Mutations in the yeast gene KRE1 lead to resistance to the K1 killer toxin of S. cerevisiae. The resistant phenotype is associated with a 40% reduction of the cell wall (1 $ to$ 6)-$ beta$-glucan fraction. Yeast cell wall (1 $ to$ 6)-$ beta$-glucan is a highly branched glucose polymer composed predominantly of linear (1 $ to$ 6)-$ beta$- sc D-linked glucopyranosyl residues. This glucan acts as a receptor for the killer toxin, leading to a concentration of active toxin on the yeast cell surface. The KRE1 gene was cloned by complementation of the kre1-1 mutant allele. The KRE1 gene encodes a serine and threonine rich protein, that is directed into the yeast secretory pathway, where it is highly modified, probably through O-linked glycosylation. Haploid strains with a kre1::HIS3 disruption appear to grow somewhat more slowly than wild type, and show an unusual wall structure when examined using electron microscopy. As with strains that carry a mutant kre1-1 allele those with a kre1::HIS3 disruption show a 40% reduced level of cell wall (1 $ to$ 6)-$ beta$-glucan. Structural comparison of the (1 $ to$ 6)-$ beta$-glucan fraction isolated from a wild type strain and a kre1 mutant, showed that the glucan obtained from the mutant had fewer (1 $ to$ 6)-linked residues and displayed a smaller average polymer size. Therefore, the KRE1 gene product appears to be required for the stepwise synthesis of cell wall (1 $ to$ 6)-$ beta$-glucan.

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.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Includes bibliographical references. / The structures of some non-cellulosic β D-Glycans from three plant sources have been investigated and each was found to be characterised by linked D-pyranosyl a main chain consisting of β -(1-44)- sugars. The polysaccharides were, however, different in structural features in a manner apparently related to their respective locations within the organs of the plants concerned. The polysaccharides were isolated and purified using standard fractionation methods including chromatographic techniques and selective precipitation methods. Structural information was obtained by employing techniques such as methylation analysis (involving use of gas liquid chromatography mass spectrometry), optical rotation measurements, mass spectrometry and n.m.r. spectroscopy on the original polysaccharides and on degraded products obtained by methods such as acid- or enzyme-catalysed hydrolysis and Smith degradation.

No description available.

No description available.

Thesis (Ph. D.)--University of Nevada, Reno, 2005. / "May 2005." Includes bibliographical references. Online version available on the World Wide Web.

Glucoamylase, a saccharifying enzyme, is applied in the brewing industry to hydrolyse the dextrins of malted barley into simple sugars which can then be fermented by brewer's yeast. In order to establish the potential of glucoamylase from Lactobacillus amylovorus for application in the brewing industry, the main objectives of this study were: (1) to determine the cultural conditions for growth and glucoamylase production, (2) to purify the enzyme to homogeneity using chromatography and electrophoretic techniques, (3) to study biochemical properties of the purified enzyme, and (4) to clone the gene coding for glucoamylase, and characterize the recombinant glucoamylase. / The actively amylolytic Lactobacillus amylovorus ATCC 33621 produced an intracellular glucoamylase activity. Conditions for growth and glucoamylase production were maximized by using dextrose free MRS medium supplemented with 1% dextrin, at pH 5.5 and 37$ sp circ$C. Enzyme production was maximal during the late logarithmic phase of growth from 16-18 h. Crude cell extract showed optimal activity at pH 6.0 and 55$ sp circ$C. / Native and SDS-PAGE of the purified enzyme showed a monomeric protein of 47 kD. Glucoamylase activity was confirmed by activity staining using a starch/polyacrylamide gel where a zone of clearing was visible on a blue/black background stained with Kl/I$ sb2.$ Optimal pH, pl and temperature of purified glucoamylase were 4.5, 4.39 and 45$ sp circ$C, respectively. The enzyme was rapidly inactivated by temperatures above 55$ sp circ$C and was inhibited by heavy metals, e.g. Pb$ sp{2+}$ and Cu$ sp{2+}$ at 1.0 mM. EDTA did not inhibit the enzyme activity at a final concentration of 10 mM. Enzyme inhibition by 1 mM of p-chloromercuribenzoic acid (pCMB) and iodoacetate suggested that a sulfhydryl group was present in the enzyme active site. Kinetic studies of glucoamylase confirmed that the enzyme reacted preferentially with polysaccharides. HPLC analyses of the end products of enzyme action showed that glucose was the major end product of enzyme action and this glucose was responsible for end product inhibition. / The gene coding for glucoamylase was cloned into Escherichia coli using the STA2 glucoamylase gene of Saccharomyces diastaticus as a probe. Three glucoamylase producing transformants were identified as the insert sizes of about 5.2 Kb, 6.4 Kb and 5.9 Kb, respectively. When the characteristics of both recombinant and purified wild type glucoamylases were compared, both enzymes showed a similar pH range of 3.0-8.0, and temperature optimum of 45$ sp circ$C. The recombinant enzyme pH profiles were broader than that of the wild type and an optimum pH of 6.0 was obtained. This study has shown that glucoamylase from Lb. amylovorus is less heat stable than other bacterial glucoamylases and thus may be suitable for application in the brewing industry. Successful cloning of this gene coding for glucoamylase in brewer's yeast, Saccharomyces cerevisiae, would reap the advantageous properties of the enzyme while eliminating the costs of adding commercial enzymes.

CAPES / O mercado mundial de polissacarídeos tem atraído grandes companhias industriais interessadas em conquistar novos e rentáveis campos de atuação. Polissacarídeos com propriedades tecnológicas e biológicas podem ser obtidos a partir de plantas, algas e de microrganismos. Dentre os polissacarídeos com propriedades biológicas, as glucanas tem se destacado por apresentarem atividade imunoestimulante e potencialidades no tratamento de doenças como câncer, hipercolesterolemia, diabetes, esclerose múltipla e doenças cardiovasculares. Recentes estudos demonstram a produção extracelular de β-glucanas por fungos filamentosos em cultivos submersos. A modificação na estrutura química das glucanas por carboximetilação é considerada uma importante rota para melhorar suas propriedades, podendo contribuir para o aumento da solubilidade da molécula, bem como atividades biológicas, especialmente aquelas associadas a mecanismos de ação antioxidante e antiproliferativa. Nesse sentido, o presente trabalho teve como objetivo a produção de β-1,6-D-glucana (lasiodiplodana) pelo fungo Lasiodiplodia theobromae MMPI em cultivo submerso, bem como a carboximetilação da molécula, caracterização e avaliação da citotoxicidade e atividade antioxidante. A carboximetilação da molécula foi confirmada através da verificação de sinais químicos específicos identificados por espectroscopia de FT-IR e RMN 13C e a molécula carboximetilada apresentou grau de substituição (DS) de 1,27. A análise térmica (TG/DTA) indicou que a amostra bruta e carboximetilada apresentaram quatro estágios de perda de massa. O primeiro estágio ocorreu em 125ºC (perda de água) e houve dois eventos consecutivos de perda de massa (200ºC-400ºC) atribuídos à degradação da molécula. O quarto estágio ocorreu entre 425ºC e 620ºC (decomposição final) com pico exotérmico em 510ºC. Análise por MEV indicou que a lasiodiplodana bruta apresenta estruturas granulares que se rompem após carboximetilação. Análise de DRX demonstrou que o polímero bruto e carboximetilado apresentam estrutura não cristalina. A carboximetilação contribuiu para melhorar a hidrossolubulidade da molécula (aumento de 60%) e para melhorar a atividade antioxidante avaliada pela capacidade de captura dos radicais ABTS, DPPH e poder redutor do íon férrico (FRAP). Não foi verificado efeito citotóxico da lasiodiplodana bruta e modificada sobre hemácias. Os resultados obtidos sugerem que a carboximetilação da lasiodiplodana pode contribuir para melhoria das propriedades biológicas e para o potencial de uso biotecnológico da molécula. / The world market of polysaccharides has attracted large industrial companies interested in gaining new and profitable fields. Polysaccharides with technological and biological properties can be obtained from plants, algae and microorganisms. Among the polysaccharides with biological properties, glucans have been highlighted by demonstrate immunostimulatory activity and potential for treating diseases such as cancer, hypercholesterolemia, diabetes, multiple sclerosis and cardiovascular diseases. Recent studies demonstrate the production of exocellular β-glucans by filamentous fungi in submerged cultivations. Modifications in the chemical structure of glucans by carboxymethylation is considered an important route to improve its properties, may contribute to the increased solubility of the molecule as well as biological activities, especially those associated with antioxidant and antiproliferative mechanisms of action. Therefore, the present work aimed the production of β-1,6-D glucan (lasiodiplodana) by the Lasiodiplodia theobromae MMPI fungus in submerged cultivation and carboxymethylation of the molecule, characterization and evaluation of cytotoxicity and antioxidant activity. The carboxymethylation of the molecule was confirmed by checking specific chemical signals identified by FT-IR and NMR and 13C spectroscopy. Carboxymethylated molecule presented degree of substitution (DS) of 1.27. Thermal analysis (TG / DTA) indicated that native and carboxymethylated samples had four stages of mass loss. The first stage was at 125 ºC (loss of water) and there were two consecutive events of weight loss (200 ºC - 400 ºC) attributed to the degradation of the molecule. The fourth stage occurred between 425 ºC and 620 ºC (final decomposition) with exothermic peak at 510 ºC. SEM analysis indicated that the raw lasiodiplodan presents granular structures which are broken after carboxymetylation. XRD analysis showed that native and carboxymethylated biopolymers have no crystalline structure. Carboxymethylation aided to improve water solubility the molecule (60% increase) and to improve antioxidant activity assessed by ability to capture the ABTS, DPPH radical scavenging and the ferric ion reducing power (FRAP). There was no cytotoxic effect of raw lasiodiplodana and modified on the erythrocytes. The results suggest that the carboxymethylation of lasiodiplodan can contribute to improved biological properties and the potential biotechnological use of the molecule.