Tomohiko Kuwabara

Purification and Characterization of a Hemolysin-Like Protein, Sll1951, a Nontoxic Member of the RTX Protein Family from the Cyanobacterium Synechocystis sp. Strain PCC 6803

The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca(2+) binding, was purified from the glucose-tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca(2+) was not chelated with EGTA; however, it was released after being heated at 100 degrees C for 1 min, and it rebound to the Ca(2+)-depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca(2+), respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca(2+). Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca(2+)-induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature.

Characterization of a prolyl endopeptidase from spinach thylakoids

A prolyl endopeptidase (PEPase, EC 3.4.21.26) that specifically cleaves the 18‐kDa protein of photosystem II was extracted from photosystem II membranes with 1 M NaCl. Proteolytic activity measured with artificial substrates was less than a quarter of that with the protein. Studies on inhibition of the proteolysis by an artificial substrate suggested that the protease recognizes the scissile prolyl bond. The protease was inhibited by CuCl2, but not by diisopropyl fluorophosphate or p‐chloromercuriphenylsulfonic acid. These findings suggest that the protease represents a new class of PEPase. The specificity of the enzyme is discussed in relation to the structure of the 18‐kDa protein.

Anaerobic Coculture of Microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C Enhances Generation of n-Alkane-Rich Biofuels after Pyrolysis

ABSTRACT We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH4 production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils.

Effective and selective recovery of gold and palladium ions from metal wastewater using a sulfothermophilic red alga, Galdieria sulphuraria.

The demand for precious metals has increased in recent years. However, low concentrations of precious metals dissolved in wastewater are yet to be recovered because of high operation costs and technical problems. The unicellular red alga, Galdieria sulphuraria, efficiently absorbs precious metals through biosorption. In this study, over 90% of gold and palladium could be selectively recovered from aqua regia-based metal wastewater by using G. sulphuraria. These metals were eluted from the cells into ammonium solutions containing 0.2M ammonium salts without other contaminating metals. The use of G. sulphuraria is an eco-friendly and cost-effective way of recovering low concentrations of gold and palladium discarded in metal wastewater.

pH‐dependent reversible inhibition of violaxanthin de‐epoxidase by pepstatin related to protonation‐induced structural change of the enzyme

The redox enzyme violaxanthin de‐epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE‐I predominated at pH 7.2. On lowering the pH of the chromatography, VDE‐I decreased and VDE‐II increased. Furthermore, re‐chromatography of either peak yielded both peaks. These results suggest that VDE‐I and VDE‐II are interconvertible depending on pH, and thus, they represent the de‐protonated and protonated forms of the enzyme, respectively. Presumably the protonation‐induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.

In Situ Ore Formation Experiment : Amino Acids and Amino Sugars Trapped in Artificial Chimneys on Deep-Sea Hydrothermal Systems at Suiyo Seamount, Izu-Bonin Arc, Pacific Ocean

The present study reports on the bio-organic composition of a deep-sea venting hydrothermal system originating from arc volcanism; the origin of the particulates in hydrothermal fluids from the Suiyo Seamount in the southern Izu-Bonin (Ogasawara) Arc is discussed with regard to amino compounds. Chimney samples on deep-sea hydrothermal systems and core samples at Suiyo Seamount were determined for amino acids, and occasionally amino sugars. Two types of chimney samples were obtained from active hydrothermal systems by submersible vehicles: one was natural chimney (NC) on a hydrothermal natural vent; the other was artificial chimneys (AC), mainly formed by the growth and deposition of sulfide-rich particulate components in a Kuwabara-type in situ incubator (KI incubator). Total hydrolyzed amino acids (THAA) and hydrolyzed hexosamines (HA) in AC ranged from 10.7 nmol/g to 64.0 nmol/g and from 0 nmol/g to 8.1 nmol/g, respectively, while THAA in hydrothermally altered core samples ranged from 26.0 nmol/g to 107.4 nmol/g-rock. The THAA was thus of the same order of magnitude in AC and core samples. The representative biochemical indicator ratios of {\beta}-alanine/aspartic acid and {\gamma}-aminobutyric acid/glutamic acid were low. This is consistent with a large microbial population and labile subterranean biogenic organic compounds. A micro energy dispersive X-ray spectrometer ({\mu}EDX) was used to show that the formation of massive pyrite- and chalcopyrite-ores, the major matrixes of the natural chimney structures, were processed during hydrothermal activity. These data suggest that labile particulate organic matter (POM) and/or dissolved organic matter (DOM) from hydrothermal fluid were continuously trapped to form concentrated organic matter in a sulfide-rich matrix.

The 60-kDa precursor to the dithiothreitol-sensitive tetrameric protease of spinach thylakoids: structural similarities between the protease and polyphenol oxidase

The 60‐kDa precursor to the 39‐kDa dithiothreitolsensitive protease was purified from photosystem II membranes of spinach. When partially purified 60‐kDa protein was stored at 4°C, the protein was degraded to fragments of 39 and 21 kDa. The 39‐kDa fragment was suggested to be identical to the 39‐kDa protease from effects of dithiothreitol on these polypeptides. The N‐terminal amino acid sequences of the 60‐kDa protein and the 39‐kDa protease were the same, APILPDVEK‐, suggesting that the latter was derived from the N‐terminal portion of the former. Immunostaining with polyclonal antibodies against the 60‐kDa protein indicated that the 60‐kDa protein represents the species that occurs in the native thylakoids. These and other structural properties suggest that the protein might be identical to polyphenol oxidase.

Evidence for the Self-Degradation of Polyphenol Oxidase

Plant polyphenol oxidase (PPO; EC 1.10.3.1) may not oxidize polyphenols in living cells, since the enzyme is located in plastids, whereas polyphenols are in vacuoles. The structural study of PPO has long been retarded due to the susceptibility of this protein to the proteolytic activity tightly associated with it. However, proteolytic events occurring on PPO were thought to be artifacts by a contaminating protease (1), and hence, the mechanism and the physiological significance of the protein degradation have not been studied in detail.