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In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation
Biomaterials 22 (2001) 1653}1658
In vitro degradation of chitosan by a commercial enzyme preparation: e!ect of molecular weight and degree of deacetylation Hua Zhang, Steven H. Neau* Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, USA Received 12 July 2000; accepted 9 October 2000
Abstract A commercially available almond emulsin -glucosidase preparation has been reported to have chitobiose activity, and can hydrolyze chitin substrates due to a chitinase present in the enzyme preparation. This -glucosidase preparation was used to investigate hydrolytic activity on "ve chitosan samples with di!erent molecular weight and degree of deacetylation. The degree of deacetylation and molecular weight of the chitosan samples were determined using a circular dichroism and a viscometric method, respectively. The hydrolytic activity of this -glucosidase preparation on chitosan was monitored viscometrically as the most convenient means of screening. Solutions of chitosan in pH 5.0 acetate bu!er were prepared using the di!erent viscosity grades of chitosan. The speci"c viscosity, measured after addition of -glucosidase to the above solutions, decreased dramatically over time in comparison to that of the respective control mixture without enzyme. Eadie}Hofstee plots established that hydrolysis of chitosan by this enzyme preparation obeyed Michaelis}Menten kinetics. Apparent Michaelis}Menten parameters and initial degradation rates were calculated and compared to determine the in#uences of the degree of deacetylation and molecular weight on the hydrolysis. The results show that higher molecular weight and higher degree of deacetylation chitosans possessed a lower a$nity for the enzyme and a slower degradation rate. Faster degradation rates, then, are expected with lower molecular weight and low degree of deacetylation chitosans. Hydrolysis of these chitosan samples con"rms the existence of a chitinase in the almond emulsin -glucosidase preparation, and further studies are warranted. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Chitosan; Almond emulsin; -Glucosidase; Degradation; Michaelis}Menten kinetics
1. Introduction Chitosan [-(1P4)-2-amino-2-deoxy-D-glucose], a functional and basic linear polysaccharide, is prepared by N-deacetylation of chitin, the most abundant natural polysaccharide on the earth after cellulose, resulting in a copolymer of glucosamine and N-acetylglucosamine linked through -(1P4) glycosidic linkages [1]. The procedure to accomplish N-deacetylation involves alkaline hydrolysis, which can result in incomplete Ndeacetylation and also results in depolymerization to varying extents. Thus, chitosan is available with di!erent molecular weights and degrees of deacetylation. Because of its potential use as a biodegradable material for drug delivery systems, as well as other applications in the
* Corresponding author. Tel.: #1-816-235-2425; fax: #1-816-2355190. E-mail address: [email protected] (S.H. Neau).
biomedical and biotechnological "elds, enzymatic hydrolysis of chitosan is of interest recently. -Glucosidase (-D-glucoside glucohydrolase; EC 3.2.1.21) has widespread sources including bacteria, animals, and plants. It has broad substrate speci"city and is capable of cleaving -glucosidic linkages of conjugated glucosides and disaccharides [2]. However, -glucosidase cannot hydrolyze polysaccharides. Any substitution on carbon atoms 2, 3, or 4 of a sugar moiety, such as the amine at the second carbon in chitosan, completely prevents hydrolysis by -glucosidase [3]. At best, its substrates include some oligosaccharides, such as cellotetraose and cellopentaose [4]. Its enzymatic properties depend on the source and conditions under which the enzyme is produced and puri"ed [5]. With the development of elegant methodologies, the speci"c catalytic residues of some -glucosidases from di!erent sources have been identi"ed [6}8], and the genes for di!erent glucosidases have been cloned and expressed [9}12]. On the basis of amino acid sequence similiaries, a large
0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 3 2 6 - 4
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number of -glucosidases are assigned to glycosyl hydrolase family 1, a large and biologically important group of enzymes [6]. Among the enzymes from that family, glucosidase from almond emulsin is one of the earliest enzymes investigated because of its simple assay procedure, its easy isolation process, as well as its commercial availability. It has also been reported that -glucosidase from almond emulsin can hydrolyze chitin substrates due to an existing chitinase in the enzyme preparation [13,14]. Chitosan can be enzymatically degraded by chitinases and chitosanases [15]. However, chitinases and chitosanases are presently enzymes that are unavailable in bulk quantities for commercial applications. Because of the properties of the almond -glucosidase preparation noted above, the readily available almond emulsin -glucosidase was studied for its hydrolytic activity on "ve samples of chitosan which di!er in molecular weight and degree of deacetylation (DD). The in#uences of DD and molecular weight on chitosan enzymatic susceptibility were examined using a viscometric technique that re#ects the e!ect of enzymatic scission at sites along the chitosan polymer chain [16]. Initial degradation rates and apparent Michaelis}Menten parameters for the degradation were also estimated.
2. Materials and method 2.1. Materials Chitosan products, labeled Sea Cure 242, 342, 442 and 443 (sample 1, 2, 3, and 4, respectively) from Protan Laboratories Inc. (Redmond, WA) were gifts from G.D. Searle (Skokie, IL). Chitosan HD (sample 5) was purchased from DCV BioNutritionals (Wilmington, DE). Almond emulsin -glucosidase was obtained from ICN Biomedical Inc. (Aurora, OH). This was the higher purity sample with a reported -glucosidase activity of 3811 unit/mg of material, where a unit represents 1.0 mol of glucose liberated from salicin at pH 5.0 and 373C. Perchloric acid (60% v/v) was purchased from Fisher Scienti"c (St. Louis, MO). N-acetylglucosamine was obtained from Sigma Chemical Co. (St. Louis, MO). 2.2. Degree of deacetylation determination The acetyl content of the chitosan samples was determined by a literature circular dichroism (c.d.) method [17]. A 0.2% (w/v) chitosan solution was prepared in 0.1 M perchloric acid. A 0.2% (w/v) N-acetylglucosamine solution prepared in the same solvent was used as the 100% N-acetylated standard. The pathlength of the cells (Hellma Inc., New York, NY) was 0.2 mm. The spectrum of dichroic absorbance was recorded on an IBM PC connected to a J-720 spectropolarimeter (Japan
Spectroscopic Co. Ltd., Tokyo, Japan). A peak located near 211 nm was characteristic of an acetylated amine chromophore on a glycosidic ring in acidic media. The existence of only a single peak near 211 nm and the peak height varying with the degree of acetylation was readily apparent. The degree of N-acetylation was calculated as follows: "161H 100/[161H #203(H !H )], G G G G where H is the height of the signal of the chitosan sample G at 211 nm on the c.d. spectrum and H is the height at the same wavelength on the c.d. spectrum of the N-acetylglucosamine. Subsequently, the degree of deacetylation of the chitosan sample can be obtained as DD"100%! . G 2.3. Molecular weight determination Molecular weight was determined by a literature viscometric method [18]. Chitosan samples were prepared in 0.2 M acetic acid/0.1 M sodium acetate aqueous solutions. The relative viscosity, , of chitosan samples was measured using a Ubbelohde capillary viscometer at 30$0.53C. Speci"c viscosity was determined using "( ! )/ Intrinsic viscosity, [], is de"ned as reduced viscosity, , extrapolated to a chitosan concentration, C, of zero: []"( /C) "( ) , ! ! where C is in g/ml. Viscosity average molecular weight was calculated based on the Mark}Houwink equation []"KM? with K"1.64;10\;DD, a"!1.02;10\;DD#1.82, where DD is the degree of deactylation of chitosan expressed as the percentage [19]. 2.4. Enzymatic hydrolysis Hydrolysis of chitosan was studied using a literature viscometric procedure [16]. Each solution with the speci"c chitosan concentration was prepared by dissolving the desired chitosan in 0.1 M acetate bu!er solution at pH 5.0. -Glucosidase was dissolved separately in the same buffer system. The 0.1 M acetate bu!er concentration provides an ionic strength high enough to eliminate interference di!erences due to di!erent ionic impurities in the various chitosan samples [15]. The hydrolytic reaction of chitosan was started by adding a certain volume of -glucosidase solution to a certain volume of the
H. Zhang, S.H. Neau / Biomaterials 22 (2001) 1653}1658
acetate bu!er solution containing chitosan to obtain 0.02% (w/v) -glucosidase and 0.1, 0.3, 0.5 or 0.7% (w/v) chitosan, respectively. Each mixture was introduced into a Ubbelohde capillary viscometer held at 30$0.53C. The hydrolyses were monitored by viscosity determination after 1}5 h. By using this procedure, the decrease in speci"c viscosity was measured as a function of time. The initial degradation rates were determined using the results from this viscometric assay.
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Table 1 Degree of deacetylation (DD), intrinsic viscosity ([]), and molecular weight (M ) for chitosan samples Chitosan
DD (%)
[] (ml/g)
M ;10\
Sample Sample Sample Sample Sample
77.8$0.97 76.0$1.46 77.0$1.72 85.6$1.25 92.4$0.90
451 529 718 743 509
6.52 8.16 10.6 8.22 4.65
1 2 3 4 5
Mean$SD, n"3.
3. Results and discussion 3.1. Characterization of chitosan samples The acetyl content in chitosan can be determined by titration [20], NMR [21], IR [22,23], and c.d. [17] analytical techniques. Among them, c.d. spectroscopy seems to be the better choice for precise measurement [24]. The degree of deacetylation of the "ve chitosan samples obtained from the c.d. measurement are listed in Table 1. Although the average molecular weight, the most di$cult parameter to be obtained precisely, can be determined by several methods, viscometry is claimed to be the simplest, most rapid, and probably the most precise determination method [24]. Thus, the viscosity average molecular weight (M ) of the "ve chitosan samples was determined by this method. Fig. 1 presents the reduced viscosity versus concentration plot for the samples. From the y-intercept, the intrinsic viscosity was obtained which was applied in the Mark}Houwink equation to calculate the M using the appropriate Mark}Houwink parameters. The intrinsic viscosities and the M for the chitosan samples are reported in Table 1.
Table 1 con"rms that data for the "ve chitosan samples cover a range of M and DD. Samples 1}3 have almost the same DD, but di!erent M . Samples 2 and 4 possess similar M but di!erent DD. Sample 5 has the lowest M and the highest DD. 3.2. Degradation of chitosan samples Degradation of some relatively rigid linear polysaccharides, with Mark}Houwink equation exponent a close to unity, can be conveniently investigated by the viscometric method [25]. The a value here is dependent upon the conformation of the polymer. Previous studies indicate that DD can change the rigidity of the chitosan chain in solution, which in turn changes the a value [19,26]. The a values of these chitosan samples are di!erent, but they are each close to unity based upon the calculation described above. Since each of the chitosan samples can be fully extended in the solutions, the change in the viscosity of their solutions can re#ect the change in the degree of polymerization of the chitosan linear chain. Thus, the course of the degradation of these chitosan samples was studied by viscosity measurement, and characterized by the decrease of the speci"c viscosity after a given time of degradation. Fig. 2 is an example of the speci"c viscosity as a function of reaction time in the presence and absence of 0.02% (w/v) -glucosidase under the reaction conditions. The plot shows that the speci"c viscosity of the reaction mixture decreased dramatically over time in comparison to that of the control without enzyme. Such viscosity reduction pro"les were seen with each of the chitosan samples. In Fig. 2, there is a slight reduction in the speci"c viscosity for the control, which is probably due to the acid hydrolysis of the chitosan sample at pH 5.0. As shown in Fig. 2, the results indicate that enzyme treatment resulted in a substantial loss in viscosity of the chitosan solution, indicating depolymerization, and such depolymerization took place very quickly during the initial 2 h. 3.3. Determination of the degradation rate (r)
Fig. 1. Plot of reduced viscosity versus concentration for the "ve chitosan samples (mean$SD, n"3): (£) sample 1; (*) sample 2; (䉭) sample 3; (*) sample 4; (䊐) sample 5.
According to the literature method, the initial rate of degradation was determined with viscosity data by
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Fig. 2. Speci"c viscosity of 0.7% (w/v) sample 3 solution as a function of time following treatment with -glucosidase at 303C (mean$SD, n"3): (䊐) without 0.02% (w/v) -glucosidase; (䉭) with 0.02% (w/v) -glucosidase.
Table 2 Initial degradation rates (r) of the "ve chitosan samples by -glucosidase Chitosan
r (g/(ml h));10
Sample Sample Sample Sample Sample
6.849 4.452 2.017 2.542 4.042
1 2 3 4 5
Concentrations of the chitosans and -glucosidase were 0.1 and 0.02% (w/v), respectively.
employing the Mark}Houwink equation to convert the viscosity reduction to the degree of scission of the glycoside linkage of the chitosan [16]. However, K and a values in the Mark}Houwink equation are dependent on DD, as described in the M determination section. They are not constant for the "ve chitosan samples, and the a value cannot be assumed to be unity. To account for the varying a values, the viscosity data were plotted as [1/( /c) ? !1/( /c) ? ] instead of [1/( /c) !1/ RR R RR ( /c) ] versus time to establish the time course of R enzyme degradation of chitosan. Such plots are presented in Fig. 3. The initial degradation rates (r) for the chitosan samples were determined from the slope of the initial linear portion of each "tted quadratic equation by using the following equation (see Table 2): r"slope K ?M C, where M is the monomer weight of the chitosan [16].
Fig. 3. Time course of degradation of "ve 0.1% (w/v) chitosan samples by 0.02% (w/v) -glucosidase (mean$SD, n"3): (£) sample 1; (*) sample 2; (䉭) sample 3; (*) sample 4; (䊐) sample 5.
It has been shown that a linear relation would be obtained for a "rst order, random depolymerization of chitosan samples treated by H O in the presence of FeCl [16]. However, the time course of degradation of chitosan by almond emulsin -glucosidase preparation was non-linear as seen in Fig. 3. The curvature observed with each of the samples might indicate a non-random depolymerization of chitosan by such an enzyme preparation. This could be due to faster reacting cleavage sites distributed along the linear chain. Fig. 3 reveals that the initial slope increased as M decreased and DD decreased. A decrease in the DD of chitosan would increase its similarity to chitin, the observed substrate of the chitinase present in almond emulsin -glucosidase preparations. 3.4. Kinetics of -glucosidase degradation of chitosan samples The initial velocity () for use in the Eadie}Hofstee plots was calculated based upon the following equation:
"r[S] M /C"slope K ?[S] M , where [S] is the molar concentration of glycosidic link age at the beginning of the reaction. The results of the initial degradation velocity () and substrate concentrations ([S]) were plotted according to the Eadie}Hofstee equation: /[S]" /K !/K
+ + as presented in Fig. 4. The plots show that the degradation of chitosan by the -glucosidase preparation obeyed Michaelis}Menten-type kinetics. Apparent Michaelis} Menten parameters were estimated using the values of
H. Zhang, S.H. Neau / Biomaterials 22 (2001) 1653}1658
Fig. 4. Eadie}Hosftee plots for the "ve chitosan samples (mean$SD, n"3): (£) sample 1; (*) sample 2; (䉭) sample 3; (*) sample 4; (䊐) sample 5.
Table 3 Michaelis}Menten parameters, apparent catalytic constants and k /K for the "ve chitosan samples + Chitosan
K + (M)
;10
(M/ min)
Apparent k ;10
k /K ;10 +
Sample Sample Sample Sample Sample
3.13 3.42 5.01 4.00 5.81
20.7 18.2 13.7 9.86 17.5
10.4 9.10 6.85 4.93 8.75
3.32 2.66 1.37 1.23 1.51
1 2 3 4 5
the slope and y-intercept. A better estimation of the catalytic e$ciency of an enzyme for a particular substrate is k /K , in which k is de"ned as /[E] , and [E] +
2 2 represents the total molar concentration of the enzyme. Since the molecular weight of the chitinase in the glucosidase preparation used is unknown, its molar concentration cannot be calculated. An apparent k was obtained using [E] "0.02% (w/v) because the 2 glucosidase preparation concentration was constant at that level with each hydrolysis sample in the degradation studies. The K , , k , and k /K values are listed + + in Table 3. 3.5. Ewect of M and DD on the enzymatic susceptibility of chitosan Fig. 5 presents the M e!ect on the initial degradation rates (r) of chitosan by almond emulsin -glucosidase
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Fig. 5. Initial degradation rate (r) of -glucosidase hydrolysis of "ve 0.1% (w/v) chitosan samples versus the molecular weight (M ) of the chitosan.
preparation. Samples 1}3 with di!erent M , but essen tially the same DD, had a di!erent susceptibility to degradation by such enzyme preparation. The results show that the initial degradation rate decreased with an increase in the magnitude of the M . To observe the DD e!ect, one can compare the results for samples 2 and 4, which have similar M but di!erent DD. The higher DD sample (sample 4) had a slower initial degradation rate than did the lower DD sample (sample 2). Such M and DD e!ects on the susceptibility of chitosan to almond emulsin -glucosidase preparation degradation can be con"rmed in the results for sample 5 which has the lowest M but the highest DD. Its reaction to enzymatic degra dation was dependent on both M and DD e!ects. Its degradation rate would be faster than samples 3 and 4 due to its lower M , but slower than samples 1 and 2 because of its higher DD. As shown in Table 3, the results again indicate that a lower M and lower DD chitosan sample possessed a higher a$nity for the enzyme (K ), + experienced a higher catalytic e$ciency (k /K ), and + would be the sample most susceptible to enzymatic degradation. However, since there are only "ve data points and limited ranges for M and DD, further study is warranted.
4. Conclusions The results indicate that this almond emulsin glucosidase preparation has the ability to degrade chitosan and that its chitosanolytic activity can be
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H. Zhang, S.H. Neau / Biomaterials 22 (2001) 1653}1658
conveniently monitored viscometrically. The degradation of chitosan obeys Michaelis}Menten kinetics. This investigation also clearly reveals that the initial degradation rate of a chitosan sample is dependent on its molecular weight and its degree of N-deacetylation. The results show that chitosan with a lower molecular weight and a lower degree of N-deacetylation is a more susceptible substrate. Since -glucosidase itself cannot hydrolyze polysaccharides such as chitosan, the degradation activity seen in this study cannot be due to the action of -glucosidase. It is reported that a -glucosidase preparation from almond emulsin contains a chitinase, and the degradation of these chitosan samples con"rms an existing chitinase in the preparation. The chitinase in this commercially available almond emulsin -glucosidase warrants further investigation.
Acknowledgements The authors acknowledge the gift of chitosan from G.D. Searle (Skokie, IL). We thank Dr. Joseph R. Mattingly for his assistance in the c.d. analysis. This project was supported in part by a grant from the University of Missouri Research Board.
References [1] Illum L. Chitosan and its use as a pharmaceutical excipient. Pharm Res 1998;15:1326}31. [2] Dale MP, Kop#er WP, Chait I, Byers LD. -Glucosidase: substrate, solvent, and viscosity variation as probes of the ratelimiting steps. Biochemistry 1986;25:2522}9. [3] Dixon MP, Webb EC. Enzyme speci"city. In: Dixon M, Webb EC, editors. Enzymes. New York: Academic Press Inc., 1964. p. 224}5. [4] Day AG, Withers SG. The puri"cation and characterization of a beta-glucosidase from Alcaligenes faecalis. Biochem Cell Biol 1986;64:914}22. [5] Shewale JG. -Glucosidase: its role in cellulase synthesis and hydrolysis of cellulose. Int J Biochem 1982;14:435}43. [6] Sanz-Aparicio J, Hermoso JA, Martinez-Ripoll M, Lequerica JL, Polaina J. Crystal structure of beta-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolyses. J Mol Biol 1998;275:419}502. [7] Hakulinen N, Paavilainen S, Korpela T, Rouvinen J. The crystal structure of beta-glucosidase from Bacillus circulans sp. alkalophilus: ability to form long polymeric assemblies. J Struct Biol 2000;129:69}79. [8] He S, Withers SG. Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identi"cation of its active site nucleophile. J Biol Chem 1997;272:24864}7.
[9] Hashimoto W, Miki H, Nankai H, Sato N, Kawai S, Murata K. Molecular cloning of two genes for beta-glucosidase in Bacillus sp. GL1 and identi"cation of one as a gellan-degrading enzyme. Arch Biochem Biophys 1998;360:1}9. [10] Bauer MW, Bylina EJ, Swanson RV, Kelly RM. Comparison of a beta-glucosidase and a beta-mannosidase from the hyperthermophilic archaeon pyrococcus furiosus. Puri"cation, characterization, gene cloning, and sequence analysis. J Biol Chem 1996;271:23749}55. [11] Paavilainen S, Hellman J, Korpela T. Puri"cation, characterization, gene cloning, and sequencing of a new beta-glucosidase from Bacillus circulans subsp. alkalophilus. Appl Environ Microbiol 1993;59:927}32. [12] Dharmawardhana DP, Ellis BE, Carlson JE. CDNA cloning and heterologous expression of coniferin beta-glucosidase. Plant Mol Biol 1999;40:365}72. [13] Grassmann W, Zechmeister L, Bender R, Toth G. UG ber die chitinspaltung durch emulsin-praK parate (III. Mitteil uK ber enzymatische spaltung von polysacchariden). Chem Berichte 1934;67:1}5. [14] Wadsworth SA, Zikakis JP. Chitinolytic activity of commercially available -glucosidase. In: Zikakis JP, editor. Chitin, chitosan, and related enzymes. New York: Academic Press Inc., 1984. p. 182}91. [15] Tracey MV. Chitinase in some basidiomycetes. Biochem J 1955;61:579}86. [16] Nordtveit RJ, Va rum KM, Smidsr+d O. Degradation of fully water-soluble, partially N-acetylated chitosans with lysozyme. Carbohydr Polym 1994;23:253}60. [17] Domard A. Determination of N-acetyl content in chitosan samples by c.d. measurements. Int J Biol Macromol 1987;9:353}8. [18] Chen RH, Hwa HD. E!ect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane. Carbohydr Polym 1996;29:253}60. [19] Wang W, Bo S, Li S, Qin W. Determination of the Mark}Houwink equation for chitosan with di!erent degrees of deacetylation. Int J Biol Macromol 1991;13:281}5. [20] Domard A, Rinaudo M. Preparation and characterization of fully deacetylated chitosan. Int J Biol Macromol 1983;5:49}52. [21] Kenne L, Lindberg B. N-deacetylation of polysaccharides. Methods Carbohydr Chem 1980;III:295}6. [22] Domszy JG, Robers GAF. Evaluation of infrared spectroscopic techniques for analyzing chitosan. Macromol Chem 1985;186:1671}7. [23] Miya M, Iwamoto R, Yoshikawa G, Mima S. IR spectroscopic determination of CONH content in highly deacetylated chitosan. Int J Biol Macromol 1980;2:323}4. [24] Rinaudo M, Domard A. Solution properties of chitosan. In: Skja k-Brvk G, Anthonsen T, Sandford P, editors. Chitin and chitosan: sources, chemistry, biochemistry, physical properties and applications. New York: Elsevier Applied Science, 1988. p. 71}86. [25] Smidsr+d O, Haug A, Larsen B. Oxidative-reductive depolymerization: a note on the comparison of degradation rates of di!erent polymers by viscosity measurements. Carbohydr Res 1987;5:485. [26] Anthonsen MW, Va rum KM, Smidsr+d O. Solution properties of chitosans: conformation and chain sti!ness of chitosans with di!erent degree of N-acetylation. Carbohydr Polym 1993;22:193}201.
In vitro degradation of chitosan by a commercial enzyme preparation: e!ect of molecular weight and degree of deacetylation Hua Zhang, Steven H. Neau* Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, USA Received 12 July 2000; accepted 9 October 2000
Abstract A commercially available almond emulsin -glucosidase preparation has been reported to have chitobiose activity, and can hydrolyze chitin substrates due to a chitinase present in the enzyme preparation. This -glucosidase preparation was used to investigate hydrolytic activity on "ve chitosan samples with di!erent molecular weight and degree of deacetylation. The degree of deacetylation and molecular weight of the chitosan samples were determined using a circular dichroism and a viscometric method, respectively. The hydrolytic activity of this -glucosidase preparation on chitosan was monitored viscometrically as the most convenient means of screening. Solutions of chitosan in pH 5.0 acetate bu!er were prepared using the di!erent viscosity grades of chitosan. The speci"c viscosity, measured after addition of -glucosidase to the above solutions, decreased dramatically over time in comparison to that of the respective control mixture without enzyme. Eadie}Hofstee plots established that hydrolysis of chitosan by this enzyme preparation obeyed Michaelis}Menten kinetics. Apparent Michaelis}Menten parameters and initial degradation rates were calculated and compared to determine the in#uences of the degree of deacetylation and molecular weight on the hydrolysis. The results show that higher molecular weight and higher degree of deacetylation chitosans possessed a lower a$nity for the enzyme and a slower degradation rate. Faster degradation rates, then, are expected with lower molecular weight and low degree of deacetylation chitosans. Hydrolysis of these chitosan samples con"rms the existence of a chitinase in the almond emulsin -glucosidase preparation, and further studies are warranted. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Chitosan; Almond emulsin; -Glucosidase; Degradation; Michaelis}Menten kinetics
1. Introduction Chitosan [-(1P4)-2-amino-2-deoxy-D-glucose], a functional and basic linear polysaccharide, is prepared by N-deacetylation of chitin, the most abundant natural polysaccharide on the earth after cellulose, resulting in a copolymer of glucosamine and N-acetylglucosamine linked through -(1P4) glycosidic linkages [1]. The procedure to accomplish N-deacetylation involves alkaline hydrolysis, which can result in incomplete Ndeacetylation and also results in depolymerization to varying extents. Thus, chitosan is available with di!erent molecular weights and degrees of deacetylation. Because of its potential use as a biodegradable material for drug delivery systems, as well as other applications in the
* Corresponding author. Tel.: #1-816-235-2425; fax: #1-816-2355190. E-mail address: [email protected] (S.H. Neau).
biomedical and biotechnological "elds, enzymatic hydrolysis of chitosan is of interest recently. -Glucosidase (-D-glucoside glucohydrolase; EC 3.2.1.21) has widespread sources including bacteria, animals, and plants. It has broad substrate speci"city and is capable of cleaving -glucosidic linkages of conjugated glucosides and disaccharides [2]. However, -glucosidase cannot hydrolyze polysaccharides. Any substitution on carbon atoms 2, 3, or 4 of a sugar moiety, such as the amine at the second carbon in chitosan, completely prevents hydrolysis by -glucosidase [3]. At best, its substrates include some oligosaccharides, such as cellotetraose and cellopentaose [4]. Its enzymatic properties depend on the source and conditions under which the enzyme is produced and puri"ed [5]. With the development of elegant methodologies, the speci"c catalytic residues of some -glucosidases from di!erent sources have been identi"ed [6}8], and the genes for di!erent glucosidases have been cloned and expressed [9}12]. On the basis of amino acid sequence similiaries, a large
0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 3 2 6 - 4
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number of -glucosidases are assigned to glycosyl hydrolase family 1, a large and biologically important group of enzymes [6]. Among the enzymes from that family, glucosidase from almond emulsin is one of the earliest enzymes investigated because of its simple assay procedure, its easy isolation process, as well as its commercial availability. It has also been reported that -glucosidase from almond emulsin can hydrolyze chitin substrates due to an existing chitinase in the enzyme preparation [13,14]. Chitosan can be enzymatically degraded by chitinases and chitosanases [15]. However, chitinases and chitosanases are presently enzymes that are unavailable in bulk quantities for commercial applications. Because of the properties of the almond -glucosidase preparation noted above, the readily available almond emulsin -glucosidase was studied for its hydrolytic activity on "ve samples of chitosan which di!er in molecular weight and degree of deacetylation (DD). The in#uences of DD and molecular weight on chitosan enzymatic susceptibility were examined using a viscometric technique that re#ects the e!ect of enzymatic scission at sites along the chitosan polymer chain [16]. Initial degradation rates and apparent Michaelis}Menten parameters for the degradation were also estimated.
2. Materials and method 2.1. Materials Chitosan products, labeled Sea Cure 242, 342, 442 and 443 (sample 1, 2, 3, and 4, respectively) from Protan Laboratories Inc. (Redmond, WA) were gifts from G.D. Searle (Skokie, IL). Chitosan HD (sample 5) was purchased from DCV BioNutritionals (Wilmington, DE). Almond emulsin -glucosidase was obtained from ICN Biomedical Inc. (Aurora, OH). This was the higher purity sample with a reported -glucosidase activity of 3811 unit/mg of material, where a unit represents 1.0 mol of glucose liberated from salicin at pH 5.0 and 373C. Perchloric acid (60% v/v) was purchased from Fisher Scienti"c (St. Louis, MO). N-acetylglucosamine was obtained from Sigma Chemical Co. (St. Louis, MO). 2.2. Degree of deacetylation determination The acetyl content of the chitosan samples was determined by a literature circular dichroism (c.d.) method [17]. A 0.2% (w/v) chitosan solution was prepared in 0.1 M perchloric acid. A 0.2% (w/v) N-acetylglucosamine solution prepared in the same solvent was used as the 100% N-acetylated standard. The pathlength of the cells (Hellma Inc., New York, NY) was 0.2 mm. The spectrum of dichroic absorbance was recorded on an IBM PC connected to a J-720 spectropolarimeter (Japan
Spectroscopic Co. Ltd., Tokyo, Japan). A peak located near 211 nm was characteristic of an acetylated amine chromophore on a glycosidic ring in acidic media. The existence of only a single peak near 211 nm and the peak height varying with the degree of acetylation was readily apparent. The degree of N-acetylation was calculated as follows: "161H 100/[161H #203(H !H )], G G G G where H is the height of the signal of the chitosan sample G at 211 nm on the c.d. spectrum and H is the height at the same wavelength on the c.d. spectrum of the N-acetylglucosamine. Subsequently, the degree of deacetylation of the chitosan sample can be obtained as DD"100%! . G 2.3. Molecular weight determination Molecular weight was determined by a literature viscometric method [18]. Chitosan samples were prepared in 0.2 M acetic acid/0.1 M sodium acetate aqueous solutions. The relative viscosity, , of chitosan samples was measured using a Ubbelohde capillary viscometer at 30$0.53C. Speci"c viscosity was determined using "( ! )/ Intrinsic viscosity, [], is de"ned as reduced viscosity, , extrapolated to a chitosan concentration, C, of zero: []"( /C) "( ) , ! ! where C is in g/ml. Viscosity average molecular weight was calculated based on the Mark}Houwink equation []"KM? with K"1.64;10\;DD, a"!1.02;10\;DD#1.82, where DD is the degree of deactylation of chitosan expressed as the percentage [19]. 2.4. Enzymatic hydrolysis Hydrolysis of chitosan was studied using a literature viscometric procedure [16]. Each solution with the speci"c chitosan concentration was prepared by dissolving the desired chitosan in 0.1 M acetate bu!er solution at pH 5.0. -Glucosidase was dissolved separately in the same buffer system. The 0.1 M acetate bu!er concentration provides an ionic strength high enough to eliminate interference di!erences due to di!erent ionic impurities in the various chitosan samples [15]. The hydrolytic reaction of chitosan was started by adding a certain volume of -glucosidase solution to a certain volume of the
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acetate bu!er solution containing chitosan to obtain 0.02% (w/v) -glucosidase and 0.1, 0.3, 0.5 or 0.7% (w/v) chitosan, respectively. Each mixture was introduced into a Ubbelohde capillary viscometer held at 30$0.53C. The hydrolyses were monitored by viscosity determination after 1}5 h. By using this procedure, the decrease in speci"c viscosity was measured as a function of time. The initial degradation rates were determined using the results from this viscometric assay.
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Table 1 Degree of deacetylation (DD), intrinsic viscosity ([]), and molecular weight (M ) for chitosan samples Chitosan
DD (%)
[] (ml/g)
M ;10\
Sample Sample Sample Sample Sample
77.8$0.97 76.0$1.46 77.0$1.72 85.6$1.25 92.4$0.90
451 529 718 743 509
6.52 8.16 10.6 8.22 4.65
1 2 3 4 5
Mean$SD, n"3.
3. Results and discussion 3.1. Characterization of chitosan samples The acetyl content in chitosan can be determined by titration [20], NMR [21], IR [22,23], and c.d. [17] analytical techniques. Among them, c.d. spectroscopy seems to be the better choice for precise measurement [24]. The degree of deacetylation of the "ve chitosan samples obtained from the c.d. measurement are listed in Table 1. Although the average molecular weight, the most di$cult parameter to be obtained precisely, can be determined by several methods, viscometry is claimed to be the simplest, most rapid, and probably the most precise determination method [24]. Thus, the viscosity average molecular weight (M ) of the "ve chitosan samples was determined by this method. Fig. 1 presents the reduced viscosity versus concentration plot for the samples. From the y-intercept, the intrinsic viscosity was obtained which was applied in the Mark}Houwink equation to calculate the M using the appropriate Mark}Houwink parameters. The intrinsic viscosities and the M for the chitosan samples are reported in Table 1.
Table 1 con"rms that data for the "ve chitosan samples cover a range of M and DD. Samples 1}3 have almost the same DD, but di!erent M . Samples 2 and 4 possess similar M but di!erent DD. Sample 5 has the lowest M and the highest DD. 3.2. Degradation of chitosan samples Degradation of some relatively rigid linear polysaccharides, with Mark}Houwink equation exponent a close to unity, can be conveniently investigated by the viscometric method [25]. The a value here is dependent upon the conformation of the polymer. Previous studies indicate that DD can change the rigidity of the chitosan chain in solution, which in turn changes the a value [19,26]. The a values of these chitosan samples are di!erent, but they are each close to unity based upon the calculation described above. Since each of the chitosan samples can be fully extended in the solutions, the change in the viscosity of their solutions can re#ect the change in the degree of polymerization of the chitosan linear chain. Thus, the course of the degradation of these chitosan samples was studied by viscosity measurement, and characterized by the decrease of the speci"c viscosity after a given time of degradation. Fig. 2 is an example of the speci"c viscosity as a function of reaction time in the presence and absence of 0.02% (w/v) -glucosidase under the reaction conditions. The plot shows that the speci"c viscosity of the reaction mixture decreased dramatically over time in comparison to that of the control without enzyme. Such viscosity reduction pro"les were seen with each of the chitosan samples. In Fig. 2, there is a slight reduction in the speci"c viscosity for the control, which is probably due to the acid hydrolysis of the chitosan sample at pH 5.0. As shown in Fig. 2, the results indicate that enzyme treatment resulted in a substantial loss in viscosity of the chitosan solution, indicating depolymerization, and such depolymerization took place very quickly during the initial 2 h. 3.3. Determination of the degradation rate (r)
Fig. 1. Plot of reduced viscosity versus concentration for the "ve chitosan samples (mean$SD, n"3): (£) sample 1; (*) sample 2; (䉭) sample 3; (*) sample 4; (䊐) sample 5.
According to the literature method, the initial rate of degradation was determined with viscosity data by
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Fig. 2. Speci"c viscosity of 0.7% (w/v) sample 3 solution as a function of time following treatment with -glucosidase at 303C (mean$SD, n"3): (䊐) without 0.02% (w/v) -glucosidase; (䉭) with 0.02% (w/v) -glucosidase.
Table 2 Initial degradation rates (r) of the "ve chitosan samples by -glucosidase Chitosan
r (g/(ml h));10
Sample Sample Sample Sample Sample
6.849 4.452 2.017 2.542 4.042
1 2 3 4 5
Concentrations of the chitosans and -glucosidase were 0.1 and 0.02% (w/v), respectively.
employing the Mark}Houwink equation to convert the viscosity reduction to the degree of scission of the glycoside linkage of the chitosan [16]. However, K and a values in the Mark}Houwink equation are dependent on DD, as described in the M determination section. They are not constant for the "ve chitosan samples, and the a value cannot be assumed to be unity. To account for the varying a values, the viscosity data were plotted as [1/( /c) ? !1/( /c) ? ] instead of [1/( /c) !1/ RR R RR ( /c) ] versus time to establish the time course of R enzyme degradation of chitosan. Such plots are presented in Fig. 3. The initial degradation rates (r) for the chitosan samples were determined from the slope of the initial linear portion of each "tted quadratic equation by using the following equation (see Table 2): r"slope K ?M C, where M is the monomer weight of the chitosan [16].
Fig. 3. Time course of degradation of "ve 0.1% (w/v) chitosan samples by 0.02% (w/v) -glucosidase (mean$SD, n"3): (£) sample 1; (*) sample 2; (䉭) sample 3; (*) sample 4; (䊐) sample 5.
It has been shown that a linear relation would be obtained for a "rst order, random depolymerization of chitosan samples treated by H O in the presence of FeCl [16]. However, the time course of degradation of chitosan by almond emulsin -glucosidase preparation was non-linear as seen in Fig. 3. The curvature observed with each of the samples might indicate a non-random depolymerization of chitosan by such an enzyme preparation. This could be due to faster reacting cleavage sites distributed along the linear chain. Fig. 3 reveals that the initial slope increased as M decreased and DD decreased. A decrease in the DD of chitosan would increase its similarity to chitin, the observed substrate of the chitinase present in almond emulsin -glucosidase preparations. 3.4. Kinetics of -glucosidase degradation of chitosan samples The initial velocity () for use in the Eadie}Hofstee plots was calculated based upon the following equation:
"r[S] M /C"slope K ?[S] M , where [S] is the molar concentration of glycosidic link age at the beginning of the reaction. The results of the initial degradation velocity () and substrate concentrations ([S]) were plotted according to the Eadie}Hofstee equation: /[S]" /K !/K
+ + as presented in Fig. 4. The plots show that the degradation of chitosan by the -glucosidase preparation obeyed Michaelis}Menten-type kinetics. Apparent Michaelis} Menten parameters were estimated using the values of
H. Zhang, S.H. Neau / Biomaterials 22 (2001) 1653}1658
Fig. 4. Eadie}Hosftee plots for the "ve chitosan samples (mean$SD, n"3): (£) sample 1; (*) sample 2; (䉭) sample 3; (*) sample 4; (䊐) sample 5.
Table 3 Michaelis}Menten parameters, apparent catalytic constants and k /K for the "ve chitosan samples + Chitosan
K + (M)
;10
(M/ min)
Apparent k ;10
k /K ;10 +
Sample Sample Sample Sample Sample
3.13 3.42 5.01 4.00 5.81
20.7 18.2 13.7 9.86 17.5
10.4 9.10 6.85 4.93 8.75
3.32 2.66 1.37 1.23 1.51
1 2 3 4 5
the slope and y-intercept. A better estimation of the catalytic e$ciency of an enzyme for a particular substrate is k /K , in which k is de"ned as /[E] , and [E] +
2 2 represents the total molar concentration of the enzyme. Since the molecular weight of the chitinase in the glucosidase preparation used is unknown, its molar concentration cannot be calculated. An apparent k was obtained using [E] "0.02% (w/v) because the 2 glucosidase preparation concentration was constant at that level with each hydrolysis sample in the degradation studies. The K , , k , and k /K values are listed + + in Table 3. 3.5. Ewect of M and DD on the enzymatic susceptibility of chitosan Fig. 5 presents the M e!ect on the initial degradation rates (r) of chitosan by almond emulsin -glucosidase
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Fig. 5. Initial degradation rate (r) of -glucosidase hydrolysis of "ve 0.1% (w/v) chitosan samples versus the molecular weight (M ) of the chitosan.
preparation. Samples 1}3 with di!erent M , but essen tially the same DD, had a di!erent susceptibility to degradation by such enzyme preparation. The results show that the initial degradation rate decreased with an increase in the magnitude of the M . To observe the DD e!ect, one can compare the results for samples 2 and 4, which have similar M but di!erent DD. The higher DD sample (sample 4) had a slower initial degradation rate than did the lower DD sample (sample 2). Such M and DD e!ects on the susceptibility of chitosan to almond emulsin -glucosidase preparation degradation can be con"rmed in the results for sample 5 which has the lowest M but the highest DD. Its reaction to enzymatic degra dation was dependent on both M and DD e!ects. Its degradation rate would be faster than samples 3 and 4 due to its lower M , but slower than samples 1 and 2 because of its higher DD. As shown in Table 3, the results again indicate that a lower M and lower DD chitosan sample possessed a higher a$nity for the enzyme (K ), + experienced a higher catalytic e$ciency (k /K ), and + would be the sample most susceptible to enzymatic degradation. However, since there are only "ve data points and limited ranges for M and DD, further study is warranted.
4. Conclusions The results indicate that this almond emulsin glucosidase preparation has the ability to degrade chitosan and that its chitosanolytic activity can be
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conveniently monitored viscometrically. The degradation of chitosan obeys Michaelis}Menten kinetics. This investigation also clearly reveals that the initial degradation rate of a chitosan sample is dependent on its molecular weight and its degree of N-deacetylation. The results show that chitosan with a lower molecular weight and a lower degree of N-deacetylation is a more susceptible substrate. Since -glucosidase itself cannot hydrolyze polysaccharides such as chitosan, the degradation activity seen in this study cannot be due to the action of -glucosidase. It is reported that a -glucosidase preparation from almond emulsin contains a chitinase, and the degradation of these chitosan samples con"rms an existing chitinase in the preparation. The chitinase in this commercially available almond emulsin -glucosidase warrants further investigation.
Acknowledgements The authors acknowledge the gift of chitosan from G.D. Searle (Skokie, IL). We thank Dr. Joseph R. Mattingly for his assistance in the c.d. analysis. This project was supported in part by a grant from the University of Missouri Research Board.
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