 1842–1848. Domard, A., 1987. Determination of N-acetyl content in chitosan samples by c.d. measurements. Int. J. Biol. Macromol. 9, 353–358. Fink, D., Boucher, I., Denis, F., Brezinski, R., 1991. Cloning and expression in Streptomyces lividans of a chitosanase encoding gene from the actinomycete Kitasatosporia N174 isolated from soil. Biotechnol. Lett. 13, 845–850. Hadwiger, L.A., Fristensky, B., Riggleman, R.C., 1984. Chitosan, a natural regulator in plant pathogen interaction,increase crop yield. In: Zikakis, J.P. (Ed.), Chitin, Chitosan and Related Enzyme. Academic Press, Orlando, FL, pp. 291–302. Kim, P.I., Kang, T.H., Chung, K.J., Kimand, I.S., Chung, K.C., 2004. Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene. FEMS Microbiol. Lett. 240, 31–39. Masahiro, K., Shou, Y., Kiyomi, N., Minako, S., Toshiaki, K., 2000. Properties of chitosanase from Bacillus cereus S1. Curr. Microbiol. 40, 6–9. Miller, G.L., 1959. The use of dinitrosalicylic acid reagent for the determination of reducing sugars. Anal. Chem. 31, 426–428. Montgomeryd, D.C., 1991. Design and analysis of experiments (3rd), third ed. John Wiley and Sons, New York. Plackett, R.L., Burman, J.P., 1946. The design of optimum multifactorial experiments. Biometals 33, 305–325. Roberts, G.A.F., 1992. Chitin Chemistry. Macmillan Press, Basingstoke, UK. Sharma, D.C., Satyanarayana, T., 2006. A marked enhancement in the production of a highly alkaline and thermostable pectinase by Bacillus pumilus dcsr1 in submerged fermentation by using statistical methods. Bioresource Technol. 97 (5), 727–733. Shimosaka, M., Nagawa, M., Ohno, Y., Okazaki, M., 1993. Chitosanase from the plant pathogenic fungus, Fusarium solani f. sp. phaseoli purification and some properties. Biosci. Biotechnol. Biochem. 57, 231–235. Simunek, J., Tishchenko, G., Rozhetsky, K., Bartonova, H., Kopecny, J., Hodrova, B., 2004. Chitinolytic enzymes from Clostridium aminovalerium: activity screening and purification. Folia Microbiol. 49, 194–198. Suzuki, K., Mikami, T., Okawa, Y., Tokoro, A., Suzuki, S., Suzuki, M., 1986. Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohydr. Res. 151, 403–408. Suzuki, S., Watanabe, T., Mikami, T., Matsumoto, T., Suzuki, M., 1992. Immuno-enchancing effects of N-acetylchitohexaose. In: Brine, C.J., Sandford, P.A., Zikakis, J.P. (Eds.), Advance in Chitin and Chitosan. Elsevier Applied Science, New York, pp. 96–105. Tokoro, A., Kobayashi, M., Tatekawa, N., Suzuki, K., Okawa, Y., Mikami, T., Suzuki, S., Suzuki, M., 1989. Protective effect of N-acetyl chitohexaose on listeria monocytogens infection in mice. Microbiol. Immunol. 33, 357–367. Tsai, G.J., Wu, Z.Y., Su, W.H., 2000. Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. J. Food. Protect. 63, 747–752. Yabuki, M., Uchiyama, A., Suzuki, K., Ando, A., Fujii, T., 1988. Purification and properties of chitosanase from Bacillus circulans MH-K1. J. Gen. Appl. Microbiol. 34, 255–270.
Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp. OU01 Yuying Sun a, Baoqin Han a, Wanshun Liu a
a,* ,
Jiquan Zhang b, Xingshuang Gao
a
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, PR China b Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, PR China Received 1 January 2006; accepted 13 June 2006 Available online 22 August 2006
Abstract The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacterium sp. OU01 in shake-flask. A significant influence of (NH4)2SO4, MgSO4 Æ 7H2O and initial pH on chitosanase production was noted with Plackett–Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0 g/L (NH4)2SO4, 1.3 g/L MgSO4 and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6 U/mL to 118 U/mL. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Chitosanase; Induction; Colloidal chitosan; Statistical optimization; Plackett–Burman; Response surface methodology
1. Introduction Chitooligosaccharides, prepared by hydrolyzing chitosan with chitosanase enzyme, have various potential applications in the fields of pharmaceutical (Tokoro et al., 1989), agricultural (Hadwiger et al., 1984), and food (Tsai et al., 2000) industry. In addition, their applications in physiological studies have been impressed (Suzuki et al., 1986, 1992). Most of the chitosanases reported so far have been inducible. In Aspergillus sp. CJ22-326 (Chen et al., 2005), Fusarium solani f. sp. Phaseoli (Shimosaka et al., 1993) and Bacillus cereus S1 (Masahiro et al., 2000), chitosanases were inducible in medium containing colloidal chitosan as carbon source. The molecular mass of colloidal chitosan varies from batch to batch, and, thus, preparation of colloidal chitosan is time consuming. Furthermore, sterilization makes colloidal chitosan caramel. These factors limit the large-scale production of chitosanase.
*
Corresponding author. Tel./fax: +86 532 82032105. E-mail address: [email protected] (W. Liu).
0960-8524/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.07.020
Optimization of fermentation has long been used in enhancing the yield of many bioprocesses. Conventionally, fermentation process was optimized by implementing the variation of one component at a time. Whereas at present, this approach has been replaced with statistical designs for screening of independent variables in generating the response and one of the statistical designs is Plackett–Burman (Plackett and Burman, 1946). This step is generally followed by a process optimization tool, e.g. the steepest ascent method, response surface methodology (RSM) (Simunek et al., 2004; Banik et al., in press; Sharma and Satyanarayana, 2006), etc. However, no investigations have yet been carried out on the statistical optimization of medium composition and culture conditions for chitosanase production. In the present study, an effort was done to maximize the chitosanase production by Microbacterium sp. OU01 in shake-flask culture. The optimization steps were performed as follows: (1) manipulating the variation of one component at a time in order to screen the optimal carbon and nitrogen source; (2) elucidating medium composition and culture conditions that affect enzyme production significantly by
Y. Sun et al. / Bioresource Technology 98 (2007) 1548–1553
Plackett–Burman, (3) accessing the optimal region of these significant variables using the steepest ascent method; (4) optimizing these significant variables by response surface methodology (RSM). 2. Methods 2.1. Materials Chitosan and chitin were purchased from Sigma– Aldrich Co. Colloidal chitosan was prepared according to the methods described by Roberts (1992), and preparation of partially deacetylated chitosan (62–100% DDA) was performed following the methods of Yabuki et al. (1988). A dichroism method (Domard, 1987) was applied in determining the acetyl content of the chitosan samples. All the other reagents were of the highest grade available commercially in China. 2.2. Microorganism and growth medium The bacterial strain was isolated in the laboratory from the soil samples collected from China and maintained on seed culture slants with 1.0% colloidal chitosan at 4 °C and also stored as glycerol stocks at 80 °C. The strain was identified as Microbacterium sp. OU01 (99.5% homology) based on 16S rDNA sequence analysis (16S rDNA sequence was deposited in the GenBank with accession number DQ118082). Seed culture medium consisted (g/L) peptone 5.0, glucose 3.0, MgSO4 Æ 7H2O 0.5, K2HPO4 Æ 3H2O 0.7, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0; and the main culture medium consisted (g/L) chitosan 10.0, NH4NO3 20.0, glucose 0.5, MgSO4 Æ 7H2O 1.0, K2HPO4 Æ 3H2O 0.7, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0. The pH of main culture medium was adjusted to 6.0 with 2 mol/L HCl before sterilization. Seed culture medium was incubated at 30 °C, 150 r/min for 20 h in a rotary shaking incubator and main culture medium was maintained at the same conditions for 96 h. After sterilization at 121 °C for 20 min, main culture medium was inoculated with 4% inoculum (V/V). Shakeflasks (500 mL) containing 80 mL of the medium were used in both seed and main cultures. All experiments were triplicated and results presented were the mean of three values. The standard deviation was within 5%. 2.3. Evaluation of carbon and nitrogen sources for the production of chitosanase In order to optimize the medium composition: a total of twelve different carbon sources, including D-Mannose, colloidal chitin, starch, chitin, Galactose, powder chitosan, sodium carboxymethyl cellulose, N-acetylglucosamine, D-Fructose, carboxymethyl chitosan, glucosamine–hydrochloride, and colloidal chitosan and four kinds of organic nitrogen sources including corn steep liquor, soybean meal, beef extract, and peptone; and two kinds of inorganic
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nitrogen salts including NH4Cl and (NH4)2SO4 were individually evaluated for their performances in chitosanase production. The experimental media and culture conditions were same as described above. For comparison of different carbon and nitrogen sources, the above different carbon (10.0 g/L) and nitrogen (20.0 g/L) sources took the place of chitosan and NH4NO3, respectively. 2.4. Analytical methods Cell growth was monitored by measuring the absorbance at 600 nm after appropriate dilution with distilled water. The supernatant obtained by centrifugation of the culture medium at 10,000g for 10 min (4 °C) was used in chitosanase assay. 2.5. Chitosanase assay Chitosan with 95% DDA was used as the substrate in the chitosanase assay. Chitosanase activity was determined by quantitative estimation of the reducing sugars produced from chitosan. The reaction mixture contained 0.9 mL of 1% soluble chitosan (pH 5.8 sodium acetate buffer), 0.1 mL of diluted enzyme solution and 1 mL of 0.2 mol/L sodium acetate buffer (pH 5.8). The reaction tubes were incubated at 50 °C for 15 min. The reducing sugars formed in the supernatant were estimated spectrophotometrically by using the modified dinitrosalicyclic acid (DNS) method (Miller, 1959), with glucosamine–hydrochloride as the calibration standard. One unit of chitosanase was defined as the amount of enzyme that could liberate l lmol of reducing sugar as GlcN per min under the conditions described above. 2.6. Experimental design data analysis 2.6.1. Optimization of medium composition and culture conditions using Plackett–Burman design The carbon and nitrogen sources, which had been screened earlier, were added to the main culture medium for medium optimization. For the selection of these factors (Table 1), ‘Design ExpertÒ 7.0’ Stat-Ease, Inc., Minneapolis, USA, was used to generate and analyze the experimental design of Plackett–Burman. The Plackett–Burman method allows evaluation of N 1 variables by N number of experiments (N must be a multiple of four). In addition to the variables of real interest, the Plackett–Burman design considers insignificant dummy variables, whose number should be one-third of all variables. The dummy variables, which were not assigned any values, introduce some redundancy required by the statistical procedure. Incorporation of the dummy variables into an experiment allowed an estimation of the variance (experimental error) of an effect (Desai et al., 2006). According to the above depiction, seven variables (X13, X14, X15, X16, X17, X18, X19) were designated as dummy variables. Each parameter
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Table 1 Range of different variables studied in the Plackett–Burman design Variables
Names
X1 (g/L) X2 (g/L) X3 (g/L) X4 (g/L) X5 (g/L) X6 (g/L) X7 (g/L) X8 (g/L) X9 (°C) X10 (mL/500 mL) X11 X12 (h) X13 X14 X15 X16 X17 X18 X19
Yeast extracts (NH4)2SO4 MgSO4 Æ 7H2O K2HPO4 Æ 3H2O KH2PO4 NaCl Glucose Power chitosan Incubation temperature Fermentation holding Initial pH Incubation time Dummy 1 Dummy 2 Dummy3 Dummy 4 Dummy5 Dummy 6 Dummy 7
Level 1
+1
3.0 20.0 1.0 0.7 0.3 5.0 0.5 10.0 30 80 2.1 96
4.0 25.0 1.5 1.4 0.7 10.0 1.0 12.5 32 100 2.6 120
was tested at two levels, high (+1) and low (1). Concentration ranges for the variables were decided by extensive literature survey (Table 1). The experiments were performed in 500 mL shake-flasks at 150 r/min following the instruction of design matrix (data not shown). Response was measured in terms of chitosanase activity. The effect of variables on enzyme production was calculated by ‘Design ExpertÒ 7.0’. 2.6.2. Steepest ascent method Variables that significantly influenced chitosanase production were optimized with respect to enzyme activity by applying a single steepest ascent experiment (Montgomeryd, 1991). 2.6.3. Response surface methodology (RSM) Once the ranges of relevant variables were selected, RSM was used to determine the optimum concentration of these variables for the elevation of chitosanase production. A central composite design (CCD) was used to optimize the concentration of the variables and an experimental design of 20 experiments was formulated using the statistical software package ‘Design ExpertÒ 7.0’. Response obtained was the measurement of chitosanase activity. The statistical software package Design ExpertÒ 7.0 (Stat-Ease, Inc., Minneapolis, MN) was used to analyze the experimental design. 3. Results and discussion 3.1. Shake-flask culture using medium reported in literature Based on the literature reports of chitosanase production, the shake-flask cultivation was performed by using main culture medium and the culture conditions described
and then the chitosanase activity was observed as 3.6 U/ mL. As glucose and yeast extract added to promote cell growth during the first period, the optimization of carbon and nitrogen source was not taken into consideration. 3.2. Effect of different carbon and nitrogen sources After extensive screening, the highest chitosanase production was obtained when (NH4)2SO4 was used as the nitrogen source (data not shown) and a high degree of enzyme activity was observed while colloidal chitosan was taken as carbon source (data not shown). Therefore, it seemed that chitosanase produced by Microbacterium sp. OU01 was inducible and the best inducer appeared to be colloidal chitosan. The effect of DDA of chitosan was also investigated (data not shown) and it revealed that 85–100% DDA of chitosan had the highest chitosanase activity. The mass of colloidal chitosan varied from batch to batch, and thus, preparation of colloidal chitosan was time consuming. Furthermore, sterilization made colloidal chitosan caramel. Thus, the pH of the medium was adjusted with 2 mol/L HCl and colloidal chitosan was observed in the medium after sterilization at 121 °C for 20 min (data not shown). An initial pH of 2.0 made the colloidal chitosan content optimal for obtaining highest chitosanase activity in the medium after sterilization than any other pH. 3.3. Plackett–Burman design Plackett–Burman design was used to analyze the effect of 19 variables (including 7 dummy) on chitosanase production by Microbacterium sp. OU01. Variability in three factors ((NH4)2SO4, MgSO4 Æ 7H2O and initial pH) significantly affected enzyme production (Table 2). The F-value Table 2 Identifying significant variables for chitosanase production by Microbacterium sp. OU01 using Plackett–Burman design Source
Sum of squares
DF
Mean square
F-value
Prob > F
Model Yeast extracts (NH4)2SO4 MgSO4 Æ 7H2O K2HPO4 Æ 3H2O K2HPO4 NaCl Glucose Power chitosan Incubation temperature Fermentation holding Initial pH Incubation time Residual Cor total
123.96 1.85 17.77 51.30 5.25 0.64 0.82 8.18 10.30 0.29
12 1 1 1 1 1 1 1 1 1
10.33 1.85 17.77 51.30 5.25 0.64 0.82 8.18 10.30 0.29
4.38 0.79 7.53 21.75 2.23 0.27 0.35 3.47 4.37 0.12
0.0296a 0.4047 0.0287a 0.0023a 0.1792 0.6173 0.5739 0.1049 0.0750 0.7381
6.53
1
6.53
2.77
0.1400
18.34 2.70 16.51 140.48
1 1 7 19
18.34 2.70 2.36
7.77 1.15
0.0270a 0.3201
a
Indicates model terms are significant.
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of 4.38 implied that the model was significant. ‘Prob > F’ of further substantiated model terms were significant (Table 2). An adequate precision of 6.587 indicates an adequate signal as it measures the signal-to-noise ratio. A ratio >4 was desirable. This model could be used to navigate the design space. According to the Plackett–Burman design, the optimum medium composition and culture conditions were as follows: medium was formulated with (g/l) chitosan powder 10.0, glucose 1.0, K2HPO4 Æ 3H2O 1.4, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0 (g/L); the main culture medium was inoculated with 4% inoculum (V/V) and incubated for 96 h at 30 °C, 150 rpm in a rotary shaking incubator. The main culture was grown in 500 mL Erlenmeyer flasks containing 100 mL medium. 3.4. The method of steepest ascent Despite the fact that Plackett–Burman proved to be a valuable tool for screening variables that significantly affected the chitosanase production, it was unable to predict the optimum levels of the variables. Based on Table 3, the path of the steepest ascent was determined to find the proper direction of changing variables by decreasing the concentration of (NH4)2SO4, MgSO4 Æ 7H2O and initial pH to improve chitosanase production. It was found that the yield plateau was reached at the second step. Then, these variables were chosen for further optimization. 3.5. Response surface methodology
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Table 5 Experimental design and results of central composite design Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Level X1
X2
X3
1 1 1 1 1 1 1 1 1.682 1.682 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 1.682 1.682 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0 0 0 0 1.682 1.682 0 0 0 0 0 0
Chitosanase activity/UmL1 94.8 86.54 91.02 85.63 91.89 82.43 86.32 75.21 85.96 95.62 88.75 95.21 75.63 89.12 116.32 113 117.36 113.21 114.25 115.24
analysis of variance (ANOVA) and following quadratic regression equations were obtained in terms of chitosanase production Chitosanase activity ¼ 114:90 þ 2:81X 1 þ 2:08X 2 þ 4:17X 3 8:53X 21 1:01X 1 X 2 0:87X 1 X 3 8:11X 22 þ 0:15X 2 X 3 11:51X 23 ;
Based on the Placket–Burman design and the method of the steepest ascent, three variables ((NH4)2SO4, MgSO4 Æ 7H2O, Initial pH), which significantly influenced the chitosanase production, were used to determine the optimum levels of these parameters. A total of 20 experiments with different combinations of (NH4)2SO4 (X1), MgSO4 Æ 7H2O (X2) and initial pH (X3) were performed (Tables 4 and 5). The results were analyzed by standard
where X1 is the coded value of (NH4)2SO4 concentration, X2 the coded value of MgSO4 concentration, X3 the coded value of initial pH.
Table 3 Experimental design of steepest ascent and corresponding response Experiment no.
(NH4)2SO4 (g/L)
MgSO4 Æ 7H2O (g/L)
Initial pH
Chitosanase activity/UmL1
0 0 + 1D 0 + 2D 0 + 3D
20.0 19.0 18.0 17.0
1.5 1.2 0.9 0.6
2.1 2.0 1.9 1.8
75.23 86.54 75.92 35.36
Table 4 Levels of the variables tested in central composite design Variables
Level 1.682
1
0
1
1.682
DXi
(NH4)2SO4 MgSO4 Æ 7H2O Initial pH
15.6 0.5 1.83
17.0 0.8 1.90
19.0 1.2 2.00
21.0 1.6 2.10
22.4 1.9 2.17
0.2 0.4 0.10
Fig. 1. Response surface plot and its contour plot of chitosanase production by Microbacterium sp. OU01 showing the interaction between (NH4)2SO4 concentration and MgSO4 concentration at X3 = 0.
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Fig. 2. Response surface plot and its contour plot of chitosanase production by Microbacterium sp. OU01 showing the interaction between (NH4)2SO4 concentration and initial pH at X2 = 0.
The regression equation obtained after ANOVA indicating that the R2 value of 0.9951 (a value of R2 > 0.75 indicated the aptness of the model) was in reasonable agreement with the adjusted R2 of 0.9907. This ensured a satisfactory adjustment of the quadratic model to the experimental data. The predicted sum of squares (PRESS), which measured how a particular model fitted each point in design, was 48.98. Values of ‘Prob > F’ less than 0.05 indicated that model terms were significant. Six model terms (X1, X2, X3, X 21 , X 22 , and X 23 ) were most significant. An adequate precision of 41.539 indicated an adequate signal as it measured the signal-to-noise ratio. A ratio >4 was desir-
Fig. 3. Response surface plot and its contour plot of chitosanase production by Microbacterium sp. OU01 showing the interaction between MgSO4 concentration and initial pH at X1 = 0.
able. This model could be used to navigate the design space. Figs. 1–3 show the response surface plots and their contour plots of chitosanase production. Evidently chitosanase yield varied significantly upon changing the initial pH, (NH4)2SO4 or MgSO4 concentration. It indicated that the molecular mass of chitosan also significantly induced chitosanase production. The optimum values of each variable was identified based on the hump in the three dimensional plot, or from the central point of the corresponding
Fig. 4. Profile of cell growth and chitosanase production in shake-flask experiments: Main culture medium consisted (g/L) chitosan powder 10, (NH4)2SO4 19.0, glucose 1, MgSO4 Æ 7H2O 1.3, K2HPO4 Æ 3H2O 1.4, KH2PO4 0.3, NaCl 5.0 and yeast extract 3.0 (g/L). The pH was adjusted to 2.0 before main culture medium was sterilized. After sterilization at 121 °C for 20 min, main culture medium was inoculated with 4% inoculum (V/V). The main culture was carried out in 500 mL Erlenmeyer flasks containing 100 mL of medium. The flasks were kept at 30 °C under shaking conditions (150 r/min) for 144 h. Samples were withdrawn at regular intervals and analyzed for chitosanase activity.
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contour plot. The results predicted by the model equation from RSM showed that a combination of adjusting the (NH4)2SO4 concentration to 19.0 g/L (X1 = 0.149), increasing the MgSO4 concentration to 1.3 g/L (X2 = 0.12), and adjusting initial pH to 2.0 (X3 = 0.176) would favor maximum chitosanase production, giving 115.6 U/mL. In order to confirm the optimization results, growth kinetics of the culture were studied using predicted medium composition and culture conditions (Fig. 4). The cell grew rapidly and reached stationary phase just after 60 h. A maximum rate of chitosanase production was observed during the exponential and stationary phase, where it attains a peak of 118.0 U/mL at 96 h, which was almost equal to the actual predicted value. By means of optimizing medium composition and culture conditions, the production of chitosanase was enhanced from 3.6 U to 118 U/ mL. In addition, in this study, the concentration of mineral nitrogen source was very high (19 g/L). Apparently it was probably the characteristic of the strain. It was noteworthy that the level of chitosanlytic activity of Microbacterium sp. OU01 (118 U/mL) was high by contrast to the cloned Streptomyces lividans TK24 (35–40 U/ mL) (Fink et al., 1991), Bacillus sp. MET 1299 (50 U/ mL) (Kim et al., 2004), Bacillus sp. KCTC 0377BP (100 U/mL) (Choi et al., 2004), and other bacteria (not higher than 3 U/mL). Therefore, Microbacterium sp. OU01 sounded good potential in the production of chitosanase. 4. Conclusions From the results it could be concluded that the enzyme production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions. The chitosanase obtained from Microbacterium sp. OU01 could be a promising biocatalyst for the production of chitooligosaccharides. Primarily, colloidal chitosan appeared to be the best inducer and carbon source for chitosanase production. However, work still needs to be done in order to understand the induction mechanism of chitosanase production and practical application of Microbacterium sp. OU01. Acknowledgements This research was supported by the National High Technology Research and Development Program of China (863 Program) in the Tenth five-year Plan (No. 2001BA708B0407). The helpful work of Mr. Bing Liu and Mrs. Xiaojuan Wei is also appreciated. References Banik, R.M., Santhiagu, A., Upadhyay, S.N., in press. Optimization of nutrients for gellan gum production by Sphingomonas Paucimobilis ATCC-31461 in molasses based medium using response surface methodology. Bioresource Technol., doi:10.1016/j.biortech.2006.03.012.
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