The glucoamylase of Coniophora cerebella

1. The major amylolytic enzyme in culture filtrates of Coniophora cerebella grown in starch-containing media has been purified and characterized as a glucoamylase (EC 3.2.1.3). 2. The activity/unit wt. of protein was increased 11-fold and the enzyme showed one major component on polyacrylamide-gel electrophoresis. 3. The glucoamylase had optimum pH4.0-4.5. 4. Hg(2+) completely inhibited the enzyme, but other ions tested had little effect on the activity at the concentration of ions used (5mm). 5. The action of the enzyme on amylopectin, amylose and maltose was studied. Hydrolysis proceeded by the stepwise removal of glucose units from the non-reducing ends of the polymer chains, and the enzyme was able to bypass or to hydrolyse the alpha-(1-->6)-glucosidic linkages at branch points in the amylopectin molecule. Glucose was the only product found in digests of these substrates. 6. At the same substrate concentration (0.1%, w/v) and enzyme concentration, the initial rates of glucose production from amylopectin, amylose and maltose were in the proportions 40:10:1. 7. K(m) values at 40 degrees and pH4.0 were calculated for the enzyme acting on amylopectin, amylose and maltose.     

The xylanase system of Coniophora cerebella

1. The culture filtrate of the fungus Coniophora cerebella grown on poplar 4-O-methylglucuronoxylan as carbon source and enzyme inducer contained an enzyme system that degraded the polysaccharide to xylose, acidic and neutral oligosaccharides and an enzyme-resistant polymer. Free uronic acid was not produced. 2. Cold ethanol fractionation of the culture filtrate yielded two active fractions, one of which had only xylanase (EC 3.2.1.8) and the other both xylanase and xylosidase (EC 3.2.1.37) activities. Further fractionation on DEAE-cellulose resolved the xylanase and xylosidase activities. 3. The xylanase degraded poplar 4-O-methylglucuronoxylan in an essentially random manner, producing oligosaccharides, but some xylose residues in the vicinity of uronic acid side groups were protected from hydrolysis, preventing a truly random attack. The xylosidase attacked the polysaccharide very slowly, releasing xylose, but the oligosaccharides produced by the action of the xylanase were much more susceptible to hydrolysis by the xylosidase. 4. The products of xylanase action were separated into neutral and acidic fractions. The neutral oligosaccharides were separated by chromatography on charcoal-Celite, and the major products were characterized as xylobiose, xylotriose, xylotetraose and xylopentaose. Some of the acidic sugars were branched, having the uronic acid residue attached to a xylose residue other than the terminal non-reducing one. 5. Gel filtration of various xylanase fractions gave values for the molecular weight of the enzyme from 34000 to 38000.     

New Structures in the Basidiomycete, Coniophora cerebella

Previously undescribed structures found in Coniophora cerebella are described. They appear like spherical bodies, about 0.5 mum in diameter, situated along the wall and septa of the hyphae. They are not membrane bound and seem to be composed of ribosomes.     

The effect of extracellular enzymes of wood-rotting fungi on wood

YKa3bIBaeMBIe B JIиTepaType IIpиeMbI биOJIOГиЧecKOЙ OбpaбOTKи ДpeBecиHbI иCnoJIb3yЮT ДeЙcTBиe pacTyЩиx ДpeBecHbIx rpибKOB. ЭTO — cЦocoб IIpocToЙ, HO, BO-IIepBbIx, MeДJIeHHbIЙ, a BO-BTOPBIX, He rapaHTиpyЮЩиЙ; paBHOMepHocTи ДeЙCTBиЯ. IIpиMeHЯЯ aKTиBHbIe npeπapaTbI; фepMeHTOB ДpeBecHbIx rpибKOB, MOжHO COKpaTиTb BeCb C0pOЦeCC ДO HeCKOJIbKиX ДecЯTKOB ЧacoB и IIpeДyπpeДиTb HepaBHOMepHoe pa3pyШneHиe CTpyKTypbI ДpeBeCиHbI.     

The extracellular proteolytic enzymes of the mosquito-parasitizing fungus Lagenidium giganteum

Lagenidium giganteum produces extracellular proteases when grown in peptone-yeast extract-glucose medium. The production of the enzymes required a suitable inducer such as protein, and was repressed by glucose. The total filtrate proteases had a pH optimum of 8.4, and were heat denatured t 60°C. The use of specific substrates and inhibitors demonstrated the presence of collagenase, trypsin-like protease, and a weak elastase. Trypsin-like activity was present in the medium after 38 h of growth, whereas collagenase appeared in 43 h. The roles of these enzymes in pathogenesis were discussed.     

Production of extracellular enzymes by aquatic hyphomycetes

Preliminary studies of the production of extracellular enzymes in eight species of aquatic hyphomycetes showed that none of the organisms contained a proteinase activity. Pyrocateehol oxidase activity was restricted toI. hamata andP. constricta. Significant cellulase and α-amylase activity was found inL. curvula. Triacylglycerol lipase activity was maximum inP. constricta.     

Production of extracellular enzymes in continuous culture

The production of bacterial enzymes in batch fermentations is compared with results obtained in continuous culture. When studying the production of α-amylase inBacillus subtilis it was found that instability of the enzyme synthesis was due to nonhomogeneity of the population rather than to “the culture’s history” (i.e. succession of several physiological states necessary for the enzyme production). The plasmid contained in the production clone was found to be the factor responsible for the α-amylase production. Predominance of the production clone or of the nonproduction one depends on the cultivation conditions used. As compared with batch cultivation the continuous production yields higher enzyme concentrations under optimal conditions and the fermentor productivity may be four to five times higher.     

Pilot-scale production of intracellular and extracellular enzymes

Optimization of pilot-scale enzyme production is described for the case of an extracellular protease and an intracellular esterase. Media optimization was conducted to reduce medium costs and to determine the effect of various defined ingredients as well as complex nitrogen sources on enzyme production. Fermentation conditions such as inoculum transfer timing, agitation rate, and cultivation temperature were evaluated for their effect on enzyme production. Broths were harvested via microfiltration, diafiltered, and in the case of the extracellular enzyme, lysed via homogenization. An improvement in enzyme titer and reduction in medium costs for the extracellular protease was realized through replacement of Sabouraud dextrose broth medium with more reasonably priced complex nitrogen sources such as N-Z-amine A. An improvement in enzyme titer and reduction in medium costs for the intracellular esterase was realized by decreasing the amount of malt extract and omitting glycerol from the medium. An improvement in the harvest conditions for both enzymes was realized by using large-lumen-diameter hollow-fiber membranes (2.7 mm) which seemed wide enough to pass clumps of fungal and filamentous bacterial fermentation broth without clogging.     

Diverse biological functions of extracellular collagen processing enzymes

Collagens are abundant proteins in higher organisms, and are formed by a complex biosynthetic pathway involving intracellular and extracellular post-translational modifications. Starting from simple soluble precursors, this interesting pathway produces insoluble functional fibrillar and non-fibrillar elements of the extracellular matrix. The present review highlights recent progress and new insights into biological regulation of extracellular procollagen processing, and some novel functions of byproducts of these extracellular enzymatic transformations. These findings underscore the notion that released propeptides and other proteolytic products of extracellular matrix proteins have important biological functions, and that structural proteins are multifunctional. An emerging concept is that a dynamic interplay exists between extracellular products and byproducts with cells that helps to maintain normal cellular phenotypes and tissue integrity.     

Production of extracellular enzymes by Antarctic fungal strains

 Thirty-three fungal strains, isolated from different sites on Victoria Land (continental Antarctica), were plate-screened for their ability to produce twelve extracellular enzymes. Lipases were generally present and in high quantities in almost all the strains. Polygalacturonase, as well as amylase and phosphatase, was common. Glucose oxidase, protease and DNAase appeared to be generally low or absent. Many strains, producing a limited number of enzymes, appeared to have a low eco-nutritional versatility while a few, such as Verticillium cfr. lecanii no. 1, V. cfr. lecanii no. 3, Aspergillus versicolor and Phoma sp. no. 2, showing a diversified enzymatic competence, are probably advantaged in extreme terrestrial environments characterized by low competition. The possibility of utilizing the enzyme-producing ability of these fungi in applied research is also discussed. Received: 3 November 1995/Accepted: 29 May 1996