Generation of alcohol for biofuels from fermentation of sugar or starch has several economic disadvantages such as high cost of sugar processing and land usage competing with staple food. The solution may reside in hydrolysis of cellulose from crop waste such as stalks of rice and corn or non-crop plants such as weeds or wood. Our goal is to identify cellulases that can degrade cellulosic biomass more efficiently. Studies of microbial Family 9 glycoside hydrolase (GH9) proteins, including both endo-glucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), have shown that they function through an inverting mechanism to cleave the 1, 4-£]-glucosidic bond between two unsubstituted Glc units. The main function of plant glycoside hydrolases are involved in polysaccharide metabolism of cell wall during cell growth. Twelve Arabidopsis thaliana (Columbia) endo-1,4-£]-glucanases that belong to the GH9, were cloned and expressed in Pichia pastoris in order to produce cellulases to facilitate efficient bio-alcohol production. The recombinant proteins do not show in vitro endo-1, 4-£]-glucanase activity, but we can detect the recombinant proteins expression in supernatant or in pellet. The lack of enzymatic activity from recombinant proteins is probably due to improper folding or glycosylation, or fast degradation resulted from the above reasons. Other bioreactor will be tested in the future. Genetic engineering to modify Arabidopsis thaliana (Columbia) endo-£]-1, 4-glucanases is another approach to produce functional cellulases with economic efficiency that can be adapted to industrial scale for alcohol generation. On the other hand, we use semi-quantitative PCR method to study the Arabidopsis GH9 genes expression level in different tissue. At4g39000 and At3g43860 were found only in flowers and inflorescence, and At1g65610 expression in roots and shoots of the amount of more. Other genes in different tissues, was no found significant difference.

Academic dissertation : Chemistry : Helsinki : 1998. / Textes et résumés en anglais. ISBN de la version électronique 951-38-5226-1. Pagination multiple pour les articles reproduits en annexe. ISSN de la version électronique : 1455-0849. Bibliogr. p. 56-72. Bibliogr. à la suite des articles.

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In this project, Cytophaga hutchinsonii, an aerobic gliding bacterium with cellulose-degrading ability, was studied, since its cellulase system was unknown and might be very different from those of other cellulose-degrading species. Only ß-1,4- endoglucanases and non-specific ß-glucosidases were found in the C. hutchinsonii genome sequence, whereas specific exoglucanases were apparently absent. Almost all putative cellulases were composed of catalytic domains only, without carbohydrate-binding modules. Samples from C. hutchinsonii cultures were analyzed by using TLC and colorimetric assays. Glucose was detected in the cellobiose grown culture, but not in cellulose-grown cultures, suggesting that cellobiose is hydrolyzed extracellularly rather than being directly assimilated, and that cellulose may not be degraded via cellobiose. Also, cellobiose-based cultures caused greater acidification of the medium than glucose or cellulose grown cultures. Nine putative cellulases were expressed in four bacterial strains. In some cases, expression was toxic to host cells. The crude lysates were tested for endoglucanase, specific exoglucanase or nonspecific ß-glucosidase activity. CHU_1280 and CHU_1842 showed apparent endoglucanase activity when expressed in Citrobacter freundii. Four putative GH family 3 ß-glucosidases with similar conserved domains were expressed in Escherichia coli JM109 and E. coli BL21(DE3)pLysS. One of these, CHU_2268, was found to possess MUC-degrading ability. This suggests that CHU_2268 may be the 'missing' exoglucanase in C. hutchinsonii. Another two ß-glucosidases, CHU_2273 and CHU_3784, possessed only MUG-degrading activity.

The enzymatic deconstruction of lignocellulosic plant biomass is performed by specialist microbial species. It is a ubiquitous process within nature and central to the global recycling of carbon and energy. Lignocellulose is a complex heteropolymer, highly recalcitrant and resistant to hydrolysis due to the major polysaccharide cellulose existing as a crystalline lattice, intimately associated with a disordered sheath of hemicellulosic polysaccharides and lignin. In this thesis I aim to transfer the highly efficient cellulolytic mechanism of the bacterium Cellulomonas fimi, to that of a suitably amenable and genetically tractable expression host, in the hopes of better understanding the enzymatic hydrolysis of lignocellulose. Using tools and concepts from molecular biology and synthetic biology, I constructed a library of standardised genetic parts derived from C. fimi, each encoding a known enzymatic activity involved in the hydrolysis of cellulose, mannan or xylan; three of the major polysaccharides present in lignocellulose. Characterization assays were performed on individual parts to confirm enzymatic activity and compare efficiencies against a range of substrates. Results then informed the rational design and construction of parts into modular devices. The resultant genetic devices were introduced into the expression hosts Escherichia coli and Citrobacter freundii, and transformed strains were assayed for the ability to utilize various forms of xylan, mannan and cellulose as a sole carbon source. Results identified devices which when expressed by either host showed growth on the respective carbon sources. Notably, devices with improved activity against amorphous cellulose, crystalline cellulose, mannan and xylan were determined. Recombinant cellulase expressing strains of E. coli and C. freundii were shown capable of both deconstruction and utilization of pure cellulose paper as a sole carbon source. Moreover, this capacity was shown to be entirely unhindered when C. freundii strains were cultured in saline media. These findings show promise in developing C. freundii for bioprocessing of biomass in sea water, so as to reduce the use of fresh water resources and improve sustainability as well as process economics. Work presented in this thesis contributes towards understanding the complementarities and synergies of the enzymes responsible for lignocellulose hydrolysis. Moreover, the research emphasizes the merits of standardizing genetic parts used within metabolic engineering projects and how adopting such design principles can expedite the research process.

Modified cellulase enzyme assay methods were developed to determine the potential of using enzyme activities to evaluate the decomposition of organic matter in carbon amended mine tailings systems. Cellulase assays are commonly performed in soil science applications, industrial production and bio-energy research to determine organic matter response to physical, chemical or temporal variation but, they have not been applied in a mine waste environment. Heavy metal content is considered to be a potential inhibitor to cellulase enzyme activity. Using samples collected from Greens Creek Mine in Juneau, Alaska, USA, the modified assay was evaluated to develop a method that provided reproducible results. It was determined that a sample mass of three grams is sufficient to provide consistent enzyme measurements. Each sample location was characterized by four replicates to ensure statistically representative data. Matrix interferences were determined to be inconsequential in this system. Despite the low organic content amended to the tailings, heavy metal content and potentially low enzyme activity, the modified enzyme assay method provided reproducible enzyme measurements. Following the development of the cellulase assays, spatial and temporal variations in cellulase enzymes were investigated from carbon amended tailings samples collected at the Greens Creek Mine. Six test cells, containing a mixture of tailings and a combination of peat, spent brewery grain and/or municipal biosolids were sampled in the fall of 2005, 2007 and 2009. Exo-(1,4)-β-D-glucanase (EC 3.2.1.91), Endo-(1,4)-β-D-glucanase (EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21) enzymes were assayed from core sections at five different depths. Enzyme activities were compared to sulfate reducing and acid producing bacterial enumerations, sulfide trends and carbon content. General trends were consistent between enzyme activity and SRB enumerations. The range of total carbon values fell between 3 and 5 wt % in each test cell while the average inorganic carbon content was 3.5 wt %. The range of organic carbon content was between 0.2 and 1.2 wt %. Total, inorganic and organic carbon values were more characteristic of test cell carbon distribution. Cellulase enzyme assays provide valuable information regarding the degradation of cellulose and hemi-cellulose. This study demonstrates that enzymes can be monitored in a tailings environment and that enzyme assays conducted for monitoring purposes may be a useful practice to indicate the sustained or declining performance of organic matter in a carbon amended remedial system.

Cellulose is the most abundant organic compound on the planet, and is found in nearly every ecosystem. Cellulose is also the most abundant waste product produced by human activity. These enormous stores of natural cellulose and cellulose-containing wastes are a potential renewable energy source. The hydrolysis of cellulose is referred to as cellulolysis and is carried out by cellulase enzymes, which are members of certain glycoside hydrolase families. For most of its history, the microbiology of organisms like those that hydrolyze cellulose was based solely on the testing of physiological and biochemical behaviour of isolated organisms in pure cultures. Despite having gained an important foundation of knowledge in the characterization of microorganisms, cultivation-based techniques introduce major bias into understanding the role that specific microorganisms play because the majority of microorganisms are not readily cultured. Two of the most powerful culture-independent approaches for accessing microbial communities are DNA stable-isotope probing (DNA-SIP) and metagenomics. Though each methodology has been used on its own, it is a combination of these two approaches that has enormous potential to generate results for industrial applications and to help characterize biogeochemical cycling. This thesis presents the first research combining DNA-SIP and metagenomics using cellulose, and the first to target glycoside hydrolase genes from Arctic tundra. For this research, two-month DNA-SIP incubations were carried out with 200 mg of 13C-labelled cellulose as a substrate. Denaturing gradient gel electrophoresis (DGGE) provided evidence indicating the successful enrichment of microorganisms able to metabolize cellulose. Multiple displacement amplification (MDA) was applied to both the bulk-soil samples and DNA-SIP samples. Following MDA, all DNA samples were subjected to Illumina sequencing, including DNA from a cellulose-degrading enrichment. Functional annotation for each Illumina library was done using the SwissProt database within MG-RAST. The DNA-SIP enrichment resulted in a ~3 fold increase in the relative abundance of glycoside hydrolases and cellulase enzymes in relation to bulk soil samples. A cellulose degrading enrichment contained the highest relative abundance of glycoside hydrolases and cellulase enzymes, with a five fold increase relative to the DNA-SIP enrichment. The enrichment culture had a much lower relative diversity, which was measured using the Shannon Index. An unrooted neighbor-joining tree constructed using Bray-Curtis similarity coefficients for each sample demonstrated that as a result of a considerably higher proportion of cellulase gene sequences and a lower diversity the enrichment culture was the most distinct library, with the DNA-SIP library most closely related to it. DGGE provided initial evidence that MDA introduced bias into the amplification of DNA from the DNA-SIP sample. This was confirmed following sequencing and annotation as the proportion of glycoside hydrolase enzymes sequences decreased 67% following MDA of DNA-SIP enriched DNA and the mean G+ C content of libraries decreased. This research provides evidence indicating that DNA-SIP enrichment prior to the construction of metagenomic libraries increases the abundance of targeted gene sequences, which should enable greater access to functional genes of active microorganisms for potential industrial applications.

Graduation date: 1992

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 7 uppsatser.