Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824.

Butanol, at sub-growth-inhibitory levels, caused a ca. 20 to 30% increase in fluidity of lipid dispersions from Clostridium acetobutylicum. When grown in the presence of butanol or into stationary phase, C. acetobutylicum synthesized increased levels of saturated acyl chains at the expense of unsaturated chains.     

Ethanol Perturbs Glycosylation and Inhibits Hypersecretion in Trichoderma reesei

The effects of ethanol and phenylethanol on the growth of and glycoprotein secretion by Trichoderma reesei were studied. Low levels (1.5%, vol/vol) of ethanol perturbed the glycosylation process, as shown by alterations in the isoelectric profile of the secreted proteins and a reduction in the rate of incorporation of mannose into oligosaccharides. In addition to these effects on posttranslational modification, ethanol drastically lowered the protein secretion level of a hypersecretory strain.     

Cellulase secretion from a hyper-cellulolytic mutant of Trichoderma reesei Rut-C30

Two strains of Trichoderma reesei, wild type QM6a and mutant Rut-C30, were grown in meida containing an inducer, insoluble crystalline cellulose (Avicel PH101), as carbon source for 11 days. The cell growth, expressed as myceliar protein content, of Rut-C30 was 4–5 times higher than QM6a. The lack of ultrastructural disorganization, and absence of intracellular enzyme release into the growth medium, indicated that none of these two strains had undergone any significant autolysis during the entire growth phase. Cellulase activities, mainly endoglucanase, cellobiase and filter paper degrading activity (disc) were enhanced in Rut-C30 cells. A major change was observed in the endoglucanase activity which was 30 times higher in Rut-C30 than QM6a, whereas, both -glucosidase and disc activities were 3 times enhanced in Rut-C30 compared to QM6a. In addition to synthesis, cellulase secretion was also enhanced in Rut-C30. Both the organisms contained same amounts of intracellular marker enzyme activities (e.g., inosine diphosphatase, thiamine pyrophosphatase, alkaline phosphatase). Finally, the enahncement of secretory activity of Rut-C30 was correlated with the proliferation of rough endoplasmic reticulum (RER) and increased phospholipid content. It appears that Rut-C30 is not only a hypercellulolytic but also a hypersecretor mutant.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Semiquantitative Plate Assay for Determination of Cellulase Production by Trichoderma viride

A plate clearing assay was devised to screen for high-producing cellulase mutants of Trichoderma viride. The method employs (i) the use of either rose bengal or oxgall to limit colony size and (ii) Phosfon D (tributyl-2, 4-dichloroben-zylphosphonium chloride) to enhance cellulase detection, in combination with acid-swollen cellulose on agar plates. The method was used to isolate constitutive cellulase mutants of T. viride and should prove useful for isolating high-producing mutants from a range of organisms. This technique has been also used to determine the concentration at which glucose and glycerol inhibit cellulase synthesis by catabolite repression in the wild-type strains.     

Cellobiose-quinone oxidoreductase —application in monitoring cellobiohydrolase purification

Summary The enzymatic saccharification of cellulose has traditionally been monitored via total reducing sugar analyses. Yet with cellobiohydrolase being a major component of fungal and actinomycete cellulases, there is a need for a more specific measurement of cellobiose besides other soluble cellulodextrin products without interference from glucose, the major product of cellulose saccharification. One approach is to utilize cellobiose: quinone oxidoreductase (CBOase) from the fungus Phanerochaete chrysosporium to monitor saccharification. We describe here methods for the production of CBQase and a specialized application to monitor the chromatographic separation of cellobiohydrolase.Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Cryptic plasmids of thermophilic actinomycetes isolated from composts

Abstract Chemotaxonomic studies were performed on some gram-positive coryneform bacteria of uncertain taxonomic position isolated from human skin. The results indicate that the cutaneous strains represent a new mycolic acid-less Corynebacterium species for which the name Corynebacterium amycolatum sp. nov. is proposed.