Anaerobic bacterial degradation for the effective utilization of biomass

Biomass is originally photosynthesized from inorgainic compounds such as CO₂, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellulosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria,Clostridium thermocellum, Clostridium stercorarium, andClostridium josui, were isolated from compost and the chitinolyticClostridium paraputrificum from beach soil andRuminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed inEscherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex,i.e., cellulosome which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced byE. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed inE. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing CO₂ reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase fromC. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated.C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol H₂/mol glucose) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene inC. paraputrficum using a modified vector ofClostridium perfringens. The hydrogen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel (RDF), were also found to be effective in converting biomass waste to hydrogen gas.     

Reconsidering the function of the xyloglucan endotransglucosylase/hydrolase family

Journal of Plant Research - Plants possess an outer cell layer called the cell wall. This matrix comprises various molecules, such as polysaccharides and proteins, and serves a wide array of...     

Inhibition by A23187 of conformational changes involved in the Ca(2+)-induced activation of sarcoplasmic reticulum Ca(2+)-ATPase.

We previously showed that A23187 in high ionophore/protein ratios almost completely inhibits the sarcoplasmic reticulum Ca(2+)-ATPase [Hara, H. & Kanazawa, T. (1986) J. Biol. Chem. 261, 16584-16590]. In an attempt to obtain information on the mechanism of this inhibition, the effects of A23187 on conformational changes involved in the Ca(2+)-induced activation of the enzyme were investigated. The purified enzyme from sarcoplasmic reticulum of rabbit skeletal muscle as well as the purified enzyme labeled with fluorescein 5-isothiocyanate (FITC) were preincubated with A23187 in the absence of Ca2+ at pH 7.0 and 0 degrees C for 45 min. The activation of the enzyme following addition of CaCl2 was assessed by determining the capacity for rapid formation of phosphoenzyme from ATP. This activation was strongly inhibited by the preincubation with A23187. This indicates that the previously observed inhibition of the Ca(2+)-ATPase is mostly due to hindrance of the Ca(2+)-induced activation of the enzyme. In the control, in which the FITC-labeled enzyme was preincubated without A23187, the fluorescence intensity of the bound FITC decreased in a biphasic manner upon addition of CaCl2. The first rapid phase of this fluorescence drop was unaffected by A23187, whereas its second slow phase was almost completely inhibited by this drug. These results show that the Ca(2+)-dependent conformational change is biphasic and that the second slow phase (but not the first rapid phase) of this conformational change is inhibited by A23187. This suggests that the observed inhibition of Ca2+ activation is attributed to hindrance of the second slow phase of the Ca(2+)-dependent conformational change.     

Dorsal spinule patch variations in the puffer Lagocephalus spadiceus from Japan; revisited evidence for the existence of “spadiceus”- and “wheeleri”-forms

Ichthyological Research - Examination of the dorsal spinule patch in Lagocephalus spadiceus specimens revealed the existence of two intraspecific forms, the “wheeleri”-form,...     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Development of a dynamic imaging method for gravitropism in pea sprouts using clinical magnetic resonance imaging system

Although magnetic resonance imaging (MRI) is a useful technique, only a few studies have investigated the dynamic behavior of small subjects using MRI owing to constraints such as experimental space and signal amount. In this study, to acquire high-resolution continuous three-dimensional gravitropism data of pea (Pisum sativum) sprouts, we developed a small-bore MRI signal receiver coil that can be used in a clinical MRI and adjusted the imaging sequence. It was expected that such an arrangement would improve signal sensitivity and improve the signal-to-noise ratio (SNR) of the acquired image. All MRI experiments were performed using a 3.0-T clinical MRI scanner. An SNR comparison using an agarose gel phantom to confirm the improved performance of the small-bore receiver coil and an imaging experiment of pea sprouts exhibiting gravitropism were performed. The SNRs of the images acquired with a standard 32-channel head coil and the new small-bore receiver coil were 5.23±0.90 and 57.75±12.53, respectively. The SNR of the images recorded using the new coil was approximately 11-fold higher than that of the standard coil. In addition, when the accuracy of MR imaging that captures the movement of pea sprout was verified, the difference in position information from the optical image was found to be small and could be used for measurements. These results of this study enable the application of a clinical MRI system for dynamic plant MRI. We believe that this study is a significant first step in the development of plant MRI technique.     

N-(3-oxobutyl)cytisine: A new lupin alkaloid from eChinosophora koreensis

A new lupin alkaloid, N-(3,-oxobutyl)cytisine, was isolated from the aerial parts of Echinosophora koreensis. Its structure was determined by s     

Lupin alkaloids from Sophora mollis

The major alkaloids of Sophora mollis are (+)-sparteine and (?)-cytisine, and the minor ones are also of the sparteine-type (lupanine and 5,6-deh     

Isokuraramine and (−)-7,11-dehydromatrine,lupin alkaloids from flowers of Sophora flavescens

Two new lupin alkaloids, isokuraramine and (?)-7, 11-dihydromatrine, were isolated from the fresh flowers of Sophora flavescens along with 16 kno