Accelerating Enzymatic Hydrolysis of Chitin by Microwave Pretreatment

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1 1648 Biotechnol. Prog. 2003, 19, Accelerating Enzymatic Hydrolysis of Chitin by Microwave Pretreatment Ipsita Roy, Kalyani Mondal, and Munishwar N. Gupta* Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi , India Response surface analysis was used to determine optimum conditions [2% (w/v) chitin, 57.5 C, 38 min] for microwave irradiation of chitin to improve its enzymatic hydrolysis. V max /K m of cabbage chitinase toward untreated and microwave-irradiated chitin was found to be 21.1 and 31.7 nmol h -1 mg -2 ml, respectively. Similar improvement was observed in the case of pectinase in its unusual catalytic activity of chitin degradation. It was found that a greater extent of chitin hydrolysis by chitinase was possible after the substrate chitin was irradiated with microwaves. * To whom correspondence should be addressed. Phone: Fax: mn_gupta@ hotmail.com. Introduction Chitin [(1f4)-2-acetamido-2-deoxy-β-D-glucan] is a polysaccharide that occurs in insect exoskeletons, crustacean shells, and fungal cell walls (1-3). A large amount of chitin is generated as a solid waste from processing of shell fish, crabs, shrimp, and krill. Hence, there is considerable interest in its enzymatic hydrolysis for obtaining value-added soluble carbohydrates: oligomers, dimers, and the monomer N-acetylglucosamine (1-6). There are two technical challenges associated with this bioconversion. The first one is that the insoluble nature of chitin makes it a poor substrate; the second one is that chitinases are relatively expensive enzymes. In the case of insoluble substrates, their pretreatment by physical and chemical methods is a well-known strategy to enhance the accessibility of the bonds susceptible to hydrolysis. For example, a large number of such pretreatments have been described for cellulose and lignocellulosic materials (7, 8). Recently, microwave pretreatment has been found to increase the conversion yields in hydrogenolysis of lignins (9). A patent for an apparatus for microwave preprocessing of cellulosic waste before enzymatic hydrolysis has been granted to NASA ( patent ). Microwave pretreatment has also been found helpful for extraction of chitin from red crab ( although the attempted microwave treatment was found not to change the susceptibility of chitin to deacetylation (10). The present work describes a pretreatment process for chitin by microwave irradiation which resulted in an increase of its enzymatic hydrolysis by cabbage chitinase. The pretreatment process optimization was carried out by response surface analysis (11-13). The microwave irradiation was carried out using a microwave equipped with a noncontact infrared feedback controller which allowed fixing a constant temperature during microwave irradiation. Regarding the cost factor of the enzyme, we have recently described the use of pectinase (for chitin hydrolysis), which is considerably less expensive (than chitinases) and is freely available as commercial preparations for applications in the food-processing industry (14). The effect of microwave pretreatment on chitin hydrolysis by pectinase was also examined. Materials and Methods Materials. Chitin (from crab shells) was purchased from Sigma Chemical Co., St. Louis, MO. Pectinex Ultra SP-L (a highly purified preparation of pectolytic enzymes from a selected strain of Aspergillus niger) was from Novo Nordisk, Denmark. Biogel P2 was a product of Bio-Rad. Cabbage was obtained from the local market. All other chemicals used were analytical grade. Methods. Design of Experiments. Response surface methodology (RSM) is an efficient approach for obtaining optimum conditions (parameters) for a process when the latter depends on two or more variable parameters (15, 16). It consists of four stages: (1) Important variable parameters are identified. This presupposes some knowledge of the process. (2) The range in which various parameters can be varied is determined. This again is decided by practical considerations and keeping in view the operation of the process. (3) Some limited sets of parameters are taken, and prediction is tested by actual experiments. The prediction utilizes a multivariate equation. (4) The response surfaces generated finally predict one optimum set of parameters. In the present case, the Box-Behnken design was chosen as it requires the minimum number of experiments to predict the best performance conditions (16, 17). The experiments were designed using the software Design Expert, version 5.0 (Stat-Ease, Minneapolis). The independent variables considered for the process were chitin concentration, temperature, and time. The maximum amount of N-acetylglucosamine formed was taken as the dependent variable or response. Preparation of the Chitin Suspension. The chitin suspension was prepared by adding 1gofchitin to 100 ml of 0.05 M acetate buffer, ph 5.0, and stirring the suspension for 3hat25 C.Thesuspension was stirred again before use. Determination of Enzyme Activity. Enzyme activity using chitin as substrate was determined as described by Chang et al. (18). One unit of enzyme activity using chitin as substrate is defined as the amount of enzyme required for the formation of 1 nmol of the product /bp CCC: $ American Chemical Society and American Institute of Chemical Engineers Published on Web 07/19/2003

2 Biotechnol. Prog., 2003, Vol. 19, No (estimated as N-acetylglucosamine) per minute, under assay conditions. The amount of reducing sugar was estimated by the dinitrosalicyclic acid method (19). Purification of Chitinase. Fresh cabbage leaves (250 g) were homogenized with 750 ml of 0.1 M acetate buffer, ph 5.2, in a mixer. After the mixture was strained through four layers of cheesecloth, the turbid filtrate was centrifuged at 10000g for 10 min (Sigma 3K-30 refrigerated centrifuge). The supernatant obtained was dialyzed against several changes of 0.1 M acetate buffer, ph 5.2, and used as the chitinase extract (18). Microwave Irradiation. Various concentrations of chitin were taken in a total volume of 20 ml and placed in the microwave oven (model RM2001, Plazmatronika, Wroclaw, Poland; operating frequency 2.5 GHz). The oven had a built-in magnetic stirrer which was used to stir the reaction mixture. The temperature of the suspension was set as desired and measured using a noncontact infrared continuous feedback temperature system. A control was heated in a water bath (under optimized conditions) in an identical fashion but without microwave irradiation. Determination of Kinetic Parameters. K m and V max values of the enzymes were determined by measurement of enzyme activity with various concentrations of chitin at 40 C (with the enzyme extracted from cabbage) and at 37 C (with the enzyme from Pectinex Ultra SP-L), for 1 h. The values of the kinetic parameters were calculated with the Leonora software program (20), using a Lineweaver-Burk plot. Hydrolysis of Chitin by Cabbage Chitinase. Chitin [2% (w/v), microwave-irradiated at 57.5 C for 38 min] was incubated with different concentrations of chitinase, and the amount of reducing sugar was monitored after 1h. In a second experiment, chitin [2% (w/v), microwaveirradiated at 57.5 C for 38 min] was incubated with chitinase, and the amount of reducing sugar was monitored at different time intervals. Results and Discussion During microwave irradiation, polar molecules such as water (in the sample) align with the continuously changing magnetic field generated by microwaves. This is supposed to accelerate various chemical, biological, and physical processes (21-23). To study the individual and interactive effects of the three parameters (chitin concentration and time and temperature of microwave irradiation) on the amount of N-acetylglucosamine formed, a Box-Behnken design with 17 experiments was performed (24). Table 1 presents the design matrix of the independent variables. The experimental and predicted values of the amount of product formed are also given in Table 1. The results were analyzed using the analysis of variance (ANOVA) (12) (Table 2). The computed F ratio ( ) is much greater than the tabular F 0.01(9,7) value (6.72) at the 1% confidence level (16). This demonstrates that the quadratic regression model is highly significant, as is also evident from a very low probability value (P model ) ). The goodness of the model was checked by the correlation coefficient, R 2. The R 2 value of 0.99 indicates a good agreement between the experimental and predicted values of product formation (16). The response surface profile of the calculated model for the product formed is shown in Figure 1. To obtain a greater amount of the product, both the chitin concentration and the temperature of irradiation should be high Table 1. Effect of Various Process Parameters on the Amount of Product Formed When Pretreated Chitin Was Hydrolyzed by Cabbage Chitinase chitin concn (%, w/v) time (min) temp amt of product formed (nmol) ( C) exptl predicted Table 2. ANOVA of the Calculated Model for the Amount of N-Acetylglucoamine Formed (nmol) Model sum of squares F ratio no. of degrees of freedom 9 probability (P model) mean squares Residual sum of squares F ratio - no. of degrees of freedom 7 probability - mean squares correlation coefficient (R 2 ) 0.99 (Figure 1a). A similar trend is observed in Figure 1b,c. The Design Expert software predicted maximum product formation of nmol under optimized conditions of 2% (w/v) chitin concentration, 57.5 C, and 38 min of exposure to microwave irradiation. Experimentally, nmol of N-acetylglucosamine (NAG) was formed under these conditions, which was in good agreement with the predicted value. Figure 2 shows the effect of this treatment on chitin as seen in scanning electron microscopy (SEM). Whereas the control sample (just heated, not irradiated) shows a continuous splattered appearance (Figure 2a), SEM of the microwave-treated sample (Figure 2b) shows a discrete granular structure. Figure 3 shows the powder X-ray diffraction patterns of these samples. The diffraction patterns, in general, are in agreement with the one described in the literature for chitin (25). There is only a slight increase in the d values (where d is the interplanar spacing) upon microwave irradiation, indicating some limited unit cell expansion. Figure 4 shows the hydrolysis of microwave-irradiated chitin by varying the concentration of cabbage chitinase in1hat40 C.Italso shows the control in which the hydrolysis was carried out in an identical manner except microwave irradiation was not used (the temperature of hydrolysis was however kept at 40 C). The hydrolysis, as monitored by production of reducing sugar equivalents, indicates the amount of change (extent of reaction) in a fixed period of time. Thus, the result in the case of nonirradiated chitin reflects substrate exhaustion. In this particular case, it was more of a case of all accessible bonds in the chitin particles having been hydrolyzed. Hence, it is understandable that microwave treatment generated more accessible bonds and the hydrolysis continued with a linear response to enzyme concentration.

3 1650 Biotechnol. Prog., 2003, Vol. 19, No. 6 Figure 2. Scanning electron micrographs of (a) control and (b) microwave-irradiated chitin at a magnification factor of The bar represents 10 µm. Figure 1. (a, top) Effect of temperature and chitin concentration on the amount of product formed at a constant time of 38 min. (b, middle) Effect of time and chitin concentration on the amount of product formed at a constant temperature of 57.5 C. (c, bottom) Effect of time and temperature on the amount of product formed at a chitin concentration of 2% (w/v). Figure 5 shows the kinetics of hydrolysis of treated and untreated chitin by chitinase and agrees well with the consequence of microwave treatment stated above. The higher rate obtained in the case of treated chitin presumably reflects reduction in mass-transfer constraints for the enzyme accessing the substrate. The addition of fresh chitin (1%) at the end of 2hinthecase of the control (nonirradiated chitin) resulted in the fresh production of NAG. Figure 6 shows the actual soluble products obtained after 30 min and 48 h of enzymatic hydrolysis of pretreated chitin (both microwave-irradiated and control). The products were NAG, (NAG) 2, and (NAG) 3 only; i.e., no higher oligomers could be detected. At 30 min, (NAG) 3 was the major product, although (NAG) 2 and NAG were also already produced in comparable amounts. After 48 h of enzymatic hydrolysis, just heat-treated chitin produced all three products in comparable amounts, with (NAG) 2 being produced in a slightly higher amount. In the case of microwave-treated chitin, there was a clearer trend in the product profile of NAG > (NAG) 2 > (NAG) 3. Thus, at least in this case, one can expect all chitin to be hydrolyzed to NAG ultimately. Figure 7 shows the Lineweaver-Burk plot, which allows calculation of kinetic parameters K m, V max, and V max /K m for treated and untreated chitin (Table 3). The catalytic efficiency of the enzyme is measured by k cat /K m (26). As k cat ) V max /[total enzyme], V max /K m also reflects the relative catalytic efficiencies, if the same amount of

4 Biotechnol. Prog., 2003, Vol. 19, No Figure 3. X-ray diffraction pattern of (a) control and (b) microwave-irradiated chitin. The samples were lyophilized and ground in an agate mortar after being mixed with an internal standard (NaCl), and the diffraction pattern was recorded on a Bruker D8 advance AXS X-ray diffractometer and Ni monochromator. The peak at 2θ ) 31 is that of the internal standard (NaCl). Figure 4. Hydrolysis of chitin with different amounts of cabbage chitinase. Different volumes of cabbage chitinase (stock solution of 38 U ml -1 ) were taken, and the volumes were increased to 0.5 ml with 0.05 M acetate buffer, ph 5.2, and incubated with microwave-treated chitin (O) and the control (b). The assay was carried out as described in the text. Each experiment was carried out in duplicate, and the difference in individual readings in each set was less than 5%. enzyme is used in the two cases. It is also well-known that this ratio can be used to compare substrate specificities (26). The higher value obtained with microwave- Figure 5. Hydrolysis of chitin by cabbage chitinase. The experiment was performed as described in the text by carrying out hydrolysis of microwave-treated chitin (O) and the control (b) by 9.5 U of chitinase. The arrow denotes the time when fresh chitin [2% (w/v)] was added to the reaction mixture in the case of the control. Each experiment was carried out in duplicate, and the difference in individual readings in each set was less than 5%.

5 1652 Biotechnol. Prog., 2003, Vol. 19, No. 6 Figure 6. Time course of the hydrolysis pattern of microwavetreated chitin (empty symbols) and the control (filled symbols) by cabbage chitinase. The experiments were carried out as described in the caption to Figure 5. The products formed after 30 min (circles) and after 48 h (triangles) of enzymatic hydrolysis were passed through a precalibrated Biogel P2 column, equilibrated with 0.05 M sodium acetate buffer, ph 5.0. The column (bed volume 196 ml, cm) was calibrated with N-acetylglucosamine (M r 221.2), maltose (M r ), and raffinose (M r 594.4). The amounts of sugars produced were detected after their reaction with dinitrosalicylic acid (16). Figure 7. Kinetic parameters for hydrolysis of chitin by cabbage chitinase. The experiment was carried out as described in the text for microwave-treated chitin (O) and the control (b). Table 3. Determination of Kinetic Parameters for Microwave-Treated Chitin and the Control Using Cabbage Chitinase and Pectinase kinetic param microwave-treated chitin control Chitinase K m (mg ml -1 ) V max (nmol h -1 mg -1 ) V max/k m (nmol h -1 mg -2 ml) Pectinase K m (mg ml -1 ) V max (nmol h -1 mg -1 ) V max/k m (nmol h -1 mg -2 ml) treated chitin (31.7 nmol h -1 mg -2 ml) as compared to the value with untreated chitin (21.1 nmol h -1 mg -2 ml) shows clearly that the former is a better substrate for the enzyme. It may be noted that, in this case, untreated chitin refers to the chitin kept at 57.5 C, so the result of microwave treatment was not due to a thermal effect. Figure 8. Kinetic parameters for hydrolysis of chitin by pectinase. The experiment was carried out as described in the text for microwave-treated chitin (O) and the control (b). Also, the temperature during microwave treatment was kept constant at 57.5 C ((0.5 C) by a nonthermal infrared feedback temperature controller. It is also interesting to note that the increase in V max /K m was solely due to reduction in K m. Figure 8 shows the Lineweaver-Burk plot when a commercial preparation of pectinase was used to hydrolyze chitin. As reported by us earlier, pectinase hydrolyzes chitin but at a slower rate as compared to chitinases (14). Kinetic consequences of using microwave-treated chitin were summarized in Table 3. The changes are less significant in this case. Nevertheless, microwave treatment of the substrate resulted in marginal reduction of K m as well as an increase of V max. The latter implies that the turnover number (since V max ) k cat [total enzyme]) also increased when the microwave-irradiated chitin was used as the substrate. Higher V max /K m reflects that a more efficient process design was possible due to better affinity of the substrate as a result of microwave pretreatment. Conclusion The results described here indicate that microwave pretreatment, at least in the case of chitin, works successfully for more efficient enzymatic hydrolysis of the macromolecule. The SEM and X-ray diffraction data indicate that this may be due to greater accessibility of the susceptible bonds in the microwave-irradiated chitin. The fact that chitin subjected to just high temperature did not give similar results shows that the effect of microwave pretreatment was of nonthermal origin. Acknowledgment The partial financial support provided by the Department of Science and Technology, Council for Scientific and Industrial Research (Extramural Division and Technology Mission on Oilseeds, Pulses and Maize), all Government of India organizations, is gratefully acknowledged. We acknowledge Dr. A. K. Ganguli (Indian Institute of Technology (IIT), Delhi) for his help in recording and interpreting the X-ray diffraction patterns of the samples. The financial support provided by IIT Delhi to K.M., in the form of a junior research fellowship, is also acknowledged. References and Notes (1) Deshpande, M. V Enzymatic degradation of chitin and its biological applications. J. Sci. Ind. Res. 1986, 45,

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