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.     

Sugar constituents of fucose-containing polysaccharides from various Japanese brown algae

Sugar constituents of the fucose-containing polysaccharides (FCPs) from 21 species of brown algae were analyzed. FCPs were extracted with hot water (100 °C, 4 h), separated by precipitation with 20% (v:v) ethanol in the presence of 0.05 M MgCl₂ to remove contaminating soluble alginate, and purified by DEAE-Sephadex column chromatography. The samples were hydrolyzed with HCI, and neutral sugar and uronic acid were separated by anion exchange chromatography. Their amounts were determined by gas-liquid chromatography. The neutral sugars in the FCPs from Ishige okamurae, Laminaria ochotensis, Myelophycus simplex, Padina arborescens and Sargassum thunbergii all contained arabinose, fucose, galactose, glucose, mannose, rhamnose and xylose residues. The FCPs from Ishige okamurae, Padina arborescens, Sargassum hemiphyllum, S. patents and S. sagamianum contained the four uronic acids, galacturonic acid, glucuronic acid, guluronic acid and mannuronic acid.     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Nitrite-reducing activity of modified cytochrome c-553 from the red alga Porphyra yezoensis Ueda

Crystalline cytochrome c-553 was obtained from Porphyra yezoensisUeda. The cytochrome in areduced form was modified to show anitrite-reducing activity after appropriate treatment with heat,hydrogen peroxide, or photooxidation using methylene blue asthe electron acceptor, but the reducing activity was far lowerthan that of the nitrite reductase isolated from this alga.The modified cytochrome c-553 was autooxidizable and showedan absorption spectrum resembling that of cytochrome c-553 inthe oxidized form except for slight shifts of the absorptionmaximumin the -band region toward shorter wavelengths. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

Incorporation of14C-radioactivity into various lipid classes at different growth-stages of culturedLaminaria japonica

A study is reported on the incorporation of¹⁴C-acetate into lipid classes from three different growth stages ofLaminaria japonica, a species long used for food in Japan. This was done because of the possible utilization of its lipids.Radioactivity incorporated into whole lipids in the three growth stages under the same experimental conditions (10 °C, 500 lux) increased with maturity of the thalli. The radioactivity was found mainly in PC, TG and 1,2-DG and subsequently distributed into other lipid classes (PG,PI,PE,MGDG,SQDG and DGDG) to a lesser extent. The incorporation patterns of the former group were similar at all stages, but those of the latter group differed slightly according to growth stage.In juvenile thalli,¹⁴C was incorporated to a much higher extent into PG, MGDG, PI and fucosterol than PE, SQDG, DGDG and MG, while the¹⁴C-incorporation into MG, SQDG, DGDG and PS in the mature growth stage was higher than into the other lipid classes. The absolute level of incorporation was higher for all these compounds in mature thalli than the thalli of other growth stages.     

Changes in M:G ratios of extracted and residual alginate fractions on boiling with water the dried brown alga Kjellmaniella crassifolia (Laminariales,Phaeophyta)

Kjellmaniella crassifolia, the edible macro-brown alga in Japan contained nearly 27% of alginates of which nearly 7% was extractable from the fronds with boiling water for 6 h and the residual alginates in the frond were almost exhaustively extracted with a dilute alkali at 60 °C for 6 h. The alginates dissolved in all these extracts with both boiling water and dilute alkali were purified by fractionation with MgCl₂ and alcohol.The content of MM blocks in the boiling water-soluble alginate sample increased remarkably during heating for 6 h while that of GG blocks from the same sample decreased. In contrast, MM blocks in the alkali-soluble alginate sample decreased during 6 h heating while GG blocks continued to increase. Since the amounts of MG blocks showed slight fluctation, the M:G ratio of alginates extracted with boiling water increased towards the end of extraction whereas the reverse is true for the alkali-soluble alginates.     

Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.