Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2014-09-29T19:48:15Z No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014RuandaSilvaRafael.pdf: 903715 bytes, checksum: 324efd3857a20f7e9735070f16b1b6f9 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2014-10-06T14:07:37Z (GMT) No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014RuandaSilvaRafael.pdf: 903715 bytes, checksum: 324efd3857a20f7e9735070f16b1b6f9 (MD5) / Made available in DSpace on 2014-10-06T14:07:37Z (GMT). No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014RuandaSilvaRafael.pdf: 903715 bytes, checksum: 324efd3857a20f7e9735070f16b1b6f9 (MD5) / A enzima β – galactosidase é reconhecida por catalisar a hidrólise da lactose e possibilitar a formação de galactooligossacarídeos. O objetivo do presente trabalho foi estudar diferentes condições de imobilização de β – galactosidases de Aspergillus oryzae e Kluyveromyces lactis utilizando suporte comercial Immobead. Ambas as enzimas foram imobilizadas nos suportes tratados e não tratados com etilenodiamina e submetidas a processos de imobilização uni e multipontual. Os derivados também foram avaliados quanto ao bloqueio dos grupamentos epóxi com glicina. As análises de estabilidade ao armazenamento sob refrigeração, estabilidade térmica, ciclos de reuso para hidrólise da lactose, determinação das propriedades cinéticas e determinação de pH e temperatura ótimos foram realizadas nos derivados obtidos. Os resultados de eficiência de imobilização variaram de 30 a 50% e os valores de rendimento variaram entre 80 e 90%. Modificações químicas no suporte foram realizadas utilizando etilenodiamina com o objetivo de gerar modificações químicas no suporte, causando a rápida adsorção de enzimas e favorecendo a formação de ligações covalentes multipontuais em tempo reduzido. Verificou-se que suportes modificados com etilenodiamina imobilizaram a mesma carga de enzimas em menor tempo, quando comparados a suportes sem modificação. Entretanto, a significativa perda de atividade verificada nesses suportes durante os ciclos de reuso sugere que a superfície do suporte possa ter sido modificada em sua totalidade, dificultando a formação de ligações covalentes e permitindo a lixiviação de enzimas para o meio reacional. Derivados não bloqueados apresentaram perda considerável de atividade enzimática durante a armazenagem, indicando a ocorrência de possíveis distorções da enzima, ocasionadas pela interação de grupamentos epóxi livres. Ensaios submetidos à imobilização multipontual apresentaram melhorias em sua estabilidade térmica. Os valores de Km para as enzimas imobilizadas de K. lactis e A. oryzae foram 49,69 e 55,29 mM, valores superiores àqueles verificados para as enzimas livres (19,11 e 17,37 mM, respectivamente), indicando possíveis alterações conformacionais na estrutura da proteína, resultantes do processo de imobilização. Os resultados indicaram que derivados não tratados com etilenodiamina, submetidos à imobilização covalente multipontual e bloqueio com glicina apresentaram os resultados mais expressivos para as condições estudadas de estabilidade ao armazenamento, estabilidade térmica e ciclos de reuso para hidrólise de lactose. Esses derivados não apresentaram distorções em relação às condições ótimas de temperatura e pH quando comparadas com as respectivas enzimas livres. As β – galactosidases de A. oryzae e K. lactis submetidas à imobilização covalente multipontual no suporte Immobead posteriormente bloqueado com glicina apresentaram as melhores propriedades para futura aplicação industrial.

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Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 173-189). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "May 2002." Typescript. Vita. Includes bibliographical references (leaves 237-245).

$ beta$-Galactosidase (E.C. 3.2.1.23) or lactase was produced by the growth of a selected Bacillus subtilis strain (KL88) which was adapted to grow at 10$ sp circ$C. The growth and enzyme production were maximal at 2% (w/v) lactose supplemented with 0.2% (w/v) yeast extract. A Fast Protein Liquid Chromatography system (FPLC) was used for $ beta$-galactosidase purification. The enzyme was purified to 44-fold over the crude extract with a recovery of $ sim$54%. Native-PAGE and SDS-PAGE using "PhastSystem" showed the presence of two isoenzymes having molecular weights of 88 and 170 kD. The purified enzyme showed high activity at low temperatures (10$ sp circ$C) and recorded an optimum pH of 7.0. The K$ sb{ rm m}$ values were found to be 2.21 mM and 28.08 mM for o-nitrophenyl-$ beta$-D-galactopyranoside (ONPG) and lactose, respectively. / $ beta$-Galactosidase from psychrotrophic Bacillus subtilis was specific to the $ beta$-D-glycosidic linkage normally present in lactose. / To investigate the possibility of producing proteinase-free $ beta$-galactosidase from this psychrotrophic microorganism, FPLC was used for the rapid separation of $ beta$-galactosidase.

Among 29 strains of bifidobacteria studied as sources of beta-galactosidase enzyme, Bifidobacterium infantis HL96 showed the highest hydrolytic and transgalactosylic activities. This strain grew well in a MRS medium containing various sugars including lactose, and produced three beta-galactosidases (termed beta-Gal I, II, III). / Two genes, beta-galI and beta-galIII, located on 4.6 and 4.4 kb DNA fragments respectively, were cloned into E. coli, and the nucleotide sequences were determined. The 3,069 by-long beta-galI, encoded a polypeptide with a Mr of 113 kDa. A putative ribosome-binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. A partial sequence of an ORF transcribing divergently from beta-galI resembled a lactose permease gene. The beta-galIII gene, which is 2,076 bp long, encoded a polypeptide with a Mr of 76 kDa. A rho-independent, transcription terminator-like sequence was found 25 bp downstream of the termination codon. / The amino acid sequences of beta-GalI and beta-GalIII were homologous to those in the LacZ and LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, containing a glutamate at residue 160. beta-GalI and beta-GalIII were over-expressed 35 and 96 times respectively in E. coli by using a pET expression system. / Both beta-GalI and beta-GalIII were specific for beta-D -anomeric linked galactosides, but beta-GalI showed more hydrolytic and synthetic activities toward lactose than beta-GalIII. The galacto-oligosaccharides (GaOS) production mediated by beta-GalI at 37°C in 20% (w/v) lactose was 130 mg/ml, which is six times higher than that of beta-GalIII. The yield of GaOS further increased to 190 mg/ml in 30% (w/v) lactose. A major tri-saccharide produced by beta-GalI was characterized as O-beta- D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)- D-glucopyranose. / beta-GalI was purified by ammonium sulphate precipitation, and anion-exchange (Mono-Q) and gel filtration (Superose 12) chromatographic steps. The enzyme appeared to be a tetramer, with a Mr of 470 kDa as estimated by native PAGE and gel-filtration chromatography. The optimum temperature and pH for ONPG and lactose as substrates were 60°C, pH 7.5, and 50°C, pH 7.5, respectively. The enzyme was stable over the pH range of 5~8.5, and was particularly active at 50°C for more than 80 min. The enzyme was significantly activated by reducing agents, especially glutathione, as well as by Na+ and K+ cations. Maximal activity required both Na+ and K+ at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, and by most bivalent metal ions. Hydrolytic activity using 20 mM lactose as substrate was significantly inhibited by 10 mM galactose. The Km and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. / The objectives of this research were to characterize beta-galactosidases of B. infantis HL96 at the molecular and biochemical levels, and to over-express the enzymes in Escherichia coli. Two beta-galactosidase isoenzymes with unique properties were genetically characterized for the first time. beta-GalI properties included a neutral pH optimum, relatively higher temperature stability and a high transgalactosylic activity that makes it very competitive for GaOS synthesis. The results were also important for the advancement of knowledge on the catalytic mechanism and the evolutionary aspect of this enzyme.

A beta-galactosidase gene from Bifidobacterium breve B24 which showed the higher hydrolytic and synthetic activity was cloned in E. coli. The complete beta-galactosidase gene contained 2076 bp nucleotides and encoded 691 amino acids which had a high homology to the other Bifidobacterium species. This beta-galactosidase was homologous to that of the LacA family. The galA gene was successfully over-expressed in E. coli ER2566. To observe any change in the recombinant enzyme, beta-galactosidases from Bifidobacterium breve B24 and recombinant E. coli ER2566 were purified to homogeneity by ion exchange chromatography (Mono-Q) and gel-filtration chromatography (Superose-12 and Superdex 200) columns. The molecular mass of both beta-galactosidases was estimated to be 75 kDa on SDS-PAGE. Activity staining on non-denaturing Native-PAGE and Superose-12 gel-filtration chromatography showed that the enzymes are composed of a dimer with a molecular mass of 150 kDa. / The optimum pHs of the native and recombinant enzymes for hydrolyzing O-nitrophenyl-beta-D-galactopyranose (ONPG) were pH 6.0 and 7.0, respectively, and they were stable over the pH range of 5-8 and 6-9, respectively. The optimum temperature of both enzymes for hydrolyzing ONPG was similar at 45 °C and they were stable over the temperature range of 20-45 °C. Both enzymes were stable up to 45 °C during 5 h of incubation at pH 6.5. The recombinant enzyme was slightly activated by bivalent metal ions, Mg2+, Mn2+, and Zn2+ at 1 mM but strongly inhibited by Hg2+ and p-chloromercuribenzoic acid (PCMB). The K m values of both native and recombinant beta-galactosidases for ONPG were 2.77 and 1.82 mM, respectively, and the Vmax values were 1.02 and 1.39 mM/min, respectively. / The two beta-galactosidase activities were also tested with lactose as substrate. The optimum pH of the native and recombinant enzymes for hydrolyzing lactose was similar at pH 6.0. Both enzymes had more than 80 % of their activity in the range of pH 6-8, indicating that the enzymes were stable at neutral pH. However, the native beta-galactosidase had around 40 % of its activity at pH 5.0, whereas the recombinant enzyme had no activity at this pH. On the other hand, the recombinant enzyme had over 50 % of its activity at pH 9.0, while the native beta-galactosidase showed lower than 5 % of its activity. The optimum temperature of both enzymes was at 45 °C. The profiles of both enzyme activities were very similar except at the temperature of 10 °C. The recombinant beta-galactosidase still had around 20 % of its enzyme activity at 10 °C, while no enzyme activity from the native enzyme was detected at this temperature.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Galactooligosaccharides are produced by the transglycosylation activity of β-galactosidases (β-gal, EC 3.2.1.23) when utilising lactose as a substrate. They have emerged as important constituents used in the food and pharmaceutical industries owing to their prebiotic properties. Although transglycosylation was discovered in 1951 (Wallenfels 1951), and a number of β-gals have had their transglycosylation activity characterised, the activities of these enzymes are not optimal for industrial use. Their tendency to favour the hydrolytic reaction over the transglycosylation reaction, coupled with the production of shorter chain oligosaccharides has driven scientists to investigate altering protein structure both to increase chain lengths and the amount of oligosaccharide produced at lower substrate concentrations. In an attempt to alter the amount of oligosaccharide produced by a metagenomically derived β-gal belonging to the glycosyl hydrolase 2 family, random and site-directed mutagenesis were used. A randomly mutagenised library was screened on SOB agar plates containing 5% (w/v) lactose which should select for clones that synthesise oligosaccharides at relatively low concentrations. No such activity was detected. Site-directed mutagenesis was also utilised to alter protein structure. It was confirmed that the β-gal utilised in this study belonged to the glycosyl hydrolase 2 family through mutation of the predicted catalytic acid/base glutamic acid to a non-catalytic residue, thus removing activity. Another mutation was utilised to investigate if it was possible to increase the degree of polymerisation of oligosaccharides produced by the β-gal. This mutation was successful in increasing the degree of polymerisation. Biochemical characterisation of the β-gal revealed that it exhibited optimal activity at pH 8.0, with a temperature optimum of 30°C. The β-gal exhibited a Km and Vmax of 54.23 mM and 2.26 μmol/minute-1/mg protein-1 respectively, similar to kinetic parameters that have been determined for a number of previously characterised enzymes. / AFRIKAANSE OPSOMMING: Galaktooligosakkariede word geproduseer deur die transglikosileering aktiwiteit van β-galaktosidase (β-gal, EG 3.2.1.23) wanneer hulle laktose as 'n substraat gebruik. Hierdie oligosakkariede het na vore gekom as 'n belangrike bestandeel vir gebruik in die voedsel en farmaseutiese bedryf as gevolg van hulle prebiotiese eienskappe. Alhoewel transglycosylation al in 1951 ontdek is (Wallenfels 1951) en 'n aantal β-gals se transglycosylation aktiwiteit gekenmerk is, is hierdie ensieme nie ideaal vir industriële toepassings nie. Die geneigdheid om die hidrolitiese reaksie oor die transglycosylation reaksie bevoordeel, tesame met die produksie van korter oligosakkariede het wetenskaplikes ondersoek genoop om die proteïenstruktuur te verander om ketting-lengte en die kwantiteit van oligosakkaried geproduseer teen laer substraat konsentrasies te verhoog. In 'n poging om die opbrengs van die oligosakkaried wat deur 'n metagenomiese β-gal wat aan die glycosyl hidrolase 2 familie behoort te verander, is lukraak en terrein gerigte-mutagenese gebruik. Die mutagenese biblioteek is op SOB agarplate met 5% (w/v) lactose gekeur, om klone wat die fenotipe wat verband hou met die produksie oligosakkaried teen relatiewe lae konsentrasies te selekteer. Geen aktiwiteit is opgemerk nie. Terrein gerigte-mutagenese is ook gebruik om die proteïenstruktuur te verander. Deur ‘n bioinformatiese voorspelling, is dit bevestig dat die β-gal wat in hiedie studie gebruik word tot die glycosyl hidrolase 2 familie behoort. Dit is gedoen deur mutasie van die voorspelde katalitiese suur/basis glutamiensuur na 'n nie-katalitiese oorskot, dus die verwydering van aktiwiteit. Nog ‘n mutasie is gebruik om te ondersoek of dit moontlik was om die ketting-lengte van die oligosakkaried wat deur die β-gal geproduseer is te verhoog. Die mutasie was suksesvol in die verhoging van die oligosakkaried wat geproduseer was. Biochemiese karakterisering van die β-gal het getoon dat hierdie β-gal optimale aktiwiteit het by pH 8.0, met 'n optimum temperatuur van 30°C. Die β-gal het 'n Km en Vmax van 54.23 mM en 2.26 μmol/minute-1/mg proteïen-1 onderskeidelik, soortgelyk aan kinetiese parameters wat bepaal word vir ensieme wat voorheen gekenmerk is.