Appl Microbiol Biotechnol (2007) 75:10151022 DOI 10.

1007/s00253-007-0907-y

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Optimization of medium composition for the production of alkaline -mannanase by alkaliphilic Bacillus sp. N16-5 using response surface methodology
Shan-shan Lin & Wen-fang Dou & Hong-yu Xu & Hua-zhong Li & Zheng-Hong Xu & Yan-he Ma

Received: 12 November 2006 / Revised: 22 February 2007 / Accepted: 24 February 2007 / Published online: 15 March 2007 # Springer-Verlag 2007

Abstract In this work, a 22 factorial design was employed combining with response surface methodology (RSM) to optimize the medium compositions for the production of alkaline -mannanase by alkaliphilic Bacillus sp. N16-5 isolated previously from sediment of Wudunur Soda Lake in Inner Mongolia, China. The central composite design (CCD) used for the analysis of treatment combinations showed that a second-order polynomial regression model was in good agreement with experimental results, with R2 = 0.9829 (P <0.05). The maximum activity was obtained at NaCl concentration (84.4 g l1) and sodium glutamate (3.11 g l1) and a high medium pH around 10.0. Under such conditions, the activity of alkaline -mannanase achieved 310.1 U/ml in the scale of 5-l fermenter, which was increased nearly twice compared with the original. Through optimization, the substrates shifted from the expensive substrates, such as locust bean gum and peptone, to the inexpensive ones such as konjac powder, soymeal, and sodium glutamate. The experiment results also suggested that the environmental conditions of high salinity and high alkalinity, as well as the inducer substrates, play very important roles in the production of the alkaline mannanase by alkaliphilic Bacillus sp. N16-5.
S.-s. Lin : Z.-H. Xu (*) The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, 170 Huihe Road, Wuxi 214036, Peoples Republic of China e-mail: zhenghxu@sytu.edu.cn S.-s. Lin : W.-f. Dou : H.-y. Xu : H.-z. Li : Z.-H. Xu School of Biotechnology, Southern Yangtze University, Wuxi 214036, Peoples Republic of China Y.-h. Ma Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, Peoples Republic of China

Keywords Alkaliphilic Bacillus sp. . Alkaline -mannanase . Optimization . Response surface methodology

Introduction As linear polymers, mannans are composed of the backbones of -1, 4-linked mannose (and glucose) units, the mannose residues often possessing -1, 6-galactose as side groups and acetylate at the O-2 and O-3 positions depending on origins. They widely exist in the locust bean gum, guar gum, Konjac mannan, and other plant polysaccharides (Hossain et al. 1996). The -mannanase (EC 3.2.1.78) is an enzyme capable of hydrolyzing 1, 4--mannosidic linkages in the main chain of -mannans, glucomannans, and galactomannans, yielding mannooligosaccharides (McCleary 1988). Those sugars can then be hydrolyzed further to mannose by the action of the -mannosidases (EC 3.2.1.25). The -mannanase has been widely applied in such areas as food, drug, feed, paper making, printing and dyeing, textile, oil exploitation, and biological research. Most of these applications of -mannanase can only be completed under extreme conditions, for example, kraft pulp (Clarke et al. 2000; Kansoh and Nagieb 2004), detergent industry (Bettiol and Showell 2002), hydraulic fracturing of oil well (McCutchen et al. 1996), where high pH processes are common. The majority of -mannanase known to the public can hardly maintain catalyzing capacity and stability in such extreme environment. However, the diversity of microorganism provides the possibility for the exploitation and application of alkaline -mannanase. The research of the microbial production for mannanase originated in the late of 1950s, with Bacillus subtilis,

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Xanthomonas campestris, Aeromonas hydrophila, Trichoderma sp. made known to the public consecutively (Ma et al. 1991). In 1987, an alkaline -mannanase was found for the first time by Akino from alkaliphilic Bacillus sp. AM001 and the possibility of the application was proposed (Akino et al. 1987, 1989). In our previous work, an alkaline -mannanase produceralkaliphilic Bacillus sp.N16-5 has been isolated from the sediment of Wudunur Soda Lake in Inner Mongolia, China (Ma et al. 1991). This enzyme was demonstrated to be a novel alkaline -mannanase (optimum pH 9.5) and has a fine prospect of application in the enzymatic production of mannooligosaccharide, which is one of the growth factors of Bifidobacterium sp. (Ma et al. 2004). Recently, Hatada et al. cloned and characterized a high-alkaline -mannanase (up to pH 10) from an alkaliphilic Bacillus sp. strain JAMB-750 (Hatada et al. 2005). It has become a bottleneck problem for industrial application of extremozymes that the extremophiles directly isolated from nature have some shortcoming for extremozyme production, such as longer generation time, lower biomass yield leading to the lower productivity of the extremozymes, and higher production costs (Adams et al. 1995; Schiradi and De Rose 2002). To overcome these problems, some approaches were proposed as follows: (1) optimization of fermentation bioprocesses and media design; (2) implementation of innovative bioreactors; and (3) overproduction in mesophilic hosts. Some attempts were made to increase the production from the wild/mutant strains by optimization of the culture conditions; however, up to now, the research in this field has not been so mature (Heck et al. 2005a). As mentioned above, some genes of alkaline -mannanase from alkaliphiles have been cloned, sequenced, and expressed in the mesophiles host Escherichia coli and B. substilis (Akino et al. 1989; Hatada et al. 2005). For their low expression level, they were hardly to be successfully applied in the industries. In our previous work, we have cloned the alkaline -mannanase gene of strain N16-5 (Ma et al. 2004), and tried to overexpress it in Pichia pastoris and B. subtilis, but the expression levels were also comparatively low (data not shown). Interestingly, the wild alkaliphilic Bacillus sp.N16-5 showed high ability to produce extracellular alkaline -mannanase (Ma et al. 1991). Therefore, improvement of the fermentation process and media design of this wild producer to enhance the capacity of alkaline -mannanase production could be another possible option. Response surface methodology (RSM), which has been extensively applied in optimization of medium composition, conditions of enzymatic hydrolysis, fermentation, and food manufacturing processes (Cui et al. 2006), is the collection of statistical techniques for experiment design, model development, evaluation factors, and optimum

conditions search. To improve the alkaline -mannanase production by alkaliphilic Bacillus sp.N16-5, Pluckett Burman (PB) design, Central composite design (CCD), and experimental factorial design can be employed to optimize the medium compositions for enzyme production, and perform a minimum number of experiments. In this paper, the objective was to optimize the medium compositions for the cost-effective production of alkaline -mannanase by alkaliphilic Bacillus sp. N16-5 isolated previously from sediment of Wudunur Soda Lake in Inner Mongolia, China. We used the sodium glutamate and soymeal, which is a cheap by-product from the soy protein industry, to replace the expensive nitrogen resources such as peptone and yeast extract used in the previously works, in the hope of improving the economics for the production of alkaline -mannanase.

Materials and methods Microorganism Alkaliphilic Bacillus sp. N16-5, a producer of extracellular alkaline -mannanases, was isolated previously from sediment of Wudunur Soda Lake in Inner Mongolia, China (Ma et al. 1991). The bacterium was maintained as a spore stock or, for short periods of time, on nutrient agar slant. Media The seed medium contained (g l1): soluble starch 10, peptone 10, yeast extract 5, NaCl 80, K2HPO4 1.5, MgSO4 0.3. Initial pH of the culture was adjusted to 7.5 8.0 before sterilization at 121 C for 20 min, then 10 g l1 Na2CO3 was added to adjust the pH to 9.5 10.0. The basal fermentation medium used for alkaline mannanase production is the modified alkaline Horikoshi-II medium (Horikoshi 1971), which contained (g l1): konjac mannan 12; soymeal 15 (provided by Genencor (Wuxi) International, Inc.), contents (%, w/w): water 12, crude protein 4447, crude fat 67, carbohydrate 1821, ash 56), NaCl 80, sodium glutamate 1, yeast extract 5, K2HPO4 1.5, and MgSO4 0.3. Initial pH of the culture was adjusted to 7.5 8.0 before sterilization at 121C for 20 min, then 10 g l1 Na2CO3 was added to adjust the pH 9.5 10.0. The single-factor experiments design: prepared according to Table 1 The fermentation medium for the PB design was prepared according to Tables 2 and 3, and the fermentation medium for the RSM design was prepared according to Tables 4 and 5.

Appl Microbiol Biotechnol (2007) 75:10151022 Table 1 Effect of different carbon and nitrogen sources on the production of alkaline -mannanase from N16-5 Factors Glucose Mannose Soluble starch Konjac powder Concentration (g l1) 10 10 10 10 12 15 10 12 15 5 10 15 20 10 15 20 30 10 15 20 30 Enzyme activity (U/ml) 2.70.1 2.20.1 2.70.1 122.84.2 167.36.1 153.25.2 105.33.7 143.64.6 151.24.9 101.23.6 132.74.3 104.23.7 75.22.6 64.52.1 93.03.1 117.14.3 87.42.6 89.72.7 144.74.3 112.73.7 93.22.9

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Enzyme production and activity assay Enzyme centrifugation was done at a temperature below 4C. The centrifugal supernatant of the culture broth was used as the enzyme source for the enzyme analysis. With locust bean gum as the substrate, alkaline -mannanase activity was assayed at 70C and pH 9.6 (0.05 mol l1 GlyNaOH buffer) by measuring the reducing sugars liberated during the hydrolysis of locust bean mannan, as described previously. One unit of activity was defined as the amount of enzyme catalyzing the production of 1 mol of the reducing sugar per minute, using mannose as the standard (Ma et al. 2004). Batch fermentation in 5-l fermenter The batch fermentation was carried out in 5-l fermenter (Biotech-2001) equipped with three six-bladed disc impeller, oxygen and pH electrodes, under the following conditions: medium volume 3 l, inoculation volume 2% (v/v), temperature 37C, initial pH 9.5 10.0, aeration rate 1.0 vvm, and agitation speed 500 rpm. The fermentation continued until the alkaline -mannanase activity reached the highest values. Experimental design

Locust bean

Peptone

Yeast powder

Soymeal

Inoculum preparation and Shake flask experiments PluckettBurman design Inoculum was prepared by transferring colonies from a 24-h slant culture into a 30-ml seed medium in a 250-ml Erlenmeyer flask, and incubated at 37C for 24 h at 230 rpm. Each flask was then inoculated with 2% (v/v) of 24-h liquid culture. The culture was incubated at 37C at 230 rpm for 34 h. All experiments were repeated in triplicate. PB design is one special type of a two-level fractional factorial design basing on incomplete equilibrium piece principle. It can pick up the main factors with the least number of experiments from a list of candidate factors (Plackett and Burman 1946; Kalil et al. 2000). The possible factors that affected the alkaline mannanase yield included konjac mannan, soymeal, NaCl, sodium glutamate, yeast extract, K2HPO4, MgSO4, and initial pH of medium. The 12-run PB design was used to study these eight factors and dummy variables were used to estimate the standard error during analysis of data. The different variables were prepared in two levels, 1 for low level and +1 for high level, and the high level was 1.5 times of the low level, as shown in Table 2. The steepest ascent experiment The steepest ascent experiment can approach the largest response area rapidly, determine the center point of the central composite design (CCD) described further, and ensure the validity and correctness of the response surface analysis results. The first-order model equation obtained from the PB test determined the ascent direction (or descent, as required) and the length of ascent pace.

Table 2 The factors, levels, and the regression analysis of the PB design Factors (g l1) Levels 1 X1 X2 X3 X4 X5 X6 X7 X8 pH NaCl konjac mannan soybean meal yeast extract sodium glutamate MgSO4 K2HPO4 9.0 60.0 10.0 12.0 4.0 2.4 0.2 1.2 +1 11.0 90.0 15.0 18.0 6.0 3.6 0.3 1.8 0.597373 3.78336 0.0181 0.41635 0.19912 6.933144 1.43007 0.16292 0.592342 0.032369* 0.986694 0.705147 0.854897 0.006153** 0.248056 0.880938 t test P > |t|

*Statistically significant at 95% of confidence level **Statistically significant at 99% of confidence level

1018 Table 3 The experiments and results of the PB design Run number Factor-coded levels X1 pH X2 NaCl 1 1 1 1 1 1 1 1 1 1 1 1 X3 Konjac mannan 1 1 1 1 1 1 1 1 1 1 1 1 X4 Soybean meal 1 1 1 1 1 1 1 1 1 1 1 1 X5 Yeast extract 1 1 1 1 1 1 1 1 1 1 1 1 X6 Sodium glutamate 1 1 1 1 1 1 1 1 1 1 1 1

Appl Microbiol Biotechnol (2007) 75:10151022

Y alkaline -mannanase yield (U/ml) X7 MgSO4 X8 K2HPO4

1 2 3 4 5 6 7 8 9 10 11 12

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1

175.18.1 160.26.3 159.16.1 256.515.2 128.24.3 206.58.2 189.67.4 169.37.1 241.79.1 246.59.7 208.410.1 172.49.2

Central composite design Based on the results obtained from the PB design and the steepest ascent experiment, the CCD was conducted to gain the optimized levels of the main factors (Oh et al. 1995; Wang et al. 2004). We can get a second-order empirical model from the experimental data about the relationship between the response value (alkaline -mannanase yield) and the variables through polynomial regression analysis. The form of the second-order polynomial model is: Y b0 X bi i XX bij ij X bii 2 ii ;

where Xi stands for the natural value of independent variable, X0 for the natural value of the independent variable at the center point, and Xi is the step change value. Software for experimental design and statistical analysis Statistical Analysis System (SAS) Version 9.0 was used for the experimental design and statistical analysis of the experimental data. All of the optimization fermentation experiments were conducted in triplicate, and the average data of alkaline -mannanase yields were further analyzed by the SAS 9.0. The quality of fit for the regression model equation was expressed by the coefficient of determination R2 and its statistical significance was determined by an F test. The significance of the regression coefficients was tested by a t test (Heck et al. 2005b).

where Y is the predicted response, bi is the regression coefficient, and xi is the coded value of the independent variables. The relationship between xi and the natural value of independent variable is: xi Xi X0 =Xi ;
Table 4 The design and results of the steepest ascent experiment Run NaCl (g l1) 100.0 95.0 90.0 85.0 80.0 75.0 70.0 65.0 60.0 Sodium glutamate (g l1) 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Alkaline -mannanase yield (U/ml) 128.15.4 189.38.1 263.211.5 307.215.3 254.410.3 237.29.5 190.56.3 204.18.6 220.39.5

Results The single-factor experiments In our previous works, we found that the production of alkaline -mannanase by strain N16-5 was induced by addition of some substrates containing -mannan into the medium (Ma et al. 1991). The most suitable carbon source was locust bean gum and the nitrogen sources were peptone and yeast extract. But in the hope of improving the economics for the production of alkaline -mannanase, we used the cheaper substrates, such as konjac powder, soymeal, and sodium glutamate to replace the locust bean gum, peptone, and yeast extract. The results showed that all tested concentrations of the konjac powder could induce the alkaline -mannanase production, while at the concentration

1 2 3 4 5 6 7 8 9

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of 12 g l1, the enzyme activity (EA) reached the highest. In the range of 10 20 g l1, the soymeal could enhance the enzyme production. In the concentration of 15 g l1, the EA is the highest. Above this concentration, the EA decreased (Table 1). Our previous experiments indicated that in addition to soymeal, sodium glutamate was another nitrogen source for effective production of alkaline -mannanase. We investigated the effects of various sodium glutamate concentrations on the enzyme production; the results are shown in Fig. 1: the sodium glutamate really played an important role in the production of the alkaline -mannanase. In the concentration of 3 g l1, the EA was 216.2 U/ml, which was 40% higher than that of the medium without sodium glutamate. NaCl plays a very important role in the alkaliphiles physiology (Ma 1999). We also found that NaCl played a very important role in the production of alkaline mannanase by alkaliphilic Bacillus sp.N16-5. The tolerant concentration of NaCl is 150 g l1 for this strain (Ma et al. 1991), while in the range of 80 100 g l1 of NaCl, the EA were comparatively high (Fig. 1). When the concentration of NaCl was too high or too low, the enzyme production sharply decreased. Main factors for the production of alkaline -mannanase The levels of the variables for the PB design were selected according to the previous experiments. As shown in Table 2, the 12-run PB design was chosen to pick up the main factors. The experiments and results of the PB design are shown in Table 3.

We can get the first-order regression equation by regression analysis of the experimental data obtained from the PB design. Y 192:4167 2:75 X1 17:41667 X2 0:08333 X3 1:916667 X4 0:916667 X5 31:91667 X6 6:583333 X7 0:75 X8 1

This fit of the model was checked by R2, which was calculated to be 95.59%, that is to say the regression equation regressed well. According to the t-test results in Table 2, we can see NaCl and sodium glutamate are the two major factors affecting the performance of the culture in terms of alkaline -mannanase yields; the reliability values were 96.76 and 99.38%, respectively, at 95 and 99% confidence levels. Optimization of the medium components for alkaline -mannanase production Validation of the central point for CCD According to the results of the PB experiment, we can see NaCl and sodium glutamate were the essential factors. From the data shown in Table 4, we can get the center point of the subsequent experiment, which was NaCl 85.0 g l1, sodium glutamate 3.0 g l1. Optimization and analysis for the CCD design for alkaline -mannanase production The optimal concentration of medium components was determined by CCD with the two variable NaCl and sodium glutamate using the central point obtained from the steepest ascent experiment. The level of konjac mannan, soymeal, yeast extract, K2HPO4, MgSO4, and pH still remained the original (konjac mannan 12, soymeal 15, yeast extract 5, K2HPO4 1.5, MgSO4 0.3. pH 9.5 10.0). To make the regression model accurate, repeat the center point five times. The asterisk arm length =1.414. The experimental design and results are in Table 5 and the statistical analysis of data are shown in Table 6. Regressing the data through SAS 9.0, we can get the following quadratic empirical model: Y 287 3:651611 X1 9:510448 X2
19:81252 X1 X1 4 X1 X2 18:06252 X2 X2 :

concentration of glutamine (g L-1)

250 -1 0 1 2 3 4 5

200

-1 EA (U mL )

150

100

50

0 0 20 40 60 80 100 120

-1 concentration of NaCl (g L ) Fig. 1 Effect of sodium glutamate and NaCl concentration on alkaline NaCl -mannanase production. Sodium glutamate,

The analysis of variance was employed for the determination of significant parameters and to estimate the alkaline -mannanase as a function of NaCl and sodium glutamate. Data are shown in Table 6. The determining coefficient R2

1020 Table 5 The experiment design and results of the CCD Run number Coded factor values X1 NaCl (g l1) 1 2 3 4 5 6 7 8 9 10 11 12 13 1 (80) 1 (80) 1 (90) 1 (90) 1.414 (78) 1.414 (92) 0 (85) 0 (85) 0 (85) 0 (85) 0 (85) 0 (85) 0 (85) Y

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X2 Alkaline Sodium glutamate mannanase (g l1) yield (U/ml) 1 (2.6) 1 (3.4) 1 (2.6) 1 (3.4) 0 (3.0) 0 (3.0) 1.414 (2.4) 1.414 (3.6) 0 (3.0) 0 (3.0) 0 (3.0) 0 (3.0) 0 (3.0) 239.110.2 261.212.3 243.47.1 249.28.4 256.310.4 241.19.2 235.39.2 269.512.2 287.413.1 286.312.1 289.311.1 285.510.6 288.111.3

Fig. 2 Response surface plot for the effects of NaCl and sodium glutamate on alkaline -mannanase production

was 98.29%, which indicated that this model congresses well with the practice and can be used to analyze and estimate alkaline -mannanase fermentation. Significance of coefficients has been reported to be directly proportional to t test and inversely to P value (Heck et al. 2005b). The smaller the P values, the bigger the significance of the corresponding coefficient (Liu et al. 2003). In our work, sodium glutamate, second-order NaCl, and second-order sodium glutamate were highly significant, statistically significant at 99% confidence level. The NaCl and the interaction of the NaCl and sodium glutamate were also very significant, and the reliability values were 97.51 and 93.65% at 95% confidence level. We can find the influences on the yield of the alkaline mannanase imposed by the factors and the reciprocity between them directly in Fig. 2. The shapes of contour curves showed that there were great effects on each other. Maximal enzyme extraction was obtained at lower NaCl and higher sodium glutamate concentration. From the quadratic empirical model, we can get the models extreme coordinate (0.120, 0.276), the corresponding NaCl (84.40 g l1), sodium glutamate (3.11 g l1), and now
Table 6 Coefficient estimates by the regression model Term X1 X2 X1 X1 X1 X2 X2 X2 SE 1.284285 1.284285 1.377244 1.816252 1.377244 t 2.8433 7.405247 14.3856 2.20234 13.115 P> F 0.024926* 0.000149** 0.0001** 0.063512* 0.0001**

forecast the largest yield of the alkaline -mannanase for 288.5 U/ml. Validation of the models The availability of the regression model (Eq. 2) of the alkaline -mannanase by alkaliphilic Bacillus sp. N16-5 was tested using the calculated optimal culture composition (g l1), viz. konjac mannan 12, soymeal 15, yeast extract 5, NaCl 84.4, sodium glutamate 3.11, K2HPO4 1.5, MgSO4 0.3, Na2CO3 10, pH 9.5 10.0, with triplicate experiments. The mean value of the alkaline -mannanase was 295.0 U/ml, which agreed well with the predicted value (288.5 U/ml). As a result, the models developed were considered to be accurate and reliable for predicting the production of alkaline -mannanase by alkaliphilic Bacillus sp. N16-5. Batch fermentation results The feasibility of the regression models in a 5-l scaled fermenter was also tested using the predicted optimal medium composition (Fig. 3). With the optimal, maximum production of alkaline -mannanase could reach 310.1 U/ml, which is nearly twice compared with the original (167.1 U/ml). From this, we can see this model can estimate the practical ferment conditions correctly and improve the yield of alkaline mannanase effectively.

Discussion Although previous research regarding alkaline -mannanase production has been reported, little information on the

*Statistically significant at 95% of confidence level ** Statistically significant at 99% of confidence level

Appl Microbiol Biotechnol (2007) 75:10151022

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Fig. 3 Time profile of fermentation in 5-l fermenter using the basal medium (a) and the optimized medium (b) on the -mannanase cell biomass (g l1), enzyme production from N16-5. pH,

activity (EA, U/ml)

optimization of its production is available (Heck et al. 2005a). In this paper, we used the method of experimental factorial design, and RSM design demonstrated that the CCD and regression analysis methods were effective to find the optimized NaCl and sodium glutamate concentrations for the production of alkaline -mannanase from alkaliphilic Bacillus sp. N16-5, a newly isolated strain growing on some economical and abundant substrate as konjac mannan and soymeal replace the expensive carbon and nitrogen resource, locust gum, peptone, and yeast extract. As we know, NaCl plays a very important role in the alkaliphiles physiology (Ma 1999). It can function as the driving force of some endergonic processes in the cell (Detkova and Pusheva 2006), so it can balance the

extracellular and intracellular pH of the cell, help the cell produce energy, and is also very important in the substrate transports (Ma 1999). The enzymes of such organisms display maximum activity in the presence of salts and, moreover, are inactivated in their absence. Our previous experiments indicated that alkaliphilic Bacillus sp. N16-5 was also a salt-tolerant bacterium, but not so much halophilic. When under high pH condition, around 9.5 10.0, the cell growth and the production of alkaline mannanase were comparatively desirable. But for the effects of various NaCl concentrations on production of alkaline -mannanase by strain N16-5, it was not so much associated with the cell growth (data not shown). Around the concentration of 80 g l1 NaCl, the production of alkaline -mannanase is comparatively high. The lower concentration of NaCl benefited cell growth, but not enzyme production. While at higher concentration, NaCl sharply decreased both cell growth and production of alkaline -mannanase. In our works, we added different amino acids to the soymeal substrate and the sodium glutamate, and the alkaline -mannanase activity increased greatly. With the RSM analysis, we found that the maximum alkaline -mannanase activity was obtained at sodium glutamate concentration (3.11 g l1), and the activity was increased nearly twice compared with the original medium. The amino acids and their analogues have been known for their stimulatory effect on the production of a number of enzymes such as amylase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase, -galactosidase, and xylanases (Ikura and Horikoshi 1987). The effect of sodium glutamate on the production of mannanase has been reported before (Ma et al. 1991), but the mechanism as to the -mannanase stimulation has not been reported. It is possible that a certain amino acid plays an important role at the start of the regulative gene of the hydrolase (Levin and Forschiassin 1998). Gupta et al. (1999) demonstrated that the carbon chain length and the position of CH3 and NH2 groups have a significant role in the stimulation of production of xylanase. Statistical optimization method for fermentation process could overcome the limitations of classic empirical methods and was proved to be a powerful tool for the optimization of the production of alkaline -mannanase from alkaliphilic Bacillus sp. N16-5. In this study, RSM model was proposed to study the combined effects of culture media compositions. Under optimal condition, the predicted production of alkaline -mannanase was 288.5 U/ml by canonical analysis. Validation experiments were also carried out to verify the availability and the accuracy of the models, and the results showed that the predict values agreed well with the experimental values. The batch fermentation results in a 5-l fermenter also showed that optimized culture medium

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Appl Microbiol Biotechnol (2007) 75:10151022 Heck JX, De Barros Soares LH, Ayub MAZ (2005a) Optimization of xylanase and mannanase production by Bacillus circulans strain BL53 on solid-state cultivation. Enzyme and Microb Technol 37:417423 Heck JX, Flores SH, Hertzm PF, Ayub MAZ (2005b) Optimization of cellulase-free xylanase activity produced by Bacillus coagulans BL69 in solid-state cultivation. Process Biochem 40:107112 Horikoshi K (1971) Production of alkaline enzymes by alkalophilic microorganisms. Part I. Alkaline protease produced by Bacillus No. 221. Agric Biol Chem 36:14071414 Hossain MZ, Abe J, Hizukuri S (1996) Multiple forms of -mannanase from Bacillus sp. KK0l. Enzyme and Microb Technol 18:9598 Ikura Y, Horikoshi K (1987) Stimulatory effect of certain amino acids on xylanase production by alkalophilic Bacillus sp. Agric Biol Chem 51:31433145 Kalil SJ, Maugeri F, Rodrigues MI (2000) Response surface analysis and simulation as a tool for bioprocess design and optimization. Process Biochem 35:539550 Kansoh AL, Nagieb ZA (2004) Xylanase and mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp. Antonie van Leeuwenhoek 85:103114 Levin L, Forschiassin F (1998) Influence of growth conditions on the production of xylanolytic enzymes by Trametes trogii. World J Microbiol Biotechnol 14:443446 Liu JZ, Weng LP, Zhang QL, Xu H, Ji LN (2003) Optimization of glucose oxidase production by Aspergillus niger in a benchtop bioreactor using response surface methodology. World J Microbiol Biotechnol 19:317323 Ma YH (1999) Alkaliphiles. Microbiology 26:309 (in Chinese) Ma YH, Tian XY, Zhou PJ, Wang DZ (1991) Production and some properties of alkaline -mannanase. Acta Microbiol Sin 31:443 448 (in Chinese) Ma YH, Xue YF, Dou YT, Xu ZH, Tao WY, Zhou PJ (2004) Characterization and gene cloning of a novel -mannanase from alkaliphilic Bacillus sp. N16-5. Extremophiles 8:447454 McCleary BV (1988) -D-Mannanase. Methods Enzymol 160:596610 McCutchen CM, Duffaud GD, Leduc P, Petersen ARH, Tayal A, Khan SA, Kelly RM (1996) Characterization of extremely thermostable enzymatic breakers (-1,6-galactosidase and 1,4-mannanase) from the hyperthermophilic bacterium Thermotoga meapolitana 5068 for hydrolysis of guar gum. Biotechnol Bioeng 52:332339 Oh S, Rheem S, Sim J, Kim S, Back Y (1995) Optimizing conditions for the growth of Lactobacillus casei YIT9018 in tyrptone-yeast extract-glucose medium by using response surface methodology. Appl Environ Microbiol 61:38093814 Plackett RL, Burman JP (1946) The design of optimum multi-factorial experiments. Biometrika 33:305325 Schiradi C, De Rose M (2002) The production of biocatalysts and biomolecules from extremophiles. Trends Biotechnol 20:515521 Wang YX, Lv FX, Lu ZX (2004) Optimization of cultivation medium Clitocybe sp.AS5.112 for the extracellular polysaccharide production and mycelial growth by response surface methodology. Journal of NanJing Agriculture University 27:8994

could improve the production of alkaline -mannanase from alkaliphilic Bacillus sp. N16-5 in a large-scale fermentation process. It strongly suggested that the production of alkaline -mannanase by wild-strain alkaliphilic Bacillus sp. N16-5 may be interesting for industrial applications. The results of this study also provided useful information for the optimization of medium composition for the other extremozymes fermentation processes.
Acknowledgment This work was supported by grants of National Basic Research Program of China (No.2007CB707804), National High-Tech Program (No. 2006AA020104), Jiangsu Planned Projects for Postdoctoral Research Funds (No.0601004A), and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0532). The authors would thank Dr. Luhong Tang for his critical reading of this manuscript.

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