A Soluble, Dye-Labelled Chitin Derivative Adapted for the Assay of Krill Chitinase

A SOLUBLE, DYE-LABELLED CHITIN DERIVATIVE ADAPTED FOR THE ASSAY OF KRILL CHITINASE

REINI-IARDSABOROWSKI*'~, FRIEDRICHBUCHHOLZ*, RALF-ACHIMH. VETTER*, STEPHAN J. WIRTH~ and GERHARD A. WOLF~ *Institut ffir Meereskunde an der Christian-Albrechts-Universit/it, Dfisternbrooker Weg 20, D-2300 Kiel 1, Germany (Tel. 0431-5973938; Fax 0431-565876); and ~Institut ffir Pflanzenpathologie und Pflanzenschutz der Georg-August Universit/it, Grisebachstrafle 6, D-3400 G6ttingen, Germany (Tel. 0551-393724; Fax 0551-394187)

(Received 5 November 1992; accepted 8 January 1993)

Abstraet--l. Carboxymethyl-Chitin-Remazol Brilliant Violet (CM-Chitin-RBV) was used for a colorimetric assay of chitinase activity in Antarctic krill, Euphausia superba. Comparison with a reductimetric method by end-product detection was carried out by measuring FPLC elution profiles of krill crude extracts with both assays. Both profiles matched significantly. 2. Krill chitinase was highly specific to CM-Chitin-RBV. The assay was characterized by easy handling and a very high sensitivity compared to that of end-product detection. Hydrolysis of CM-Chitin-RBV by N-acetyl-fl-D-glucosaminidase, fl-glucosidase, fl-galactosidase and N-acteyl-muraminidase was negligible. 3. The enzyme characteristics ofchitinase from Antarctic krill using CM-Chitin-RBV were: PHopt = 7.5, Top t = 50-55°C, E a = 52.1 kJ' mole -I, KM = 0.07 + 0.01 mg. ml -l.

INTRODUCTION enzyme system consisting of an endochitinase (poly-fl;1,4;(2-acet-amido-2-deoxy)-D-glucoside glu- Chitin (poly-N-acetyl-fl-o-glucosanfine) is one of the conohydrolase = chitinase, EC 3.2.1.14) and an exo- most abundant organic materials in nature, and this chitinase (N-acetyl-fl-D-glucosaminidase = NAGase, is particularly so in marine environments where chitin EC 3.2.1.29). The endochitinase hydrolyses the is the dominant material of crustacean exoskeletons. chitin to oligomers, mainly dimers (Powning and Chitin also occurs in spines of some centric diatoms Irzykiewicz, 1965), which are subsequently degraded (Montgomery et al., 1990). As a result of its chemical to aminosugars by NAGase. composition (carbon and nitrogen in a ratio of 8:1), NAGase activity can be measured easily and chitin has a high nutritive value. Consequently, it is rapidly using the artificial substrate p-nitrophenyl-N- of ecological importance in both the carbon and the acetyl-fl-D-glucosaminide (Kimura, 1974). In con- nitrogen cycle. trast, the measurement of endochitinase is more The role of chitin in nutrient cycles should not only complex. The most commonly used methods are the be elucidated by the determination of the amount of detection of the end-product aminosugars (reducti- present chitin but should also include a measurement metric assay) according to Morgan and Elson (1934) of the chitinolytic degradation potential. as modified by Boden et al., (1985), and the degra- Chitin can be degraded by bacteria, fungi and also dation of tritiated chitin as a substrate (Molano et al., by many invertebrates and vertebrates (Jeuniaux, 1977). However, these methods require long incu- 1966). High activities of chitinolytic enzymes are bation times (up to 24 hr) or large-scale laboratory present in the stomach and integument of Euphausia equipment. They are not suitable for rapid and easy superba and Meganyctiphanes norvegica, where they detection of a large number of samples. act as digestive and moulting enzymes, respectively Wirth and Wolf (1990) developed the soluble, (Buchholz, 1989; Saborowski and Buchholz, 1991). dye-labelled substrate CM-Chitin-RBV for the detec- We also found chitinolytic activity in Euphausia tion of endochitinase of microbial origin. It consists triacantha, the isopod Serolis polita and the Antarctic of carboxymethylated chitin (Trujillo, 1968; Hirano, salp Salpa tompsoni (unpublished data). Spindler- 1988) labelled covalently with Remazol Brilliant Barth et al. (1990) investigated chitinolytic enzymes Violet 5R. Because of high sensitivity and low costs in Palaemon serratus. it is of interest for routine procedures. Natural degradation of chitin to N-acetyl-fl-D- In our study we investigated the application of glucosamine (NAG) is performed by a two- CM-Chitin-RBV for the detection of crustacean chiti- tPresent address: Biologisehe Anstalt Helgoland, Meerssta- nase. The linearity of the assay with increased enzyme tion, D2192 Helgoland, Germany (Tel. 04725 819 322; amounts and incubation times was determined. Fur- Fax 04725 819 369). thermore, fractions of FPLC elution profile of an

673 674 REINHARD SABOROWSKIet aL

Euphausia superba crude extract were tested and the was carried out at 15,000g for 10min (Heraeus, activity pattern was compared to that determined by Biofuge A). the end-product measurement according to Boden et al. (1985). The substrate specificity of other hydro- Chromatography iases such as NAGase, fl-glucosidase, fl-galactosidase Extracts were fractionated by Fast Protein Liquid and N-acetyl-muraminidase was compared to that of Chromatography (FPLC, Pharmacia) using a pre- chitinase, using CM-Chitin-RBV. Finally, enzyme packed Q-Sepharose HP column (16/10, Pharmacia, characteristics of Euphausia superba chitinase, such as 17-1064-01). The extracts were applied to the column pH- and temperature optimum, activation energy and at a rate of 3 ml.min -~. Elution was effected by a KM-values, were determined. gradient from 0 to 0.85 M NaC1 in 0.1 M Imida- zole-HC1 buffer, pH 6.8. Per run, 95 fractions were

MATERIAL AND METHODS collected with a volume of 4.3 ml each. Tubes con- taining the fractions were cooled on ice. Enzyme Origin of samples analysis was performed on each second fraction. Specimens of Antarctic krill, Euphausia superba, were caught west of the Antarctic Peninsula Analysis of enzyme activity (61°$44'95 57°W53'80) during the cruise 11/4 Chitinase activity by end-product detection was (21.12.1989-18.01.1990) of the research vessel FS determined reductimetrically according to Boden Meteor. Sampling was done using a Rectangular et al. (1985) as modified by Spindler and Buchholz Midwater Trawl RMT 1 + 8 (Roe and Shale, 1979). (1988) using purified crustacean chitin. Mesh width of the RMT was 4.5 mm and operation Chitinase activity with CM-Chitin-RBV as sub- depth was from 60 m to the surface. strate was essentially carried out as described by The samples were frozen immediately after the Wirth and Wolf (1990), but applied to 1.5 ml reaction catch and maintained at -80°C during transfer to tubes (Eppendorf, 3810) with the following modifi- Kiel, Germany, where they were stored at the same cations: 200pl 0.2 M CPB, pH 6.0 was added to temperature until analysis in July 1990. 100 #1 of an aqueous solution of CM-Chitin-RBV (2 mg-ml -l, Loewe Biochemica GmbH, Otterfing, Extract preparation Germany). After pre-incubation for 5 min at 35°C the Extracts for FPLC were carried out on whole enzymatic reaction was started by adding 100#1 of animals in 2 ml 0.2 M Na-citrate-phosphate buffer enzyme solution. Following 30 min of incubation at (CPB), pH 5.5 per g animal by homogenization on the same temperature, the reaction was stopped by ice using an Ultra-Turrax (Janke & Kunkel, TP adding 100/11 of 1 N HCi. The reaction caps were 18/10). Homogenization times were 3 x 15 sec, each cooled in an ice-water bath for at least 10 min to interrupted by a break of 20 sec. After centrifuging ensure complete precipitation of the non-degraded (80,000 g, 30 min, 4°C, Christ Omikron) the super- substrate at pH < 3.0 After centrifuging (15,000g, natants were desalted and rebuffered to 0.1 M 5min; Heraeus Biofuge A) the absorbance of Imidazole-HCI buffer, pH 6.8, using Sephadex G25- the supernatant was measured photometrically at PDI0 columns (17-0851-01; Pharmacia, Uppsala, 550 nm against air, in duplicate. Blanks were run in Sweden). Finally, extracts were filtered (0.45#m, parallel for which enzyme and HCI were added, Schleicher & Schfill, FP 030/2). For the detection of simultaneously. enzyme characteristics, extracts of krill thoraces NAGase activity was detected by degradation of were prepared. The abdomen, the anterior region of the artificial substrate p-nitrophenyl-N-acetyl-[3-D- the cephalothorax, including the eyes, and the glucosaminide = pNpNAG (Sigma, N-9376), as pre- pereiopods were removed. The remaining material viously described by Spindler (1976) and Spindler and was coarsely chopped, transferred to 1.5 ml reaction Buchholz (1988). In alkaline media, the released caps (Eppendorf, 3810) and CPB, pH 5.5, was added p-nitrophenol developed a yellow colour which was in the ratio 1:2. Because of the small volume here measured photometrically at 410 nm against air. The homogenization was performed by sonication on ice incubation time was 15min at 35°C. Using 5-ml (Bronson Sonifier B12, microstick 101-148-063) for reaction tubes, 50/t 1 of enzyme solution was added to 3 x 15 sec at 30W, interrupted by 20 sec pauses. 50 #1 0.2 M CPB, pH 5.5. After preincubation, the After centrifuging (15,000 g, Heraeus Biofuge A) reaction was started by adding 50 #1 of a 0.3% (w/v) supernatants of two to three samples were pooled solution of pNpNAG in 0.2 M CPB, pH 5.5. The to provide a sufficient volume for the analytical reaction was terminated by adding 2.5 ml of 0.01 M series. NaOH. Samples were run in duplicate with a parallel Investigation of the KM-value of krill chitinase blank. were performed on homogenates of the stomach fl-Glucosidase was detected by a two step assay and the midgut gland (hepatopancreas) separately. using a 1% (w/v) solution of salicine (2-[hydroxy- Both organs were removed from deep frozen animals methyl]phenyl-fl-D-glucopyranoside; Sigma, S-0625) and homogenized in a total volume of I ml 0.2 M in 0.2 M CPB, pH 5.5. Incubation was carried out for CPB by sonication as described above. Centrifuging 60 min at 35°C with 450#1 of this salicine solution Chitinase assay with CM-Chitin-RBV 675 and 50#1 of enzyme solution. The reaction was crude extract, 250#1 of buffer and 100#l of an stopped by heating for 10 min at 100°C. Blanks were aqueous solution of CM-Chitin-RBV (2mg.ml-~). run in parallel with the heat-degraded enzyme. After The pH dependence of krill chitinase activity centrifuging (15,000g, 5m in; Heraeus Biofuge A), was measured using CPB (0.2M) for the range 100 #1 of the supernatant was removed for glucose pH 3.0-7.5 and borate buffer (Boden et al., 1985) for determination using a glucose UV-test (B6hringer, pH 8.0-12.0. Incubation temperature was 35°C and 716251). the incubation time was 30 min. The temperature-dependence was measured within Specificity the range of 0-70°C at pH 6.0, using 0.2 M CPB. In order to confirm the specificity of krill chitinase Thermal stability measurements were carried out to CM-Chitin-RBV, incubation assays were per- on preincubated enzyme solutions (60 rain) in the formed with commercial enzyme preparations of range 0-60°C. The subsequent assays were run at chitinase, fl-N-acetyl-glucosaminidase, fl-glucosi- 35°C for 30 rain in 0.2 M CPB, pH 6.0. dase, fl-galactosidase and N-acetyl-muraminidase Activation Energy (Ea) for krill chitinase was (all Sigma). Activities given by the producers were calculated from the data of the thermal optimum recalculated for each enzyme as 1 U = l #mol sub- curves, applying the Arrhenius equation. strate degradation, min- ~. KM-values of stomach and midgut gland extracts In the case of chitinase, one unit of activity was were measured using substrate concentrations of described as releasing of 1 mg NAG from chitin 0.025, 0.0375, 0.045, 0.0575, 0.065, 0.0825, 0.125, 0.25 within 48 hr. The activity was calculated by measur- and 0.Smg.ml -t CM-Chitin-RBV. Incubation was ing reducing sugars (Sigma, personal communi- carried out for 30min at 35°C and pH 6.0 (0.2 M cation). This reported activity was recalculated as a CAB). molar turnover of 1.569nmol.min -~ and thus as 1.569 mU instead of 1 U. RESULTS In the case of N-acetyl-muraminidase it was not possible to apply the producer's activity definition to Linearity of the assay the common unit definition because the activity was The investigations showed a linear increase of defined as increase of absorption at 600 nm per min activity with respect to enzyme concentration up to while degrading cell walls of Streptomyces faecalis. an absorbance of 0.500 at 550 nm. Above 0.5 the The assays were run in quadruplicate according to curve became non-linear (Fig. l a), probably due to the method for chitinase detection, using CM-Chitin- limiting substrate concentrations. Furthermore, a RBV as reported in the Present paper. The activities linear relationship was demonstrated between of enzymes applied, expressed in common unit defi- incubation time and absorbance (Fig. l b). nitions, were 2.SU.m1-1 (Chitinase, EC 3.2.1.14), 0.25 and 0.5 U.ml -~ (NAGase, EC 3.2.1.29), 5.0 and Elution profiles 2.5U.ml -t (fl-glucosidase, EC 3.2.1.21), 12.5 and The elution profiles of krill chitinase were deter- 25.0U.m1-1 (fl-galactosidase, EC 3.2.1.23). N- mined with both assays, using CM-Chitin-RBV and acetyl-muraminidase was applied with 125 and the reductimetric assay. In order to obtain an optimal 250 U.ml -l (here, the activity description was used comparison of both assays, each of the data sets of according to the producer). measured activities was averaged. The single values were then recalculated in relation to this average Enzyme characteristics (Fig. 2). The assays described as follows were carried out in Both profiles showed a high grade of correspon- 1.5 ml reaction caps (Eppendorf, 3810) with 50 #1 of dence. In both cases, the first activity peak eluted

1.2 0.6 o) b) 1.0 ~0 ~ 0.5 /

/- 6 / 0.8 / 0.4 o -/ tn J° tO < 0.6 0.,.3 <

0.4 0.2 10,998 0.2 0.1

O.C 0.0 20 40 60 80 100 0 10 20 30 40 50 60 70 Sample [/~1] Incubation time [mini

Fig. I. Linearity of the chitinase assay using CM-Chitin-RBV as substrate. Error bars represent SD (N = 3). (a) Hydrolysis of CM-Chitin-RBV as a function of crude extract concentration. (b) Hydrolysis of CM-Chitin-RBV as a function of incubation time. 676 RE[NHARD SABOROWSKIet al.

between fractions 30 and 40 where the NaCl-gradient 100 o was 0.1-0.25M. A further broad activity peak was detected between fraction 55 and 85 (NaC1- ~' 8O uoif * Xo\.o gradient = 0.5-0.75 M). Within fractions 40-55, the 6O O/ 1 "~) activity profiles showed slight differences. While the .> chitinase activities measured by end-product detection remained nearly constant, with a slight decrease o/ a~ 20 from fraction 40 to fraction 50, the activity detected f % using CM-Chitin-RBV as substrate showed a peak in fraction 48 (Fig. 2). 3 4 5 6 7 8 9 10 11 12 13 In order to obtain a statistical comparison of these pH data, an analysis of regression was performed. Fig. 3. pH dependence of chitinase activity in crude extracts The regression parameters and the correlation co- of E. superba. Within the range of pH 3.0-7.5 CPB (0.2 M), efficient, including all measured fractions were: and between pH8.0 and 12.0 borate buffer was used. Incubation: 30 min; 35°C. Error bars represent SD (N = 3). y = 0.943x + 3.322, r = 0.865, P<0.001 (N = 35). distinct maximum at pH 7.5 (Fig. 3). Fifty percent of The parameters, excluding fractions 39-53, were maximal activity occurred at pH 5.0 and 10.0. Lowest calculated as: activity was observed at pH 3.0 and pH 12.0. y = 0.999x - 1.377, r = 0.946, Temperature optimum and thermal stability. The highest chitinase activity was found between 50 and P <0.001, (N = 24). 55°C. Between 0 and 45°C the activity increased exponentially. Above 55°C, a sharp activity decrease Specificity investigations led to minimal activity at 70°C (Fig. 4). Krill chitinase In order to test possible interactions with other showed a high thermal stability (Fig. 4). Up to a hydrolases that cleave fl-glycosidical links, elution preincubation temperature of 40°C and a preincu- profiles of N-acetyl-fl-D-glucosidase (NAGase) and bation time of 60 min, the activity decreased only fl-glucosidase were determined. However, no distinct slightly, and did not sink below 90% of the initial activity peaks were found for these enzymes within activity at 0°C. Above 45°C, a sharp decrease of the fractions 40-55 (results not shown), indicating activity occurred. At a preincubation temperature of no influence on CM-Chitin-RBV hydrolysis. 60°C, only 4% of the initial activity remained. Additionally, the hydrolysis of CM-Chitin-RBV by Activation energy. The Arrhenius plot showed NAGase, fl-glucosidase, fl-galactosidase and mu- two different linear dependencies, between 0 and tanolysin (N-acetyl-muraminidase) was investigated. 20°C and 20 and 50°C, respectively (Fig. 5). The Based on a standardized unit definition, the highest calculated activation energy for the first range was turnover of CM-Chitin-RBV was found using chiti- 128.5 kJ .mole -I and for the second range (> 20°C) nase, and was set to be 100%. NAGase and fl-glu- 52.1 kJ. mole- ~. cosidase caused a turnover of 0.06 and 0.12%, Kinetic properties. Krill chitinase was characterized respectively, compared to that of chitinase. No degra- by typical Michaelis-Menten kinetics. The K}a-value dation of CM-Chitin-RBV could be observed using was at 0.07 ___ 0.01 mg CM-Chitin-RBV-ml-l(N = 5). fl-galactosidase and mutanolysin. Up to a concentration of 0.5rag CM-Chitin- RBV. mi-~ no inhibition of activity was detected. A Enzyme characteristics of krill chitinase pH-optimum. The activity of crude extracts of E. superba incubated with CM-Chitin-RBV showed a 100~ o/m --°---0---o-0-6.,~/ o o Stability ~ 8o --oOptimum 140 o--o CM-Chitin-RBV /: O -- O GE~dpire°dU ct - d ' t~9 n >, ~120 8o ~ 60 g >, 100 o /:/! 60 o 40 "~ 80 to ~ 60 40 ~o a~ 2o 2 ~ 4o O ~0 ~ , , 2O 2O 10 20 30 40 50 60 70 80 IncubGtion temperGture [°C~ 0 0 10 20 30 40 50 60 70 80 90 Fig. 4. Temperature dependence of chitinase activity and Froction thermal stability of krill chitinase, using CM-Chitin-RBV as Fig. 2. Chitinase FPLC elution profiles of a crude extract of substrate. Incubation: 30min; pH6.0; 0-70°C. Thermal E. superba using the method by end-product detection and stability was detected after preincubation of extracts for CM-Chitin-RBV as substrate. 60 min. Error bars represent SD (N = 3). Chitinase assay with CM-Chitin-RBV 677 typical curve of activity of midgut gland chitinase is 0.14 shown in Fig. 6 (including the Lineweaver-Burk O. 12 o/°~°~"~/~'120 plot). Separate extracts of the stomach and the 0.10 I 11~ midgut gland of the krill gave identical results. / / o 008 g I _.J 41o5

CBP(B)105-3/4--Q 678 REINHARD SABOROWSKIet al.

activity increase, reaching 50% of the maximal ac- Acknowledgements--We are grateful to Dipl. Biol. Jutta tivity at 22°C. In comparison, the assay with CM- Heimann (Institute of Forest Botany, University of Grttin- gen) who initiated contact between both working groups Chitin-RBV showed very low activity at 0°C. With and Dr Rory Wilson (Institute for Marine Research, Kiel) higher incubation temperatures, the activities in- for correcting the English. This work was supported by a creased more slowly. Fifty percent of activity was grant from the German Research Council (DFG Bu 548/2). reached at 38°C. Also in this case a specific property of CM-Chitin-RBV became noticeable. The sub- REFERENCES strate--viscosity probably increased at low incubation Boden N., Sommer U. and Spindler K.-D. (1985) Demon- temperatures or the substrate did not reach its stration and characterization of chitinases in the entire solubility. The enzyme was denaturated in Drosophila K c cell line. Insect Biochem. 15, 19-23. both assays at about 40 and 45°C, respectively. Buchholz F. (1989) Moult cycle and seasonal activities of The Arrhenius plot was divided into two linear chitinolytic enzymes in the integument and the digestive tract of the Antarctic krill, Euphausia superba. Polar Biol. parts. For the low temperature range, the resulting 9, 311-317. activation energy was 128.8kJ.mol -l and much Buchholz F. and Vetter R.-A. (1990) Enzyme kinetics in higher than that of the upper temperature range cold water' studied on chitin degrading enzymes of the (52.6kJ.mol-l). Neither value corresponds to the Antarctic krill, Euphausia superba. Verh. Dtsch. Zool. Ges. 83, 527. uniform Arrhenius plot and activation energy of Hackman R. H. and Goldberg M. (1964) New substrates 27.6kJ.mol -l reported by Spindler and Buchholz for use with chitinases. Analyt. Biochem. 8, 397-401. (1988), carried out on krill chitinase by end-product Hirano S. (1988) Water-soluble glycol chitin and car- detection. Here, the turnover of substrate at low boxymethylchitin. In Methods in Enzymology (Edited by temperatures probably decreased due to incomplete Wood W.-A. and Kellogg S. T.), Vol. 161, pp. 408-416. Academic Press, New York. solution, as reported above. In contrast, maximal Jeuniaux C. (1966) Chitinases. In Methods in Enzymology solubility is reached at temperatures above 20°C, (Edited by Nuffield E. F. and Ginsberg V.), Vol 8, resulting in a lower value of Ea. pp. 644-650. Academic Press, New York. The investigation of the kinetic properties showed Kimura S. (1974) The fl-N-acetylglucosaminidase of Bom- byx mori L. Comp. Biochem. Physiol. 49B, 345-351. a typical Michaelis-Menten curve. In contrast to Molano J., Duran A. and Cabib E. (1977) A rapid and NAGase from Euphausia superba (Buchholz and sensitive assay for chitinase using tritiated chitin. Analyt. Vetter, 1990) and NAGase from Meganyctiphanes Biochem. 83, 648-656. norvegica (Spindler and Buchhoiz, 1988), the chiti- Montgomery M. T., Welschmeyer N. A. and Kirchman nase reaction was not inhibited at higher substrate D. L. (1990) A simple assay for chitin: application to sediment trap samples from the subarctic Pacific. Mar. concentrations. The average KM-value of 0.07 mg Ecol. Prog. Ser. 64, 301-308. CM-Chitin-RBV.m1-1 was very low compared Morgan W. T. J. and Elson L. A. (1934) A colorimetric to results reported by Spindler and Buchholz method for the determination of N-acetylglucosamine (1988). For chitinase from M. norvegica these and N-acetylchondrosamine. Biochem. J. 28, 988-995. Powning R. F. and Irzykiewicz H. (1965) Studies on the authors found values of 1.6 and 5.2 mg chitin, ml-1, chitinase system in bean and other seeds. Comp. Biochem. respectively, using end-product detection. The low- Physiol. 14B, 127-133. ered half-saturation concentration measured here Roe H. S. J. and Shale D. M. (1979) A new multiple could also be explained by the different characteristics rectangular midwater trawl (RMT 1+ 8 M) and some of the soluble substrate compared to that of crys- modifications to the Institute of Oceanographic Sciences, RMT 1 + 8. Mar. Biol. 50, 283-288. talline chitin. The soluble substrate is better available Saborowski R. and Buchholz F. (1991) Induction of en- for enzymatic attack compared to insoluble chitin zymes in the digestive tract of the Antarctic krill, Euphau- which can only be attacked from the surface. Accord- sia superba. Verh. Dtsch. Zool. Ges. 84, 422. ingly, the affinity of the enzyme to the soluble sub- Spindler K.-D. and Buchholz F. (1988) Partial characterization of chitin degrading enzymes from two euphausiids, strate may be increased and the apparent KM-value Euphausia superba and Meganytiphanes norvegica. Polar thus lowered. Biol. 9, 115-122. Summarized, the chitinase assay using CM-Chitin- Spindler K.-D. (1976) Initial characterization of chitinase RBV is distinguished by high sensitivity and easy and chitobiase from the integument of Drosophila hydei. handling. A satisfactory application of CM-Chitin- Insect Biochem. 6, 663-667. Spindler-Barth M., van Wormhoudt A. and Spindler K.-D. RBV as a substrate for the detection of krill endo- (1990) Chitinolytic enzymes in the integument and chitinase activity can be carried out over the range of midgut-gland of the shrimp Palaemon serratus during the optimal temperature and pH conditions. moulting cycle. Mar. Biol. 106, 49 52. However, for comparative investigations of eco- Trujillo R. (1968) Preparation of carboxymethylchitin. Car- bohydr. Res. 7, 483-485. physiological parameters, dealing with extreme pH- Wirth S. J. and Wolf G. A. (1990) Dye-labelled substrates values and low temperature, other chitinase detection for the assay and detection of chitinase and lysozyme methods are recommended. activity. J. Microbiol. Meth. 12, 197-205.