In the first part of this thesis, I describe the expression of C. fimi cellulase genes in the closely related Brevibacterium lactofermentum by generating a shuttle vector able to replicate selectively in the latter and carrying full length cellulase-encoding genes. The expression of those genes apparently originated from some unpredicted regulatory sequences, possibly located within the vector itself. The enzymatic activity was mostly found in the culture medium in B. lactofermentum indicating that the organism secreted the enzymes. The putative C. fimi promoter sequences did not function in B. lactofermentum, making difficult the analysis of their roles in expression of C. fimi cellulase genes. In the second part of this thesis, I describe the characterization of a recombinant C. fimi exo-ϐ-1,4-glucosidase (CbgA) expressed in E. coli. The purified enzyme had a Mr of 183 kDa and hydrolysed various ϐ-glucosides with a preference for cello-oligosaccharides in the order C5>C4>C3>C2. The intact CbgA polypeptide was not required for enzymatic activity since removal of about 700 residues from the amino terminus did not reduce activity. The purified enzyme was used to raise polyclonal antibodies which in turn were used to identify the corresponding enzyme in C. fimi. During the fractionation of C. fimi ϐ-glucosidases, several enzymes hydrolyzing various ϐ-glucosides were isolated together with the native CbgA, which was present in the culture medium as part of a protein aggregate. Part of the nucleotide sequence of the 7.2 kb insert was determined. Alignments of the N-terminal amino acid sequences of the purified CbgA and truncated polypeptides with the partial nucleotide sequence of the cloned C. fimi DNA showed that precise excision was responsible for the appearance of a truncated form of CbgA. Alignment of the amino-terminal sequence of a CbgA:CexCBD fusion peptide indicated that the pre-mature CbgA starts with a putative leader sequence of 49 amino acids which is followed by a region rich in Pro and Ala residues. Two GTG translational initiation codons followed by sequences resemblingprokaryotic ribosome binding sites and separated by a large open reading frame were identified from data obtained after in vitro site-directed mutagenesis of the most upstream initiation codon suggesting that internal re-initiation may occur and that upstream regulatory sequences had not been isolated. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Orientador: Helia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-01T14:14:34Z (GMT). No. of bitstreams: 1 Ferro_LilianAparecida_D.pdf: 39343182 bytes, checksum: 85dd4ff7fee79e9940cf96d45fae535d (MD5) Previous issue date: 2002 / Resumo: Este trabalho objetivou o isolamento de microrganismos capazes de produzir enzimas que lisam a parede celular de leveduras, a produção, purificação e caracterização de 13-1,3-glucanase lítica. As bactérias líticas foram isoladas de Iodo da usina de açúcar e álcool Santa Helena, Piracicaba, SP. As bactérias líticas isoladas aderiram-se às células viáveis de Saccharomyces cerevisiae 701 e lisaram-nas. A linhagem YLM-B191-1, selecionada para o estudo, foi identificada através das características bioquímicas e fisiológicas como Cellulomonas cellulans. Para a produção da 13-1,3-glucanase lítica, a linhagem de C. cellulans YLM-B191-1 foi cultivada em meio composto de 15 9 de levedura seca; 2,0 9 de (NH4hSO4; 13,6 9 de KH2PO4; 4,2 9 de KOH; 0,2 9 de MgSO47H2O; 0,001 9 de Fe2(SO4h 6H2O; 1 mg de biotina e 1 mg de tiamina por litro. A 13-1,3-glucanase foi purificada do sobrenadante do meio de cultura através de ultrafiltração e cromatografia em coluna de CM-Sepharose CL-6B. A enzima purificada apresentou atividade ótima a 55°C e na faixa de pH entre 4,5 a 6,5. A 13-1,3- glucanase purificada apresentou estabilidade na faixa de pH 5,5 a 6,5 e foi inativada em temperaturas superiores a 55°C. A massa molecular da 13-1,3- glucanase purificada foi estimada em 17,1 kDa através de SDS-PAGE. A 13-1,3- glucanase purificada hidrolisou as ligações 13-1,3-glicosídicas da laminarina atuando como uma endoenzima. Através de microscopia eletrônica de varredura, observou- se que as enzimas líticas da linhagem de C. cellulans YLM-B191-1 foram capazes de alterar a superfície celular de leveduras / Abstract: The objective of this research was to isolate microorganisms which produced yeast cell walllytic enzymes and to study the production, purification and characterization of a Iytic ~-1,3-glucanase. The yeast-Iytic bacterium was isolated from the sludge of the Santa Helena sugar and alcohol factory in Piracicaba, SP. The isolated yeast-Iytic bacterium adhered to viable cells of Saccharomyces cerevisiae701 and Iysed them.The yeast-IyticbacteriumYLM-8191-1, selected for this study, was identified as Cellulomonas cellulans, from its biochemical and physiological characteristics.The strain C. cellulansYLM 8191-1 was cultivated in a medium containing (per liter) 2.0 9 of (NH4hSO4; 13.6 9 of KH2PO4;4.2 9 of KOH; 0.2 9 of MgSO47H2O; 0.001 9 of Fe2(SO4h6H2Oand 1 mg each of biotin and thiamin being supplemented with 15 9 of dried yeast as the carbon source for the production of ~-1,3-glucanase. The ~1 ,3-glucanase was purified from the culture fluid of C. cellulans YLM-8191-1 by ultrafiltration and CM-Sepharose CL-68 column chromatography. The purified enzyme showed greatest activity at 55°C and between pH 4.5 - 6.5. The purified ~-1,3-glucanase was stable in the range from pH 5.5 to 6.5 and was inactivated by heating at temperatures above 55°C. The molecular weight of purified ~-1,3-glucanasewas estimated at about 17.1 kDa by SDS-PAGE. The ~-1,3-glucanase hydrolyses the ~-1,3-glucosidic linkages of the laminarin acting as an endoenzyme. Scanning electron microscopy showed that Iytic enzymes from C. cellulans YLM-8191-1 were able to modify the cellular surface of yeast / Doutorado / Doutor em Ciência de Alimentos

In Cellulomonas fimi the cenB gene encodes a secreted endoglucanase (EngB) involved in the degradation of cellulose. The cenB gene carried on a 5.6 kb C fimi DNA fragment encodes a polypeptide of Mr 110,000 in Escherichia coli. The level of expression of the gene was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. The intact EngB polypeptide is not required for enzymatic activity: active polypeptides of Mr 95,000 and 82,000 also appear in E. coli and a deletion mutant of cenB encodes an active polypeptide of Mr 72,000. The intact and truncated EngB both bind to microcrystalline cellulose. A simple, rapid affinity chromatography procedure on Avicel was developed for the purification of intact EngB and of the 72,000 deletion derivative. Alignment of the amino-terminal amino acid sequence of the purified intact EngB from E. coli with the partial nucleotide sequence of the cloned C. fimi DNA showed that the mature EngB is preceded by a sequence encoding a putative signal polypeptide of 32 amino acids, a translational initiation codon and a sequence resembling an E. coli ribosome binding site 4 nucleotides before the initiation codon. The signal peptide functions and is correctly processed in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of β-galactosidase. The truncation of EngB does not affect its export to the periplasm of E.coli. In the intact EngB, 25% of the residues are hydroxyamino acids. It displays features common to endo-β-1 ,4-glucanases, since it has a high activity on carboxymethylcellulose. The kinetic parameters for carboxymethylcellulose hydrolysis of both intact and truncated EngB are not significantly different. C. fimi protease cleaves intact EngB, in a specific manner, to generate two polypeptides of Mr 65,000 and 43,000; the former has the capacity to bind Avicel. A polyclonal antibody raised against the purified intact EngB recognizes a C. fimi extracellular protein of M 110,000 as well as 5 polypeptides of lower molecular weight. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Transcriptional analysis was used to investigate the molecular mechanisms which effect cellulase gene expression in the gram-positive bacterium Cellulomonas fimi strain ATCC 484 and the gram-negative bacterium Agrobacterium sp. strain ATCC 21400. The cenA, cex and cenB genes of C. fimi encoding the extracellular β-1,4-endoglucanase, EngA (EC 3.2.1.4; Mr 48,700), the extracellular β-1, 4-exoglucanase, Exg (EC 3.2.1.91; Mr 47,300) and the extracellular β-1,4-endoglucanase EngB (EC 3.2.1.4; Mr 110,000) respectively, were characterised. By northern blot analysis, cenA mRNA was detected in C. fimi RNA prepared from glycerol- and carboxymethylcellulose (CMC)-grown cells but not in RNA from glucose-grown cells. The cex mRNA was found only in RNA from CMC-grown cells. The cenB mRNA was found in all three preparations of RNA. Therefore, the expression of these genes is subject to regulation by the carbon source provided to C. fimi. High resolution nuclease SI protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, were used to map the 5' termini of cenA and cex mRNAs. Two cenA mRNA 5' ends, 11 bases apart, mapped 51 and 62 bases upstream of the cenA start codon, suggesting that in vivo, cenA transcription was directed from two promoters in tandem. The cex mRNA 5' end was found to map 28 bases upstream of the cex start codon. Using SI mapping with unlabeled DNA probes and C. fimi RNA which had been isolatedin vivo but which had been 5'-labeled in vitro with vaccinia virus capping enzyme confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. The SI mapping revealed mRNA 3' termini 1,438, 1,449, and 1, 464 bases from the major cenA start site, and one 3' terminus 1,564 bases from the major cex mRNA start site, in good agreement with the northern blot data. High resolution SI studies were also used to show that abundant mRNA 5' ends mapped upstream of the cenB start codon in RNA prepared from CMC-grown cells, while less-abundant species mapped 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results seem to indicate a tandem promoter arrangement with an ATG-proximal promoter directing low-level constitutive cenB transcription and a more distal promoter directing higher levels of cenB transcription as a result of C. fimi growth on cellulosic substrate. Steady- state levels were determined for cenA, cex and cenB mRNAs with RNA prepared from glycerol-, glucose-, and CMC-grown cultures of C. fimi in slot-blot hybridisations with radiolabeled oligodeoxyribonucleotide probes. A cex-linked gene (clg) was identified by sequence inspection and SI mapping. Transcripts of the abg gene encoding the β-glucosidase (Abg, EC 3.2.2.21/ Mr 50,000) of Agrobacterium sp. strain ATCC 21400 were also characterised. Northern blot analysis of Agrobacterium RNA revealed the size of the in vivo abgmRNA was approximately 1,500 bases in length. High resolution SI mapping determined abg mRNA 5' ends 22 bases upstream of the abg ATG codon and 3' ends 71 bases downstream of the abg stop codon. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The world stocks of fossil fuels are dwindling and may be all but out before the end of the century. Despite this there is increasing demand for them to be used for transport, and the ever increasing green house gases which their use produces. Renewable and less environmentally damaging forms of fuel are needed. Biofuels, particularly bioethanol, are a possibility to subsidise or replace fossil fuels altogether. Ethanol produced from fermentation of starch sugars from corn are already in wide use. As this bioethanol is currently produced from crops such as corn and sugar cane, that puts fuel crops in direct competition for space and resources with food crops. This has led to increases in food prices and the search for more arable land. Hydrolysis of lignocellulosic biomass, a waste by-product of many industries, to produce the sugars necessary for ethanol production would ease many of the problems with current biofuels. Degradation of lignocellulose is not simple and requires expensive chemical pre-treatments and large quantities of enzymes usually from fungal species making it about 10 times more expensive to produce than corn starch bioethanol. The production of a consolidated bioprocessor, an organism able to degrade, metabolise and ferment cellulosic material to produce ethanol or other useful products would greatly reduce the cost currently associated with lignocellulosic biofuel. Cellulomonas fimi ATCC 484 is an actinomycete soil bacterium able to degrade efficiently cellulosic material. The US Department of Energy (DOE) released the genome sequence at the start of 2012. In this thesis the released genome has been searched, for genes annotated as encoding polysaccharide degrading enzymes as well as for metabolic pathways. Over 100 genes predicted to code for polysaccharide hydrolysing enzymes were identified. Fifteen of these genes have been cloned as BioBricks, the standard synthetic biology functional unit, expressed in E. coli and C. freundii and assayed for endo β-1,4-glucanase activity using RBB-CMC, endo β-1,4-xylanase activity using RBB-xylan, β-D-xylosidase activity using ONPX, β-D-cellobiohydrolase activity using ONPC and α-L-arabinofuranosidase activity using PNPA. Eleven enzymes not previously reported from C. fimi were identified as active on a substrate with the strongest activities being for 2 arabinofuranosidases (AfsA+B), 4 β-xylosidases (BxyC, BxyF, CelE and XynH), an endoglucanase (CelA), and 2 multifunctional enzymes CelD and XynF, active as cellobiohydrolases, xylosidases and endoxylanases. Four enzymes were purified from E. coli cell lysates and characterised. It was found that AfsB has an optimum activity at pH 6.5 and 45ºC, BxyF has optimum activity at pH 6.0 and 45ºC and XynH has optimum activity at pH 9.0 and 80ºC. XynF exhibited different optima for the 3 substrates with pH 6.0 and 60ºC for ONPC, pH 4.5 and 50ºC for ONPX and pH 5.5 and 40ºC for RBB-xylan. Searching the genome and screening genes for activities will help genome annotation in the future by increasing the number of positively annotated genes in the databases. The BioBrick format is well suited for rapid cloning and expression of genes to be classified. Searching and screening the genome has also given insights into the complex and large network of enzymes required to fully hydrolyse and metabolise the sugars released from lignocellulose. These enzymes are spread across many different glycosyl hydrolase families conferring different catalytic activities. The characterisation of these novel enzymes points towards a system adapted to not only a broad specificity of substrate but also environmental factors such as high temperature and pH. Genomic analysis revealed gene clusters and traits which could be used in the design of a synthetic cellulolytic network, or for the conversion of C. fimi into a consolidated bioprocessor itself.