Visando dar continuidade ao estudo de incorporação de N-acil-asparagina a derivados de aminoácidos e a peptídeos mediante catálise por termolisina, foi estudada a viabilidade de síntese dos peptídeos Z-Asn-Cys(S-Bzl)-OBut e Moz-Asn-Cys(S-Bzl)-Pro-Leu-Gly-NH2. Durante a síntese do pentapeptídeo Moz-Asn-Cys(S-Bzl)-Pro-Leu-Gly-NH2 foi constatada a formação de um subproduto cuja estrutura foi elucidada a partir da comparação ao padrão autêntico Moz-Asn-Leu-Gly-NH2. Para a obtenção deste padrão, sintetizamos o tripeptídeo Moz-Asn-Leu-Gly-OEt a partir de Moz-Asn-OH e H-Leu-Gly-OEt na presença de termolisina. A recristalização deste peptídeo em MeOH/H2 O levou à transformação do mesmo em Moz-Asn-Leu-Gly-OMe, confirmada pelo isolamento e caracterização do produto por espectrometria de massa e ressonância magnética protônica, análise elementar e de aminoácidos e por determinação do tempo de retenção por HPLC. Estudos posteriores demonstraram o envolvimento do íon Ca(II) no processo e nos permitiram sugerir um modelo da ligação deste íon ao peptídeo e da sequência de reação da transesterificação estudada. Também foi investigada a possibilidade de diversos ésteres de peptídeos e de peptidilresinas de Merrifield sofrerem transesterificação quando incubados em metanol na presença de Ca(II). Para tanto, os tripeptídeos Z-Asn-Leu-Gly-OEt, Boc-Asn-Leu-Gly-OEt, Moz-Asn-Leu-Gly-OBzl, Moz-Asn-Leu-Gly-OBu Moz-Gln-Leu-Gly-OEt, Moz-Asn-Ile-Gly-OEt e Moz-Asn-Leu-Ala-OEt foram sintetizados mediante catálise por termolisina. Também foram testados vários outros peptídeos disponíveis no laboratório e Boc-Leu-Gly-Res e Moz-Asn-Leu-Gly-Res. Com exceção dos ésteres terc-butílicos, todos sofreram transesterificação em soluções metanólicas de acetato de cálcio. / The main obJective was the study of thermolysin catalyzed incorporation of N-acyl-asparagine into amino acid and peptide derivatives. Both Z-Asn-Cys(S-Bzl)-OBut and Moz-Asn-Cys(S-Bzl)-Pro-Leu-Gly-NH2 syntheses have been studied. The formation of a by-product occurred during the synthesis of the later pentapeptide. By comparison with an authentic standard this by-product was identified as Moz-Asn-Leu-Gly-NH2. The standard Moz-Asn-Leu-Gly-NH2 was obtained by aminolYSlS of Moz-Asn-Leu-Gly-OEt. This peptide was synthesized by coupling Moz-Asn-OH with H-Leu-Gly-OEt using thermolysin as catalyst. During the recrystallization in MeOH/H2O, the transformation of Moz-Asn-Leu-Gly-OEt to its methyl-ester was obtained. This observation has been confirmed by purification and caracterization of the peptide by: Mass Spectroscopy, Nuclear Magnetic Ressonance, Amino Acid and Elemental Analysis and HPLC. Later studies have shown that the Ca(II) ion participates in the process. A model for the binding of this ion to the peptide and for the transesterification reaction has been suggested. Further studies have been performed with (1) the newly synthesized peptides Z-Asn-Leu-Gly-OEt, Boc-Asn-Leu-Gly-OEt, Moz-Asn-Leu-Gly-OBzl, Moz-Asn-Leu-Gly-OBut, Moz-Gln-Leu-Gly-OEt, Moz- Asn-Ile-Gly-OEt and Moz-Asn-Leu-Ala-OEt; (2) several other peptides available in our laboratory; and (3) Boc-Leu-Gly-Res and Moz-Asn-Leu-Gly-Res. With the exception of t-butyl-esters, all peptides tested have transesterified in methanolic calcium acetate solutions.

Acrylamide is a carcinogenic substance generated in heat processed food stuff, where the temperature increases over 120 °C. The main acrylamide precursors are carbonyl compounds and the amino acid asparagine. In this diploma work, possible elimination strategies were observed in a bread matrix with an addition of inorganic salts into the basic powder formulation for home bread making. Samples were analyzed from the point of view of acrylamide content as well as their physico-chemical properties and sensory evaluation too. Results show, that the addition of inorganic salts hasn`t expected considerable elimination impact as in the case of model samples. It has been shown the importance of fermentation process, whereas was the amount of acrylamide in bread without yeast added almost 14times higher. The addition of CaCl2 in bread mixture improved the organoleptic quality of final product, as proved in sensory evaluation with simultaneus positive effect on acrylamide reduction in bread.

La déamidation est la modification post-traductionnelle de l'asparagine (Asn) et de la glutamine (Glu). Elle est communèment observée dans les peptides et les protéines. Il a été démontré que la déamidation limite la durée de vie de ces macromolécules. Dans ce travail, la déamidation de l'asparagine dans des petits peptides et dans l'enzyme triosephosphate isomérase a été modélisée. La déamidation dans la triosephosphate isomérase de mammifères a été observée sur deux sites distincts: Asn15 et Asn71. Asn71 a une vitesse de déamidation plus élevée que Asn15 et moins grande que pour un petit peptide. Il a été suggéré que la déamidation de Asn15 se produit sous l'influence de la déamidation de Asn71. Pour expliquer ces résultats expérimentaux, des simulations de dynamiques moléculaires classiques à l'échelle de la microseconde et des calculs d'énergie libre, de type umbrella sampling, à l'aide de méthodes combinées mécanique quantique/mécanique moléculaire ont été réalisés. Nous montrons que la déamidation séquentielle dans la triosephosphate isomérase est due à la fois à des effets locaux et globaux. Ces résultats apporte une nouvelle perspective sur l'impact de l'ordre structurel sur la vitesse de déamidation Nous avons également déterminé la voie la plus plausible de cette reaction ainsi que l'influence de la variation du pKa, dans la chaîne principale, de la partie amide du résidu adjacent de l'asparagine sur la vitesse de déamidation. En regard de l'importance des variations de pKa dans l'environnement protéique, nous avons élaboré un protocole informatique permettant d'évaluer de manière rapide et précise des pKa . Ce protocole a été appliqué à des petites molécules organiques et nous avons montré qu'il était également applicable à des études relatives à la prédiction de pKa dans les protéines / Deamidation is the posttranslational modification of asparagine (Asn) and glutamine (Glu) residues, which is observed in several proteins and peptides. It has been shown that deamidation limits the lifetime of these macromolecules. In this work, deamidation of asparagine in small peptides and in the enzyme triosephosphate isomerase has been modeled. Deamidation in mammalian triosephosphate isomerase has been observed at two distinct deamidation sites: Asn15 and Asn71. Asn71 deamidates faster than Asn15 and slower than a small peptide. It has been suggested that, deamidation at Asn15 occurs with the influence of deamidated Asn71. In order to explain these experimental findings, microsecond long classical molecular dynamics simulations and free energy calculations using quantum mechanics/molecular mechanics tools combined with umbrella sampling technique have been performed. The sequential deamidation in triosephosphate isomerase has been shown to be related with both global and local effects. These results bring a new perspective to the impact of the high-order structure on deamidation rate. The most plausible route of this reaction was also determined. The pKa shift of backbone amide of the residue adjacent to asparagine has been found to be one of the most crucial factor determining the rate of deamidation. Considering the importance of pKa shifts in protein environment, a computational protocol was suggested in order to obtain accurate and fast pKa predictions. This protocol was applied to small organic molecules, and it has been shown to be applicable to studies concerning aminoacid pKa predictions

A commercial valve-assisted micromixer, manufactured by Ehrfeld (Germany), was tested for its use to precipitate L-asparagine from an aqueous solution using isopropanol as antisolvent. In a first part the mixing quality provided by the micromixer was studied by means of a competitive/parallel set of reactions following the approach of Baldyga, Bourne and Walker, Canadian J. Chem. Eng. 76 (1998) 641-649. Different experiments have been implemented and interpreted considering the average of Reynolds number of the inlet streams. Results show a good mixing quality that is comparable, in terms of absolute values of conversion, with other works present in literature. The precipitation experiments that followed revealed the limitation of the micromixer. The system was instable and particles adhesion occurred inside the mixing chamber. Improvements have been realized by changing the spring tension of the valve and introducing a commercial surfactant TRITON X-100.

No description available.

L'acrylamide, connu comme étant une molécule cancérigène, apparait lors de traitement thermique à haute température (>120°C) d'aliments riches en asparagine et en sucre. La friture profonde génère ce composé à des teneurs proche de 0,44 ppm pour les chips de plantain. Or les aliments à base de plantain sont largement consommés dans les pays du sud. Ces travaux de recherche ont pour objectif de mieux comprendre l'accumulation de l'acrylamide lors de l'élaboration de produits frits à base de plantain. Pour y parvenir la teneur en précurseurs (asparagine) a d'abord été obtenue dans différentes variétés de bananes, mais aussi durant le mûrissage d'une variété de plantain d'exportation (Harton). Ces résultats ont permis d'identifier l'asparagine comme étant limitant pour la formation d'acrylamide dans le plantain. Ensuite, une étude cinétique du couplage entre la température et l'activité en eau a été entreprise en système fermé. Le domaine d'étude consistait en 4 températures de traitement (140, 160, 180 et 200°C) et 3 activités en eau (0,43 ; 0,9 ; 0,97). La cinétique de formation et d'élimination de l'acrylamide a été traduite par la somme de 2 cinétiques d'ordre 1. Les paramètres cinétiques ont été identifiés et montrent que la température est le paramètre qui influence le plus la cinétique de l'acrylamide. Le modèle cinétique a dans une autre étape été couplé à un modèle traduisant les transferts d'énergie et de vapeur durant la friture. Le modèle a bien simulé les teneurs en eau et en acrylamide de deux produits frits à base de plantain « tajadas » (produit épais) et « tostones » (produit mince). Les coulages entre transfert et réaction sont discutés en vue d'imaginer des stratégies de réduction de l'acrylamide. Il a été démontré que la formation de l'acrylamide se fait presque exclusivement dans la zone hygroscopique périphérique (croûte). De ce fait, la conduite de la friture en températures variables est recommandée et permet de réduire d'au moins 50% la teneur en acrylamide. La sélection variétale et l'état de maturité du plantain sont les moyens en amont les plus souples pour contrôler la teneur en acrylamide dans le produit final. Des prétraitements par immersion se sont avérés efficaces induisant une réduction significative de l'acrylamide. En revanche, la formulation de molécules antioxydantes ne semble pas avoir d'effet réducteur. / Acrylamide, a molecule known as a carcinogen, appears during heat treatment of asparagine and sugars rich foods at high temperature (> 120 ° C). Deep frying produces this compound at levels close to 0.44 ppm for plantain chips, these foods being widely consumed in developing countries. The aim of the present work is to better understand the accumulation of acrylamide during the process of plantain-based products. In this context, the level of precursors (asparagine) was obtained firstly in different varieties of bananas and also during ripening of an export plantain variety (Harton). These results allowed identifying asparagine as a limiting factor of acrylamide formation in plantain. Afterwards, a kinetic study of the coupling between temperature and water activity was undertaken in a closed system. The field of study consisted of four temperatures of treatment (140, 160, 180 and 200 ° C) and three water activities (0.43, 0.9, 0.97). The kin etics of formation and elimination of acrylamide was translated by the sum of 2 order 1 kinetics. The kinetic parameters were identified and showed that temperature is the parameter that influences the most acrylamide kinetic. The kinetic model, in a further step, was coupled to a model describing energy and steam transfers during frying. The model simulated well water and acrylamide content in two plantain-based fried products: "Tajada" (produced thick) and "tostones" (thin product). The coupling between transfers and reaction were discussed in order to identify strategies to reduce acrylamide. It was demonstrated that acrylamide formation occurs almost exclusively in the hygroscopic and peripheral area (crust). Thus, conducting frying process at varying temperatures is recommended and can reduce by at least 50% the acrylamide content. The varietal selection and the ripeness degree of plantain are more flexible ways to control upstream the levels of acrylamide in the final product. Immersion pretreatments were also effective to reduce significantly acrylamide. However, the formulation of antioxidant molecules did not appear to have reduction effect.

A bactéria Listeria monocytogenes é reconhecida como um importante patógeno humano estando amplamente distribuída no ambiente e é responsável pela contaminação de alimentos crus e processados. O mecanismo de patogenicidade é determinado pela presença de genes no cromossomo da bactéria e entre eles estão os genes iap e hly que são essenciais para o mecanismo de invasão e atividade hemolítica do microorganismo, respectivamente. O obejtivo do presente estudo foi confirmar as cepas de L. monocytogenes usando a ténica de PCR para o gene hly e análise da variação nucleotídica do domínio central do gene iap, que é caracterizado pela presença de seqüências repetidas dos aminoácidos treonina e asparagina. Vinte e seis cepas de L. mnocytogenes, previamente isoladas de produtos lácteos e identificdas por métodos clássicos, se mostraram positivas para a PCR espécie-específico e então submetidas à determinação da seqüência nucleotídica. Os resultados mostraram variações na seqüência nucleotídica contendo substituições, inserções, deleções e um número de seqüências similares entre as cepas isoladas e a cepa controle EGD-e. Vinte e três cepas exibiram a mesma deleção que compreende 24 pares de bases dentro da seqüência de repetição e alterações similares na proteína traduzida. Apenas três cepas mostraram tamanhos diferentes de deleções e diferentes alterações na proteína. De acordo com estes resultados, a maioria das cepas apresentou uma característica molecular comum, diferentes da cepa padrão e este perfil predominante pode ser considerado como a característica das L. monocytogenes isoladas de produtos lácteos no Sul do Brasil. / The bacterium Listeria monocytogenes is recognized as an important human pathogen being omnipresent in the environment and is responsible for contamination in raw and processed foods. The mechanism of pathogenicity is established by presence of some genes on chromosome of bacteria between them, iap and hly genes that are essential to the invasion mechanism and hemolytic activity of microorganism, respectively. The aim of present study was confirm the strain L. monocytogenes using PCR to hly gene and analysis the nucleotide variability of central domain of iap gene characterized by the presence of similar sequences of threonine and asparagine amino acids. Twenty-six strains previously isolated from dairy products and classified by classic methods to L. monocytogenes were positive to specie-specific PCR and than submitted to nucleotide sequence determination. The results showed a sequence variation containing nucleotide substitutions, insertions, deletions and a number of repeated sequences among the isolates and control strain EGD-e. Twenty-three strains exhibited the same gap that includes a deletion of 24 base pairs inside of the repeated sequence and similar alterations in the translated protein. Just three strains showed different sizes of gaps and different protein alterations. According to these results, the majority strains displayed a common molecular characteristic different from the strain pattern and this predominant profile can be considerate as characteristic to L. monocytogenes isolated from dairy products in South Brazil.

A bactéria Listeria monocytogenes é reconhecida como um importante patógeno humano estando amplamente distribuída no ambiente e é responsável pela contaminação de alimentos crus e processados. O mecanismo de patogenicidade é determinado pela presença de genes no cromossomo da bactéria e entre eles estão os genes iap e hly que são essenciais para o mecanismo de invasão e atividade hemolítica do microorganismo, respectivamente. O obejtivo do presente estudo foi confirmar as cepas de L. monocytogenes usando a ténica de PCR para o gene hly e análise da variação nucleotídica do domínio central do gene iap, que é caracterizado pela presença de seqüências repetidas dos aminoácidos treonina e asparagina. Vinte e seis cepas de L. mnocytogenes, previamente isoladas de produtos lácteos e identificdas por métodos clássicos, se mostraram positivas para a PCR espécie-específico e então submetidas à determinação da seqüência nucleotídica. Os resultados mostraram variações na seqüência nucleotídica contendo substituições, inserções, deleções e um número de seqüências similares entre as cepas isoladas e a cepa controle EGD-e. Vinte e três cepas exibiram a mesma deleção que compreende 24 pares de bases dentro da seqüência de repetição e alterações similares na proteína traduzida. Apenas três cepas mostraram tamanhos diferentes de deleções e diferentes alterações na proteína. De acordo com estes resultados, a maioria das cepas apresentou uma característica molecular comum, diferentes da cepa padrão e este perfil predominante pode ser considerado como a característica das L. monocytogenes isoladas de produtos lácteos no Sul do Brasil. / The bacterium Listeria monocytogenes is recognized as an important human pathogen being omnipresent in the environment and is responsible for contamination in raw and processed foods. The mechanism of pathogenicity is established by presence of some genes on chromosome of bacteria between them, iap and hly genes that are essential to the invasion mechanism and hemolytic activity of microorganism, respectively. The aim of present study was confirm the strain L. monocytogenes using PCR to hly gene and analysis the nucleotide variability of central domain of iap gene characterized by the presence of similar sequences of threonine and asparagine amino acids. Twenty-six strains previously isolated from dairy products and classified by classic methods to L. monocytogenes were positive to specie-specific PCR and than submitted to nucleotide sequence determination. The results showed a sequence variation containing nucleotide substitutions, insertions, deletions and a number of repeated sequences among the isolates and control strain EGD-e. Twenty-three strains exhibited the same gap that includes a deletion of 24 base pairs inside of the repeated sequence and similar alterations in the translated protein. Just three strains showed different sizes of gaps and different protein alterations. According to these results, the majority strains displayed a common molecular characteristic different from the strain pattern and this predominant profile can be considerate as characteristic to L. monocytogenes isolated from dairy products in South Brazil.

A bactéria Listeria monocytogenes é reconhecida como um importante patógeno humano estando amplamente distribuída no ambiente e é responsável pela contaminação de alimentos crus e processados. O mecanismo de patogenicidade é determinado pela presença de genes no cromossomo da bactéria e entre eles estão os genes iap e hly que são essenciais para o mecanismo de invasão e atividade hemolítica do microorganismo, respectivamente. O obejtivo do presente estudo foi confirmar as cepas de L. monocytogenes usando a ténica de PCR para o gene hly e análise da variação nucleotídica do domínio central do gene iap, que é caracterizado pela presença de seqüências repetidas dos aminoácidos treonina e asparagina. Vinte e seis cepas de L. mnocytogenes, previamente isoladas de produtos lácteos e identificdas por métodos clássicos, se mostraram positivas para a PCR espécie-específico e então submetidas à determinação da seqüência nucleotídica. Os resultados mostraram variações na seqüência nucleotídica contendo substituições, inserções, deleções e um número de seqüências similares entre as cepas isoladas e a cepa controle EGD-e. Vinte e três cepas exibiram a mesma deleção que compreende 24 pares de bases dentro da seqüência de repetição e alterações similares na proteína traduzida. Apenas três cepas mostraram tamanhos diferentes de deleções e diferentes alterações na proteína. De acordo com estes resultados, a maioria das cepas apresentou uma característica molecular comum, diferentes da cepa padrão e este perfil predominante pode ser considerado como a característica das L. monocytogenes isoladas de produtos lácteos no Sul do Brasil. / The bacterium Listeria monocytogenes is recognized as an important human pathogen being omnipresent in the environment and is responsible for contamination in raw and processed foods. The mechanism of pathogenicity is established by presence of some genes on chromosome of bacteria between them, iap and hly genes that are essential to the invasion mechanism and hemolytic activity of microorganism, respectively. The aim of present study was confirm the strain L. monocytogenes using PCR to hly gene and analysis the nucleotide variability of central domain of iap gene characterized by the presence of similar sequences of threonine and asparagine amino acids. Twenty-six strains previously isolated from dairy products and classified by classic methods to L. monocytogenes were positive to specie-specific PCR and than submitted to nucleotide sequence determination. The results showed a sequence variation containing nucleotide substitutions, insertions, deletions and a number of repeated sequences among the isolates and control strain EGD-e. Twenty-three strains exhibited the same gap that includes a deletion of 24 base pairs inside of the repeated sequence and similar alterations in the translated protein. Just three strains showed different sizes of gaps and different protein alterations. According to these results, the majority strains displayed a common molecular characteristic different from the strain pattern and this predominant profile can be considerate as characteristic to L. monocytogenes isolated from dairy products in South Brazil.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-06-26T18:28:31Z No. of bitstreams: 2 Dissertação - Rafael Carvalho Couto - 2013.pdf: 41193945 bytes, checksum: 74a020dad23640afb84a085b841b91aa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2017-07-07T20:26:09Z (GMT) No. of bitstreams: 2 Dissertação - Rafael Carvalho Couto - 2013.pdf: 41193945 bytes, checksum: 74a020dad23640afb84a085b841b91aa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-07T20:26:10Z (GMT). No. of bitstreams: 2 Dissertação - Rafael Carvalho Couto - 2013.pdf: 41193945 bytes, checksum: 74a020dad23640afb84a085b841b91aa (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-04-15 / This work focuses on the development of the software MGA, which aims to determine the lowest energy structures of a given molecular system, using Genetic Algorithm (GA). The GA is a method of artificial intelligence that was developed to work with finding the best solutions of the specified conditions, ie, an algorithm that seeks the best answer desired, an optimal result. The MGA uses three techniques: Random Search (RS), Noninclusive Genetic Algorithm (NGA), Inclusive Genetic Algorithm (IGA). The last one is characterized by a new type of evolutionary strategy that allows in a single calculation and a single cycle, obtain several minimum of the potential energy surface. For optimum operation of the algorithm, was made an optimization of the parameters used in MGA, through response surface methodology. Using the techniques RS, IGA and NGA, were determined 141 distinct molecular structures of the amino acid asparagine. In the electronic structure calculations were considered the semi-empirical methods PM3, AM1 and RM1; and DFT potentials, with basis sets 6-311G ** and PC1. The RS determined the Global Minimum (GM) with ease, for the different potentials used, and proved that it’s quite useful in determining molecular geometries where there is no accuracy in the determination of local minima in order of energy. The NGA is efficient in determining the GM, performing in a shorter time, if compared to RS and IGA. The IGA proved to be a more robust method than the others, because in addition to determining the GM, it can find the local minima in order of energy. Performing calculations on an intermediate time of RS and NGA, the IGA determined the GM as the NGA, and found structures that were not founded using RS. The GM’s of asparagine determined using the potentials PC1, PM3, AM1 and RM1 have a large structural difference. This demonstrates that different potencials used in the electronic structure calculations may lead to different results. By analyzing the structures obtained for potentials PC1, PM3, AM1 and RM1, using the IGA, it appears that there is a difference in the topology of the potential energy surface of these potentials. / O presente trabalho é focado no desenvolvimento do software MGA, que tem como objetivo a determinação das estruturas de menor energia de um dado sistema molecular, utilizando o Algoritmo Genético (AG). O AG é um método de inteligência artificial que foi desenvolvido para trabalhar com a procura de soluções que melhor atendam as condições especificadas, isto é, um algoritmo que procura a melhor resposta desejada, um resultado ótimo. O MGA utiliza três técnicas: Busca Aleatória (RS), Algoritmo Genético Não-inclusivo (NGA), Algoritmo Genético Inclusivo (IGA). Este último é caracterizado por um novo tipo de estratégia evolutiva que permite em um único cálculo e um único ciclo evolucionário obter diversos mínimos da superfície de energia potencial. Para o melhor funcionamento do algoritmo, foi feita uma otimização dos parâmetros utilizados do MGA, através da metodologia de superfície de resposta. Utilizando as técnicas RS, NGA e IGA, foram determinadas 141 estruturas moleculares distintas do aminoácido asparagina. Nos cálculos de estrutura eletrônica foram considerados os métodos semi-empíricos PM3, AM1 e RM1; e potenciais DFT, com os conjuntos de base 6-311G** e PC1. O RS determinou o Mínimo Global (GM) com facilidade, para os diferentes potenciais utilizados, e se mostrou bastante útil na determinação de geometrias moleculares onde não há um rigor na determinação de mínimos locais em ordem de energia. O NGA é eficiente na determinaçãoao do GM, realizando em um menor tempo, se comparado ao RS e IGA. O IGA mostrou-se um método mais robusto que os outros, pois além de determinar o GM é possível encontrar os mínimos locais em ordem de energia. Realizando cálculos em um tempo intermediário ao RS e NGA, o IGA determinou o GM assim como o NGA, e encontrou estruturas que não foram possíveis utilizando o RS. Os GM’s da asparagina determinados utilizando os potenciais PC1, PM3, AM1 e RM1 possuem uma grande diferença estrutural. Isto demonstra que diferentes potencias utilizados nos cálculos de estrutura eletrônica podem levar a diferentes resultados. Ao analisarmos as estruturas obtidas para os potenciais PC1, PM3, AM1 e RM1, utilizando o IGA, constata-se que há uma diferença na topologia de suas superfícies de energia potencial.