Purification and some properties of two β-N-acetylhexosaminidases from the hepatopancreas of Northern shrimp, Pandalus borealis

Comp. Biochem. PhysioLVol. 101B,No. 4, pp. 513-517, 1992 Printed in Great Britain

0305-0491/92$5.00+ 0.00 © 1992PergamonPress plc

PURIFICATION AND SOME PROPERTIES OF TWO fl-N-ACETYLHEXOSAMINIDASES FROM THE HEPATOPANCREAS OF NORTHERN SHRIMP, PANDALUS BOREALIS MARGRETHEESAIASSEN,BJORNARMYRNESand RAGNARL. OLSEN Norwegian Institute of Fisheries and Aquaculture, P.O. Box 677, N-9001 Troms6, Norway (Tel: 47-83-29000; Fax: 47-83-29100) (Received 16 September 1991) Abstract--1. Two fl-hexosaminidases were obtained from the hepatopancreas of the Northern shrimp Pandalus borealis by ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. According to the substrate specificities, both enzymes are ~-N-acetylhexosaminidases (EC 3.2.1.52), designated forms I and II. 2. The two enzyme forms have an absolute requirement for the N-acetylated sugar moiety and the fl-glycosidic linkage, and showed no significant difference in the preference for the 4-C-epimer of glucose over galactose. 3. Form II is more efficient hydrolyzing the di-, tri- and tetramer of N-acetyl-fl-n-glucosamine than form I. 4. fl-N-Acetylhexosaminidase form I has a slightly lower pH-optimum than form II.

INTRODUCTION The exoskeleton of the Northern shrimp Pandalus borealis has a high chitin content and, as a member of the phylum Arthropoda, it is believed that shrimps have a chitin metabolism similar to insects (Cobb, 1976). In order to grow, both shrimps and insects have to molt, i.e. shed the old, and build a new, larger, exoskeleton. During this process, parts of the chitin present in the old exoskeleton are recycled for the synthesis of the new one (Chen, 1987). For this purpose, both chitin-degrading and chitin-synthesizing enzymes are needed (Jeuniaux, 1961; Hackman, 1971). The Northern shrimp feeds on small bottom animals and detritus, and probably also algae (Allen, 1959). It is therefore reasonable to believe that chitin degrading enzymes are also a part of the digestive system of P. borealis since chitin may well be present in its diet. Biological degradation of chitin has been shown to be brought about by a chitinolytic enzyme system (Goodrich and Morita, 1977). The first step in this process is hydrolysis of chitin by chitinases (EC 3.2.1.14) which degrade the polysaccharide to small oligosaccharides, mainly the dimer. These oligosaccharities are converted into N-acetylglucosamine (GlcNAc) by fl-N-acetylhexosaminidase (EC 3.2.1.52) or fl-N-acetylglucosaminidase(EC 3.2.1.30) (Cabib, 1987). The difference between these two enzymes is that fl-N-acetylhexosaminidase is able to use both N-acetylglucosamine- and N-acetylgalactosamine-based substrates (Cabezas, 1989). fl-N-Acetylhexosaminidase activity, often referred to as fl-N-acetylglucosarninidase, has been detected in the alimentary canal, molting fluid and heronlymph from numerous insect species (Kimura, 1976; Spindler, 1976; Mommsen, 1980; Dziadik-Turner

et al., 1981; Koga et al., 1982, 1986, 1987, 1989; Fukamizo and Kramer, 1985; Spindler-Barth, 1986; Kramer and Aoki, 1987). In contrast to the welldocumented chitin degradation in insects, much less is known about this in the crustaceans. Chitindegrading enzymes have been studied in some crustacean species such as lobster and crab (Brun and Wojtowicz, 1976; Lynn, 1990), krill (Spindler and Buchholz, 1989), and recently in shrimps (Funke and Spindler, 1989; Kono et al., 1990; Koga et al., 1990). In the last two references, an endo-type chitinolytic enzyme (chitinase, EC 3.2.1.14) is described, while Funke and Spindler (1989) have, in addition, also detected two activities described as fl-N-acetylglucosaminidase during the purification of a chitinase, Recently, chitin-degrading enzymes have been detected in wastewater from the commercial processing of the Northern shrimp Pandalus borealis (Olsen et al., 1990). As part of a study to elucidate chitin metabolism in the cold water Norther shrimp, this work describes the purification and some properties of two fl-Nacetylhexosaminidase found in the hepatopancreas. MATERIALSAND METHODS Materials Q Sepharose Fast Flow, Sephacryl S-200 HR, phenyl Sepharose and standard proteins for gel filtration were purchased from Pharmacia (Uppsala, Sweden). Dispersed, high molecular chitin, N,N'-diacetylchJtobiose, N,N',N"triacetylchitotriose, N,N',N',N".tetraacetylchitotetraose, p-nitrophenyl-N-acetyl-~-D-glucosaminide, p-nitrophenylN-acetyl-~-D-glucosaminide, p -nitrophenyl-N-acetyl-fl-Dgalactosaminide, p -nitrophenyl-N -acetyl-,,-D-galactosaminide, p-nitrophenyl-~-D-glucopyranoside, p-nitrophenyl-~D-glucopyranoside, p-nitrophenyl-fl-D-galactopyranoside

513

514

MARGRETI-IEESAIAS~Net al.

and p-nitrophenyl-a-D-galactopyranoside were all obtained from Sigma Chemical Company (St. Louis MO). Nortbern shrimps (Pandalus borealis) were caught by trawling in the Barents Sea (71°5YN, 31°5YE) in November. The hepatopancreas (digestive gland) was immediately recovered and frozen at -30°C. After landing, the samples were stored at -50°C.

Determination of enzyme activity The activity of p-N-acetylbexosaminidase was measured at 37°C using p-nitrophenyl-N-acetyl-~-D-glucosaminide as substrate at pH 4.6 as described by Olsen et al. (1990). Enzyme activity expressed as described by Armstrong et al. (1966). The activity of chitinases was measured at 37°C. The method is based on a procedure by Reissig et al. (1955) by using dispersed, high molecular chitin as substrate. The reaction mixture consisted of 200 #1 substrate solution (5 mg chitin/ml H20 ), 100 #10.2 M sodium acetate buffer containing 0.6M NaC1 pH 5.5, 50/~1 of shrimp fl-N-acetylbexosaminidase (6 U/ml) isolated by gel filtration in a preliminary study, and the enzyme solution (50/zl). After incubation for 30 min, the reaction was stopped by adding 50/~1 50% TCA. Then 50/zl of 3.4M NaOH and 100/zl 0.8 M K2B, O7 were added. The samples were then placed in a boiling water bath for 3 rain and subsequently cooled in ice water. Finally, 3 ml of 10% dimethylaminobenzaldehyde in 9:1 HAc:HC1 was added. After 3rain the absorbance at 586 nm was measured on a Shimadzu UV-150 spactrophotometer. One unit of enzyme activity will produce 1 nmol of GlcNAc per min. Quantity of released GlcNAc was determined according to a standard curve. For determining the substrate specificity of the fl-Nacetylhexosaminidases, the corresponding p-nitropbenyl (pNP) glycosides were used as substrates, and the activity was measured in the same way as above. The enzymatic activity with N,N'-diacylchitobiose, N,N',N"-triacetylchitotriose and N,N',N",N"-tetraacetylchitotetraose as substrates was measured as with dispersed chitin, except that the concentration of the substrate solution was changed to 2.5 mg/ml H 20. For determination of the pH-optima at different temperatures, the enzymes were incubated with p-nitrophenyl-Nacetyl-/~-v-glucosaminide at pH from 3.0 to 8.0. The buffers used were 0.02 M citrate buffer (pH 3.0-6.4), 0.02 M citric acid-phosphate buffer (pH 6.5-7.4) and 0.02 M phosphate buffer (pH 7.5-8.0). Purification of ~-N-acetylhexosaminidases All steps in the purification were carried out at 4-8°C. Frozen hepatopancreas (22 g) were thawed and homogenized with 220ml 10raM Tris-HCl pH 7.4 by magnetic stirring for 45 min. The homogenate was then centrifuged for 30rain at 35,000g. The supernatant was mixed with 0.3 vol n-butanol, stiried for 45 rain and centrifuged for 30min at 35,000g. The aqueous phase was dialysed overnight against 10 mM Tris-HC1 pH 7.4, and applied to a Q Sepharose Fast Flow column (1.7 × l 1 cm) equilibrated with the same buffer. After washing the column with 500 mi

of this buffer containing 0.15 M NaC1, the enzymes were eluted with 0.6 M NaCI in the same buffer. Fractions containing the p-N-acetylhexosaminidase activity were pooled and concentrated by ultrafiltration in an Amicon Diaflo stirred cell using a PM 10 membrane. The concentrated enzyme solution from the Q Sepharose chromatography was gel filtrated on a Sephacryl S-200 HR column (2.6x93em) equilibrated with 0.15M NaCI in 10mM Tris-HC1 pH 7.4. Fractions containing fl-N-acetylbexosaminidase activity from the gel chromatography were rechromatographed on a Q Sepharose Fast Flow column ( l . 5 x 3 e m ) equilibrated with 0.15M NaCI in 10raM Tris-HC1 pH 7.4. The column was eluted with a linear gradient (total volume I00 ml) of NaCI (0.15-0.60 M) in the same buffer, and two separated p-N-acetylhexosaminidase activities were obtained and designated forms I and II. Sodium chloride was added to the pooled enzymecontaining fractions to a final concentration of 4 M. Both enzyme forms were further purified using separate phenyl Sepharose columns (1.5 × 3 cm) equilibrated with 4 M NaC1 in 10 mM Tris--HCl pH 7.4. The columns were eluted with linear gradients (total volumes I00 ml) of NaC1 (4-0 M) in the same buffer. Fractions containing ~8-N-acetylhexosaminidases were pooled and concentrated by ultrafiltration as previously described. The purified fl-N-acetylhexosaminidases were stored in aliquots of 50/~1 at -18°C.

Protein determination Protein concentration was estimated by the method of Bradford (1976) using the BioRad protein assay kit with bovine serum albumin as standard. RESULTS AND DISCUSSION

Enzyme purification Two ~-N-acetylhexosaminidase (EC 3.2.1.52) enzymes, designated forms I and II have been isolated from the hepatopancreas of the Northern shrimp. The purification procedure is summarized in Table 1. The initial n-butanol extraction of homogenized hepatopancreas tissue produces a defatted and clear aqueous enzyme solution. The first ion-exchange step removes the bulk of foreign proteins and makes the enzyme solution suitable for concentration and gel filtration, while the gel filtration step almost completely separates the ~-N-acetylhexosaminidase from the chitinase activity. The elution profile of the gel filtration column is shown in Fig. 1. Similar separations have been reported with enzymes from krill (Spindler and Buchholz, 1988) and from insects (Spindler, 1976; M o m m s e n , 1980). By comparison with globular marker proteins (run in separate experiments), ]]-N-acetylhexosaminidase eluted at a position corresponding to a globular protein of molecular mass 150 k D a (data not shown). When compared with other similar enzymes from Crustacea

Table I. Purification of two fl-N-acetylhexosaminidases(forms I and lI) from Pandalusboreali~ Yield Total protein Total activity specific activity Purification (%) (rag) (unit) (unit/mg) (fold) I II I II I II I II I II Step Crude extract 936 2231 2 1 100 Butanol extract. 506 2128 4 2 95 Q Scpharose 203 1615 8 4 72 Gel filtration 35 1350 43 22 67 Q Sepharose b 3.2 12.4 151 1141 47 92 24 46 7 51 Phenyl Seph. 0.4 0.5 44 610 ! |0 1220 55 610 2 27 The phenyl Scpharos¢ chromatography was run separately for each of the two fl-N-acctylhexosaminidase activities appearing in the last Q Sepharos¢ step.

P. borealis ~-N-acetylhexosaminidases 140

120

100

gO

6O

4O

10

20 Fraction n u m b e r

Fig. 1. Separation of chitinase ( - - O - - ) and ~-N-acetylhexosaminidase (--C)--) from the initial Q Sepharose step of the Pandalus hepatopancreas extract on a Sephacryl S 200 HR gel filtration column equilibrated with 0.15 M NaCI in 10 mM Tris-HCl pH 7.4

to results reported for B-N-acetylglucosaminidases (chitobiases) from lobster (Lynn, 1990), but comparable with results from the tobacco hornworm Manduca sexta (Dziadik-Turner et al., 1981). Finally, these two separated activities were purified by phenyl Sepharose columns. Form II of the ~-N-acetylbexosaminidase elutes already at 2.0M NaCI (Fig. 3A), while form I has a stronger affinity for the phenyl Sepharose gel and is obtained at approximately 0.9 M NaCI (Fig. 3B). Such difference in hydrophobicity between two ~-N-acetylhexosaminidase activities in the same tissue from Inseeta and Crustacea has apparently not been reported previously. This final chromatography step was mainly introduced to remove traces of the other enzyme form within each enzyme fraction. This was successfully carried out, and a small quantity of the predominant enzyme form II was removed from the fraction containing form I. Substrate specificity studies The substrate specificity of the two forms of B-Nacetylhexosaminidase were tested by using several

and Insecta, the molecular weight of these two enzymes are among the highest reported (Spindler, 1976; Mommsen, 1980; Spindler and Buchholz, 1988; Lynn, 1990). The ~-N-acetylhexosaminidase from the gel filtration step was separated into two activities by a second Q Sepharose column (Q Sepharose b) eluted with a linear gradient of NaC1 at pH 7.4 (Fig. 2). The results show that the ~-N-acetylhexosaminidase activity present in minor amounts (form I) is eluted with 0.25 M NaC1, while the predominant form (form II) is obtained with a concentration of approximately 0.50 M NaC1. This indicates that both enzyme forms have a relatively acid isoelectric point, and that form II has a more acidic pI than form I. This is in contrast

1oo

60 •

4o

"~

40 5

i

6O

4O

J

515

60

~0 ,;~

80

100

12U

J

<

30

40

50

60

"~0

80

90

10G

Fraction numlxx Fraction n u m b e r

Fig. 2. Chromatography on Q Sepharose. The shrimp ~-N-acetylhexosaminidase activity from the gel filtration step was applied to the Q Sepharose column in I0 mM Tris-HCl buffer pH 7.4, containing 0.15 M NaC1. After washing the gel with this buffer, the enzyme was eluted using a salt gradient (0.15-0.60 M NaC1) in I0 mM Tris-HCl pH 7.4. The arrows point to the start and the end of the gradient. The enzymes were further purified separately on phenyl Sepharose. ( ) Protein absorbance 280 nm; (--C)--) p-N-acetylhexosaminidase activity with pNP-GlcNAc as substrate.

Fig. 3. Chromatography on phenyl Sepharose. The shrimp p-N-a~tylhexosamimdase form I (B) and form II (A) from the Q Sepharose step was, after adjusting the sodium chloride concentration in the fractions to 4 M NaCI, applied to the column equilibrated with 10 mM Tris-HC1 buffer pH 7.4, containing 4 M NaC1. After washing the gel with this buffer, the enzyme was eluted using a salt gradient (4-0 M NaCl) in 10 mM Tris-HC1 pH 7.4. The arrows point to the start and the end of the gradient. ( ) Protein absorbance 280 nm; (--C)--) ~-N-acetylhexosaminidase activity with pNP-GIcNAc as substrate.

M A R O R ~ ' I ~ E S A t ~ & ~ e t al.

516

Table 2. Substrate specificities for ~-N-acetylhexosaminidase form I and II Substrate Form I Form II pNP-N-Acetyl-jS-D-glucosaminide 1.4 20.0 pNP-N-Acetyl-,, -I~glucosaminide 0 0 pNP-N-Acetyl-~-D-galactosaminide 1.0 29.0 pNP-N-Acetyl-~-D-galactosaminide 0 0 pNP-/~-v-Glucopyranoside 0 0 pNP-~-v-Glucopyranoside 0 0 pNP-/~-v-Galactopyranoside 0 0 pNP-u-v-Galactopyranoside 0 0 pNP-~ -D-N,N'-Diacetylchitobioside 0 1.9 pNP-~-v-N,N',N"-Triacetylchitotriose 0 0 N,N'-Diacetylchitobiose 4.0 11,100 N,N',NO-Triaoetylchitotriose 1.3 6200 N,N',N",N"-Tetraacetylchitotetraose 1.0 3800 The activities are relative according to the lowest activity appearing within each group of substrates.

p-nitrophenylated substrates and reduced N-acetylchitooligosaccharides (Table 2). In accordance with a similar study of/~-N-acetylhexosaminidases from the insect Manduca sexta (Dziadick-Turner et al., 1981), no activity was exhibited with the nitrophenylated forms of ~-N-acetylglucosamine, ~-N-acetylgalactosamine, /~-glucose, a-glucose, /~-galactose, or ~-galactose. The only nitrophenylated substrates cleaved by both enzyme forms were p-nitrophenyl-Nacetyl-/~-D-glucosaminide and its galactosamine epimer. Since the enzymes have an absolute requirement for the N-acetylated sugar moiety and the ~-glycosidic linkage, and since they can use both derivatives of N-acetylglucosamine and N-acetylgalactosamine as substrates, they should be classified as /~-N-acetylhexosaminidases. In addition, /~-Nacetylhexosaminidase form II was able to cleave p-nitrophenyl-p-D-N,N'-diacetylchitobioside. Comparing the relative activities of the two enzyme forms shows that form II has approximately 550-fold higher activity with N,N'-diacetylchitobiose than with pnitrophenyl-N-acetyl-fl-D-glucosaminide as substrate, while the corresponding ratio for form I is approximately 3. The activities with the p-nitrophenylated substrates and the reduced N-acetylchitooligosaccharides were measured at pH 4.6 and 5.5, respectively. At least some of the observed differ-

ences between form I and form II might be due to the fact that form I shows relatively lower activity at pH 5.5 than form II (Fig. 5).

General properties The temperature optimum of the /~-N-acetylglucosaminidase activity of enzyme forms I and II was measured using p-nitrophenyl-N-acetyl-~-v-glucosaminide as described in the Materials and Methods section. The apparent temperature optimum is 55°C for form I and 50°C for form II (Fig. 4). It has to be noticed that at pH 5.5, the temperature optimum is 45°C and 50°C for forms I and II, respectively (not shown). The temperature optima for the p-N-acetylglucosaminidases from this cold water shrimp are similar to the values described for ~-N-acetylglucosaminidases from the polar krill species Euphausia superba and the boreal species Meganyctiphanes norvegica (Spindler and Buchholz, 1988). Since the temperature optima for enzymes are obviously influenced by pH, the pH optima for the p-N-acetylglucosaminidase activity of the two enzymatic forms was measured with p-nitrophenyl-Nacetyl-/~-v-glucosaminide as substrate at various temperatures. Generally, the pH optimum of the

6

A

0

. 2

i 3

.

J 4

•

I

.

$

i 6

.

?

-

g

pH

10o

B

120

8O

too

°i

.i ° 20

0

° o

'

31o

'

.Io

'

,o

60

70

T ~ Fig. 4. Effect o f the temperature on the p-N-acetylgluco~minidase activity at p H 4.6 o f form I ( - - I 1 ~ ) and form

II (

) of the shrimp p oNoacetylh©xosamimdases.

$

4

5

6

?

$

pH Fig. 5. pH activity curves for the p-N-acetylglucosaminidase activity of form I (A) and form II (B) o f the shrimp ~-N-

acetylhexosaminidase as different temperatures. (--O--) 25°C, (--O--) 37°C, (--A--) 50°C, ( - - a - - ) (,0°C.

P. borealis ~-N-acetylhexosaminidases

predominant enzyme (form II) is between pH 4 and 6, while it is between 3.5 and 5 for form I. At temperatures above 25°C, /~-N-acetylhexosaminidase form I (Fig. 5A) has a more acidic p H - o p t i m u m than form II (Fig. 5B): at any temperature, the shrimp ~-N-acetylhexosaminidase form I exhibits no activity at pH values above 7.0, while form II still retains activity. At pH 3.0 form II shows no activity when measured at 50°C, while form I still exhibits 40% of maximum activity. The pH-optima for both enzyme forms gets more narrow with in reasing temperatures, and the pH optimum at 37°C is 3.5-5 and 4 - 6 for enzyme forms I and II, respectively. This is about the same pH values as the pH optima for similar enzymes from different insects acting on the same substrate (Mommsen, 1980; Koga et al., 1986; Kramer and Aoki, 1987). At room temperature, the activity of both enzymes is little influenced by the pH within the range of pH 3.0-7.0. In this work we have separated two acid ~-Nacetylhexosaminidase from the hepatopancreas of Northern shrimp (Pandalus borealis). The enzymes are clearly different, and it is possible that the pred o m i n a n t form which is very active towards Nacetylchitooligomers, is part of a chitinolytic enzyme system, similar to that proposed by Dziadik-Turner et al. (1981) for such enzymes in insects. Acknowledgements--This work was supported by grants from the Norwegian Fisheries Research Council. We would also like to thank the crew on M/S Remifisk for allowing one of the authors to stay on board, and for their assistance during the sample collection. REFERENCES

Allen J. A. (1959) On the biology of Pandalus borealis Kr6yer with reference to a population off the Northumberland coast. J. mar. biol. Ass. U.K. 38, 189-220. Armstrong J. McD. (1966) Purification and properties of human erythrocyte carbonic anhydrases. J. biol. Chem. 241, 5137-5149. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-due binding. Analyt. Biochem. 72, 248-254. Brun G . L. and Wojtowicz M. B. (1976) A comparative study of the digestive enzymes in the hepatopancreas of Jonah crab (Cancer borealis) and Rock crab (Cancer irroratus). Comp. Biochem. Physiol. 53B, 387-391. Buchholz F. (1989) Moult cycle and seasonal activities of chitinolytic enzymes in the integument and digestive tract of the Antarctic krill, Euphausia superba. Polar Biol. 9, 311-31"/. Cabezas J. A. (1989) Some comments on the type references of the official nomenclature (IUB) for ~-N-acetylglucosaminidase, /~-N-acetylhexosaminidase and /]-N-acetylgalactosaminidase. Biochem. J. 261, 1059-1060. Cabib E. (1987) The synthesis and degradation of chitin. Adv. Enzymol. 59, 59-101. Chen A. C. (1987) Chitin metabolism. Arch. Insect Biochem. Physiol. 6, 267-277. Cobb B. F. (1976) Biochemistry and physiology of shrimps. Effect on use as food. Proc. Trop. Subtrop. Fish. Technol. Conf. I, 141-165. Dziadik-Turner C., Koga D., Mai M. S. and Kramer K. J. (1981) Purification and characterization of two p-Nacetylhexosaminidases from the tobacco hornworm,

517

Manduca sexta (L.). Archs Biochem. Biophys. 212, 546-560. Fukamizo T. and Kramer K. J. (1985) Mechanism of chitin and oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem. 15, I-7.

Funke B. and Spindler K.-D. (1989) Characterization of chitinase from the brine shrimp Artemia. Comp. Biochem. Physiol. 94B, 691-695. Goodrich T. D. and Morita R. Y. (1977) Incidence and estimation of chitinase activity associated with marine fish and other estuarine samples. Mar. Biol. 41, 349-360. Hackman R. H. (1971) The integument of Arthropoda. In Chemical Zoology Vol. VI B (Edited by Florkin M. and Scheer B. T.), pp. 1-62. Academic Press, New York. Jeuniaux C. (1961) Biochimie de la mue chez Arthropods. Bull. Soc. Zool. France 86, 590-599. Kimura S. (1976) The chitinase system in the cuticle of the silkworm Bombyx mori. Insect Biochem. 6, 479-482. Koga D., Mai M. S., Dziadik-Turner C. and Kramer K. J. (1982) Kinetics and mechanism of exochitinase and ~-N-acetylbexosaminidase from the tobacco hornworm, Manduca sexta (L.). Insect Biochem. 12, 493-499. Koga D., Nakashima M., Matsukara T., Kimura S. and Ide A. (1986) Purification and properties of fl-N-acetylglucosaminidase from alimentary canal of the silkworm, Bombyx mori. Agric. Biol. Chem. 50, 2357-2368. Koga D., Shimazaki C., Yamamoto K., Inoue K., Kimura S. and Ide A. (1987) ~-N-Acetylglucosaminidases from integument of the silkworm Bombyx mori: comparative biochemistry with the pupal alimentary canal enzyme. Agric. Biol. Chem. 51, 1679-1681. Koga D., Fujimoto H., Funakoshi T., Utsumi T. and Ide A. (1989) Appearance of chitinolytic enzymes in integument of Bombyx mori during the larval-pupal transformation. Evidence for zymogenic forms. Insect Biochem. 91, 123-128. Koga D., Mizuki K., Ide A., Kono M., Matsui T. and Shimizu C. (1990) Kinetics of a chitinase from a prawn, Penaeus japonicus. Agric. Biol. Chem. 54, 2505-2512. Kono M., Matsui T., Shimizu C. and Koga D. (1990) Purification and some properties of chitinase from the liver of a prawn, Penaeusjaponicus. Agric. Biol. Chem. 54, 2145-2147. Kramer K. J. and Aoki H. (1987) Chitinolytic enzymes from pupae of the red flour beetle, Tribolium castaneum. Comp. Biochem. Physiol. 86B, 613-621. Lynn K. R. (1990) Chitinase and chitobiases from the American lobster (Homarus americanus ). Comp. Biochem. Physiol. 96B, 761-766. Mommsen T. P. (1980) Chitinase and ~-N-acetylgiucosaminidase from the digestive fluid of the spider, Cupiennius salei. Biochem. biophys. Acta 612, 361-372. Olsen R. L., Johansen A. and Myrnes B. (1990) Recovery of enzymes from shrimp waste. Process Biochem. 4, 67-68. Reissig J. L., Strominger J. L. and Leloir L. F. (1955) A modified colorimetric assay for the estimation of Nacetylamino sugars. J. biol. Chem. 217, 959-966. Spindler K.-D. (1976) Initial characterization of chitinase and chitobiase from the integument of Drosophila hydei. Insect Biochem. 6, 663-667. Spindler K.-D. and Buchholz F. (1988) Partial characterization of chitin degrading enzymes from two Euphausiids, Euphausia superba and Meganyctiphanes norvegica. Polar Biol. 9, 115-122. Spindler-Barth M., Shaaya E. and Spindler K.-D. (1986) The level of chitinolytic enzymes and ecdysteroids during larval-pupal development in Ephestia cautella and their modifications by a juvenile hormone analogue. Insect Biochem. 16, 187-190.