A group of Escherichia coli clones harbouring recombinant pBR322 plasmid, containing Cellulomonas fimi DNA inserts, that reacted with antiserum to C.fimi culture supernatant, was screened for their ability to hydrolyze carboxymethyl cellulose (CMC) and 4-methylumbeliferyll-β-D-cellobioside (MUC). A clone, pEC62, hydrolyzed MUC but did not hydrolyze CMC. The recombinant enzyme encoded by pEC62 was shown to be a β-glucosidase (cellobiase). C.fimii itself was shown to encode an extracellular β-glucosidase in C.fimi. This is the first report of an extracellular β-glucosidase from a bacterium. Deletion analysis localized the cloned gene (cbg)to the tet promoter proximal region of the 7.0 kilobase insert of pEC62. Further analysis and sequence data showed a highly active derivative of pEC62 contained a translational gene fusion between lacZ of pUC13 and cbg. From this data, a location for the cbg start site was proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 96-113).

As nascent secretory and membrane proteins are inserted into the endoplasmic reticulum (ER), they are maintained in folding and/or assembly competent states by molecular chaperones including the Hsp70 and Hsp90 homologues, BiP and GRP94, and the lectin-like chaperones calnexin (CNX) and calreticulin (CRT). Folding is catalyzed by protein disulfide isomerase (PDI), its CNX (and CRT) associated homologue, ERp57, and protein prolyl isomerase (PPI). Moreover, N-linked glycoproteins benefit from a lectin-based "quality control apparatus" that ensures their correct folding or oligomeric assembly. Binding to these lectins occurs through oligosaccharide trimming from Glc 3Man9GlcNAc2 to the monoglucosylated form (Glc 1Man9GlcNAc2). Release and subsequent rebinding occurs though the hydrolysis and reglucosylation of the innermost glucose by glucosidase II and UDP-glucose glycoprotein:glucosyltransferase, respectively. This cyclical process, termed the "Calnexin Cycle", continues until their correct conformation is achieved. / The cloning and characterization of human glucosidase II is reported here. cDNAs for two splice variants of the catalytic alpha subunit and the beta subunit were isolated. Expression of the beta subunit was shown to be required for enzymatic activity, solubility and/or stability, and ER retention of the enzyme. Detailed kinetic analysis on recombinant alpha1/beta and alpha2/beta isoforms, using p-nitrophenyl alpha-D-glucopyranoside as a substrate, reveals that both exhibit kinetic profiles of a two binding site model, and share properties of catalysis and inhibition on this substrate. Moreover, similar rates of hydrolysis of the oligosaccharide substrates rules out the possibility that the two binding site kinetic model, first proposed by Alonso et al. (1999, Biochem J. 278:721--7), is the result of co-purified isoforms of glucosidase II that have different substrate specificities. / Also, an ER protein two-hybrid system, based on Ire1p and the unfolded protein response (UPR) pathway in Saccharomyces cerevisiae, was developed to examine and map the interactions between CNX/CRT and ERp57. Ire1p fusions with CNX and CRT were shown to interact specifically with ERp57, and as expected, PDI did not. Through deletion analysis, new roles were assigned to the proline-rich loop domains of CNX and CRT, and the non-catalytic B thioredoxin domain of ERp57 in mediating their heterodimerization.

The β-glucosidase (Abg) from ATCC 21400, an Agrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed phase high pressure liquid chromatography. The partial amino-acid sequences for three CNBr peptides, CNBr1, CNBr2 and CNBr3, were determined by automated Edman degradation. A sequence from CNBr2 was used to synthesize a mixture of oligonucleotides which was used as a hybridization probe to identify a recombinant DNA clone carrying the gene for β-glucosidase. A single clone was isolated which expressed an enzymatic activity that hydrolyzed several β-glucosides. The enzymatic activity produced by this clone could be adsorbed by rabbit antiserum raised against the Agrobacterium enzyme. The direction of transcription of the β-glucosidase gene was determined by verifying the DNA sequence 3' to the oligonucleotide probe binding site. After subcloning the gene a high level of expression was obtained in the plasmid vector pUC18 using the lacZ gene promoter. The nucleotide sequence of the 1599 bp insert in pABG5 was determined using the chain terminator method. The start of the protein coding region was determined by aligning the amino terminal sequence of the protein with the predicted amino acid sequence of the cloned gene. The open reading frame was 1387 nucleotides and contained 458 codons. The molecular weight calculated from the deduced amino acid sequence agreed with that observed from both the native and recombinant enzymes. The predicted amino acid composition from the open reading frame matched with that determined for the native β-glucosidase. The stop codon of this coding region was followed by a potential stem loop structure which may be the transcriptional terminator. There was a region of the deduced Abg sequence which had homology to a region from two other β-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Cheung Wai-chit. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004 / Includes bibliographical references (p. 135-140) / 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.

*Please refer to dissertation for diagrams. / In this thesis, a review concerning enantiospecific syntheses and structure activity relationship of N-linked carbadisaccharides from 1994 to 2005 is presented. / Valienamine was isolated from microbial degradation products of validoxylamine A with Pseudomonas denitrificans. It showed alpha-glycosidase inhibitory activity and antibiotic activity. Isopropylidene protected allylic chlorides, 2-epi-valienamine and 4-amino-6-deoxy-alpha- D-pseudomannopyranose were synthesized from (-)-quinic acid. The acetonide blocking groups were shown to be the best hydroxyl protecting groups for coupling precursors, compatible with palladium-catalyzed coupling reaction which afforded high yields of the 1,1'- and 1,4'-N-linked carbadisaccharides 83-88 and 89-91, respectively, with a minimum amount of an elimination diene side-product. 2-epi-Valienamines 92 and N-alkyl 2-epi-valienamine 93 and 94 were also prepared. The key palladium-catalyzed coupling reaction was shown to be a regio- and stereospecific reaction. Acid hydrolysis was used to obtain free pseudoaminosugars as glycosidase inhibitors. The inhibitory activities of these pseudoaminosugars were evaluated by the Biochemistry Department of the Chinese University of Hong Kong.* / Kwong Suk Kwan. / "January 2006." / Adviser: Tony K. M. Shing. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6409. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 146-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The argument of whether to use food for biofuel (bioethanol) production prompted the search for an alternative non-food biomass, such as lignocellulose, as feedstock for bioethanol production. However, a hindrance in producing bioethanol from lignocellulose on an industrial scale is the cost associated with hydrolysing the lignocellulose to its respective sugar monomers. Improving enzyme production and enhancement of enzyme cocktails for efficient lignocellulose hydrolysis is, therefore, a necessary prerequisite. In this study, a yeast culture collection from the Wine and Fermentation Technology Division (ARC Infruitec- Nietvoorbij, Stellenbosch, South Africa), isolated from fruit from various regions in South Africa, was screened for β-glucosidase and β-xylosidase enzyme activities. β-glucosidases catalyse the hydrolysis of cellobiose and by doing so prevents end-product inhibition of cellobiohydrolases and endoglucanases during cellulose degradation. Similarly, β-xylosidases hydrolyse xylobiose and prevents end-product inhibition of endoxylanases during hemicellulose degradation. After initially screening 2180 non- Saccharomyces yeasts, two yeast isolates were selected that could potentially serve as enzyme source for lignocellulose hydrolysis; one as a producer of a β-glucosidase and another as a β-xylosidase producer. The yeasts were identified as a β-glucosidase producing Rhodotorula slooffiae-like yeast isolate 131B2 and a β-xylosidase producing Aureobasidium pullulans isolate 23B25, respectively. The production of β-glucosidase by Rhodotorula slooffiae-like yeast isolate 131B2 and of β-xylosidase by Aureobasidium pullulans isolate 23B25 was optimised using response surface methodology according to a central composite design. Subsequently, the crude and partially purified enzymes were characterised based on molecular mass, pH optima and stability, temperature optima and stability and inhibition by lignocellulose hydrolysis end-products, such as glucose, xylose and ethanol. The crude β-glucosidase from Rhodotorula slooffiae-like yeast isolate 131B2 was also compared to the commercial Aspergillus niger βglucosidase preparation (Novozyme 188) based on the characteristics mentioned above and as βglucosidase supplement during Avicel (microcrystalline cellulose) hydrolysis by the commercial cellulase preparation (Celluclast). The crude β-xylosidase by Aureobasidium pullulans isolate 23B25 could not be compared to a commercial β-xylosidase as none was available at the time of the study. During the study, the crude β-glucosidase 131B2 and β-xylosidase 23B25 showed potential as lignocellulose hydrolytic enzymes. Attempts were made to obtain the β-glucosidase and β-xylosidase genes from the respective yeast isolates using PCR-based approaches and by constructing cDNA libraries. However, cloning the β-glucosidase and β-xylosidase genes using these methods proved after several attempts to be unsuccessful, although, during this section of the study valuable information was obtained about the obstacles involved with using these approaches when the desired gene sequence is unknown and novel. / AFRIKAANSE OPSOMMING: Die debat oor die toepaslikheid van voedsel vir bio-brandstofproduksie (bio-etanol), het daartoe gelei dat alternatiewe nie-voedsel grondstowwe, soos lignosellulose, as voermateriaal vir bio-ethanol ondersoek word. Die koste geassosieer met die hidrolise van lignosellulose na die onderskeie suiker monomere belemmer industriële-skaal toepassing van lignosellulose vir bio-etanolproduksie. Verbeterde ensiemproduksie en verhoogde doeltreffendheid van ensiemmengsels vir lignosellulose hidrolise is dus ‘n noodsaaklik voorvereiste. In hierdie studie is 'n giskultuurversameling geisoleer vanaf vrugte van verskillende streke in Suid-Afrika deur die Wyn en Fermentasie Tegnologie Afdeling (ARC Infruitec-Nietvoorbij, Stellenbosch, Suid-Afrika) vir β-glukosidase en β-xilosidase ensiemaktiwiteite gesif. β-glukosidases wat die hidrolise van sellobiose kataliseer voorkom eindprodukinhibisie van sellobiohidrolases en endoglukanases tydens sellulose afbraak. β-xilosidases, op hul beurt, hydroliseer xilobiose en voorkom eindprodukinhibisie van endoxilanases tydens hemisellulose afbraak. Na afloop van die aanvanklike sifting van 2180 nie-Saccharomyces giste, is twee giste wat potensiëel as 'n ensiembron vir lignosellulose hidrolise kan dien geselekteer; een vir β-glukosidase en ‘n ander vir β-xilosidase produksie. Die giste is as ʼn β-glukosidase-produserende Rhodotorula slooffiaeagtige gisras 131B2 en ʼn β-xilosidase-produserende Aureobasidium pullulans gisras 23B25 onderskeidelik geïdentifiseer. Die Rhodotorula slooffiae-agtige gisras 131B2 se produksie van β-glukosidase en die Aureobasidium pullulans gisras 23B25 produksie van β-xylosidase was geoptimiseer met behulp van “response surface methodology” volgens 'n “central composite design”. Daarna was die gedeeltelik-gesuiwerde kru-ensieme volgens molekulêre massa, pH optima en stabiliteit, temperatuur optima en stabiliteit, en inhibisie deur lignocelluloses hidrolise end-produkte soos glukose, xylose en etanol, gekarakteriseer. Die kru βglukosidase van die Rhodotorula slooffiae-agtige gisras 131B2 is ook met die kommersiële Aspergillus niger β-glukosidase (Novozyme 188) volgens die eienskappe vroeër genoem vergelyk en as β-glukosidase aanvulling tydens die kommersiële sellulase (Celluclast) se hidrolise van Avicel (mikrokristalline sellulose). Die kru β-xylosidase van die Aureobasidium pullulans gisras 23B25 kon nie vergelyk word met 'n kommersiële β-xylosidase nie, aangesien daar nie een beskikbaar was tydens die studie nie. Gedurende die studie het altwee, die kru β-glukosidase 131B2 en β-xylosidase 23B25, potensiaal getoon as lignosellulose hidrolitiese ensieme. Pogings was aangewend om die β-glukosidase en β-xilosidase gene vanuit die onderskeie gis isolate met behulp van PKR-gebaseerde tegnieke en die opstel van cDNA biblioteke te kloneer. Hierdie klonering strategieë was egter na verskeie pogings onsuksesvol, maar waardevolle inligting oor die struikelblokke betrokke by die gebruik van hierdie benaderings wanneer die gewenste geen se DNS basispaarvolgorde onbekend en uniek is, was verkry.

No abstract available. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

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.