Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-25T19:29:24Z (GMT). No. of bitstreams: 1 Silveira_RodrigoLeandro_D.pdf: 21325666 bytes, checksum: e2332474509730ff297050a513a97a70 (MD5) Previous issue date: 2014 / Resumo: A produção de bioetanol a partir da biomassa lignocelulósica integra processos físico-químicos e enzimáticos que comprometem sua viabilidade econômica. A biomassa possui uma estrutura recalcitrante composta de celulose, hemicelulose e lignina. Tal estrutura, bem como os mecanismos das enzimas, não são bem compreendidos. Nesta tese, simulações de dinâmica molecular e a teoria mecânico-estatística 3D-RISM foram utilizada para investigar aspectos moleculares da degradação de biomassa, incluindo: (1) dinâmica estrutural de celulases; (2) base molecular da termofilicidade de laminarinaes; (3) disrupção não-hidrolítica de biomassa por expansinas; (4) nanoarquitetura da parede ceular primária; e (5) forças termodinâmicas da parede celular secundária. No tópico (1), observou-se que a acessibilidade ao substrato em celulases pode ser modulada por alterações na estrutura primária, com consequências para a atividade enzimática. Observou-se também que a inibição por produto está relacionada a alterações conformacionais de resíduos próximos ao sítio de ligação. Adicionalmente, alterações na dinâmica intrínseca das enzimas ocorrem conforme a etapa do processo de hidrólise. No tópico (2), os resultados mostraram que a conformação do sítio de ligação ao substrato de laminarinases é sensível a variações de temperatura. No tópico (3), observou-se que a expansina pode transladar sobre a superfície da celulose e induzir torções em cadeias de glucano, sugerindo a possibilidade de romper ligações de hidrogênio celulose-celulose e/ou celulose/xiloglucano como um zíper. No tópico (4), observou-se que a agregação de nanofibrilas de celulose se dá através de suas faces hidrofílicas e que a presença de hemicelulose estabiliza tal agregação. No tópico (5), os resultados mostraram que as forças de coesão da parede celular secundária são de natureza entrópica e que a composição química da lignina modula as interações lignina-lignina e lignina-hemicelulose / Abstract: Biofuel production from lignocellulosic biomass involves physico-chemical and enzymatic processes that challenge its economic viability. The lignocellulosic biomass is recalcitrant against degradation and is made up of cellulose, hemicellulose and lignin. This structure and the enzyme mechanisms are not fully understood. In this thesis, molecular dynamics simulations and the statistical mechanical theory 3D-RISM were employed to assess molecular aspects of the biomass degradation, including: (1) structural dynamics of cellulases; (2) molecular basis of the thermophilicity of laminarinases; (3) non-hydrolytic disruption of biomass by expansins; (4) primary cell wall nanoarchitecture; and (5) thermodynamic forces of the secondary cell wall. In the topic (1), we observed that cellulase substrate accessibility can be modulated through changes in its primary structure, with consequences to the enzymatic activity. Moreover, the product inhibition is related to conformational changes of residues located close to the substrate binding site. In addition, changes of the intrinsic dynamics allow cellulases change their function according to the hydrolysis step. In the topic (2), we show that the substrate binding site conformation of laminarinases is sensitive to temperature variations. In the topic (3), we observed that the expansin can translade over the cellulose surface and induce torsions in free glucan chains, suggesting the possibility of disruption of cellulose-cellulose and cellulose-xyloglucan hydrogen bonds as a ziper. In the topic (4), the results showed that the aggregation of cellulose nanofibrils takes place through their hydrophilic face and that hemicellulose plays roles in stabilizing such aggregation. In the topic (5), we observed that the cohesion forces within the secondary cell wall are of entropic origin and that the lignin chemical composition modulates the lignin-lignin and lignin-hemicellulose interactions / Doutorado / Físico-Química / Doutor em Ciências

Glykosidhydrolaser (GHs) är enzymer som katalyserar hydrolys av glykosidbindningar i polysackarider och fungerar på endo- eller exo-sätt, beroende på om de riktar sig mot mitten eller änden av en glykan-kedja. De är viktiga i kolcykeln och i olika industrier som använder biomassa som substrat. GHs är fördelaktiga i många industriella processer på grund av deras höga specificitet, omsättningsgrad och biologiska nedbrytbarhet, men de kan vara instabila och är ofta dyra att producera. De varierar i specificitet och har ibland flera katalytiska domäner eller icke-katalytiska tillbehörsdomäner, vilket hjälper till att bryta ner polysackarider och/eller främjar enzymets livslängd. Många GHs kan ha kolhydratbindande moduler (CBMs) som ökar deras termostabilitet och/eller katalytiska aktivitet. CBMs är kopplade till andra domäner i multimodulära domäner av inter-domän länkar (IDLs), vilket är polypeptidkedjor som ger strukturell flexibilitet och låter CBMs nå önskade mål på ett substrat, men den fulla funktionen av IDLs i enzymstabilisering har inte dokumenterats. Kitinaser är en grupp av GHs som riktar sig mot det motsträviga polysackaridet kitin, vilket finns i både marina och markbundna miljöer. De finns i organismer såsom insekter med kitinhaltiga exoskelett och i svampar eller andra mikrober med kitininnehållande cellväggar, men de finns även i organismer som inte syntetiserar eller ens metaboliserar kitin, på grund av deras andra relevanta funktioner inom patogenicitet, immunförsvar, etc. Kitin och dess oligosackarid-derivat har flera funktioner i biomass-industrier och kan användas för medicinska ändamål. Många GHs innehåller icke-katalytiska CBMs, varav många är kitinbindande, och spelar därför en roll i att främja kitinbindning och hydrolys av deras enzympartners. Detta projekt fokuserar på ett modulärt GH18-kitinas kodat av genen Cpin_2580. Kitinasdomänen är flankerad av två CBMs. Tidigare forskning har visat att dessa inte är kitinbindande men föreslog att de påverkar enzymets termostabilitet. Däremot undersöktes inte IDL:ernas påverkan i den tidigare studien. För att bestämma rollen av IDLs designades primers för att klona nya genvarianter av Cpin_2580 för att producera nya proteiner med varierande längder av länkar för att bestämma vad för effekt längden har på enzymets termostabilitet. Dessa primers användes till PCR för att skapa gensekvenser med den befintliga Cpin_2580-18s-plasmiden som mall, följt av kloning, proteinproduktion, rening och analys med hjälp av fluoroforbindningsanalys. Nya proteinvarianter kunde genereras och produceras i liten skala, men produktionen upplevde problem, vilket ledde till att IDLs roll inte kunde fastställas fullt ut. / Glycoside hydrolases (GHs) are enzymes that catalyse the hydrolysis of glycosidic bonds in polysaccharides, functioning in endo- or exo-manners, depending on whether they target the middle or the end of a glycan chain. They are crucial in the carbon cycle and various industries that utilise biomass as substrate. GHs are advantageous in many industrial processes due to their high specificity, turnover rates, and biodegradability, but they can be unstable and are often costly to produce. They vary in specificity and sometimes carry multiple catalytic domains or non-catalytic accessory domains, aiding in polysaccharide breakdown and/or promoting the longevity of the enzyme. Many GHs can have carbohydrate binding modules (CBMs) attached that can be considered accessory domains, which increases their thermostability and/or catalytic activity in many cases. CBMs are attached to other domains in multi-modular enzymes by inter-domain linkers (IDLs), which are polypeptide chains that give structural flexibility and allow the CBMs to reach desired targets on a substrate, but the full function of IDLs in enzyme stabilisation has not been documented. Chitinases are a group of GHs that targets the recalcitrant polysaccharide chitin, which exists in both marine and terrestrial environments. They exist in organisms such as insects that have chitinous exoskeletons and in fungi or other microbes with chitin-containing cell walls, but they are also found in organisms that do not synthesise or even metabolise chitin, due to their other functions of relevance in pathogenicity, immune defence, etc. Chitin and its oligosaccharide derivatives have multiple functions in biomass industries, and can be used for medical purposes. Many chitinases contain non-catalytic CBMs, many of which are often chitin-binding, and therefore have a role in promoting chitin attachment and hydrolysis by their enzyme partners. This project focuses on a modular GH18 chitinase encoded by the gene Cpin_2580. The chitinase domain is flanked by two CBMs. Previous research has shown that these are not chitin-binding but suggested they do influence the thermostability of the enzyme. However, the impact of the IDLs was not explored in that previous study. To determine the role of the IDLs, primers were designed with the purpose of cloning new gene variants of the gene Cpin_2580 to produce novel proteins with varying lengths of linkers to determine the effect the length has on the thermostability of the enzyme. These primers were used for PCR to create novel gene sequences using the pre-existing Cpin_2580-18s plasmid as a template, followed by cloning, protein production, purification, and analysis using fluorophore binding assay. Novel protein variants could be generated and produced at small scale, but scaled-up protein production experienced problems, which led to the role of IDLs not being fully determined.

Tesis por compendio / [ES] Los oligosacáridos de la leche humana (OLHs) y la parte glicana de los glicoconjugados son hidrolizados por las glicosil hidrolasas (GHs), las cuales son expresadas por la microbiota intestinal de niños lactantes promoviendo el establecimiento de una microbiota intestinal con beneficios para su salud. El objetivo de esta Tesis Doctoral consistió en la caracterización funcional de GHs de la microbiota intestinal de niños lactantes capaces de metabolizar OLHs y glicoconjugados, y el estudio de su relevancia biológica y su potencial biotecnológico. Se aislaron cepas bacterianas a partir de heces de niños lactantes.Solo las cepas del género Bifidobacterium metabolizaron alguno de los OLHs testados. Bifidobacterium infantis Y538 consumió eficientemente todos los OLHs testados, mientras que las dos cepas de Bifidobacterium dentium Y510 y Y521 solo metabolizaron lacto-N-tetraosa (LNT) y lacto-N-neotetraosa (LNnT). Se caracterizaron dos ß-galactosidasas de B. dentium Y510; Bdg42A mostró la mayor actividad en LNT, hidrolizándolo en galactosa y lacto-N-triosa (LNTII), mientras que Bdg2A mostró actividad contra lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina y LNnT. También se aislaron cepas bacterianas con actividad glicosidasa extracelular de las heces de lactantes. B. infantis E17 y E18, y Enterococcus faecalis E8 y E41 exhibieron actividad de endo-ß-N-acetilglucosaminidasa, liberando N-glicanos de glicoproteínas. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 deglicosiló eficientemente la proteína S1 del coronavirus 2 del síndrome respiratorio agudo severo (SARS-CoV-2). Tanto la EndoE silvestre como un mutante catalíticamente inactive mostraron actividad lectina frente a la proteína S1 y actividad neutralizante frente a la infección de un virus pseudotipado que presenta la proteína S de SARS-CoV-2 expresada. También se identificaron GHs putativas a través del análisis metagenómico de las heces de niños lactantes, pertenecientes a los géneros Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola y Streptococcus. Se seleccionaron diez ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 y Fuc5372). Las ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 y Fuc1584 mostraron actividad hidrolítica frente a enlaces de fucosa ¿-1,3/4 y Fuc35A, Fuc193 y Fuc2358 mostraron actividad enlaces de fucosa ¿-1,2/3/4/6. Fuc30 mostró actividad sobre la enlaces de fucosa ¿-1,6 mientras que Fuc5372 mostró preferencia por los enlaces ¿-1,2. Fuc2358 mostró actividad frente a glicoconjugados con lacto-N-fucopentaosa II, lacto-N-fucopentaosa III y contra la mucina. Fuc18, Fuc19A y Fuc39 eliminaron fucosa de neoglicoproteínas y de la glicoproteína ¿-1 ácida. Las ¿-L-fucosidasas aisladas fueron evaluadas por su capacidad para sintetizar fucosil-oligosacáridos (FUS) a través de reacciones de transfucosilación. Fuc2358 produjo rendimientos del 35% de 2'-fucosillactosa (2'FL) y también 3'-fucosillactosa (3'FL) y 1-fucosillactosa (1FL). Fuc5372 sintetizó 2'FL, 3'FL y 1FL, con una proporción más alta de 3'FL. Se llevó a cabo mutagénesis dirigida para aumentar los rendimientos de transfucosilación. Los mutantes Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R y Fuc5372-R230K mostraron una mayor relación entre la 2'FL producida y el pNP-Fuc hidrolizado que sus respectivas enzimas silvestres. Además, se observó que los residuos F184 de Fuc2358 y W151 de Fuc5378 afectan a la regioselectividad de la transfucosilación, la fenilalanina aumentando la selectividad por los enlaces ¿-1,2 y el triptófano aumentando la selectividad por los enlaces ¿-1,3. Los resultados presentados muestran la diversidad de GHs presentes en la microbiota intestinal de niños lactantes y expanden el conocimiento sobre su especificidad, contribuyendo al conocimiento del posible papel de las GHs en la colonización bacteriana del tracto gastrointestinal y, además, muestra su potencial biotecnológico / [CA] Els oligosacàrids de la llet humana (OLHs) i la part glicana de glicoconjugats són hidrolitzats per les glicosil hidrolases (GHs), les quals són expressades per la microbiota intestinal de xiquets lactants, promovent l'establiment d'una microbiota intestinal amb beneficis per a la seua salut. L'objectiu d'aquesta Tesi Doctoral va ser la caracterització funcional de GHs de la microbiota intestinal de xiquets lactants capaços de metabolitzar OLHs i glicoconjugats i l'estudi de la seua rellevància biològica i el seu potencial biotecnològic. Es van aïllar soques bacterianes a partir de les femtes de xiquets lactants. Només els soques del gènere Bifidobacterium van metabolitzar algun dels OLHs testats. Bifidobacterium infantis Y538 va consumir eficientment tots els OLHs testats. Les dos soques de Bifidobacterium dentium Y510 i Y521 sol van metabolitzar lacto-N-tetraosa (LNT) i lacto-N-neotetraosa (LNnT).Es van caracteritzar dos ß-galactosidasas de B. dentium Y510; Bdg42A va exhibir la major activitat enfront de LNT, hidrolitzant-la en galactosa i lacto-N-triosa (LNTII), mentre que Bdg2A va mostrar activitat enfront de lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina i LNnT. També es van aïllar soques bacterianes amb activitat glicosidasa extracelul·lar. B. infantis E17 i E18, i Enterococcus faecalis E8 i E41 van exhibir activitat endo-ß-N-acetilglucosaminidasa, alliberant N-glicans de glicoproteïnes. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 va deglicosilar eficientment la proteïna S1 del coronavirus 2 del síndrome respiratori agut greu (SARS-CoV-2).Tant la EndoE salvatge com un mutant catalíticament inactiu van mostrar activitat lectina enfront de la proteïna S1 i activitat neutralitzador enfront de la infecció d'un virus pseudotipat que presenta la proteïna S de SARS-CoV-2 expressada. També es van identificar GHs putatives a través de l'anàlisi metagenómic de la femta de xiquets lactants, pertanyents als gèneres Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola i Streptococcus. Es van seleccionar deu ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 i Fuc5372). Les ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 i Fuc1584 van mostrar activitat hidrolítica enfront d'enllaços de fucosa ¿-1,3/4 i Fuc35A, Fuc193 i Fuc2358 van mostrar activitat enllaços de fucosa ¿-1,2/3/4/6. Fuc30 va mostrar activitat enfront d'enllaços de fucosa ¿-1,6 mentre que Fuc5372 va mostrar preferència pels enllaços ¿-1,2. Fuc2358 va mostrar activitat enfront de glicoconjugats amb lacto-N-fucopentaosa II, lacto-N-fucopentaosa III i contra la glicoproteïna de la mucina. Fuc18, Fuc19A i Fuc39 van eliminar fucosa de neoglicoproteïnes i de la glicoproteïna ¿-1 àcida. Les ¿-L-fucosidasas aïllades van ser avaluades per la seua capacitat per a sintetitzar fucosil-oligosacàrids (FUS) mediant reaccions de transfucosilació. Fuc2358 va produir rendiments del 35% de 2'-fucosillactosa (2'FL) i també 3'-fucosillactosa (3'FL) i 1-fucosillactosa (1FL). Fuc5372 va sintetitzar 2'FL, 3'FL i 1FL, amb una proporció més alta de 3'FL. Es va dur a terme mutagénesis dirigida per a augmentar els rendiments de transfucosilación. Els mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R i Fuc5372-R230K van mostrar una major relació entre la 2'FL produïda i el pNP-Fuc hidrolitzat que els seus respectius enzims salvatges.A més, els residus F184 de Fuc2358 i W151 de Fuc5378 afecten la regioselectivitat de la transfucosilación; la fenilalanina augmenta la selectivitat pels enllaços ¿-1,2 i el triptòfan augmenta la selectivitat pels enllaços ¿-1,3. Els resultats presentats mostren la diversitat de GHs presents en la microbiota intestinal de xiquets lactants i expandixen el coneixement sobre la seua especificitat, contribuint al coneixement del possible paper de les GHs en la colonització bacteriana del tracte gastrointestinal i, a més, mostra el seu potencial biotecnològic. / [EN] Human milk oligosaccharides (HMOs) and the glycan portion of glycoconjugates are hydrolyzed by glycoside hydrolases (GHs) that are expressed by the neonatal intestinal microbiota, promoting the establishment of an intestinal microbiota with health benefits for infants. The objective of this Doctoral Thesis consisted of the functional characterization of GHs from the intestinal microbiota of breastfed infants capable of metabolizing HMOs and glycoconjugates and the study of their biological relevance and their biotechnological potential. Bacterial strains were isolated from breastfed infant faeces, showing that only Bifidobacterium genus strains metabolized any of the HMOs tested. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs, while the two strains isolated from Bifidobacterium dentium Y510 and Y521 only metabolized lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). Two ß-galactosidases from B. dentium Y510 were characterized; Bdg42A exhibited the highest activity on LNT, hydrolyzing it into galactose and lacto-N-triose (LNTII), while Bdg2A displayed activity against lactose, 6'-galactopyranosyl-N-acetylglucosamine, N-acetyllactosamine and LNnT. Bacterial strains with extracellular glycosidase activity were also isolated from breastfed infant faeces. B. infantis E17 and E18, and Enterococcus faecalis E8 and E41 exhibited endo-ß-N-acetylglucosaminidase activity, releasing N-glycans from glycoproteins. The endo-ß-N-acetylglucosaminidase EndoE from E. faecalis E8 efficiently deglycosylated the spike S1 protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Both the EndoE wild-type and a catalytically inactive mutant exhibited lectin activity towards the S1 protein and neutralizing activity against SARS-CoV-2 S pseudotyped virus infection. Putative GHs were also identified through metagenomic analysis of breastfed infant faeces, belonging to Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola, and Streptococcus genera. Ten ¿-L-fucosidases GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358, and Fuc5372) were selected. The ¿-L-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on ¿-1,3/4-linked fucose and Fuc35A, Fuc193 and Fuc2358 showed activity on ¿-1,2/3/4/6-linked fucose. Fuc30 displayed activity only on ¿-1,6-linked fucose, and Fuc5372 showed a preference for ¿-1,2-linked fucose. Fuc2358 displayed activity against glycoconjugates carrying lacto-N-fucopentaose II, lacto-N-fucopentaose III and against the mucin glycoprotein. Fuc18, Fuc19A, and Fuc39 removed fucose from neoglycoproteins and human ¿-1 acid glycoprotein. The isolated ¿-L-fucosidases were evaluated for their capacity to synthesize fucosyl-oligosaccharides (FUS) through transfucosylation reactions. Fuc2358 produced 35 % yields of 2'-fucosyllactose (2'FL) and also 3'-fucosyllactose (3'FL) and 1-fucosyllactose (1FL). Fuc5372 synthesized 2'FL, 3'FL and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis was conducted to increase the transglycosylation yields. Mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R and Fuc5372-R230K showed a higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes. The transfucosylation activity results also showed that the residues F184 of Fuc2358 and W151 of Fuc5378 affect transfucosylation regioselectivity, with phenylalanine increasing the selectivity for ¿-1,2 linkages and tryptophan for ¿-1,3 linkages. The results presented in this doctoral thesis illustrate the diversity of GHs in the intestinal microbiota of breastfed infants and have expanded our knowledge of their specificities, which could contribute to a better understanding of the possible role of GHs in the bacterial colonization of the infant gastrointestinal tract and presents significant biotechnological potential. / This work is part of the Grant PID2020-115403RB (C21 and C22) funded by the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI)/10.13039/501100011033. The study was also supported by Valencian Government grant AICO/2021/033. EMM-G was supported by the Grant PRE2018-085768 funded by MICIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. / Moya Gonzálvez, EM. (2024). Isolation, Functional Characterization and Biotechnological Applications of Glycoside Hydrolases from the Intestinal Microbiota of Breastfed Infants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203171 / Compendio

No description available.