Kouji Miyauchi

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)2) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)n, n = 2-6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)4-6 and produced (GlcNAc)2-4, it was judged to be an endo-type chitinase. Meanwhile, an increase in beta-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)5 to produce (GlcNAc)2 (79.2%) and (GlcNAc)3 (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)n (n = 2-4) was pNp-(GlcNAc)2 >> pNp-(GlcNAc)4 > pNp-(GlcNAc)3. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)n. The chitinase also showed wide substrate specificity for degrading alpha-chitin of shrimp and crab shell and beta-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.

Purification and characterization of a 56 kDa chitinase isozyme (PaChiB) from the stomach of the silver croaker, Pennahia argentatus.

A 56 kDa chitinase isozyme (PaChiB) was purified from the stomach of the silver croaker Pennahia argentatus. The optimum pH and pH stability of PaChiB were observed in an acidic pH range. When N-acetylchitooligosaccharides ((GlcNAc)n, n=2 –6) were used as substrates, PaChiB degraded (GlcNAc)4 –6 and produced (GlcNAc)2,3. It degraded (GlcNAc)5 to produce (GlcNAc)2 (23.2%) and (GlcNAc)3 (76.8%). The ability to degrade p-nitrophenyl N-acetylchitooligosaccharides (pNp-(GlcNAc)n, n=2 –4) fell in the following order: pNp-(GlcNAc)3 ≫ pNp-(GlcNAc)2 > pNp-(GlcNAc)4. Based on these results, we concluded that PaChiB is an endo-type chitinolytic enzyme, and that it preferentially hydrolyzes the third glycosidic bond from the non-reducing end of (GlcNAc)n. Activity toward crystalline α- and β-chitin was activated at 124%–185% in the presence of 0.5 M NaCl. PaChiB exhibited markedly high substrate specificity toward crab-shell α-chitin.

Purification and Characterization of Chitinase Isozymes from a Red Algae, Chondrus verrucosus

Three seaweed chitinase isozymes (Chi-A, B, and C) were purified from a red algae, Chondrus verrucosus. The molecular weights and isoelectric points were 24.5 kDa and 3.5 for Chi-A, 25.5 kDa and 4.6 for Chi-B, and 24.5 kDa and <3.5 for Chi-C. Optimum pH and temperature were observed at pH 2.0 at 80 °C for Chi-A and Chi-C, and at pH 1.0 and 70 °C for Chi-B. Toward N-acetylchitooligosaccharide (GlcNAcn) (n=2 to 6), Chi-A, B, and C hydrolyzed GlcNAc5 and GlcNAc6 and produced GlcNAcn (n=2 to 4). GlcNAcn (n=3, 4) with the reducing end-side of β anomer was detected in the hydrolysis products. These results indicate that the reactions of Chi-A, B, and C for GlcNAcn were a retaining mechanism similar to that of family 18 chitinase. Toward crystalline chitins, Chi-A, B, and C degraded squid pen β-chitin more than crab shell or shrimp shell α-chitin.

Purification and characterization of lysozyme from purple washington clam Saxidomus purpurata

Lysozyme was purified from purple washington clam Saxidomus purpurata by sequential procedures using Chitopearl Basic BL-01 affinity and TSKgel ODS-120T column chromatographies. Molecular mass of the purified enzyme was estimated to be 12 kDa by SDS-PAGE. Optimum pH of the enzyme was 5.2 toward Micrococcus lysodeikticus cells. The optimum temperature was 50°C. The enzyme was stable in the range of pH 4.8–6.8 and 20–90°C. Further, the N-terminal amino acid sequence of the enzyme showed similarity to lysozymes from invertebrates. However, the specific activity of the enzyme toward M. lysodeikticus cells and p-nitrophenyl penta-N-acetyl-β-chitopentaoside was 143 times and 12 times higher than that of hen egg white lysozyme, respectively.

Substrate specificity and partial amino acid sequence of a Chitinase from the stomach of coelacanth Latimeria chalumnae

Chitin, an insoluble polysaccharide consisting of b-1,4-linked N-acetyl-D-glucosamine (GlcNAc) units, is the second most important natural polymer in the world. It is a major structural component in the exoskeleton of arthropods and in the cell walls of fungi and yeast. Chitinase (EC 3.2.1.14), which cleaves the b-1,4-glycosidic bonds of chitin, is distributed widely in living organisms. Chitinases play various important physiological roles in nutrition, morphogenesis, defense and aggression. Chitinase activities are detected from the digestive juice of many fish species feeding preys, especially which possess chitin exoskeleton. Chitinases are also used to produce N-acetylchitooligosaccharides (GlcNAc)n and GlcNAc, which have biological activities. Most of the chitin present in nature has either an aor a b-crystalline structure, with the a-form being predominant. Therefore, study of the substrate specificity of chitinases, especially toward crystalline chitin, is important not only to reveal physiological roles but also in order to degrade chitin to generate novel products with industrial applications. Recently, Matsumiya et al. reported that chitinases from two species of fish (Actinopterugii), Scomber japonicus and Hexagrammos otakii, and from an insect, Manduca sexta, possess substrate specificities that are correlated with their physiological roles in the digestion of food or cuticle. Chitinase from a fish known as a living fossil is also of interest. This study investigated the substrate specificity and partial amino acid sequence of a chitinase from the coelacanth Latimeria chalumnae (Sarcopterygii) and compared the results with those of other chitinases. Coelacanth Latimeria chalumnae chitinase (46 kDa, LcChi) was purified from the stomach by the method of Matsumiya et al. Chitinase activity was assayed using various substrates. When glycol chitin (Seikagaku Kogyo, Tokyo, Japan) was used as the substrate, the reducing sugar produced was measured by the method of Imoto and Yagishita. When colloidal chitin, a-chitin or b-chitin, and p-nitrophenyl N-acetylchitooligosaccharides (pNp-(GlcNAc)n, n = 1–4) were used as the substrate, enzyme activity was assayed in the previous study. Protein concentration was measured by the method of Bradford using bovine serum albumin (Bio-Rad, Hercules, CA, USA) as the standard protein. To identify the anomeric form of the product in the enzymatic reaction of (GlcNAc)5, 100 mL of 0.11 mM (GlcNAc)5 (final concentration, 0.1 mM) dissolved in 50 mM of sodium acetate buffer (pH 4.0) was reacted with 10 mL of the enzyme at 25°C for 10 min. The reaction mixture was immediately cooled in an ice bath in order to delay the equilibrium reaction between a and b anomers, and a 20-mL portion was analyzed by high-pressure liquid chromatography (HPLC) using a Tosoh TSK-Gel amide-80 column (Tokyo, Japan) (4.6 ¥ 250 mm) by the method of Koga et al. The N-terminal amino acid sequences of LcChi were analyzed using a protein sequencer (PE Applied Biosystems 447/120A, Foster City, CA, USA). *Corresponding author: Tel: 81-466-84-3684. Fax: 81-466-84-3684. Email: matsumiya@brs.nihon-u.ac.jp Received 19 April 2007. Accepted 14 September 2007. FISHERIES SCIENCE 2008; 74: 1360–1362

Characterization of lysozymes from three shellfish

Lysozymes (EC 3.2.1.17) are known to be widely distributed in life as lytic enzymes that hydrolyze the coupling of b-1,4 between N-acetylmuramic acid and N-acetylglucosamine in a bacterial cell wall. Lysozymes have been classified by primary structure and substrate specificities, mainly into five families (chicken, goose, phage, bacterial and plant lysozymes). Recent studies have clarified the complete primary structures of Ruditapes philipplnarum (Tapes japonica) and several other invertebrate lysozymes. According to these reports, some invertebrates have a new type of lysozyme (type-i lysozyme) not belonging to the above five lysozyme families. The authors purified a 12 kDa lysozyme from Corbicula japonica and examined some of its properties. The amino acid sequences in the N-terminal part showed high homology with type-i lysozymes. When Micrococcus lysodeikticus was used as substrate, the lysozyme showed approximately 250 times greater specific activity than hen egg white lysozyme (HEWL). These indicate that invertebrates, especially shellfish, may have type-i lysozymes of strong bacteriolysis activity. Obtaining information about these enzyme characteristics is considered significant both in enzymology and molecular biology. Therefore, in this study, lysozymes were purified from three kinds of shellfish and their general properties and resolving power toward M. lysodeikticus and p-nitrophenyl penta-N-acetyl-b-chitopentaoside (PNP-(GlcNAc)5) as substrate were compared. The Liolophura japonica, Monodonta labio and Omphalius pfeifferi used in the present study were sampled from the reef belt in Tanoura Bay, off Shimoda City, Shizuoka Prefecture, in July 2005. The samples were stored at -20°C and provided for the experiments as required. The authors used HEWL and dried M. lysodeikticus (Sigma-Aldrich, St. Louis, MO, USA), b-N-acetylhexosaminidase (Wako Pure Chemical Industries, Osaka, Japan) and PNP-(GlcNAc)5 (Seikagaku Kogyo, Tokyo, Japan).Whole internal organs from L. japonica and midgut glands from M. labio and O. pfeifferi were homogenized each with five volumes of 10 mM phosphate buffer (pH 7.2) at 20 000 r.p.m. for 2 min, using a Ultra-talax T25 Homogenizer (Janke & Kunkel GmbH & Co. KG – IKA-Labortechnik, Staufen, Germany). The homogenates were centrifuged by 10 000 g for 20 min and the supernatants were used as crude extract. For the L. japonica lysozyme, the crude extract was loaded onto CHITOPEARL BASIC BL-01 (Fuji Spinning, Tokyo, Japan) and CM-Toyopearl 650S (Tosoh, Tokyo, Japan) column chromatographies, as previously reported. Next, the lysozyme active fraction was applied to gel-filtration high-pressure liquid chromatography (HPLC) on a Superdex75 (Amersham Biosciences, Piscataway, NJ, USA) using 10 mM phosphate buffer (pH 7.0) with 0.15 M NaCl. Finally, the lysozyme peak was put onto reversedphase HPLC on a TSKgel ODS-120T (Tosoh, Tokyo, Japan) using acetonitrile with a gradient from 0 to 70% for elution. The M. labio and O. pfeifferi lysozymes were purified by a method similar to the L. japonica lysozyme. Regarding the lysozyme activity, the turbidity decrease was measured with dried M. lysodeikticus as substrate, as in the previous report. The absorbance data obtained by the 10 min reaction in 10 mM phosphate buffer (pH 6.2) at 37°C was applied to a precreated calibration curve of HEWL and represented as mg of HEWL. In addition, the activity toward *Corresponding author: Tel: 81-466-84-3356. Fax: 81-466-84-3683. Email: miyauchi@brs.nihon-u.ac.jp Received 24 March 2006. Accepted 7 July 2006. FISHERIES SCIENCE 2007; 73: 1404–1406