Thesis (M.S. in chemical engineering)--Washington State University. / Includes bibliographical references.

The catalytic domain is separated from the cellulose-binding domain in Cellulomonas fimi endoglucanase CenA by a proline-threonine rich sequence called the Pro-Thr box. To study the function of the Pro-Thr box region, a deletion mutant, cenAAPT, was made from cenA by an oligonucleotide directed in vitro mutagenesis. The truncated enzyme, CenAAPT, was purified to homogeneity by affinity chromatography on cellulose and characterized. Comparing CenAAPT to CenA, the following characteristics were observed: 1) the Pro-Thr box affected the migration of CenA on SDS-PAGE; 2) the deletion of the Pro-Thr box altered the high affinity interaction with cellulose; 3) the truncated enzyme showed 40-50% reduction in catalytic activity towards both microcrystalline and amorphous cellulose; 4) the truncated enzyme was as sensitive as CenA to a C.fimi protease, and both enzymes were cleaved at the same site adjacent to the binding domain. The Pro-Thr box is not essential for the catalytic activity of CenA or its binding to cellulose, but it does contribute to both functions. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Lignocellulose is one of the most abundant carbon sources in nature. This naturally-occuring substance is an underutilized source of bioenergy. A major bottleneck in biofuel processing is the enzymatic hydrolysis of lignocellulose into its ultimate fermentable product, glucose. Cellulomonas fimi is a well-studied soil organism known for its capabilities to efficiently hydrolyze cellulose. Recently sequenced genomes of Cellulomonas fimi and Cellulomonas flavigena have allowed analysis to reveal previously unidentified cellulases from several glycoside hydrolase (GH) families. This study also includes the expression of secreted cellulases from families GH 5, 6, and 9 at the protein level by the native organism after growth in media supplemented with carboxymethylcellulose or soluble xylan. In order to find enzymes with novel qualities, the cloning and expression of these newly identified cellulases from C. fimi and C. flavigena were done. One of these enzymes is Celf_1230 (Cel6C), a putative cellobiohydrolase from the glycoside hydrolase family 6. Using substituted cellulose derivatives as substrates, we have characterized Celf_1230 to be a thermostable enzyme with endoglucanase activity.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 102-112). Also available in electronic version. Access restricted to campus users.

Lignocellulose is one of the most abundant carbon sources in nature. This naturally-occuring substance is an underutilized source of bioenergy. A major bottleneck in biofuel processing is the enzymatic hydrolysis of lignocellulose into its ultimate fermentable product, glucose. Cellulomonas fimi is a well-studied soil organism known for its capabilities to efficiently hydrolyze cellulose. Recently sequenced genomes of Cellulomonas fimi and Cellulomonas flavigena have allowed analysis to reveal previously unidentified cellulases from several glycoside hydrolase (GH) families. This study also includes the expression of secreted cellulases from families GH 5, 6, and 9 at the protein level by the native organism after growth in media supplemented with carboxymethylcellulose or soluble xylan. In order to find enzymes with novel qualities, the cloning and expression of these newly identified cellulases from C. fimi and C. flavigena were done. One of these enzymes is Celf_1230 (Cel6C), a putative cellobiohydrolase from the glycoside hydrolase family 6. Using substituted cellulose derivatives as substrates, we have characterized Celf_1230 to be a thermostable enzyme with endoglucanase activity.

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

Recombinant DNA techniques were used to clone and isolate Cellulo- monas fimi cellulase genes. A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant plasmids carrying cellulase genes. The screening procedure is based on binding cellulases and other proteins released from lysed clones to CNBr-acti- vated paper. The paper is treated with anti-cellulase antibody and the antigen-antibody complex is detected by autoradiography using ¹²⁵I-labeled protein A from Staphylococcus aureus. This- immunoassay, was used to identify recombinant plasmids containing strains, carrying at least two different cellulase genes. The enzymes present in extracts of E. coli cellulase clones were active in catalysing the hydrolysis of carboxymethy1 eel 1ulose as indicated by the production of reducing sugars. Osmotic shock treatment of one E. coli cellulase clone revealed that the majority of the cellulase enzyme synthesized by this clone was transported to the periplasmic space. Cellulase encoding plasmids were characterized by the presence of either a 6.6 or a 5.0 kilobase C. fimi DNA gene fragment. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In addition to substrate-associated cellulases, Cellulomonas fimi secretes endoglucanases ( endo-1, 4-β-D-glucan glucanohydrolases, EC 3.2.1.4. ) which are recovered from the cellulose-free culture supernatant of cells grown on microcrystalline cellulose. Two such enzymes, C3.1 and C3.2 with Mrs of 130'000 and 120'000, respectively, were purified to homogeneity. The two endoglucanases were shown to share the same N-terminal amino acid sequence and to hydrolyze carboxymethylcellulose ( CMC ) with similar efficiencies ( 236u/mg protein for C3.1 and 367u/mg protein for C3.2 ). The recombinant lambda vector L47.1-169 was identified from a C.fimi DNA-lambda library on the basis of hybridization with C3.1/2-specific oligonucleotide probes. The subclone pTZ18R-8 only moderately expressed CMCase activity. The 5'-terminus of cenC ( the gene coding for C3.1/2 ) was localized in the insert by Southern transfer experiments and nucleotide sequence analysis. Results from total C.fimi RNA-DNA hybrid protection analyses defined the boundaries of cenC in pTZ18R-8 and led to the tentative identification of -10 and -35 promoter sequences. To improve the expression of cenC, its entire coding sequence, except for the start codon GTG, was fused in frame to the ATG codon of a synthetic ribosomal binding site ( PTIS ) and placed under the transcriptional control of the lac p/o system. Induction of the resulting clone ( JM101[pTZP-cenC] ) led to impaired growth in liquid cultures because overproduction of CenC inhibited cell division'" and eventually led to cell death. Analysis of cell fractions by SDS-PAGE revealed a dominant ( >10% of total cell extract proteins ), clone-specific protein with a Mr of approximately 140'000 which was found exclusively in the cytoplasmic fraction. Conversely, 60% of the total CMC-hydrolyzing activity was localized in the periplasmic fraction indicating that the export of CenC is required for maximal expression of endoglucanase activity. Isolation of the cellulolytic activities from an osmotic shockate led to the purification to homogeneity of two recombinant cellulases, CenC1 and CenC2, with Mr of 130'000 and 120'000, respectively. Both enzymes hydrolyzed CMC with similar efficiencies ( 278u/mg protein for CenC1 and 390u/mg protein for CenC2 ). In addition, amino acid sequence analyses showed the two enzymes to have the same N-termini as the native enzymes and proved furthermore that the CenC leader peptide was functional in Escherichia coli. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

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.