Fine substrate specificities of four exo-type cellulases produced by Aspergillus niger, Trichoderma reesei, and Irpex lacteus on (1-->3), (1-->4)-beta-D-glucans and xyloglucan.

To investigate the fine substrate specificities of four highly purified exo-type cellulases (Exo-A from Aspergillus niger, CBHI and CBHII from Trichoderma reesei, and Ex-1 from Irpex lacteus), water-soluble substrates such as barley glucan, xyloglucan from tamarind (Tamarindus indica L.), and their oligosaccharides were employed. Four exo-type cellulases immediately hydrolyzed 3-O-beta-D-cellotriosylglucose to produce cellobiose and laminaribiose. In contrast, CBHII showed no hydrolytic activity towards 3(2)-O-beta-D-cello-biosylcellobiose, which was hydrolyzed to cellobiose by the other exo-type cellulases. These cellulases hydrolyzed the internal linkages of barley glucan and lichenan in an endo-type fashion to produce cellobiose and mix-linked oligosaccharides as main products. The DP-lowering activities of the four exo-type cellulases on barley glucan were in the order of Ex-1, CBHII, Exo-A, and CBHI. Based on gel permeation chromatography analysis of the hydrolysates, Ex-1 seemed to attack the internal cellobiosyl unit adjacent to beta-1,3-glucosidic linkages in barley glucan molecule more frequently than did the other cellulases. Xyloglucan was hydrolyzed only by CBHI and CBHII, and produced hepta-, octa-, and nona-saccharides. In addition, a xyloglucan tetradecasaccharide (XG14) was split only to heptasaccharide (XG7) by CBHI and CBHII.

Hydrolysis of cotton cellulose by Exo- and endo-type cellulases from Irpex lacteus: differential scanning calorimetric study.

The mode of hydrolysis of cotton cellulose by two highly purified exo- and endo-type cellulases from Irpex lacteus was investigated by differential scanning calorimetry, to measure changes in the size of the amorphous region in cotton fibers with the enzymatic reaction. The cellulases induced entirely different changes in the size of the amorphous region, particularly at earlier stages of reaction. Exo-type cellulase gradually reduced the amorphous region with release of cellobiose from the initial stage of hydrolysis, but began to increase the amorphous region at more advanced stages of hydrolysis. By contrast, endo-type cellulase caused no liberation of reducing sugar at the initial stage of hydrolysis but caused a sharp increase in the amorphous region, and it thereafter caused a rapid decrease of the amorphous region, accompanied with the production of various kinds of cellooligosaccharides. The rate of size reduction of the amorphous region caused by endo-type cellulase was much higher than that by exo-type cellulase. Convergence of the decrease in the size of amorphous region during hydrolysis by endo-type cellulase is followed by the increase in this region being influenced by further hydrolysis of remained crystalline region. Substantial changes in the morphology of cotton occurred with the two cellulases after the hydrolysis stages at which the size of the amorphous region was minimum.

Mode of action of exo- and endo-type cellulases from Irpex lacteus in the hydrolysis of cellulose with different crystallinities.

The mode of action of two highly purified cellulases of exo- and endo-types from Irpex lacteus was investigated by using pure cellulosic substrates with different crystallinities derived from cellulose I. Exo-type cellulase saccharified all celluloses more effectively than endo-type enzyme, and the saccharification activities of both cellulases similarly increased with decreasing crystallinity of cellulose. The DP-lowering activity of exo-type cellulase remained similar for celluloses with higher crystallinity, while this cellulase showed a degradation mode resembling that of the endo-type enzyme for the substrates with lower crystallinity. Compared with exo-type cellulase, endo-type cellulase remarkably decreased the DP of cellulose with higher crystallinity, while this activity was abated for cellulose with lower crystallinity. Thus, the effects of both cellulases became similar in the degradation of amorphous substrates such as H3PO4-treated cellulose. Endo-type cellulase produced several kinds of cellooligosaccharide from all kinds of cellulose used, while the product of the exo-type enzyme was only cellobiose from crystalline cellulose such as cotton and cotton linter even after a 12-h incubation period. The results indicate that each cellulase shows a typical mode of action (exo or endo) for crystalline cellulose, but that their characteristic modes of attack may change with decreasing crystallinity of cellulose.

Electron microscopic observation of cotton cellulose degradation by exo- and endo-type cellulases from Irpex lacteus.

The interaction of two highly purified cellulases, exo- and endo-type cellulases from Irpex lacteus, with pure cotton and amorphous cellulose was investigated by electron microscopy. The morphological observations indicated that exo- and endo-type cellulases are both strongly adsorbed on the internal microfibril of cotton fiber before enzymatic hydrolysis, and then initiate their action toward the internal cellulose microfibrils with retention of the original shape. The two cellulases, however, caused considerably different morphological changes in cotton cellulose, and each cellulase seems to degrade native cellulose with a distinct mode of action. In the hydrolysis of cotton with exo-type cellulase, deep transverse cracks were produced and they extended from the fiber surface to the lumen structure located inside the fibers. In contrast, it was found that there were no deep cracks on fibers treated with endo-type cellulase, but severe internal erosion and cavitation occurred along fibril or microfibril layers inside the fibers. Thus, the degradation of cotton by exo- and endo-type cellulases yielded quite different morphological patterns, while little difference was found for regenerated celluloses. The mode of enzymatic hydrolysis of cellulose shown by cellulases with different degrees of randomness (exo and endo types) appears to be markedly affected by the fine structure of cellulose fibers.

Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus tulipiferae) on cellulose with different crystallinities.

The adsorption mode of two highly purified cellulases, exo- and endo-type cellulases, from Irpex lacteus (Polyporus tulipiferae) was investigated by using pure cellulosic materials with different crystallinity as substrates. Adsorption of the two enzymes on the substrates was found to fit the Langmuir-type adsorption isotherm. Maximum amount of adsorbed enzyme obtained from the Langmuir plots showed an inverse correlation to the crystallinity of the substrate with both enzymes, and this value of endo-type cellulase was less dependent on the degree of crystallinity of substrates than that of exo-type cellulase, whose isotherms reached saturation in the range of low enzyme concentrations. The two enzymes showed relatively high affinities for all the substrates and their affinities increased with increasing crystallinity, but this tendency was less marked with endo-type cellulase than with exo-type one. In addition, large negative values of free energy change were observed on the adsorption of both enzymes, and the values became more negative with increasing crystallinity. Consequently, both cellulases showed high adsorption on crystalline cellulose and the adsorption process became smoother with increasing crystallinity. The adsorption of the two types of cellulases was endothermic with an increase in entropy, especially for amorphous cellulose, suggesting the occurrence of water release from the substrates during enzyme adsorption. In addition, the changes in thermodynamic parameters (delta H, delta S, and delta G) in adsorption of exo-type cellulase were larger than in that of endo-type enzyme.

Substrate specificities of exo- and endo-type cellulases in the hydrolysis of beta-(1----3)- and beta-(1----4)-mixed D-glucans.

An exo-type cellulase (Ex-1) was extracted from Irpex lacteus (Polyporus tulipiferae) and purified essentially to homogeneity. This cellulase attacked cellulosic substrates in an exo-wise fashion to produce almost exclusively cellobiose. In contrast, Ex-1 was found to attack beta-glucans having beta-(1----3)- and beta-(1----4)-mixed linkages in a way similar to an endo-type cellulase. The products formed from barley glucan by Ex-1 were 3(2)-O-beta-D-cellobiosyl-cellobiose much greater than 3(2)-O-beta-D-glucosyl-cellobiose greater than cellobiose much greater than or equal to cellotriose much greater than glucose in the early stage, but no laminaribiose was produced. An endo-type cellulase (En-1) obtained from the same fungus also hydrolyzed beta-glucans but in a typical endo-wise fashion and the products from barley glucan were 3(2)-O-beta-D-glucosyl-cellobiose much greater than 3(2)-O-beta-D-cellobiosyl-cellobiose greater than cellobiose much greater than laminaribiose; no glucose or cellotriose was produced. Thus, it seems likely that En-1 can attack any intramolecular linkage of beta-glucan, while Ex-1 requires the presence of at least cellobiosyl residues adjacent to a beta-(1----3)-D-linked glucosyl residue. This finding, together with the mode of hydrolysis of cellulosic substrates by Ex-1, suggests that the stereochemical structure of successive beta-(1----4)-cellobiosyl residues inserted by beta-(1----3)-D-glucosidic linkage is permissible in the action of Ex-1, although this enzyme prefers the beta-(1----4)-linked cellobiosyl sequence.

Purification and properties of two endo-1,4-beta-xylanases from Irpex lacteus (Polyporus tulipiferae).

Two different endo-1,4-beta-xylanases [1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8], named Xylanases I and III, were purified to homogeneity by gel filtration and ion exchange column chromatography from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The purified enzymes were found to be homogeneous on polyacrylamide disc electrophoresis and their specific activities toward xylan were increased approximately 28.7 and 19.8 times, respectively. The activities of each enzyme were considerably inhibited by Hg2+, Ag+, and Mn2+. Their molecular weights were estimated to be approximately 38,000 and 62,000 by gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, respectively. Their carbohydrate contents were 2.5% and 8.0% as glucose, and their amino acid composition patterns resembled each other, showing high contents of acidic amino acids, serine, threonine, alanine, and glycine. Both enzymes were most active at pH 6.0 but Xylanase I was more stable as to pH. Their optimum temperatures were 60 degrees C and 70 degrees C, respectively. Xylanase I split up to 34.5% of larchwood xylan whereas Xylanase III split only 18.9% of it. The products with the former were mainly xylose (X1), xylobiose (X2), and xylotriose (X3), whereas X2 and X3 were the main products with the latter. Both enzymes did not hydrolyze X2. Xylanase I produced almost equal quantities of X1 and X2 from X3, while Xylanase III did not attack this substrate. Both enzymes showed no activity toward glycans, other than xylan, such as starch, pachyman and Avicel (microcrystalline cellulose), except the almost one twentieth activity of Xylanase III toward sodium carboxymethyl cellulose (CMC).

Purification and characterization of a cellulase from Dolabella auricularia.

A highly purified cellulase [EC 3.2.1.4] preparation (B beta) was obtained from a crude extract of gastric teeth of Dolabella auricularia by successive chromatographies on Sephadex G-100, DEAE-Toyopearl and CM-Toyopearl. The purified cellulase showed a single protein band on disc electrophoresis, and its isoelectric point was at pH 8.6. It contained relatively large amounts of basic amino acids and its molecular weight was estimated to be approximately 44,000 by sodium dodecyl sulfate (SDS) electrophoresis. The highest activity of this enzyme was attained at pH 6.3, but the enzyme was rather labile to heat. The activity of this enzyme was strongly inhibited by Hg2+, Mn2+, Zn2+, and Cu2+, whereas Ca2+ and Mg2+ showed no significant effect on the activity. The purified cellulase hydrolyzed sodium carboxymethyl cellulose (CMC) and phosphoric acid-swollen cellulose (swollen cellulose), as well as cellooligosaccharides and their reduction products, in an endowise fashion. It produced higher cellooligosaccharides effectively from swollen cellulose. Cellooligosaccharides with degrees of polymerization of 4-6 (G4-G6) were also hydrolyzed by the purified cellulase, but the modes of hydrolysis of these oligosaccharides were different from each other. The enzyme did not effectively attack cellooligosaccharides lower than G4. It produced G4 and G2 from G6, and G4 and glucose from G5. When these oligosaccharides were modified by reduction with sodium borotritide, the second linkage from the reducing end became, in each case, significantly susceptible to the enzyme and was preferentially cleaved.

Cellulase production from immobilized growing cell composites prepared by radiation polymerization.

The cellulase production from immobilized Trichoderma reesei composites prepared by radiation polymerization at low temperature was studied. The production of cellulase from the cells irradiated by radiation was slightly retarded at the initial stage of the culture, but was immediately recovered. The production of cellulase resulting from the growth of the immobilized cells proceeds efficiently in the composite having a porous polymer matrix, in which the productivity of cellulase varied with the hydrophilicity, the shape of the composite, monomer, and cell concentration. It was found that cellulase produced by immobilized growing cell composites effectively hydrolyzed cellulosic wastes such as newspaper and chaff which are pretreated by irradiation and crushing.

Transglycosylation activities of exo- and endo-type cellulases from Irpex lacteus (Polyporus tulipiferae).

Two highly purified cellulases, Ex-1 [exo-type, exo-cellobiohydrolase, EC 3.2.1.91] and En-1 [endo-type, EC 3.2.1.4] obtained from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae), were used in this work. Both cellulases produced 14C-cellooligosaccharides such as 14C-G2 and 14C-G3 by transglycosylation when G3, G5, or beta-PNPC was used as a donor and 14C-G1 as an acceptor. However, the transglycosylation activity of Ex-1 was far higher than that of En-1. When Ex-1 or En-1 was incubated with beta-PNPG only, no p-nitrophenol was released, but it was readily released when G3 was added to the reaction mixture. In this reaction, the optimal donor (G3) concentration for Ex-1 was 1.0 mM, and the optimal pH values of Ex-1 were at 2.7 and 3.7 for beta-PNPG and beta-PG as acceptors, respectively, these values being far lower than the ordinary optimal pH values of the cellulase (4.0-5.0).

Purification and properties of a lower-molecular-weight endo-cellulase from Irpex lacteus (Polyporus tulipiferae).

A new endo-cellulase component of carboxymethyl cellulase (CMCase) type (En-1) was obtained by gel filtration and column chromatography from Driselase, a commerical enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The enzyme behaved as a single protein on polyacrylamide disc electrophoresis in the presence of sodium dodecyl sulfate (SDS). Its molecular weight was estimated to be 15,500 and it contained only 0.73% carbohydrate as glucose. The pattern of its amino acid composition is similar to those of other cellulases in respect of high contents of acidic amino acids, glycine, serine, and threonine. The cellulase was most active at pH 4.0 and was very stable in the pH range of 3.0 to 6.0, but was completely inactivated by heating at 70 degrees C for 10 min. A series of cellooligosacharides, including cellobiose, was formed by this enzyme from sodium carboxymethyl cellulose (CMC) as well as from water-insoluble celluloses. In the hydrolysis of CMC, the increase in the fluidity of the substrate was relatively large as compared with the simultaneous increase in reducing power. From this result and the pattern of hydrolysis products, En-1 was elucidated to be an endo-cellulase, and it showed the highest randomness among the cellulase components obtained so far from Irpex lacteus.

Modes of action of exo- and endo-cellulases in the degradation of celluloses I and II.

Cotton and Valonia-cellulose (cellulose I) were readily attacked by endo-cellulase of a highly endowise-hydrolysis type, with a sharp decrease in the degree of polymerization, while the simultaneous production of reducing sugar was low. In contrast, viscose rayon and alkali-cellulose (cellulose II) showed little lowering of the degree of polymerization by endo-cellulase, though the effect was slightly greater than with an exo-cellulase, while the simultaneous production of reducing sugar was very high. The synergistic effect of exo- and endo-cellulases on the hydrolysis of cellulose was much higher with cellulose I than with cellulose II. These results can be explained in terms of differences in the polarity of cellulose chains and in the crystallinity and/or ultrastructure of the cellulose fibers.

Purification and properties of an exo-cellulase of Avicelase type from a wood-rotting fungus, Irpex lacteus (Polyporus tulipiferae).

A cellulase component of Avicelase type was obtained from Driselase, a commercial enzyme preparation from a wood-rotting fungus Irpex lacteus (Polyporus tulipiferae). It showed a single band on SDS-polyacrylamide electrophoresis. The amino acid composition of this cellulase resembled those of cellulase components of endo-type from the same fungus. However, it produced exclusively cellobiose from CMC as well as from water-insoluble celluloses such as Avicel or cotton at earlier stages of hydrolysis. In addition, the hydrolysis of CMC practically stopped after an initial rapid stage. The cellulase showed a strong synergistic action with an endo-cellulase of higher randomness (typical CMCase-type) in the hydrolysis of CMC as well as Avicel. In contrast to cellotriose and -tetraose, cellopentaose and -hexaose were attacked very rapidly, and only cellobiose was produced. These results suggest that the cellulase is an exo-type component. However, it mutarotated the products from cellopentaitol in the same direction as endo-cellulases. it represented a relatively large portion of the total cellulase activity, and may play an important role in the degradation of native cellulose in vivo.

Mutarotation of hydrolysis products by different types of exo-cellulases from Trichoderma viride.

Mutarotation of products from p-nitrophenyl beta-D-cellobioside and cellopentaitol by two different types of exo-cellulases from Trichoderma viride was investigated. It was found that an exo-cellulase of glucosidase type produced from the former substrate D-glucose which was mutarotated in a downward direction, while another exo-cellulase of Avicelase type produced from the latter substrate cellobiose which was mutarotated in an upward direction.

Multiple components of endo-polyguluronide lyase of Pseudomonas sp.

A lyophilized alginate lyase preparation obtained from dialyzed extract of sonicated Pseudomonas sp. cells was fractionated by gell filtration on a Sephadex G-150 column, and three alginate lyase [EC4.2.2.3] fractions, peaks I, II, and III, were obtained. They were remarkably thermolabile. The lyase fractions degraded two kinds of alginate fragments, a polygluuronide (SG), and a polyuronide consisting of both mannuronic and guluronic acid residues (SMG), as well as commerical alginate, but were virtually inactive toward polymannuronide fragment (SM). The modes of degradation of these substrates by the lyase fractions were endowise with different degrees of randomness. Attack by the peak I fraction was more random than those by peaks II and III. The main lysis products formed from SG and SMG by these layses were identified as mixtures of unsaturated tri- and monouronides. The unsaturated triuronide from SG was deltatugg and SMG yielded a mixture of deltaUGG and a poorly characterized unsaturated trimer, possibly deltaUMG. However, the patterns of monomer and trimer production by theselyase fractions changed in different ways during incubation.

Fine structure of SMG alginate fragment in the light of its degradation by alginate lyases of Pseudomonas sp.

An alginate fragment named SMG, consisting of mannuronic (M) and guluronic acid residues (G)(DP=25), was prepared from the partial acid hydrolysate of a commercial alginate. Two subfractions, SMG-ppt (DP=52) and SMG-sup (DP=18) were obtained from SMG by fractionation with MgC12 and CaC12. The M/G ratios of these alginate fragment were 1.4-1.9. Their lysis products by a pseudomonad alginate lyase [EC 4.2.2.3] preparation were fractionated by gel filtration, giving similar patterns. The major products in their digests were unsaturated monouronides (53-50%) and triuronides (30-35%). The former was identified as a delta4,5-hexuronic acid (deltaU) and the latter was identified as a mixture of delta4,5-hexuronosyl-(1 leads to 4)-beta-D-mannuronosyl-(1 leads to 4)-L-guluronic acid (deltaUMG) and delta4,5-hexuronosyl-(1 leads to 4)-alpha-L-guluronosyl-(1 leads to 4))L-guluronic acid (deltaUGG). The two unsaturated triuronides were present in roughly equal amounts. The presence of 4-O-alpha-L-guluronosyl-L-guluronic acid (GG) and 4-O-beta-D-mannuronosyl-L-guluronic acid (MG) or 4-O-beta-L-guluronosyl-D-mannuronic acid (GM) was also demonstrated inthe digest. Moreover, indirect evidence suggested nonreducing terminal deltaU residue and free deltaU in the digest to be derived more from M than G of the original SMG. Thus, it was concluded that more than one-third of uronic acid residues of SMG molecules may be composed of almost equal amounts of MG and GG sequences, most of which may be connected by M to form MMG and MGG sequences, respectively.

Substrate specificity of endo-polyguluronide lyases from Pseudomonas sp. on the basis of their kinetic properties.

Two endo-alginate lyases [EC 4.2.2.3] differing in their mode of degradation of substrates and practically free of polymannuronide lyase activity were partially purified from Pseudomonas sp. cells. Their substrate specificities were investigated for two different kinds of alginate fragments; a polyguluronide (SG) and a polyuronide consisting of mannuronic (M) and guluronic (G) acid residues (SMG). The effects of various salts and some organic compounds such as EDTA and p-chloromercuribenzoate on the degradation of the two substrates were similar. High concentrations of the substrates similarly inhibited the action ofthe lyases, giving a bell-shaped plot. A polymannuronide alginate fragment (SM) which was a substrate for polymannuronide lyase but was not attacked by these guluronide lyases also inhibited the degradation of SG and SMG. The overall degradation velocities of a mixture of SG and SMG by both lyases coincided with those calculated from the Michaelis-Menten formula. Based on the above results, it was concluded that SG and SMG are attacked by the same endo-polyguluronide lyase.

Synergistic action of two different types of endo-cellulase components from Irpex lacteus (Polyporus tulipiferae) in the hydrolysis of some insoluble celluloses.

The substrate specificities of three endo-cellulase [EC 3.2.1.4] components, F-1, F-2, and S-1, obtained from the culture filtrate of Irpex lacteus (Polyporus tulipiferae), were investigated in detail. It was confirmed that the former is of a more random type, belonging to the carboxymethyl-cellulase (CMCcase) group, and the latter two are of a less random type, beloning to the Avicelase group. It was found that a mixture of CMCase and Avicelase shows a remarkable synergistic action in the degradation of cotton and Avicel and that CMCase lowers the degree of polymerization of both cotton and CM-cellulose faster than Avicelases, relative to the production of reducing sugar. Thus, it was assumed that cotton and similar cellulosic substrates were degraded mainly by the synergistic action of these cellulase components produced by this celluloytic fungus.