Masahiro Matsumiya

Properties of catalytic, linker and chitin-binding domains of insect chitinase

Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the Km for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.

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.

Substrate Specificity of Chitinases from Two Species of Fish, Greenling, Hexagrammos otakii, and Common Mackerel, Scomber japonicus, and the Insect, Tobacco Hornworm, Manduca sexta

Three chitinase isozymes, HoChiA, HoChiB, and HoChiC, were purified from the stomach of the greenling, Hexagrammos otakii, by ammonium sulfate fractionation, followed by column chromatography on Chitopearl Basic BL-03 and CM-Toyopearl 650S. The molecular masses and pIs of HoChiA, HoChiB, and HoChiC are 62 kDa and pH 5.7, 51 kDa and pH 7.6, and 47 kDa and pH 8.8, respectively. Substrate specificities of these chitinases were compared with those of another fish stomach chitinase from the common mackerel, Scomber japonicus (SjChi), as well as two from the tobacco hornworm, Manduca sexta (MsChi535 and MsChi386). The efficiency parameters, k cat⁄K m, toward glycolchitin for HoChiA and SjChi were larger than those for HoChiB and HoChiC. The relative activities of HoChiA and SjChi toward various forms of chitin were as follows: shrimp shell or crab shell α-chitin > β-chitin >> silkworm cuticle α-chitin. On the other hand, the relative activities of HoChiB and HoChiC were β-chitin >> silkworm α-chitin > shrimp and crab α-chitin. MsChi535 preferred silkworm α-chitin to shrimp and crab α-chitins, and no activity was observed toward β-chitin. MsChi386, which lacked the C-terminal linker region and the chitin-binding domain, did not hydrolyze silkworm α-chitin. These results demonstrate that fish and insect chitinases possess unique substrate specificities that are correlated with their physiological roles in the digestion of food or cuticle.

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.

Quantitative Nuclear Magnetic Resonance Spectroscopy Based on PULCON Methodology: Application to Quantification of Invaluable Marine Toxin, Okadaic Acid.

ERETIC2 (Electronic Reference To access In vivo Concentrations 2) based on PULCON (Pulse Length–based Concentration determination) methodology is a quantitative NMR (qNMR) using an external standard. The performance of the PULCON method was assessed using maleic acid (MA). Quantification of the diarrhetic shellfish toxin and okadaic acid by PULCON was successfully consistent with that obtained by a conventional internal standard method, demonstrating that the PULCON method is useful for the quantification of invaluable marine toxins without any contaminations by an internal standard.

Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker

Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)8–fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

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.

Biochemistry of fish stomach chitinase

Fish are reported to exhibit chitinase activity in the stomach. Analyses of fish stomach chitinases have shown that these enzymes have the physiological function of degrading chitinous substances ingested as diets. Osteichthyes, a group that includes most of the fishes, have several chitinases in their stomachs. From a phylogenetic analysis of the chitinases of vertebrates, these particular molecules were classified into a fish-specific group and have different substrate specificities, suggesting that they can degrade ingested chitinous substances efficiently. On the other hand, it has been suggested that coelacanth (Sarcopterygii) and shark (Chondrichthyes) have a single chitinase enzyme in their stomachs, which shows multiple functions. This review focuses on recent research on the biochemistry of fish stomach chitinases.

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