ANALYTICAL BIOCHEMISTRY

Analytical Biochemistry 363 (2007) 128134 www.elsevier.com/locate/yabio

Natural substrate assay for chitinases using high-performance liquid chromatography: A comparison with existing assays
Inger-Mari Krokeide, Bjrnar Synstad, Sigrid Gseidnes, Svein J. Horn, Vincent G.H. Eijsink, Morten Srlie
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway Received 7 November 2006 Available online 4 January 2007

Abstract The determination of kinetic parameters of chitinases using natural substrates is diYcult due to low Km values, which require the use of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)4, we have developed an assay for the determination of kcat and Kmvalues of chitinases. Product concentrations as low as 0.5 M were detected using normal-phase high-performance liquid chromatography (HPLC) with an amide 80 column (0.20 25 cm) using spectrophotometric detection at 210 nm. By means of this assay, kcat and Kmvalues for chitinases A (ChiA) and B (ChiB) of Serratia marcescens were found to be 33 1 s1 and 9 1 M and 28 2 s1 and 4 2 M, respectively. For ChiB, these values were compared to those found with commonly used substrates where the leaving group is a (nonnatural) chromophore, revealing considerable diVerences. For example, assays with 4-methylumbelliferyl-(GlcNAc)2 yielded a kcat value of 18 2 s1 and a Km value of 30 6 M. For two ChiB mutants containing a Trp ! Ala mutation in the +1 or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)2 assays yielded rather similar Km values (5-fold diVerence at most) but showed dramatic diVerences in kcat values (up to 90-fold). These results illustrate the risk of using artiWcial substrates for characterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research. 2007 Elsevier Inc. All rights reserved.
Keywords: Enzymatic assay; Chitinase; Natural substrate; HPLC

Chitin, a -1,4-linked polymer of N-acetylglucosamine (GlcNAc), is an abundant biopolymer in nature. It is the most important nonplant structural biopolymer, occurring in, e.g., the exoskeletons of invertebrates, the cell walls of fungi, and the digestive tracts of insects. Chitin is easily derived from waste products such as shrimp shells. So far, chitin is primarily used as a source for chitosan, a partially deacetylated soluble form of chitin, and for glucosamine. Fragments of chitin or chitosan (chitooligosaccharides) may inhibit certain chitin-degrading enzymes, giving them potential as fungicides [1], insecticides [24], and antimalarials [5,6]. Chitooligosaccharides are environmentally friendly because of their fast degradation in nature and, since chitin does not occur in humans, chitin metabolism is
*

Corresponding author. Fax: +47 64965901. E-mail address: morten.sorlie@umb.no (M. Srlie).

an interesting target area for development of drugs and pesticides. Chitin does not accumulate in nature because the polymer is eVectively degraded by diVerent chitinases belonging to the glycoside hydrolase enzyme families 18 and 19 [7]. Serratia marcescens has an eYcient chitinolytic machinery and, when grown on chitin, three family 18 chitinases are expressed: chitinases A (ChiA), B (ChiB), and C (ChiC) [8]. ChiA and ChiB are processive chitinases that digest the chitin polymer in opposite directions producing mainly (GlcNAc)2, while ChiC is a nonprocessive endochitinase that hydrolyzes the polymer randomly, yielding longer chitooligosaccharides [9,10]. The S. marcescens chitinases have been characterized in several studies [811], using a variety of substrates. Kinetic analysis of chitinases is usually conducted with artiWcial substrates such as 4-methylumbelliferyl-(GlcNAc)2

0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.12.044

Natural substrate assay for chitinases using HPLC / I.-M. Krokeide et al. / Anal. Biochem. 363 (2007) 128134

129

((GlcNAc)2-4MU), (GlcNAc)3-4MU, and p-nitrofenyl(GlcNAc)2 ((GlcNAc)2-pNP). These substrates are not optimal because they present the enzyme with nonnatural leaving groups and because of substrate inhibition [12]. Because of the length and the nature of the leaving groups, non-natural substrates are of very limited use when assessing the eVects of mutations meant to aVect natural substrate degradation. In addition, the leaving groups 4-MU and pNP are corresponding bases to weak acids with pKa values of 7.8 [13] and 7.2 [14], respectively, while acid dissociation constants for sugars are V 12 [15]. At high pH (GlcNAc)2-4MU and (GlcNAc)2-pNP yield negatively charged leaving groups while this will not be the case for a sugar leaving group. Kinetic characterization with natural substrates is diYcult because (1) low Km values require detection of low concentrations of substrate and product, (2) most longer substrates contain more than one hydrolysable glycosidic bond, (3) degradation products longer than dimers are also substrates, and (4) some chitinases degrade longer substrates processively. With respect to the latter two problems, (GlcNAc)4 is an exception since most chitinases convert this compound exclusively to two dimers, which are not degraded any further. We present a chitinase assay based on the use of the natural substrate (GlcNAc)4 and the use of a sensitive HPLC setup to monitor substrate and product concentrations. We have compared the natural substrate assay with existing assays using wild-type ChiA and ChiB from S. marcescens and two engineered variants of ChiB. The engineered enzymes are ChiB-W97A and ChiB-W220A. Trp97 is located in the +1 subsite of the active site of ChiB while Trp220 is located in the +2 subsite [16,17]. Mutation of these residues will thus aVect the enzymes interaction with the leaving group. Materials and methods Chemicals Tetra-N-acetylchitotetraose, 4-methylumbelliferyl-di-Nacetylchitobiose, 4-methylumbelliferyl-tri-N-acetylchitotriiose, para-nitrophenyl-di-N-acetylchitobiose, and acetonitrile were purchased from SigmaAldrich. Production and puriWcation of chitinases ChiA [18], ChiB [19], and mutants of ChiB were puriWed from periplasmatic extracts of the producer strains by hydrophobic interaction chromatography, as described previously [12]. Enzyme purity was veriWed using SDS/ PAGE and was above 95% in all cases. Protein concentrations were determined using the Bradford assay kit provided by Bio-Rad (Hercules, CA, USA). Chromatography of chitooligosaccharides Mixtures of (GlcNAc)4 and (GlcNAc)2 were separated by normal-phase HPLC using a Tosoh TSK Amide 80

column (0.20 25 cm) with an amide 80 guard column. The sample size was 50 L and the chitooligosaccharides were eluted isocratically at 0.25 mL/min with 70% (v/v) acetonitrile at room temperature. The chitooligosaccharides were monitored by measuring absorbance at 210 nm and the (GlcNAc)4 concentrations were quantiWed by measuring peak areas and by comparing these to those of standard samples with known concentrations of (GlcNAc)4. Using these standard samples, it was established that there was a linear correlation between the peak area and the analyzed (GlcNAc)4 concentration within the concentration range 0.5300 M used in this study. (GlcNAc)4 assay Reactions were started by adding 0.5 nM ChiA or ChiB or 1.0 nM ChiB-W220A or 2.0 nM ChiB-W97A (Wnal concentrations) to 1-mL solutions containing 100, 80, 60, 40, 20, 10, 5, or 2 M (for ChiA and ChiB), 500, 400, 300, 200, 100, 50, or 25 M (for ChiBW220A), and 1000, 600, 400, 200, 100, 50, or 25 M (for ChiB-W97A) (GlcNAc)4 in 20 mM NaAc buVer, pH 6.1, and 0.1 mg/mL bovine serum albumin (Wnal concentrations). After enzyme addition, 50L aliquots were transferred to a HPLC vial containing 150 L of acetonitrile at appropriate time points. Reaction conditions and timing were such that the (GlcNAc)4 concentration in the sample would not go below 80% of the starting concentration. An aliquot taken before enzyme addition was used as the t D 0 sample. The slopes of the plots of substrate concentrations vs time were taken as the hydrolysis rate. Then the hydrolysis rates were plotted vs the substrate concentration in a MichaelisMenten plot and the experimental data were Wtted to the Michaelis Menten equation using nonlinear Wtting in Origin 7. Alternatively, substrate concentrations divided by the hydrolysis rates were plotted vs substrate concentrations in a Hanes plot using a standard spreadsheet program to obtain kcat and Km values. The derived values using the two diVerent approaches were the same within experimental errors. Assays with nonnatural substrates The kinetic parameters of ChiB variants for the (GlcNAc)2-4MU substrate at pH 6.1 were determined as thoroughly described elsewhere [12,20] with an enzyme concentration of 0.2 nM and substrate concentrations in the 5 to 50- M range. The kinetic parameters of wild-type ChiB toward (GlcNAc)2-pNP were determined in the same way, except that the substrate concentration range was adapted to the much higher Km for this substrate. ChiB converts the (GlcNAc)3-4MU substrate exclusively to (GlcNAc)2 and (nondetectable) GlcNAc-4MU. The kinetic parameters of wild-type ChiB toward the (GlcNAc)3-4MU substrate were determined using a two-step assay based on detection of substrate depletion, a ChiB concentration of 0.2 nM, and substrate concentrations in the 1 to 10- M range [21]. In short, reactions were conducted as usual but,

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Natural substrate assay for chitinases using HPLC / I.-M. Krokeide et al. / Anal. Biochem. 363 (2007) 128134

before adding the 0.2 M Na2CO3 stop solution, the reaction mixtures were incubated for less than 1 min with a 300-fold excess of ChiA to convert all remaining substrate to (detectable) free 4MU. In all cases, the substrate concentrations divided by the hydrolysis rates were plotted vs substrate concentration in Hanes plots using a standard spreadsheet program to obtain kcat and Km values. Results (GlcNAc)4 as substrate (GlcNAc)4 productively binds in subsites 2 to +2 of ChiA and ChiB of S. marcescens, yielding two (GlcNAc)2 molecules as products that are not subject to further hydrolysis [8,12,21]. Longer substrates have several productive binding modes and yield products that in turn are substrates for the enzymes, complicating kinetic analysis of the overall reaction. Productive binding of (GlcNAc)3 yields only products that are not further hydrolyzed, (GlcNAc)2 and GlcNAc. However, this substrate is less suitable than (GlcNAc)4 because it occupies fewer subsites and because there are two productive binding modes, at least in some chitinases such as ChiA (binding in 2 to +1 and in 1 to +2) [21]). HPLC analysis of chitooligosaccharides Normal-phase HPLC-based quantitative analysis of saccharides with an amide 80 column has been used previously [2124] but, so far, the use of this method for determination of kinetic parameters (implying measurement of very low concentrations of sugar) has not been reported. In the present study, the necessary increase in sensitivity was achieved through a combination of adjustments. First, we used a column with a considerably reduced diameter (0.20 cm vs 0.48 cm used in previous studies). A smaller column diameter yields a smaller degree of sample dilution compared to a larger column diameter and thus results in sharper peaks. Second, injection volumes were 50 L. Since water elutes the chitooligosaccharides, the samples were diluted to 75% (v/v) acetonitrile before application to allow for retardation on the column of the chitiooligosaccharides before elution with 70% (v/v) acetonitrile. Together these adjustments permitted detection of (GlcNAc)4 concentrations down to 0.5 M. Fig. 1 shows the HPLC chromatograms resulting from enzymatic hydrolysis of (GlcNAc)4 by ChiB. Determination of kcat, Km, and eYciency constants (kcat/Km) for wild-type ChiB with diVerent substrates Enzymatic hydrolysis of (GlcNAc)4 by ChiB follows MichaelisMenten kinetics as depicted by Fig. 2A. Kinetic data for hydrolysis of (GlcNAc)4 and three diVerent artiWcial substrates are shown in Table 1. For (GlcNAc)4 hydrolysis, the nonlinear Wt of theoretical data to experimental data yielded kcat and Km values of 28 2 s1 and 4 2 M,

respectively. These values diVer considerably from values obtained with the often-used substrate (GlcNAc)2-4MU, which are 18 2 s1 and 31 6 M, respectively [20]. Even larger diVerences were observed with the (GlcNAc)2-pNP substrate which yielded kcat and Km values of 1.4 0.5 s-1 and 181 35 M, respectively. For (GlcNAc)3-4MU (which is converted to (GlcNAc)2 and GlcNAc-4MU), the kcat value is 57 4 s1 and the Km value is 7 1 M and both are about two times larger than those for (GlcNAc)4. The diVerences between the substrates are most apparent when using eYciency constants (kcat/Km; Table 1). ChiB has a 700 times greater eYciency toward (GlcNAc)4 compared to (GlcNAc)2-pNP and a 12 times higher eYciency compared to (GlcNAc)2-4MU. Results obtained with the (GlcNAc)3-4MU substrate were similar to those obtained with (GlcNAc)4. Determination of kinetic parameters for ChiA with (GlcNAc)4 The (GlcNAc)2-4MU susbtrate is often used because its product is easy to detect at low concentrations and because it tends to display seemingly natural Km values, i.e., in the lower micromolar range. However, for some enzymes with extended substrate binding clefts this short substrate may be suboptimal because of the occurrence of multiple binding modes and cooperativity between these. This is for example observed for ChiA [12]. With the (GlcNAc)4 substrate, ChiA displayed normal MichaelisMenten kinetics (Fig. 2B), yielding kcat and Km values of 33 1 s1 and 9 1 M, respectively (Table 2).

25

20

Absorbance /mV

15

10

0 8 10 12 14 16 18 20

Time /min
Fig. 1. HPLC analysis of (GlcNAc)4 hydrolysis by ChiB. The starting concentration of (GlcNAc)4 was 40 M, and chromatograms obtained after hydrolysis of 0% (bottom), approximately 10% (middle), and 20% (top) of the substrate are shown. Both the tetramer and the dimer peak are split into two peaks due to the anomer equilibrium of the chitooligosaccharides. The anomers (approximately 60%) elute before the anomers (approximately 40%; see, e.g., [21]). Due to the small diameter of the column and the relatively large sample volume, the anomeric forms are only partly separated. The Wrst 8 min of the chromatograms were omitted for clarity.

Natural substrate assay for chitinases using HPLC / I.-M. Krokeide et al. / Anal. Biochem. 363 (2007) 128134

131

A
Rate/(M/s)

0,020

C 0,04
0,03

0,015

0,010

Rate/(M/s)
0 20 40 60 80 100

0,02

0,005

0,01

0,000

0,00 0 100 200 300 400 500

(GlcNAc)4/M

(GlcNAc)4/M

B
Rate/(M/s)

0,020

D 0.15
Rate/(M/s)
0.10

0,015

0,010

0,005

0.05

0,000 0 20 40 60 80 100

0.00 0 200 400 600 800 1000 1200

(GlcNAc)4/M

(GlcNAc)4/M

Fig. 2. MichaelisMenten plots from HPLC analyses of (GlcNAc)4 hydrolysis with ChiB (A), ChiA (B), and the engineered enzymes ChiB-W220A (C) and ChiB-W97A (D) at pH 6.1, 37 C. Solid symbols are experimentally determined data for the rate of substrate disappearance vs substrate concentrations and the solid lines are the best nonlinear Wt using the MichaelisMenten equation.

Determination of kcat and Km values for engineered chitinases using (GlcNAc)4 and (GlcNAc)2-4MU as substrates Activity assays based on natural substrates are particularly important for the analysis of the properties of engineered enzymes carrying site-directed mutations near the catalytic center. To analyze and illustrate this, we have characterized two ChiB variants with mutations in subsite +1 (W97A) and +2 (W220A) (Fig. 3) with both (GlcNAc)4 and (GlcNAc)2-4MU (Table 2). When (GlcNAc)4 was used, kcat and Km values of 126 2 s1 and 807 2 M were obtained for ChiB-W97A, while the same values were 8 1 s1 and 175 57 M for the (GlcNAc)2-4MU substrate. For ChiBW220A, the kcat and Km values were 45 1 s1 and 71 1 M and 0.5 0.1 s1 and 82 4 M when using (GlcNAc)4 and (GlcNAc)2-4MU, respectively. Interestingly, while the diVerence between the substrates is modest with respect to Km values, it is as large as 90-fold with respect to kcat values. Discussion Comparison of the various substrates Up to now, (GlcNAc)2-4MU, which produces the Xuorescing 4MU leaving group upon hydrolysis, has been the most used substrate in chitinase assays [12,20,22,25]. Obvious advantages of the use of (GlcNAc)2-4MU are the time window and simplicity of the assay and its sensitivity.

Product formation is readily and quickly detected using Xuorescence spectroscopy. There are also several and serious disadvantages such as substrate inhibition, a nonnatural leaving group, and nonlinear kinetics, [12,22,26]. An alternative for (GlcNAc)2-4MU is (GlcNAc)2-pNP, which yields the yellow chromophore pNP. This substrate has the same disadvantages as (GlcNAc)2-4MU. The data of Table 1 show that the kinetic parameters determined with (GlcNAc)2-pNP are very diVerent from the presumably more realistic parameters obtained with (GlcNAc)4. Another alternative is (GlcNAc)3-4MU, which may be converted either to (GlcNAc)2 and GlcNAc-4MU (as does ChiB) or to (GlcNAc)3 and 4MU (as does ChiA) [12]. In the latter case, product formation can readily be determined, but the method has most of the disadvantages described above for (GlcNAc)2-4MU. For ChiB, we have been able to develop a method for measuring the conversion of (GlcNAc)3-4MU to (GlcNAc)2 and GlcNAc-4MU under conditions that permit determination of kinetic parameters [21]. The method is based on measuring product disappearance, which is achieved by converting remaining product quantitatively to 4MU through a short incubation with a large excess of ChiA. This assay has the advantage that the +1 subsite is occupied by a natural leaving group (i.e., a sugar unit). Not unexpectedly, the results obtained with (GlcNAc)3-4MU for wild-type ChiB resembled those obtained with (GlcNAc)4. A serious disadvantage of this assay is the need to purify two enzymes, especially large amounts of ChiA, which is time consuming, and it works

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Natural substrate assay for chitinases using HPLC / I.-M. Krokeide et al. / Anal. Biochem. 363 (2007) 128134

Table 1 kcat and Km of ChiB from Serratia marcescens for substrates with diVerent leaving groups, at 37 C, pH 6.1
O OH NH HO HO OH O O O HO O R NH OH O H2O NH HO HO O O O HO NH OH O OH

RH

Leaving group (RH) Di-N-acetylchitobiose

O OH NH HO HO O OH O O HO O OH NH

kcat (s1)

Km ( M)

kcat/Km (s1 M1)

28 2

42

4-Methylumbelliferyl-N-acetylglucosamine O
O NH HO HO O O O

57 4a

7 1a

OH 4-Methylumbelliferol HO O O

18 2b

31 6b

0.6

para-Nitrophenol HO
N O
a b

1.4 0.5
O

181 35

0.01

Ref [21]. Ref [20].

only for enzymes that exclusively convert (GlcNAc)3-4MU to (GlcNAc)2 and GlcNAc-4MU. Although the rate-limiting step in the reaction mechanism of family 18 chitinases is not known [17,20,27], it is clear that a change in chemical properties of the leaving group may aVect catalytic eYciency with regard to both the pKa and the ability to interact with the enzyme. At pH 6.1, about 2% of 4-methylumbelliferol and about 7% of paranitrophenol will be negatively charged while virtually all of the (GlcNAc)2 and GlcNAc-4MU leaving groups will be protonated and free of charge. It is interesting to note that the substrate yielding the by far lowest kcat value ((GlcNAc)2-pNP) has the most acidic and the smallest leaving group. ChiB is more eVective toward (GlcNAc)2-4MU whose leaving group is slightly less acidic and considerably larger. Several studies have shown that enzymesubstrate interactions in subsites +1 and +2 are important for

Table 2 Kinetic parameters for engineered and natural chitinases from Serratia marcescens at 37 C, pH 6.1, using (GlcNAc)4 and 4MU-(GlcNAc)2 as substrates Chitinase (GlcNAc)4 kcata Km
b

(GlcNAc)2-4MU kcat/Km 7 4 0.2 0.6

c

kcata 18 2 n.d.d 81 0.5 0.1

K mb 31 6 n.d.d 175 57 82 4

kcat/Kmc 0.6 n.d.d 0.03 0.01

ChiB 28 2 42 ChiA 33 1 91 ChiB-W97A 126 4 807 40 ChiB-W220A 45 2 71 3

a b

(s1). ( M). c (s1 M1). d The combination of sigmoidal behavior and substrate inhibition precludes determination of kinetic parameters [12].

catalytic eYciency [17,22,28,29]. The present data do not permit discrimination between the roles of the acidity and

Natural substrate assay for chitinases using HPLC / I.-M. Krokeide et al. / Anal. Biochem. 363 (2007) 128134

133

Fig. 3. Crystal structure of the active site of ChiB with (GlcNAc)5 bound [17]. Trp97 stacks with the sugar moiety in subsite +1. Similarly, Trp220 stacks with the sugar moiety in subsite +2. Note that the 1 sugar has a non-chair conformation that resembles a sofa conformation [27], the occurrence of which is crucial during the catalytic cycle [17,27]. Clearly, mutations that aVect substrate binding may also aVect this crucial structural deformation of the sugar. Note. The complex was determined with an inactive E144Q mutant of ChiB. For illustration purposes, the picture shows a glutamate.

the interaction potential of the leaving group. The data in Table 1 do suggest though that both the occupancy of the +1 and +2 subsites and a high pKa of the leaving group are beneWcial for catalytic eYciency because the longer, sugarlike substrates yielded lower Km and higher kcat values. The present data show that the use of (GlcNAc)4 to circumvent the problems connected to other substrates is fully feasible. Both ChiA and ChiB showed straightforward MichaelisMenten kinetics without substrate inhibition and the range of accessible substrate concentrations was such that kinetic parameters could be determined with reasonable accuracy. The results show that ChiA and ChiB have similar activities toward soluble substrates, in line with the rather similar architectures of their active sites and substrate-binding grooves [11,16,17,30]). Analysis of mutants The W97A and W220A mutants displayed large increases in Km with both substrates, as might be expected upon mutating important interaction partners in subsites that are known to make a dominant positive contribution to ligand binding. The increase in Km was larger for the natural substrate (GlcNAc)4, presumably because the wildtype subsites are optimized for sugar binding rather than binding of the 4MU group. Not unexpectedly, mutation of Trp220, which primarily aVects subsite +2, had only a modest eVect on the Km for (GlcNAc)2-4MU. Mutational eVects on kcat showed a dramatic dependence on the substrate used (Table 2). With the natural substrate, both mutations lead to an increase in kcat (4.5 and 1.6 times higher for ChiB-W97A and W220A, respectively) whereas the kcat is reduced from 18 to 8 s1 for ChiB-W97A and to 0.5 s1 for ChiB-W220A with (GlcNAc)2-4MU. For the ChiB-W220A mutant, the two substrates yield a 90-fold diVerence in kcat and a 60-fold diVerence in the eYciency

constant kcat/Km. Apart from illustrating the diVerences between the substrates, these observations also lead to questions concerning the mechanism and rate-limiting step of catalysis. For example, the dramatic eVect of the ChiBW220A mutation on the kcat obtained with (GlcNAc)24MU must mean that Trp220 is involved in catalysis, even in the case of short substrates. It is not known how the 4MU group binds to the enzyme, but it is clear that the group is large enough (i.e., larger than a single sugar) to interact with parts of the +2 subsite, including Trp220. Another puzzling issue concerns the fact that mutation of the two tryptophans increased the kcat for the natural substrate. Interestingly, Watanabe et al. [31] found that mutation of analogous tryptophans in chitinase A1 from Bacillus circulans led to increased speciWc activity toward chitopentaose. Further kinetic and mutational studies are necessary to Wnd possible explanations for these observations [32]. In conclusion, the results obtained with wild-type ChiB and, particularly, with the two mutants clearly show that the use of easy-to-handle artiWcial substrates for detailed characterization of family 18 chitinases may lead to wrong conclusions. Thus, such characterization should be based on a natural substrate assay, such as the one described here. Acknowledgments We are grateful for the help of Dr. Gustav VaajeKolstad for the making of Fig. 3. Part of this work was funded by The Norwegian Research Council, Grants 140497 and 140440. References
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