Takahisa Kanda

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.

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.

Improvement in the productivity of xylooligosaccharides from waste medium after mushroom cultivation by hydrothermal treatment with suitable pretreatment

The effective xylooligosaccharides (XOs) production from the waste medium after mushroom cultivation (WM) was investigated. The WM contains rich nutrients (protein, etc.) which induce Maillard reaction with reducing sugars under hydrothermal conditions. To improve the productivity of XOs, the suitable pretreatment combined with washing and grinding was investigated, and subsequently hydrothermal treatment was demonstrated with batch type and continuous flow type reactor. The washing pretreatment with hot water of 60 degrees C was effective to remove nutrients from the WM, and it led to prevent brownish discoloration on the hydrothermal treatment. On the basis of experimental data, industrial XOs production processes consisting of the pretreatment, hydrothermal treatment and purification step was designed. During the designed process, 2.3 kg-dry of the purified XOs was produced from 30 kg-wet of the WM (15% yield as dry basis weight). Theoretical yield of XOs attained to 48% as xylan weight in the WM.

Reactivities of cellulases from fungi towards ribbon-type bacterial cellulose and band-shaped bacterial cellulose

We have investigated the reactivities of various cellulases onribbon-type bacterial cellulose (BC I) and band-shaped bacterial cellulose (BCII) so as to clarify the properties of different cellulases. BC I waseffectively hydrolyzed by exo-type cellulases from different fungi from twicetofour times as much as BC II, but endo-type cellulases showed little differencein reactivity on those substrates. One of the endo-type cellulases, EG II fromTrichoderma reesei, degraded BC II more rapidly thanexo-type cellulases even in the production of reducing sugars. The degree ofpolymerization (DP) of BC II was rapidly decreased by endo-type cellulases atanearly stage, while exo-type cellulases did not cause the decrease of DP atthe initial stage, though the decrease of DP was observed after an incubation of24 h. All exo-type cellulases adsorbed on BC I and BC II,whileendo-type cellulases except for EG II adsorbed slightly on both substrates. Itwas interesting to observe EG II adsorbed on BC I but not on BC II. It issuggested that the adsorption of enzyme on cellulose is important for thedegradation of BC I, but not for BC II. It is proposed that the ratio of aspecific activity of each enzyme between BC I and BC II represents thedifference in the mode of action of cellulase. Furthermore, the KRW value, which we can calculate from thedecrease of DP/reducing sugar produced, is effective for discriminating themode of action of cellulase, especially the evaluation of randomness in thehydrolysis of cellulose by endo- and exo-type cellulases.

Isolation and transcriptional analysis of a cellulase gene (cell) from the basidiomycete Irpex lacteus

A gene (named cell) homologous to the cellobiohydrolase I gene (cbhl) of Trichoderma reesei was isolated and sequenced from the white rot basidiomycete Irpex lacteus MC-2. The cell open reading frame consists of 1551 bp, which is interrupted by two introns, encoding a polypeptide of 517 amino acid residues with a calculated molecular mass of 54,522 Da. The deduced amino acid sequence showed that CEL1 (the protein encoded by cell) has a modular structure consisting of a catalytic domain of 449 amino acids and a C-terminal cellulose-binding domain (CBD) of 36 amino acids separated by a proline-, serine-, threonine-rich linker region of 32 amino acids. The CEL1 catalytic domain is homologous with fungal cellobiohydrolases (CBHs) belonging to family 7 of the glycosyl hydrolases. The transcription of cell was induced in the presence of various cellulosic substrates and repressed by glucose. It was therefore concluded that the reported sequence represents the first cellulase gene isolated from the basidiomycete Irpex.

Structure and Characteristics of an Endo-β-1,4-glucanase, Isolated from Trametes hirsuta, with High Degradation to Crystalline Cellulose

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-β-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.