Bioscience Technology Center, RIKEN and Japan Science and Technology Corporation Saitama, JAPAN

mohkuma@mailman.riken.go.jp

　

A relationship between termites and cellulolytic protists in their hindgut is one of the fascinating examples of symbiosis. After the findings of a cellulase of termite origin, the cellulolytic activity of the symbiotic protists is considered still important for termites to live on wood or cellulose. Ingested cellulose that is probably digested partially by the endogeneous cellulase and traverses to the hindgut is incorporated by the protists and degraded completely inside the protists cells. Until recently, however, almost nothing is known about the cellulolytic system of the symbiotic protists because they are difficult to cultivate. We have been studying the cellulase genes of the gut symbiotic protists without cultivation of them.

　

The consensus PCR with using a primer set against the conserved regions of known cellulases successfully amplified the genes encoding cellulase-homologs that belong to the glycosyl hydrolase family (GHF) 45 from the symbiotic protists. We identified diverse full-length cDNA sequences of the GHF45, which represented a monophyletic but a distinct lineage from known members of GHF45.

　

A cDNA library constructed from the mixed population of the symbiotic protist species in the hindgut was expressed heterologously in Escherichia coli and screened cellulase activity to isolate genes encoding GHF5 cellulase. The recombinant GHF5 cellulase was purified and characterized its enzymatic property.

　

Meanwhile, we determined partial DNA sequences of more than 900 clones of a full-length cDNA library from the symbiotic protists and found that approximately 10% of the clones encoded GHF-related enzymes. These were cellobiohydrolase-homologs of GHF5 and GHF7, endoglucanase-like GHF5, GHF7, and GHF45. Homologs of GHF3-glucosidase, GHF 10- and GHF 11-xylanases, and GHF48-xylosidase. The GHF7-like cellobiohydrolases were the most abundant clones in the library.

　

The culture-independent approaches reveal, in the symbiotic protists, the presence of diverse cellulases, some of which perhaps work simultaneously in the cellulolytic process in the termite hindgut. The results also indicate that the symbiotic protists are rich reservoirs of novel cellulases. We are currently studying their protist origins in terms of specifying the set of cellulases expressed in single protists species.

　

　

¹Kitasato University, School of Fisheries Sciences Sanriku-cho Ofunato Iwate, JAPAN

r.sakai@kitasato-u.ac.jp

　

²Department of Pharmacology and Toxicology University of Texas Medical Branch Galveston, Texas, USA

　

Excitatory amino acids activate a family of neuronal receptors called ionotropic glutamate receptors (iGluRs). iGluRs are highly divergent receptors, and more than twenty different subtypes have been identified. Several naturally occurring excitatory amino acids are known, and some are used widely in neurobiological research because they not only selectively activate iGluRs but also induce epilepsy like convulsion in model animals. Selective iGluR agonists such as kainic acid and domoic acid, marine algae-derived compounds, have been used as indispensable tools in neuroscience. Yet, more subtype selective drugs are required to understand complex natures of neuronal GluRs.

　

A goal of our research is to discover and characterize novel marine organism-derived excitatory amino acids which might have unique subtype selectivity toward GluRs. During our effort to discover such compounds from marine benthic organisms, we found dysiherbaine (DH), a novel excitatory amino acid from a Micronesian sponge Dysidea herbacea. DH can selectively activate non-NMDA type iGluRs in rat hippocampal neuron. It also induces epilepsy-like convulsion in mice upon intracerebroventricular injection at exceptionally low doses (ED50= 13 pmol/mouse). In the presentation chemical and biological aspects of DH and other marine derived excitatory amino acids will be discussed.

　

　

¹National Institute of Agrobiological Sciences Ibaraki, JAPAN

hinabe@affrc.go.jp

　

²Center of Molecular Biosciences, University of the Ryukyus Nishihara, Okinawa, JAPAN

　

Since our finding of an endogenous cellulase from a termite in 1998, we have been exploring cellulases of animal origin. We isolated more than 40 cellulase CDNAS from termites and cockroaches (glycoside hydrolase family; GHF9), two from a beetle (GHF5) and a pill bug (GHF18), and another two from decapods (American crayfish and a land crab, GHF9). All GHF9 members of arthropods form a single phylogenetic clade independent of other origins, and other animal cellulases also appear to have been present in early metazoans like other digestive enzymes. All of these arthropod cellulases consist only of a catalytic domain (no other domains or complex structures like cellulosomes), and they formed cellulolytic systems only with additional b-glucosidases. This common aspect of arthropod cellulases is apparently viable due to the presence of masticating organs which break down the ingested wood into fine particles before enzymatic processing, and their cellulolytic systems perhaps seem more relevant to the human application of cellulases than fungal and bacterial systems.

　

A shuttle vector construct of native form RsEG (endoglucanase cDNA from a termite, Reticulitermes speratus) with an alpha yeast expression signal sequence gave viability on crystalline cellulose as a sole carbon sauce to a non cellulolyiic host S. cerevisiae, and demonstrating its potential applicability. Following this demonstration, we developed a cDNA shuffling system between two animal cellulases showing less than 80% homology to each other using long chain (60-120 mer) synthetic DNA, and a cellulase-activity clone-selection system using yeast to establish fundamentals for applied studies of the arthropod cellulases.

　

We will report our on-going projects of exploration and engineering of animal cellulases supported by the Bio-oriented Technology Research Advancement Institution (BRAIN) and by the Pioneer Research Project Fund (AFFRC, Japan)

　

　

Department of Cell Biology and Molecular Genetics College Park, Maryland, USA

RW19@umail.umd.edu

　

Estimates of global warming and population related food pressures have focused attention on the importance of the carbon cycle. The rate limiting reaction is the depolymerization, usually hydrolysis, of recalcitrant complex polysaccharides (RCP). Marine RCP production amounts to 25 billion tons/year or 31% of the planet's total annual production. Marine RCPs include chitin, the second-most abundant RCP after cellulose, agar, alginic acid, laminarin and pullulan. Relatively little is known about degradation of marine RCP, even though carbon recycling in marine habitats [71% of the earth's surface area] makes a significant impact on the total carbon budget.

　

Many bacterial species secreted degradative enzymes into the environment. However, to de-polymerize RCP, often containing a number of different bonding arrangements, degrees of acylation and quaternary structure [e.g. crystalline], a consortium of enzymes are often involved in this process. Maximum efficiency is maintained when the enzymes remain close to the cell, conserving the enzyme and increasing substrate availability. Taking advantage of these factors, one group of closely related clostridia, and also some fungi, synthesize exocellular enzyme arrays that organize the catalytic components into surface-borne structures, termed cellulosomes. Cellulosomes maintain maximum contact with the RCP and enhance efficiency of its depolymerization.

　

We have found that a newly described and classified marine bacterium [proposed name, Microbulblfer degradens, strain 2-40 (2-40)] is unusually versatile, degrading at least 11 different RCP including agar, chitin and alginic acid by a clostridial-1ike mechanism. Most marine bacteria have been shown to degrade chitin or other marine RCP by exporting essentially naked enzymes to the extracellular medium. 2-40 appears instead to organize these cognate enzyme systems into substrate-specific, multi-enzyme exocellular arrays. We termed the exocellular arrays in 2-40, hydrolysomes. Perhaps highlighting the advantage of this arrangement, agar is degraded 5x more rapidly by 2-40 than by species that have been shown to simply excrete agarases. The 2-40 enzyme arrays are thought to assemble upon a large protein, termed a scaffoldin. This protein has potential commercial value in protein/protein applications, such as protein purification or drug delivery systems. Hydrolysomes also have great potential value in the rapid degradation of chitin or its conversion to chitosan and in the recycling of other marine RCP.