Yoshihiko Amano

Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification

Embryogenic cells of asparagus (Asparagus officinalis L.) were successfully cryopreserved by vitrification and subsequently regenerated plants. The cells were cryoprotected with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C for 10 min and then transferred to 1.8-ml plastic cryotubes. The cryoprotected cells were dehydrated with a new vitrification solution (designated PVS3) at 0°C for 20 min prior to a plunge into LN2. PVS3 contains 50% (w/v) glycerol and 50% (w/v) sucrose in water. The vitrified cells in LN2 were rapidly warmed in a water bath at 40°C and then expelled into LS medium supplemented with 1.2 M sucrose. The cell survival evaluated by fluoresceine diacetate and phenosafranin amounted to 80–90% of untreated, unfrozen controls. Revived cells resumed growth within 3 days after plating and regenerated plantlets via embryogenesis. Plant regeneration efficiency was nearly the same as that of the untreated, unfrozen control. Even an 80% concentration of PVS3 diluted with water produced 80% survival.

Design of Cellulose Dissolving Ionic Liquids Inspired by Nature

Cellulose is an attractive and important renewable resource for the production of biocomposites and biofuel alcohols. However, as it consists of linear glucose polymer chains that form a very tight hydrogen-bonded supramolecular structure, cellulose resists enzymatic degradation. Therefore, there has been a growing interest in the development of a means to modify the cellulose structure in such a way that it can be easily biodegraded. Rogers and coworkers first reported a breakthrough by using ionic-liquid (IL) technology in 2002: Cellulose dissolved in an ionic liquid, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), and the cellulose regenerated from the IL solution was less crystalline and easily digested by a cellulase. Since then, extensive investigations have been performed to develop an IL that possesses the capability to dissolve cellulose and reduce its crystallinity. Most reported ILs are imidazoliumbased or alkyloxyalkyl-substituted ammonium salts with chloride, formate, acetate, propionate, or phosphate as counter anion. 4] An increased reaction rate of cellulase-mediated hydrolysis was realized when cellulose regenerated from the ionic liquid solution was subjected to the enzymatic reaction because of reduced crystallinity. However, no rational design of ILs dissolving cellulose has yet been established. 6] Conversely, hydrolysis of solid celluloses can be achieved by using cellulases, such as endoglucanases (EGs) and cellobiohydrolases (CBHs). The former can hydrolyze internal b-1,4-glycosidic bonds in a cellulose polymer in the amorphous regions within the cellulose micro-fibril, and the latter can act on the free ends of cellulose polymer chains. Both types of cellulases have cellulose-binding modules that facilitate their adsorption onto crystalline cellulose, bringing the catalytic domains physically close to their site of action. 8] Inspired by this, we hypothesized that the key point of designing ILs might be to increase the affinity between ILs and cellulose by using information of the cellulase protein. We examined the protein sequences of several cellulases that make up the substrate-binding cleft and determined that glucosyl-binding sites of cellulases were frequently formed on the exposed surface of aromatic side-chains. Three of the four binding sites making up the enclosed cellulose-binding tunnel contain the tryptophan residue side-chains of Trichoderma reesei Cel A (CBH II). We expected that ILs made from amino acids might have an affinity toward a certain part of cellulose and that this may cause dissolution of the cellulose in the liquids. Ohno and coworkers prepared ILs with amino-acid moieties as anions. Based on this idea, we initially tested the dissolution of cellulose in 1-butyl-3-methylimidazolium tryptophan ([C4mim][Trp]) by using microcrystalline cellulose (Avicel) as a model compound. However, the cellulose did not dissolve in this IL. We then prepared tryptophan salts with ammonium, phosphonium, or pyrridinium cations, and found that N,N-diethyl-N-(2-methoxyethyl)-N-methylammonium tryptophan ([N221ME][Trp]) dissolved cellulose (5 wt %) at 100 8C (Table 1, Entry1). Encouraged by the result, we prepared various types of amino acid-based ILs with the [N221ME] cation, and evaluated their dissolution properties against the model cellulose (Table 1). [N221ME][Ala] was the best solvent for cellulose (Table 1, Entry 2): 12 wt % of cellulose was dissolved in the IL after just 10 min of stirring at 100 8C. More importantly, the cellulose regenerated from this solution was only of the Type II form. The X-ray diffraction patterns of the microcrystalline cellulose film (Avicel) and that of the regenerated film are shown in Figure 1. The regenerated cellulose exhibits the typical diffraction patterns of Type II cellulose at 2 q= 20.16 and 21.768. The results indicate that the transformation of cellulose from Type I to Type II occurred after the dissolution and regeneration in [N221ME][Ala] . The second best solvent was [N221ME][Lys] (Entry 3) and the third best was [N221ME][Orn] (Entry 4). [N221ME][Thr] and [N221ME][Ile] also showed similar solubility against the cellulose (Entries 5 and 6). Interestingly, most amino acid-based ILs showed solubility against cellulose (Entries 7–20), except for the glutamic acid-based IL (Entry 21). We expected that the amino acids might have an affinity with a specific part of the cellulose, which might cause dissolution of cellulose in the amino acid-based ILs. The results were as expected, but details were slightly different. However, we had anticipated one of these differences, as there was no alanine residue near the entrance to the cellulose-binding tunnel of the cellulases. Although 10 wt % of cellulose was dissolved in [N221ME]Cl (Entry 22), a higher temperature (over 120 8C) and a long mixing time was required to dissolve the cellulose, and a slight decomposition of IL was observed under the conditions used. No dissolution of cellulose was observed when [N221ME]Br was [a] K. Ohira, Dr. Y. Abe, Dr. M. Kawatsura, Prof. Dr. T. Itoh Department of Chemistry and Biotechnology Graduate School of Engineering, Tottori University 4-101 Koyama Minami, Tottori 680-8552 (Japan) Fax: (+ 81) 857-31-5259 riso-u.ac.jp E-mail : titoh@chem.totto [b] K. Suzuki Idemitsu Kosan Co. , Ltd. 1280 Kami-izumi Sodegaura, Chiba 299-0293 (Japan) [c] Dr. M. Mizuno, Prof. Dr. Y. Amano Department of Materials Sciences Faculty of Engineering, Shinshu University 4-17-1 Wakasato, Nagano 380-8553 (Japan) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cssc.201100427.

Synergistic actions of exo-type cellulases in the hydrolysis of cellulose with different crystallinities

The synergistic actions of pairs of seven different exo- and endo-type cellulases produced by Irpex lacteus (Ex-1 and En-1), Trichoderma reesei (CBH I, CBH II, and Endo-2), and Aspergillus niger (Exo-A and EG-1) were investigated using pure cellulosic materials with different crystallinities as substrates. A marked synergism was observed with certain combinations of exo-type cellulases, namely, CBH II and the other exo-type cellulases (Ex-1, CBH I, or Exo-A), when saccharification activity against crystalline and amorphous celluloses was investigated. Differences between the modes of action of each exo-type cellulase alone and in combination are not clearly observed; the production of cellobiose was accelerated during the hydrolysis of crystalline and amorphous celluloses by the combinations of exo-type cellulases, and no effective synergistic actions of the combinations of exo-type cellulases were noted in the depolymerization of crystalline cellulose. However, the synergistic actions of exo-type cellulases with CBH II caused specific changes in the surface features of highly crystalline celluloses. Characteristic morphological changes in cotton fibers (erosion and cracking of fiber surfaces) were induced at increasing rates by the combination of CBH II and Ex-1 or Exo-A. In contrast, destruction and subdivision of fibrils, which resulted in the pronounced fibrillation of cotton fibers, were observed after the treatment of cotton fibers with CBH II and CBH I. It was suggested that CBH II may be one of the key enzymes involved in the synergistic actions during cellulose hydrolysis.

Development of continuous flow type hydrothermal reactor for hemicellulose fraction recovery from corncob.

The semi-pilot scale of continuous flow type hydrothermal reactor has been investigated to separate hemicellulose fraction from corncob. We obtained the effective recovery of hemicellulose using tubular type reactor at 200 degrees C for 10 min. From constituent sugar analysis of corncob, 82.2% of xylan fraction was recovered as mixture of xylose, xylooligosaccharides and higher-xylooligosaccharide which has more than DP 10. During purification of solubilized fraction by hydrothermal reaction such as ultrafiltration and ion exchange resin, higher-xylooligosaccharide was recovered as the precipitate. This precipitate was identified as non-blanched xylan fraction which has from DP 11 to DP 21 mainly. In this system, only a small amount of furfural has been generated. This tubular reactor has a characteristic controllability of thermal history, and seems to be effective for sugar recovery from soft biomass like corncob.

Hydrophilic Domains of Scaffolding Protein CbpA Promote Glycosyl Hydrolase Activity and Localization of Cellulosomes to the Cell Surface of Clostridium cellulovorans

CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.

Mode of action of cellulases on dyed cotton with a reactive dye

Cotton woven fabrics which were previously dyed with a reactive dye were treated with a commercial cellulase preparation. Dyeing with a reactive dye for cotton apparently inhibited the weight loss activity and saccharification activity of cellulase. In addition, dyed cotton was treated with highly purified cellulases which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type cellulases were inhibited more than endo-type cellulase by dyeing in the case of saccharification activity. CBH I was severely inhibited by dyeing as compared with CBH II or EG II from the viewpoint of morphological changes in the fiber surface. Dyes on the cellulose substrates severely influenced CBH I in spite of the rare modification, because CBH I hydrolyzed cellulose with true-processive action. The change in the activity of each cellulase component on dyed cotton can affect the synergistic action of cellulases.

Gene Cloning of Cellobiohydrolase II from the White Rot Fungus Irpex lacteus MC-2 and Its Expression in Pichia pastoris

A gene (cel4) coding for a cellobiohydrolase II (Ex-4) was isolated from the white rot basidiomycete, Irpex lacteus strain MC-2. The cel4 ORF was composed of 452 amino acid residues and was interrupted by eight introns. Its deduced amino acid sequence revealed a multi domain structure composed of a cellulose-binding domain, a linker, and a catalytic domain belonging to family 6 of glycosyl hydrolases, from the N-terminus. cel4 cDNA was successfully expressed in the yeast Pichia pastoris. Recombinant Ex-4 showed endo-processive degrading activity towards cellulosic substrates, and a synergistic effect in the degradation of Avicel was observed when the enzyme acted together with either cellobiohydrolase I (Ex-1) or endoglucanase (En-1) produced by I. lacteus MC-2.

Gene Cloning of an Endoglucanase from the Basidiomycete Irpex lacteus and Its cDNA Expression in Saccharomyces cerevisiae

A gene (cen1) coding for an endoglucanase I (En-1) was isolated from white rot fungus Irpex lacteus strain MC-2. The cen1 ORF was comprised of 399 amino acid residues and interrupted by 14 introns. The deduced amino acid sequence of the cen1 ORF revealed a multi-domain structure composed of a cellulose-binding domain, a Ser-/Thr-rich linker, and a catalytic domain from the N-terminus. It showed a significant similarity to those of other endoglucanases that belong to family 5 of glycosyl hydrolases. cen1 cDNA was inserted into a yeast expression vector, YEpFLAG-1, and introduced into Saccharomyces cerevesiae. The resulting S. cerevisiae transformant secreted a recombinant En-1 that had enzymatic properties similar to the original En-1. A strong synergistic effect for a degradation of Avicel and phosphoric acid swollen cellulose was observed when recombinant En-1 was used together with a major exo-type cellobiohydrolase I of I. lacteus MC-2.

Purification, characterization and gene analysis of exo-cellulase II (Ex-2) from the white rot basidiomycete Irpex lacteus.

A new exo-type cellulase, named exo-cellulase II (Ex-2), was purified from the crude enzyme preparation of Irpex lacteus. Ex-2 was very similar to the previously characterized exo-cellulase I (Ex-1) with respect to enzymatic features such as optimal pH, temperature, heat stability, and catalytic activity. However, Ex-2 exhibited greater pH stability than Ex-1. The molecular mass and carbohydrate content of Ex-2 (56,000, 4.0%) were different from those of Ex-1 (53,000, 2.0%). A cellulase gene (named cel2) encoding both Ex-2 and Ex-1 was isolated from an I. lacteus genomic library. The cel2 gene was found to consist of 1569 bp with an open reading frame encoding 523 amino acids, interrupted by two introns. The deduced amino acid sequences revealed that cel2 ORF has a modular structure consisting of a catalytic domain and a fungal-type cellulose-binding domain (CBD) separated by a serine-rich linker region. The catalytic domain was homologous to those of fungal cellobiohydrolases belonging to family 7 of the glycosyl hydrolases. Northern blot analysis showed that expression of the cel2 gene was induced by various cellulosic substrates and repressed by glucose, fructose, and lactose.

Blood pressure-lowering peptides from neo-fermented buckwheat sprouts: a new approach to estimating ACE-inhibitory activity.

Neo-fermented buckwheat sprouts (neo-FBS) contain angiotensin-converting enzyme (ACE) inhibitors and vasodilators with blood pressure-lowering (BPL) properties in spontaneously hypertensive rats (SHRs). In this study, we investigated antihypertensive mechanisms of six BPL peptides isolated from neo-FBS (FBPs) by a vasorelaxation assay and conventional in vitro, in vivo, and a new ex vivo ACE inhibitory assays. Some FBPs demonstrated moderate endothelium-dependent vasorelaxation in SHR thoracic aorta and all FBPs mildly inhibited ACE in vitro. Orally administered FBPs strongly inhibited ACE in SHR tissues. To investigate detailed ACE-inhibitory mechanism of FBPs in living body tissues, we performed the ex vivo assay by using endothelium-denuded thoracic aorta rings isolated from SHRs, which demonstrated that FBPs at low concentration effectively inhibited ACE in thoracic aorta tissue and suppressed angiotensin II-mediated vasoconstriction directly associated with BPL. These results indicate that the main BPL mechanism of FBP was ACE inhibition in living body tissues, suggesting that high FBPs bioavailability including absorption, tissue affinity, and tissue accumulation was responsible for the superior ACE inhibition in vivo. We propose that our ex vivo assay is an efficient and reliable method for evaluating ACE-inhibitory mechanism responsible for BPL activity in vivo.