The primary purpose of this study was to (1) develop methods for the analysis of and (2) provide information on the chemical nature of reducing ends in typical cellulose substrates used for the study of cellulolytic enzymes. The studies were designed such that values obtained for cellulose substrates were compared with those obtained for a series of soluble cellooligosaccharides. The initial phase of the study tested the validity of using established colorimetric reducing sugar assays, developed for the measurement of reducing sugars in solution, for the quantification of reducing ends on insoluble substrates. The results demonstrate that published methods give widely differing values for the number of reducing ends per unit weight cellulose. The Cu⁺⁺-based assay, using bicinchoninic acid (BCA) as a color yielding chelator of Cu⁺, is shown to provide values that appear most consistent the properties of the substrates. A method was developed using the Cu⁺⁺-BCA reagent, following a mild sodium borohydride treatment, to provide an estimate of the number of solvent accessible reducing ends on insoluble substrates. The kinetics of sodium borohydride reduction of reducing ends on crystalline cellulose, amorphous cellulose and soluble cellooligosaccharides were compared in order to ascertain the relative reactivity of these reducing ends. The apparent second order rate constants for the reduction of reducing ends associated with the crystalline celluloses were significantly lower than those for the reduction of reducing ends associated with either the insoluble amorphous celluloses or the soluble cellooligosaccharides. These results indicate the reducing ends associated with crystalline celluloses are not extended out from the surface as though mimicking solution phase reducing ends. The relevance of this, as well as the other results, to the behavior of cellulolytic enzymes is discussed. The final phase of the study was the demonstration of both a reducing sugar-based and a viscositybased assay for the detection of a prototypical polysaccharide depolymerizing glycosyl hydrolase, polygalacturonase. / Graduation date: 2004

The influence of substrate and enzyme concentration on the rate of saccharification of two defined, insoluble, cellulose substrates, Avicel and Solka-Floc, by the cellulase enzyme system of Trichoderma viride has been evaluated. Assays utilized enzyme concentrations ranging from 0.014 to 0.056 filter paper unit per mL and substrate concentrations up to 10% (w/v). Analysis by initial velocity methods found the maximum velocity of the enzyme to be nearly equivalent for the two substrates and the km for the two substrates to be of similar magnitude, i.e., 0.20% for Solka-Floc and 0.63% for Avicel (w/v). Studies utilizing relatively high substrate concentrations (greater than 15 times the Km) demonstrated that the enzyme exhibits very different apparent substrate inhibition properties for the two substrates. The rate of saccharification of Avicel at relatively high substrate concentrations was up to 35% lower than the maximum rate which was obtained at a lower substrate concentration. The Avicel concentration corresponding to the maximum rate of saccharification was dependent on enzyme concentration. In contrast to the results with Avicel, the enzyme did not exhibit substrate inhibition with the Solka-Floc substrate. Potential differences in the degree of substrate inhibition with different substrates, as reported in this paper, is particularly relevant to the experimental design of comparative studies. / Graduation date: 1990

Bibliography: pages 215-244. / Clostridium acetobutylicum P262 is an endospore forming Gram-positive obligate anaerobe which has been used for the industrial production of acetone and butanol. Strains of C. acetobutylicum have been reported to exhibit some activity towards cellulosic and hemicellulosic substrates. The aim of this thesis was to establish a genebank of C. acetobutylicum P262 DNA in Escherichia coli and to isolate and characterise genes encoding enzymes which show activity towards hemicellulose and cellulose.

The purpose of the study was to set up an in vitro model of the colon which would permit the analysis of cellulose fermentation by human colonic microflora. Studies of the degradation of polysaccharides by colonic bacteria may help to explain the observed physiological consequences of consuming dietary fiber common in foods. This study resulted in the use of a simple anaerobic batch fermentation system. It is assumed that the bacteria in fresh feces are representative of colonic bacteria. This batch culture system consists of the culture medium, the food fiber and the fecal inoculum. The fecal inoculum is prepared from freshly voided feces from a single individual. The food fiber is prepared from the vegetable/fruit starting material by repeated extraction with 90% ethanol, resulting in an alcohol insoluble residue(AIR). Extents of cellulose fermentation were measured after 4, 8, 12 and 24 hour fermentation periods at 37°C. The cellulose content of the samples before and after fermentation was determined by measuring the glucose yield (glucose oxidase assay) from an acid hydrolysate of the residue remaining after repeated acid detergent extractions. The extent of cellulose fermentation was then estimated by difference. The susceptibility to intestinal fermentation of the cellulose component of acorn squash and red beets was investigated using this model system. The cellulose content of squash and beet AIR was 26.71% ± 0.95% and 23.22% ± 0.89%, respectively. The extent of cellulose of fermentation of squash cellulose after 4, 8, 12 and 24 hrs incubation was 6.04% ± 0.69%, 10.58% ± 2.10%, 17.11% ± 6.37% and 96.18% ± 1.36%, respectively. The extent of fermentation of beet cellulose after 4, 8, 12 and 24 hrs incubation was 17.52% ± 1.83%, 23.52% ± 1.44%, 30.53% ± 4.12% and 96.06% ± 0.39%, respectively. The results indicate that the cellulose component of both vegetables is susceptible to considerable degradation within the human intestinal tract. / Graduation date: 1991

Graduation date: 1992

Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.

M.S.

Sandra Jane Chapman. / "October 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 163-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Orientador: Silvana Artioli Schellini / Banca: Erika Hoyama / Banca: Roberta Lilian Fernandes de Souza Meneghim / Banca: Patricia Mitiko Santello Araishi / Banca: Ana Estela Besteti Pires Ponce Sant'Anna / Resumo: A Nanoskin® é uma película de celulose bacteriana produzida pela bactéria Acetobacter xylinum,por meio de um processo biotecnológico. É composta por uma rede de nanofibrilas cuja estrutura cria uma extensa superfície, a qual permite a retenção de grande quantidade de água e importantes modificações em seu formato, sem perder suas características estruturais. Objetivo: visando acrescentar novas possibilidades para a reconstrução da pálpebra, em seus folhetos profundo ou superficial, o presente estudo foi desenvolvido com o objetivo de avaliar se a película de Nanoskin® poderia ser uma opção. Método: foram utilizadas 40 cobaias, do sexo masculino, que receberam fragmentos de Nanoskin® na região dorsal, em dois estudos, um direcionado para a utilização da Nanoskin nos tecidos profundos, quando o biomaterial foi colocado no subcutâneo e outro no qual a Nanoskin foi colocada na superfície da pele. Em ambos os estudos foram utilizados dois tipos de Nanoskin: grupo 1 (G1), no qual foi utilizada película de Nanoskin® (2X2 cm) sem recobrimento de gelatina no subcutâneo ou na superfície da pele e o grupo 2 (G2), que recebeu implante de Nanoskin® (2X2 cm) com revestimento de gelatina no subcutâneo e na superfície da pele. O grupo controle foi obtido com colocação de enxerto de pele de espessura total no tamanho de 2X2 cm, contíguo ao implante de Nanoskin®, em todos os animais de G1 e de G2 do experimento que estudou o biomaterial na superfície da pele. Cinco animais de cada grupo foram eutanasiados em quatro momentos experimentais: 7 dias (M1), 30 dias (M2), 90 dias (M3) e 180 dias (M4). Foram realizadas avaliações morfométricas do implante das lâminas histológicas, exame histológico e exame ultraestrutural. Resultados: a Nanoskin® quando implantada no subcutâneo não foi encontrada em um animal de M3 e em cinco animais de M4. Nos momentos M3, M4 e M5, houve separação entre as suas lamelas. Houve... / Abstract: The Nanoskin® is a bacterial cellulose film produced by the bacteria Acetobacter xylinum by means of a biotechnological process. It is composed by a network of nanofibrils whose structure creates a large surface area, which allows the retention of a large amount of water and significant changes in its shape without losing its structural characteristics. Purpose: aiming to add new possibilities for the reconstruction of the eyelid, in its superficial or deep lamellae, this study was developed in order to assess whether the Nanoskin® film could be an option. Method: 40 male guinea pig were used, that received Nanoskin® fragments in the dorsal region, in two studies, one directed to the use of Nanoskin® in deep tissue when the biomaterial was placed subcutaneously and another in which the Nanoskin® was placed on skin surface. In both studies we used two types of Nanoskin: group 1 (G1), which was used Nanoskin® film (2x2 cm) without gelatin coating on the subcutaneous or on the skin surface, and Group 2 (G2), which received Nanoskin® implant (2X2 cm) with gelatin coating on the subcutaneous and on the skin surface. The control group was obtained with full-thickness skin graft placement on the size of 2X2 cm Nanoskin® adjacent to the implant in all animals in G1 and G2 experiment that studied the biomaterial on the surface of the skin. Five animals from each group were euthanized at four experimental times: 7 days (M1), 30 days (M2), 90 days (M3) and 180 days (M4). Morphometric assessments of the implant and the histological slides were held, histological and ultrastructural examination. Results: the Nanoskin® when implanted subcutaneously was not found in one animal from M3 and in five animals from M4. In moments M3, M4 and M5, there was separation between their lamellae . There was a significant inflammation at the beginning of the experiment which reduced the following times, and formation of a pseudocapsule around the ... / Doutor

Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2013 / The production of cellulase is a key factor in the hydrolysis of cellulosic materials and it is essential to make the process economically viable. Cellulases are the most studied multi- enzyme complex and comprise of endo-glucanases (EG), cellobiohydrolases (CBH) and β- glucosidases (BGL). The complete cellulase system; comprising CBH, EG and BGL components thus acts synergistically to convert crystalline cellulose to glucose. Cellulases are currently the third largest industrial enzyme worldwide. This is due to their wide applications in cotton processing, paper recycling, juice extraction, as detergent enzymes and additives in animal feed. In this study, production of cellulase by five fungal isolates (BTU 251-BTU 255) isolated from mushrooms, was investigated and optimised. Internal transcribed spacer regions (ITS1 and ITS4) were applied to identify the five fungal microorganisms. Isolates were identified as follows: BTU 251 as Aspegillus niger,BTU 253 as Penicillium polonicum, and BTU 255 as Penicillium polonicum. Cellulase was produced in shake flask cultures using Mandel’s mineral solution medium and Avicel as a carbon source. Cellulase activity was tested using 3, 5-Dinitrosalicylic acid assay and zymography, A. niger BTU 251 showed five activity bands ranging from 25- 61 kDa had an average nkat of 7000. Cultures from BTU 252 were the least active with an average nkat/ml of 200 and one activity band of 25 kDa. P. polonicum BTU 253 showed three activity bands ranging between 45 and 60 kDa and had an average nkat/ml of 2200. BTU 254 showed five activity bands ranging from 22- 116 kDa and had average nkat of 350. P. polonicum BTU 255 produced the highest cellulase activity of 8000 nkat/ml and with three activity bands estimated at 45-60 kDa on zymography. The optimal temperature for activity of the cellulases was between 55-70°C and enzymes were most active within a pH range of 4-6. Optimal pH for production of cellulases by P. polonicum BTU 255, P. polonicum BTU 253 and A. niger BTU 251 was 4 while optimal temperature for production of the cellulases was between 50-55°C. Total cellulase activity was determined using Whatman No.1 filter paper as a substrate and β- glucosidase production was determined in polyacrylamide gels using esculin as a substrate. In the hydrolysis of crystalline cellulose (Avicel), a combination of A. niger BTU 251 and P. polonicum BTU 255 (1:1), (1:9), (1:3), and (1:2) produced maximum glucose as follows: 1:1 (0.83g/L), 1:9 (10.4g/L), 1:3 (0.77g/L) and 1:2 (0.73g/L). Cellulases from P. polonicum BTU 255 were partially purified using affinity precipitation and analysed using MALDI- TOF/TOF. Peptide sequences of P. polonicum obtained from MALDI-TOF/TOF analysis were aligned by multiple sequence alignment with C. pingtungium. Conserved regions were identified using BLAST anaylsis as sequences of cellobiohydrolases. More research is required in producing a variety of cellulases that are capable of hydrolysing crystalline cellulose, the current study contributes to possible provision of locally developed combinations of cellulases that can be used in the production of bioethanol.