Texte remanié de: Th.--Sci. nat.--Université d'Aix-Marseille I, 1985. / Bibliogr. p. 171-186. Résumé en espagnol, anglais et français.

Thesis (M.S.)--University of Wisconsin--Madison, 1969. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (doctoral) -- Helsinki University of Technology, 1997. / Includes bibliographical references. Also availble on the World Wide Web.

Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.

Cellulomonas fimi secretes several cellulase activities as well as protease activity into the culture medium. In contrast, few activities are bound to the cellulose in the culture. To characterize the cellulase system and to Identify cloned gene products, it was necessary to purify native, intact cellulases. We hypothesized that the cellulose-bound cellulases would be protected from proteolysis, and therefore represent the intact enzymes. Two cellulases were purified from Cellulomonas fimi. Avicel was recovered from cultures and the proteins were eluted from it with guanidine-HCl (Gdn-HCl). The Gdn-HCl extract was fractionated by Concanavalin A-Sepharose affinity column chromatography and by Mono Q anion exchange column chromatography. The cellulases purified by this procedure were an endoglucanase, EngA, and an exoglucanase, Exg. The purified enzymes were characterized. EngA has Mr 57,000, pi 8.2, and is 10 % mannose by weight. Exg has Mv 56,000, pi 5.8, and is 8 % mannose by weight. Two recombinant DNA plasmids were identified as encoding EngA and Exg. The recombinant gene products were not glycosylated. The role of glycosylation was studied by comparing some properties of the recombinant EngA and Exg with the native EngA and Exg. Both glycosylated and unglycosylated forms bound to Avicel. Sensitivity to the C. fimi protease was also compared. The glycosylated enzymes were protected from proteolysis when bound to cellulose. In contrast, the unglycosylated forms were processed to yield active, truncated products with greatly reduced affinity for cellulose. The cleavage site was predicted based on size of the products and reactivity with anti-PT serum. The N-terminal region of EngA and the C-terminal of Exg show 50 % conservation of sequence (Warren et al., 1986). This region appears to be the cellulose-binding domain and is not required for the hydrolysis of soluble substrates. The C. fimi protease can partially degrade glycosylated EngA when it is not bound to cellulose. Some of the multiple CMCase activities in culture supernatants are derived from EngA by partial proteolysis. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / In nature, saccharification is done by a variety of microorganisms, secreting a variety of cellulase in addition to other proteins. Co-culturing enables the production of more efficient enzyme preparations that would mimic the natural decomposition of lignocelluloses. During the decay of banana (Musa paradisiaca) pseudostem, a potential feedstock for second-generation biofuels, there may be a number of microorganisms producing cellulolytic enzymes, and other factors, which in combination might decompose the lignocelluloses more efficiently. The aim of the study was to establish a microbial co-culture for the production of highly active cellulase preparations. Banana pseudostems (BPS) and microbial samples from decaying banana pseudostems were collected in the Mopani District Allesbeste Nursery, Limpopo Province, South Africa. Fungi and bacteria were isolated using CMC agar plates. The best cellulase producing fungi and bacteria were tested for cellulase activity in monocultures and in various combinations (fungi-fungi, fungi-bacteria, bacteria-bacteria, fungi-live bacterial cells and fungi-dead bacterial cells) in submerged fermentation, using Avicel™ as a carbon source. Solid-state fermentation was also performed using banana pseudostem as a carbon source. Zymography was done in studying the variety of cellulase in the secretions from co-cultures/ mixed cultures. Identification of the bacterial and fungal isolates from decomposing banana pseudostems was also done using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) or DNA sequencing. A mixed culture of fungi in combination with dead bacterial cells was the best combination to produce higher levels of endoglucosidase and β-glucosidase activities in both submerged fermentation and solid-state fermentation. During SmF, endoglucosidase was (0.229 after 144 h) and β-glucosidase (4.519 after 96 h) activities and SSF, endoglucosidase (12.793 after 48 h) and β-glucosidase (37.45 after 144 h). Endoglucosidase zymography showed that monocultures and co-cultures produced four active bands for endoglucanase, except for the monoculture Trichoderma longibrachiatum 1B that produced a faint or unclear band. The current study demonstrated that three fungal strains namely, T longibrachiatum 1B, Aspergillus fumigatus 5A, and Aspergillus flavus 2A and one bacterial strain Enterobacter asburiae 1 are capable of producing a variety of endoglucanases. It seems that a combination of fungi with dead cells could significantly improve endoglucosidase and v β-glucosidase activities. The use of A. fumigatus in mixed cultures is highly recommended in order to produce high levels of β-glucosidases, no matter the combination used. / Foodbev Seta

The production of second generation biofuels -- cellulosic ethanol from renewable lignocellulosic biomass has the potential to lead the bioindustrial revolution necessary to the transition from a fossil fuel-based economy to a sustainable carbohydrate economy. Effective release of fermentable sugars through biomass pretreatment followed by enzymatic hydrolysis is among the most costly steps for emerging cellulosic ethanol biorefineries. In this project, two pretreatment methods (dilute acid, DA, and cellulose solvent- and organic solvent-lignocellulose fractionation, COSLIF) for corn stover were compared. It was found that glucan digestibility of the corn stover pretreated by COSLIF was much higher, along with faster hydrolysis rate, than that by DA- pretreated. This difference was more significant at a low enzyme loading. Quantitative measurements of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were established to find out the cause. The COSLIF-pretreated corn stover had a CAC nearly twice that of the DA-pretreated biomass. Further supported by qualitative scanning electron microscopy images, these results suggested that COSLIF treatment disrupted microfibrillar structures within biomass while DA treatment mainly removed hemicelluloses, resulting in a much less substrate accessibility of the latter than of the former. It also concluded that enhancing substrate accessibility was the key to an efficient bioconversion of lignocellulose. A simple method for determining the adsorbed cellulase on cellulosic materials or pretreated lignocellulose was established for better understanding of cellulase adsorption and desorption. This method involved hydrolysis of adsorbed cellulase in the presence of 10 M of NaOH at 121oC for 20 min, followed by the ninhydrin assay for the amino acids released from the hydrolyzed cellulase. The major lignocellulosic components (i.e. cellulose, hemicellulose, and lignin) did not interfere with the ninhydrin assay. A number of cellulase desorption methods were investigated, including pH adjustment, detergents, high salt solution, and polyhydric alcohols. The pH adjustment to 13.0 and the elution by 72% ethylene glycol at a neutral pH were among the most efficient approaches for desorbing the adsorbed cellulase. For the recycling of active cellulase, a modest pH adjustment to 10.0 may be a low-cost method to desorb active cellulase. More than 90% of cellulase for hydrolysis of the pretreated corn stover could be recycled by washing at pH 10.0. This study provided an in-depth understanding of biomass saccharification for the production of cellulosic ethanol for cellulose hydrolysis and cellulase adsorption and desorption. It will be of great importance for developing better lignocellulose pretreatment technologies and improving cellulose hydrolysis by engineered cellulases. / Master of Science