The traditional filter paper assay for saccharifying cellulase originally described by M. Mandels et al (1976) has been modified to make possible low activity determinations of Trichoderma cellulases. The enzymatic activity appears to decline during a prolonged incubation period if no precautions have been taken. By means of adding bovine serum albumin and potassium chloride as protein stabilizers and sodium azide as an antimicrobial agent filter paper activities in the range from 0.02 to 0.37 (IUPAC assay, 1987) can be estimated by extending the incubation time up to 20 hours. Filter paper activity values obtained by this method may be compared to those obtained by the IUPAC assay by using a conversion factor from 1.4 to 1.7. Acid whey and sweet whey permeate have been investigated as media for growth and metabolite production by Trichoderma reesei QM 9414 using shake flask cultures and spore inocula. In the case of acid whey the mycelial growth after 2 weeks is 13 mg dry weight /ml substrate. The specific growth rate is 0.29/day. The fungus appears to metabolize the whey protein the first 2 weeks. The alkalinity of acid whey rises continuously over a three week period up to a pH of 8.5. In the case of whey permeate the maximal mycelial weight gain is 4.4 mg/ml which appears after 8 days. A rise in net soluble protein level comes after 3-5 days and reaches a maximum value of 0.23 mg/ml after 2 weeks. The pH of whey permeate rises continuously to 7.5 after 3 days and then slowly declines. The net production of cellulases is low on both media. Dilution 1:6 of the acid whey, supplementation with ammonium sulfate and pHadjustments did not enhance the production of cellulases. Acid whey supports a significant growth and sweet whey permeate shows potential for extracellular protein production. A literature review surveys the composition and uses of acid whey, environmental aspects of whey wastes, the fungus Trichoderma reesei, the mode of action of the Trichoderma reesei cellulase system and the structure of cellulose in cotton and wood. / Graduation date: 1994

In this research, the interfacial behavior of Trichoderma reesei CBHI and Thermomonospora fusca E��� cellulases were studied at synthetic surfaces. For this purpose, colloidal silica and polystyrene particles were used to prepare cellulase-particle suspensions that were analyzed by several solution-phase techniques. These included circular dichroism spectroscopy, size exclusion chromatography and filtration, and a spectrophotometric assay for cellulase activity. All techniques were performed in the presence and absence of particles. Circular dichroism spectroscopy (CD) and size exclusion chromatography showed, however, that binding did not occur between either cellulase and silica, presumably because silica is hydrophilic and negatively charged. Binding did occur between each cellulase and polystyrene, most likely mediated through hydrophobic associations. Cellulase-polystyrene complexes were not analyzed using CD because of high light absorption by the polystyrene nanoparticles. Upon adsorption to polystyrene, the activity of the E��� dropped about 95% relative to that of the free enzyme. While this substantial loss in activity may have been the result of binding being mediated through the catalytic domain, strong evidence supporting the thought that adsorption occurs through hydrophobic associations, mediated through the binding domain, suggests that structural or steric factors were partly responsible for the loss. / Graduation date: 1999

The efficiency of cellulose hydrolysis under straight saccharification and simultaneous saccharification and fermentation (SSF) conditions was evaluated using three lignocellulosic materials (switchgrass, cornstover, and poplar), which had been pretreated with dilute sulfuric acid under conditions which optimized xylose concentrations in the prehydrolysate liquid. Yields of glucose, cellobiose and ethanol obtained from the pretreated feedstocks were measured over 168 hrs. The final theoretical conversions of cellulose from pretreated switchgrass, cornstover, and poplar in straight saccharification were 85-100% (average 94%), 84-100% (average 96%), and 75-100% (average 87%), respectively, while in SSF the conversions were 84-90% (average 87%), 91-96% (average 90%), 72%-82% (average 76%), respectively. The conversion rates of poplar in straight saccharification and SSF were significantly lower than those of switchgrass and cornstover. The effects of reaction parameters such as enzyme activity, cellulose availability, and yeast cell viability on the extent of hydrolysis in straight saccharification and SSF were also studied. Results indicate that the lower glucose or ethanol yields associated with some of the poplar were due to the recalcitrant nature of its cellulose. To compare accurately the efficiencies between straight saccharification and SSF, a direct method for determining the cellulose content of the feedstocks residues resulting from SSF experiments has been developed and evaluated. The method improves on classical cellulose assays by incorporating a yeast lysing enzyme to remove yeast glucans from the feedstocks residue prior to acid hydrolysis and subsequent quantification of cellulose derived glucose. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzyme lysis. The method was applied to the analysis of the cellulose and yeast-glucan content of SSF residues from the three pretreated feedstocks. Cellulose assays employing the lysing enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucan were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to the subsequent chemical and physical analysis of SSF residues. / Graduation date: 1996

Graduation date: 1992

Thesis (doctoral)--University of Helsinki, 2007. / Includes bibliographical references. Also available on the World Wide Web.

Thesis (doctoral)--University of Helsinki, 2004. / Includes bibliographical references. Also available on the World Wide Web.

Thesis (doctoral)--University of Helsinki, 2004. / Includes bibliographical references. Also available on the World Wide Web.

Diss.Teknillinen korkeakoulu, 2006.

No description available.

An endo-1,4-β-D-glucanase (E.C. 3.2.1.4) and two exo-1,4-β-D-glucanases (exo-cellobiohydrolases I (D) and II, E.C. 3.2.1.91) have been purified to electrophoretic homogeneity from the extracellular culture filtrate of the imperfect fungus Trichoderma reesei QM 9414 grown on microcrystalline cellulose (Avicel). These three glycoprotein enzymes are the principal components of the cellulase system and constitute <95% of the extracellular protein produced by this organism when grown on cellulose or when incubated in the presence of sophorose (Q-β-D-glucopyranosyl (1->2) α-D-glucopyranose). The glucanases have been characterized with respect to a number of structural and enzymic properties. Neutral carbohydrate, predominantly mannose with some glucose, contributes 5, 21.2 and 14.1 percent of the weight of cellobiohydrolase I (D), cellobiohydrolase II and the endoglucanase, respectively. All three enzymes have a high proportion of acidic and hydroxylated amino acids, but much less of basic amino acids. Sedimentation equilibrium studies yielded the following molecular weights for the glucanases: cellobiohydrolase I (D) 53,220±1479; cellobiohydrolase II 54682±2683; endoglucanase 45,215±1483. Cellobiohydrolase I (D) is the most acidic (pH_I <3.8) of the three enzymes, whereas the endoglucanase and cellobiohydrolase II have isoelectric points of 4.7 and 5.6, respectively. All three lose activity when exposed to alkaline conditions, and the extent of alkali lability is directly related to carbohydrate content. This instability, as well as the low amino sugar content of the enzymes, indicates that the carbohydrate may be linked to the polypeptide via O-glycosyl bonds to serine or threonine residues. Among the three enzymes, the endoglucanase is most resistant to thermal inactivation retaining 70% of its initial activity on swollen cellulose after a 20 min preincubation at 70°. The optimum pH for activity is ca.4.9 for the endoglucanase and cellobiohydrolase II, whereas cellobiohydrolase I (D) has a broader pH range for activity, between pH 5.2 to 5.6. The mechanism of action of each glucanase was investigated by quantitative high performance liquid chromatographic analysis of the products arising from various cellulosic substrates as a result of enzymic action. Cellobiohydrolases I (D) and II produce predominantly (>90%) cellobiose from either oligosaccharides or cellulose. Cellobiohydrolase I (D) is unique among the three glucanases in its ability to cleave cellotriose. The endoglucanase reduces the viscosity of carboxymethylcellulose solutions with a specific activity of 116 (expressed as the change in specific fluidity/min/mg protein). This enzyme also possesses significant transglycosylation activity. When the three glucanases are combined in the proportion 60:25:15, cellobiohydrolase I (D): cellobiohydrolase II: endoglucanase (w/w) respectively, the resulting mixture exhibits activity identical to that of the crude enzyme preparation, with either swollen or crystalline cellulose as the substrate. All three glucanases are essential for the degradation of crystalline cellulose. Cellobiohydrolase I (D) crossreacts immunologically with cellobiohydrolases I forms A, B and C, but not with cellobiohydrolase II, justifying the designation of CBH I and II as isozymes rather than forms of the same enzyme. An enriched β-glucosidase preparation from the culture filtrate of Trichoderma reesei QM 9414 formed a single precipitin line when allowed to react with antiserum to the β-glucosidase previously purified from a commercial T. viride cellulase preparation. The absence of any spurs of nonidentity indicates that these two enzymes are structurally very similar. Lack of crossreactivity is observed between cellobiohydrolase I (D) and the endoglucanase purified during this investigation, but the latter crossreacts weakly with antiserum to cellobiohydrolase II. / Ph. D.