Application of phospholipase A2 of serpents' poisons in biocatalysis

Orientador: Jose Augusto Rosario RodriguesDissertação ( mestrado) - Universidade Estadual de Campinas, Instituto de QuimicaResumo: O projeto explora o potencial catalítico de fosfolipases A2, isoladas de venenos de serpentes brasileiras para efetuar resolução enzimática de substratos com relevância científica, visto que nenhum trabalho anterior foi feito analisando-se sua enantiosseletividade. Foram feitos estudos sobre a resolução do Binol, do a-tetralol, do 1-feniletanol, do para-nitro-1-feniletanol, do ácido 3-(2-bromo-hexanoiloxi)-4-nitrobenzóico e ácido 3-(2-metil-hexanoiloxi)-4-nitrobenzóico.Devido a dificuldade de obtenção e purificação das fosfolipases, a enzima foi imobilizada utilizando a formação de um agregado com ligações cruzadas (Cross-Linked Enzyme Aggregate . CLEA). Os agregados foram produzidos com quatro tipos de precipitantes (solução 55 % de sulfato de amônio, polietilenoglicol 600 Da, dimetoxietano e acetona) e dois adicionantes (TRITON-X100 e polietilenodiimina). Com os testes, observou-se que o CLEA formado com sulfato de amônio, sem adicionante apresentou os melhores resultados, sendo utilizado nas reações de biocatálise. A resolução dos substratos foi feita com a esterificação dos álcoois, formando-se ésteres (acetatos, propanoatos e hexanoatos), e posterior hidrólise com a enzima não-imobilizada e CLEA da fosfolipase A2, para comparação. Alíquotas das reações foram e analisadas por GC/FID com fase estacionária quiral para estudo dos excessos enantioméricos. As reações foram feitas a temperatura ambiente e a 45 °C. Os resultados indicam atividade enzimática sendo possível obter o tetralol com 16% de e.e. utilizando-se o CLEA e o p-nitro-1-feniletanol com 19% de ee usando-se a PLA2 livre. Os outros álcoois foram obtidos com baixos ee. O ácido 3-(2-bromo-hexanoiloxi)-4-nitrobenzóico não pode ser analisado por sofrer hidrólise química completa no meio reacional, e com a hidrólise do ácido 3-(2-metil-hexanoiloxi)-4-nitrobenzóico foi possível a obtenção do ácido 2-metil-hexanóico com 9 % utilizando-se CLEA e 7 % com a enzima livre. A baixa enantiosseletividade foi interpretada como decorrente da fraca interação dos substratos com o sítio ativo da enzimaAbstract: This project explores the catalytic potential of fosfolipases A2, isolated from poisons of brazilian serpents to effect enzymatic substrate resolution with scientific relevance, since no previous work was made analyzing its enantioselectivity. Studies on the resolution of several compounds had been made, including Binol, a-tetralol, 1-phenylethanol, para-nitro-1- phenylethanol, 3-(2-bromohexanoiloxy)-4-nitrobenzoic acid and 3-(2-methylhexanoiloxy)-4-nitrobenzoic acid.Due to difficulty of attainment and purification of the phospholipase, the enzyme was immobilized using the formation of an aggregate with cross-links (Cross-Linked Enzyme Aggregate ¿ CLEA). These aggregates had been produced with four types of precipitation agents (ammonium sulphate solution 55%, polietileneglycol 600 Da, dimethoxyethane and acetone) and two additives (TRITON-X100 and poliethylenediimine). With the tests, it was observed that the CLEA formed with ammonium sulphate, without additives presented the best results, being used in the reactions of biocatalysis.The resolution of substrates was made with the alcohol¿s esterification, forming different (acetates, propanoates and hexanoates) followed by hydrolysis with the free enzyme and CLEA, for comparison. Aliquots of the reactions had been made and analyzed with GC/FID with quiral stationary phase for study of the enantiomerics excesses. The reactions had been made at ambient temperature and 45 °C.The results indicate enzymatic activity and was possible to get tetralol with 16% of ee using CLEA and p-nitro-1-phenylethanol with 19% of ee. The other alcohols had been gotten with low ee. The 3- (2-bromohexanoiloxy) - 4-nitrobenzoic acid cannot be analyzed by suffering complete chemical hydrolysis during the reaction, and with hydrolysis of acid the 3- (2-metilhexanoiloxi) - 4-nitrobenzoic the attainment of the acid 2-metilhexanoic with 9% was possible using CLEA and 7% with the free enzyme. The low enantioselectivity was explained due to the weak interaction of substrates with the active site of enzymeMestradoQuimica OrganicaMestre em Químic

N-glycan utilization by bifidobacterium gut symbionts involves a specialist β-Mannosidase

Bifidobacteria represent one of the first colonizers of human gut microbiota, providing to this ecosystem better health and nutrition. To maintain a mutualistic relationship, they have enzymes to degrade and use complex carbohydrates non-digestible by their hosts. To succeed in the densely populated gut environment, they evolved molecular strategies that remain poorly understood. Herein, we report a novel mechanism found in probiotic Bifidobacteria for the depolymerization of the ubiquitous 2-acetamido-2-deoxy-4-O-(β-d-mannopyranosyl)-d-glucopyranose (Man-β-1,4-GlcNAc), a disaccharide that composes the universal core of eukaryotic N-glycans. In contrast to Bacteroidetes, these Bifidobacteria have a specialist and strain-specific β-mannosidase that contains three distinctive structural elements conferring high selectivity for Man-β-1,4-GlcNAc: a lid that undergoes conformational changes upon substrate binding, a tryptophan residue swapped between the two dimeric subunits to accommodate the GlcNAc moiety, and a Rossmann fold subdomain strategically located near to the active site pocket. These key structural elements for Man-β-1,4-GlcNAc specificity are highly conserved in Bifidobacterium species adapted to the gut of a wide range of social animals, including bee, pig, rabbit, and human. Together, our findings uncover an unprecedented molecular strategy employed by Bifidobacteria to selectively uptake carbohydrates from N-glycans in social hosts4314732747FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2015/26982-0; 2016/00740-2We would like to thank Brazilian Synchrotron Light Laboratory (LNLS) and Brazilian Biosciences National Laboratory (LNBio) for the provision of time on the MX2 and SAXS1 beamlines, and ROBOLAB facility. We thank Esther Lorizolla Cordeiro for providing illustrations for figure preparation. This work was supported by Grant No. 2015/26982-0 from São Paulo Research Foundation (FAPESP) (to M.T.M.). RLC was supported by FAPESP Grant No. 2016/00740-

The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans

Abstract Background Arabinoxylan is an abundant polysaccharide in industrially relevant biomasses such as sugarcane, corn stover and grasses. However, the arabinofuranosyl di-substitutions that decorate the xylan backbone are recalcitrant to most known arabinofuranosidases (Abfs). Results In this work, we identified a novel GH51 Abf (XacAbf51) that forms trimers in solution and can cope efficiently with both mono- and di-substitutions at terminal or internal xylopyranosyl units of arabinoxylan. Using mass spectrometry, the kinetic parameters of the hydrolysis of 33-α-l-arabinofuranosyl-xylotetraose and 23,33-di-α-l-arabinofuranosyl-xylotetraose by XacAbf51 were determined, demonstrating the capacity of this enzyme to cleave arabinofuranosyl linkages of internal mono- and di-substituted xylopyranosyl units. Complementation studies of fungal enzyme cocktails with XacAbf51 revealed an increase of up to 20% in the release of reducing sugars from pretreated sugarcane bagasse, showing the biotechnological potential of a generalist GH51 in biomass saccharification. To elucidate the structural basis for the recognition of internal di-substitutions, the crystal structure of XacAbf51 was determined unveiling the existence of a pocket strategically arranged near to the − 1 subsite that can accommodate a second arabinofuranosyl decoration, a feature not described for any other GH51 Abf structurally characterized so far. Conclusions In summary, this study reports the first kinetic characterization of internal di-substitution release by a GH51 Abf, provides the structural basis for this activity and reveals a promising candidate for industrial processes involving plant cell wall depolymerization