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K Nisizawa

Publications and source records attributed to K Nisizawa.

At least 19 recordsLinked to original sources
PubMed1992

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.

Adsorption↗
PubMed1989

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.

Cellulase↗
PubMed1985

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).

Amino Acids↗
PubMed1984

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.

Amino Acids↗
PubMed1983

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).

Basidiomycota↗
PubMed1978

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.

Animals↗
PubMed1978

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.

Amino Acids↗
PubMed1977

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.

Alginates↗
PubMed1977

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.

Alginates↗
PubMed1977

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.

Alginates↗
PubMed1976

Partial proteolysis of some cellulase components from Trichoderma viride and the substrate specificity of the modified products.

An endo-cellulase component [EC 3.2.1.4] or random type, F II, was obtained from "Cellulase Onozuka," a commercial product from Trichoderma viride, and was subjected to partial proteolysiats with a protease preparation of the same fungal origin. The resulting modified cellulase was fractioned by two steps of column chromatography, and the resulting patterns, together with the substrate specificity expressed in terms of the randomness of CMC hydrolysis and the immunological properties against anti-F II-rabbit se-um, were examined. The chromatographic patterns were very similar to those of cellulase subfractions without proteolytic treatment. Moreover, the immunological response of the modified cellulases from F II was mostly positive and their randomness of CMC hydrolysis was generally lower, compared with subfractions of F II which were not subjected to proteolysis. The subfractions of Peak III, which were obtained from F II by proteolysis, showed mostly negative immunological response and higher randomness of CMC hydrolysis compared with subfractions of Peak III which were not subjected to proteolysis. Thus, some limited proteolysis of cellulase components may, at least in part, be responsible for its multiplicity in vivo.

Amino Acids↗
PubMed1976

Purification and properties of an endo-cellulase of avicelase type from Irpex lacteus (Polyporus tulipiferae).

A culture filtrate of Irpex lacteus (Polyporus tulipiferae) was fractionated initially by salting out with ammonium sulfate, and a cellulase [EC 3.2.1.4.] fraction with high Avicel-hydrolyzing activity (formerly called Avicelase) was extensively purified by a series of column chromatography procedures. This purified endo-cellulase showed a less random hydrolytic mechanism, and was obtained in a yield of 0.04% with respect to the starting material. Its specific activity was enhanced approximately 30 times over that of the starting material. The cellulase component showed a single peak on both ultracentrifugal and acrylamide disc electrophoretic analyses. Its molecular weight was estimated to be 56,000. It contained 12.2% carbohydrate; the major sugar constituents were glucose and mannose. Regarding the amino acid composition, the contents of aspartic acid and glycine were highest, followed by those of glutamic acid, serine, and theonine. The cellulase component was not markedly inhibited by most metal ions tested excepted for Hg2+. This purified endo-cellulase attacked a series of cellooligosaccharides, beta-cellobioside, CM-cellulose, and insoluble, cellulosic substrates. In the digests from insoluble substrates, glucose, cellobiose, cellotriose, and cellotetraose were detectable, but the amount of cellobiose was the largest by far. In constrast, cellobiose and glucose were produced in almost equal amounts from beta-cellobioside.

Amino Acids↗
PubMed1976

Xylanase activity of an endo-cellulase of carboxymethyl-cellulase type from Irpex lacteus (Polyporus tulipiferae).

An endo-cellulase [EC 3.2.1.4.] of carboxymethyl-cellulase type (F-1) which was fractionated from culture filtrate of Irpex lacetus and purified to electrophoretic and ultracentrifugal homogeneity, was found to show xylanase [EC 3.2.1.8.] activity. The activity was not removed from any of the intermediate fractions during the purification of the initial F-I peak, and the radio of xylanase to cellulase activity remained almost unchanged through the purification processes. The xylanase activity of F-I showed not only the same optiomal pH, heat stability, and pH stability as its cellulase activity, but also the same mobility as the cellulase activity upon cellulose acetate film and starch zone electrophoreses. The overall rates of hydrolysis of mixtures of variouis concentrations of CM-cellulose and xylan by F-1 coincided well with those calculated from the Michaelis-Menten treatment of two substances competing for the same active site of the enzyme. These results indicate that the xylanase activity of F-1 is intrinsic to the cellulase itself.

Basidiomycota↗
PubMed1976

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.

Basidiomycota↗
PubMed1975

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.

Animals↗