Two H2O2‐dependent oxidases found in the extracellular medium of the white rot fungus Phanerochaete chrysosporium were separated by chromatography on blue agarose. The first enzyme fraction to elute from the column generated ethylene from 2‐keto‐4‐thiomethylbutyric acid (KTBA) in the presence of veratryl alcohol, and catalyzed the α,β cleavage of the diarylpropane 1‐(3,4‐diethoxyphenyl)1,3‐dihdyroxy‐(4‐methoxyphenyl)propane (I). During the diarylpropane cleavage, 18O from 18O2 was incorporated specifically into the α‐position of the product 1‐(4‐methoxyphenyl)1,2‐dihdyroxyethane (III), suggesting that this enzyme is an H2O2‐dependent oxygenase. The second enzyme which binds to blue agarose is an Mn2+‐dependent, lactate‐activated peroxidase. The enzyme catalyzed the oxidation of phenol red, o‐dianisidine, Poly R, and a variety of other dyes. It was also capable of decarboxylating vanillic acid.

No abstract

No abstract

A novel selection marker gene for transformation of the white-rot basidiomycete Pleurotus ostreatus was developed by introducing a point mutation in a gene which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase. The mutant gene, CbxR, encodes a modified Ip subunit with an amino-acid substitution (His239 to Leu) and confers resistance to the systemic fungicide, carboxin. The DNA sequence was integrated ectopically in the chromosome of the transformants. This is the first report of a homologous marker gene which is available for the molecular breeding of an edible mushroom.

No abstract

No abstract

Basidiomycetes were screened for lignin peroxidase (LPO) genes using a DNA probe prepared from the LPO restriction fragment of Phanerochaete chrysosporium. Southern blot analysis showed restriction fragments of chromosomal DNA of Bjerkandera adusta and Coriolus consors hybridized with the probe. Bjerkandera adusta produced LPO in a glucose-peptone medium. Ion-exchange chromatography showed that this fungus produced multiple molecular forms of LPO. One of the enzymes, LPO-2, was purified and characterized. The molecular weight of LPO-2 was 41,000 with a pI of 4.2. Spectral analysis demonstrated that LPO-2 is a haem protein. The enzyme cleaved lignin model dimers mainly at the C alpha-C beta position of the side chain. The LPO-2 exhibited close similarity to LPOs of P. chrysosporium with respect to their basic properties.

j-Glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-his-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specificically P-glucosidic bonds and catalyzed transglucosylation of the p-glucosyl group of cellobiose to yield 4-0-j-gentiobiosylglucose in the presence of organic solvents or under neutral conditions. The fungus Aspergillus niger excretes a wide range of carbohydrate-hydrolyzing enzymes. Various techniques, including size-exclusion, hydroxyapatite and ion-exchange chromatography, have been applied to the fractionation and purification of each enzyme from the multicomponent enzyme system. However, only limited success has been achieved with the purification of /Y-glucosidase from culture filtrates [l -71. So far, it has been thought that the substrate specificity of A . niger j-glucosidase was not strict for the fl-D-gluco configuration. For Instance, Tavobilov reported that p-glucosidase from A . niger hydrolyzed not only P-D-glucosides, but also j-D-xylosides, P-D-galactosides and r-L-arabinosides [l]. Later, McCleary et al. purified A . niger j-glucosidase 66.7-fold by gel-filtration and ion-exchange chromatography and reported that the enzyme hydrolyzed P-glucosidic bonds almost specifically but contaminating 8-xylosidase and /Igalactosidase activities still remained in the enzyme preparation [2]. Thus, the substrate specificity of A. niger p-glucosidase has differed depending on the extent of purification. Regarding the transglycosylation of this enzyme, Barker et al. reported that culture filtrates of A . niger catalyzed transglycosylation of the /I-glucosyl group of cellobiose to another cellobiose to form trisacchandes, and the principal linkages synthesized were Pl-6. However, Pl-2, j l -3 and pl-4 linkages were also formed, suggesting that j-glucosidase catalyzed the transglucosylation of p-glucosyl group [8]. However, catalytic activities of the purified enzyme have not been reported. In this study, we aimed at characterizing the substrate specificity and transglycosilation of A . niger P-glucosidase using a highly purified enzyme. We have, therefore, developed an affinity chromatography on a methacrylamide-N-methylene-bismethacrylamide copolymer bearing cellobiamine which interacts with subsites of P-glucosidases. Purification and properties of the P-glucosidase are reported here.Correspondence to T. Watanabe, Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto, Japan, 611Ahhreviutions. pNpp, p-nitrophenyl; CH,CN, acetonitrile; MeOH, methyl alcohol; EtOH, ethyl alcohol; Me2S0, dimethglsulroxide; DMF, N,N-dimethylformamide.Enzyme. &glucosidase (EC 3.2.1.21). MATERIALS AND METHODS MaterialsSephadex G-75 and PBE-94 were obtained from Pharmacia. SP-Toyopearl was obtained from Toso. Cellulosin AC-40 was obtained from Ued...