Thesis (Ph. D.)--Massachusetts Institute of Technology, 1993. / "February 1993." Includes bibliographical references (p. 215-218).

No description available.

Following the successful detection of novel exo-1,3-β-glucanase activity from marine bacterium Pseudoalteromonas sp. BB1 that was isolated from brown algae Durvillaea sp. and its partial gene identification, the exo-glucanase (ExoP) has been purified to homogeneity. Full gene identification of exoP was achieved by Southern hybridisation using a derived probe. In total, 7612 bp of the partial Pseudoalteromonas gDNA sequence was obtained, in which 6 coding regions including the full exoP sequence were identified. The exoP gene consists of 2523 nucleotides, which is translated into 840 amino acids. The first 27 amino acids are predicted to be a signal peptide in agreement with obtained N-terminal sequence of native ExoP. The molecular weight of the mature ExoP portion was calculated to be 89320.5 Da (813 amino acids), consistent with its mobility in SDS-PAGE (87 kDa). Interestingly a putative lichenase gene (licA) whose enzymatic function is related to ExoP was located only 50 bp upstream of exoP suggesting these two genes work co-ordinately. ExoP is classified as a glycosyl hydrolase GH3 family member and is homologous with a group of bacterial and plant enzymes of which barley ExoI is the best characterised. Following the complete gene identification, exoP was successfully cloned, over-expressed in E. coli and purified. Biochemical characterisation of ExoP revealed that the native and recombinant proteins were identical with optimal temperature 30�C and optimal pH 7.0 for hydrolase activity. ExoP showed substrate specificity towards both 1,3-β- and 1,4-β-glucans but did not hydrolyse aryl substrates unlike other glucosidases in the GH3 family. The ExoP was designated as an exo-1,3/1,4-β-glucanase (EC. 3.2.1-.). The crystal structure of ExoP was successfully solved at 2.45 Å resolution using a two step molecular replacement procedure. ExoP was found to consist of a distinctive three domain structure: an (α/β)₈ barrel domain A, an (α/β)₆ sheet domain B and a β-sandwich domain X. The catalytic pocket is formed by domains A and B and this two domain structure is highly similar to that of barley exo-1,3/1,4-β-glucanase ExoI. Three potential subsites were observed in the structure: the -1 subsite that is identical to that of barley ExoI, the +1 subsite that contains an antiparallel tryptophan clamp formed by W294 and W436, and a putative +2 subsite that involves W494. This observation agreed with the prediction by subsite mapping. The function of domain X remains unknown. However it was discovered that this domain is common in marine bacterial GH3 enzymes, and that marine bacteria also produce an independent protein that consists of the C-terminal half of this domain. The analysis of ExoP structure showed not only the conserved features of the -1 and +1 subsites of GH 3 family enzymes but new insights such as the hinge action between domains A and X, mobility of a flexible loop near the catalytic site and a possible role of domain X contributing to the enzyme fidelity. The second part of this project focused on glycosynthase activity generated by active site mutation. While ExoP showed no such activity the Glu to Ser mutant of exo-1,3-β-glucanase (Exg) from Candida albicans was functional. Albeit native Exg shows high specificity towards 1,3-β-glucans, using a donor 1-fluoro-α-D-glucose (1FG) and an acceptor p-nitrophenyl β-D-glucopyranoside (pNPG) the mutant E292S-Exg glycosynthase preferentially forms a 1,6-β-linked product. In this study, the crystal structure of E292S-Exg complexed with p-nitrophenyl β-gentiobioside (pNPgent), E292S-Exg/pNPgent (the product formed by the above reaction) was solved at 1.60 Å. Comparison of this structure with the previously solved complexed structure, E292S-Exg/1FG/pNPG did not explain why the 1,6-linkage was favoured by this enzyme but surprisingly revealed movement of glucose at the -1 subsite, which is organised by a complex hydrogen bond network, but did not show movement of glucose in the phenylalanine clamp (the +1 subsite). The presence of an aromatic clamp (Phe-Phe in Exg or Trp-Trp as seen in ExoP) in an exo-glucanase is seen to contribute to specificity but not explain it. Glycosynthesis using other acceptor oligosaccharides was also explored in this study. Exg glycosynthase showed broad specificity using p-nitrophenyl derivatised mono-saccharides. However, it remains unknown whether this broad specificity is acceptor dependent or intrinsically due to the mutation created in Exg.

Following the successful detection of novel exo-1,3-β-glucanase activity from marine bacterium Pseudoalteromonas sp. BB1 that was isolated from brown algae Durvillaea sp. and its partial gene identification, the exo-glucanase (ExoP) has been purified to homogeneity. Full gene identification of exoP was achieved by Southern hybridisation using a derived probe. In total, 7612 bp of the partial Pseudoalteromonas gDNA sequence was obtained, in which 6 coding regions including the full exoP sequence were identified. The exoP gene consists of 2523 nucleotides, which is translated into 840 amino acids. The first 27 amino acids are predicted to be a signal peptide in agreement with obtained N-terminal sequence of native ExoP. The molecular weight of the mature ExoP portion was calculated to be 89320.5 Da (813 amino acids), consistent with its mobility in SDS-PAGE (87 kDa). Interestingly a putative lichenase gene (licA) whose enzymatic function is related to ExoP was located only 50 bp upstream of exoP suggesting these two genes work co-ordinately. ExoP is classified as a glycosyl hydrolase GH3 family member and is homologous with a group of bacterial and plant enzymes of which barley ExoI is the best characterised. Following the complete gene identification, exoP was successfully cloned, over-expressed in E. coli and purified. Biochemical characterisation of ExoP revealed that the native and recombinant proteins were identical with optimal temperature 30�C and optimal pH 7.0 for hydrolase activity. ExoP showed substrate specificity towards both 1,3-β- and 1,4-β-glucans but did not hydrolyse aryl substrates unlike other glucosidases in the GH3 family. The ExoP was designated as an exo-1,3/1,4-β-glucanase (EC. 3.2.1-.). The crystal structure of ExoP was successfully solved at 2.45 Å resolution using a two step molecular replacement procedure. ExoP was found to consist of a distinctive three domain structure: an (α/β)₈ barrel domain A, an (α/β)₆ sheet domain B and a β-sandwich domain X. The catalytic pocket is formed by domains A and B and this two domain structure is highly similar to that of barley exo-1,3/1,4-β-glucanase ExoI. Three potential subsites were observed in the structure: the -1 subsite that is identical to that of barley ExoI, the +1 subsite that contains an antiparallel tryptophan clamp formed by W294 and W436, and a putative +2 subsite that involves W494. This observation agreed with the prediction by subsite mapping. The function of domain X remains unknown. However it was discovered that this domain is common in marine bacterial GH3 enzymes, and that marine bacteria also produce an independent protein that consists of the C-terminal half of this domain. The analysis of ExoP structure showed not only the conserved features of the -1 and +1 subsites of GH 3 family enzymes but new insights such as the hinge action between domains A and X, mobility of a flexible loop near the catalytic site and a possible role of domain X contributing to the enzyme fidelity. The second part of this project focused on glycosynthase activity generated by active site mutation. While ExoP showed no such activity the Glu to Ser mutant of exo-1,3-β-glucanase (Exg) from Candida albicans was functional. Albeit native Exg shows high specificity towards 1,3-β-glucans, using a donor 1-fluoro-α-D-glucose (1FG) and an acceptor p-nitrophenyl β-D-glucopyranoside (pNPG) the mutant E292S-Exg glycosynthase preferentially forms a 1,6-β-linked product. In this study, the crystal structure of E292S-Exg complexed with p-nitrophenyl β-gentiobioside (pNPgent), E292S-Exg/pNPgent (the product formed by the above reaction) was solved at 1.60 Å. Comparison of this structure with the previously solved complexed structure, E292S-Exg/1FG/pNPG did not explain why the 1,6-linkage was favoured by this enzyme but surprisingly revealed movement of glucose at the -1 subsite, which is organised by a complex hydrogen bond network, but did not show movement of glucose in the phenylalanine clamp (the +1 subsite). The presence of an aromatic clamp (Phe-Phe in Exg or Trp-Trp as seen in ExoP) in an exo-glucanase is seen to contribute to specificity but not explain it. Glycosynthesis using other acceptor oligosaccharides was also explored in this study. Exg glycosynthase showed broad specificity using p-nitrophenyl derivatised mono-saccharides. However, it remains unknown whether this broad specificity is acceptor dependent or intrinsically due to the mutation created in Exg.

Thesis (M.S.)--University of Wisconsin--Madison, 1971. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Adelaide, 1989. / Includes bibliographical references.

Thesis (M.S.)--Duquesne University, 2007. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 101-104) and index.