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Etude moléculaire et structurale d'une intégrase rétrovirale pour le développement de nouveaux antirétroviraux et étude cristallographique d' -galactosidases thermostables issues du microorganisme Geobacillus stearothermophilus / Molecular and structural study of a retroviral integrase for the developemnt of new antiretroviral compounds and structural study of thermostable ɑ-galactosidases from Geobacillus stearothermophilus microorganismMerceron, Romain 04 November 2013 (has links)
L'intégrase (IN) est une protéine clé du cycle de réplication des rétrovirus et constitue une cible thérapeutique importante. Nous avons découvert par cristallographie aux rayons X, une nouvelle possibilité d'assemblage dimérique du domaine central catalytique de l'intégrase du Rous associated virus type I (RAV-1 IN). Dans le cadre de mon travail de thèse, un protocole de surproduction et de purification d'un mutant du domaine catalytique isolé (H103C) a été optimisé, afin de démontrer l'existence de cet assemblage en solution grâce à un pont disulfure inter-moléculaire. Différentes méthodes ont été mises au point, afin de tester la ca pacité de petites molécules d'intérêt à se lier et à stabiliser ce "nouvel" assemblage. Un protocole de surproduction et de purification de l'IN entière du RAV-1 a également été développé et mis au point. Des études structurales ont été réalisées. Un mutant H103C de la protéine entière a été produit, afin de vérifier la formation de la "nouvelle" interface sur la protéine entière. Le microorganisme Geobacillus stearothermophilus produit deux ɑ-galactosidases, AgaA et AgaB, qui appartiennent à la famille GH36 des glycosides hydrolases. Ces deux isoenzymes partagent 97 % d'identité de séquence, mais ont des activités catalytiques différentes. Les structures cristallines d'AgaA et AgaB ont été résolues ainsi que la structures du mutant AgaA et AgaB ont été résolues ainsi que la structure du mutant AgaA A355E, qui présente des caractéristiques enzymatiques similaires de AgaB. L'analyse de ces trois structures montre que la substitution A355E entraîne un déplacement significatif du tryptophane du sous-site catalytiques -1. Ce mouvement peut expliquer les spécificités catalytiques des deux isoenzymes. / Integrase (IN) is a key protein in the retrovirus life cycle and constitutes an important therapeutic target for the development of antiretroviral compounds. This enzyme is involved in the early phase of theretroviral replication cycle and catalyses the retrotranscribed viral DNA integration into the host cell genome.The teams of BioCrystallography and Retrovirology of Lyon Gerland demonstrated by X ray crystallography, the existence of a new dimeric assembly of the central catalytic domain (CCD) of Rous Associated Virus type 1integrase or RAV 1 IN. As part of my thesis work, a protocol of overproduction and purification of the H103Cisolated catalytic domain mutant was developed to demonstrate the existence of this dimeric assembly insolution stabilized by an inter molecular disulfide bond. Biochemical and biophysical methods were developed to test the ability of small molecules of interest to bind and stabilize this "new" assembly. A protocol ofoverproduction and purification of full length RAV1 integrase was developed. Crystallization trials and SAXSstudies were undertaken. The H103C mutant of the entire protein was produced to verify the formation of the"new" interface on the full length protein.The microorganism Geobacillus stearothermophilus produces two thermostable ɑ-galactosidases named AgaA and AgaB, which belong to theGH36 glycoside hydrolase family. These two isoenzymes share97% sequence identity, but have different catalytic properties. A collaborative study was initiated with theInstitute of Industrial Genetics, University of Stuttgart (Germany),to better understand the catalytic specificity of these two isoenzymes. The crystal structures of AgaA and AgaB were solved in two different crystal systems.The crystal structure of the mutant AgaA A355E, which has catalytic properties similar of those of AgaB, wasalso determined. These three structures show that the A355E substitution results in a signifiant displacement of the W336 tryptophan residue from the catalytic subsite -1. This could explain the catalytic specificities of the two isoenzymes
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Producción del dominio c-terminal del factor de iniciación IF2 de Escherichia coli y Geobacillus stearothermophilus como blanco de posibles aptámerosPerona, Francisco, Timoteo Prado, Adriana 13 December 2017 (has links)
Introduction: The initiation factor IF2 has different functions in the initiation of protein translation. Its C-terminal domain interacts strongly with fMet-tRNA. The main objective of this study is to isolate a specific aptamer for IF2 CTD Escherichia coli and Geobacillus stearothermophilus. Methods: A protein expression system based on E. coli BL21 was used. E. coli BL21 was transformed with InfB CTD sequence. Protein induction was performed under different temperatures and IPTG concentration. His-tagged affinity chromatography was used for protein purification since a hexa-His coding sequence was added to the InfB genes. Results: IF2 CTD was purified in high yields and purity. E. coli IF2 fragment protein concentration (558.0 ng/uL) was three times larger than that of G. stearothemophilus (144.6 ng/uL). Purity of both proteins were >95%. A specific aptamer for IF2 CTD G. stearothermophilus was obtained through SELEX. Conclusion: A rapid and efficient protocol for IF2 CTD protein purification has been developed, allowing the production of thermophilic and mesophilic IF2 fragments. The obtained aptamer may serve as a novel antibiotic mechanism in further studies. / Tesis
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La cisteina desulfurasa (IscS) de Geobacillus stearothermophilus V confiere a Escherichia Cole resistencia a la telurito de potasioTantalean Vásquez, Juan Carlos January 2003 (has links)
Doctor en Ciencias con mención en Microbiología
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Proposta metodológica e avaliação da inativação de endósporos de Geobacillus stearothermophilus no tratamento de resíduos de serviços de saúde por autoclavagem / Methodological proposal and evaluation of the inactivation of endospores of Geobacillus stearothermophilus in the treatment of healthcare waste by autoclavingOliveira, Amanda Borges Ribeiro de 27 January 2017 (has links)
Resíduos de Serviços de Saúde (RSS), ainda que tratados e dispostos em aterros sanitários, podem ser causas de impactos ambientais por apresentarem algum indicador de periculosidade. Segundo o apêndice IV da Resolução da Agência Nacional de Vigilância Sanitária (Anvisa) RDC nº 306/2004, para as tecnologias de tratamento desses resíduos é necessário que se atinja pelo menos o Nível III de inativação microbiana. Perante a ausência de dados na literatura que revelassem o tempo de exposição, temperatura e pressão ideais para a inativação microbiana no tratamento de RSS por autoclavagem e se a fração ocupada dos resíduos na autoclave poderia interferir na eficiência da desinfecção definiu-se o objetivo do trabalho para otimização do processo. Para os testes foram utilizados endósporos de Geobacillus stearothermophilus como bioindicadores e instalados cinco termopares na autoclave para aferição da temperatura dentro de todo o espaço da câmara. O RSS foi sintetizado diante das caracterizações como composição gravimétrica, distribuição granulométrica, densidade específica aparente, massa específica aparente e teor de umidade. Apresentou-se uma proposta metodológica para a avaliação do tratamento de RSS frente às dificuldades encontradas. Realizou-se ensaios com 116ºC, 125ºC e 134ºC, observando a fração de inativação em seis tempos de exposição diferentes. Sabendo-se que é padronizada em estabelecimentos de saúde a temperatura ideal para esterilização de materiais de 121ºC, apesar de não terem sido encontrados trabalhos científicos que comprovem a eficiência da esterilização nessa circunstância, foram realizados testes a 50 e 60 minutos de exposição para avaliar essa condição. Inoculou-se 106 endósporos nas amostras e a recuperação foi feita com filtração após a lavagem dos resíduos, sendo realizada a técnica de pour plate para contagem das unidades formadoras de colônias (UFC). O mesmo procedimento foi feito com a amostra retirada da autoclave. A fração de inativação dos endósporos atingiu 100% no tempo de 30 minutos de exposição à temperatura máxima de 134ºC e pressão absoluta de 2,3 kgf/cm2, resultado obtido através da relação do número de micro-organismos recuperados considerados como inoculados e o número de micro-organismos sobreviventes ao tratamento. A 121ºC houve recuperação de UFC após tratamento. A fração de ocupação não foi um fator delimitante para a inativação de endósporos, pois a temperatura se manteve a mesma independentemente da quantidade de resíduo submetido à autoclavagem. Um outro objetivo era avaliar experimentalmente a reprodução dos micro-organismos frente às condições operacionais de autoclavagem e do tempo de permanência do rejeito em condições ambientais. Esse estudo comprovou que a inativação nessas condições otimizadas esteriliza o resíduo, sendo que nenhum micro-organismo voltou a se reproduzir após dias expostos em temperatura ambiente, ou seja, não haveria riscos de contaminação em aterro sanitário quando depositados os rejeitos. Ter encontrado essas condições ideais e avaliado o processo de autoclavagem pode significar um grande avanço nas próprias unidades de tratamento, que terão um parâmetro estabelecido para trabalho. / Healthcare waste, even when treated and disposed of in landfills, can be causes of environmental impacts because they present some hazard indicator. According to appendix IV of Resolution of the National Health Surveillance Agency of Brazil (Anvisa) RDC nº 306/2004, for technologies of healthcare waste treatment, it is necessary to achieve at least Level III of microbial inactivation. The purpose of this research to optimize the process was defined taking into account the absence of data in the literature that revealed the optimal exposure time, temperature and pressure for microbial inactivation through the healthcare treatment by autoclaving and whether the fraction occupied by waste in the autoclave could interfere with the effectiveness of the disinfection. For the tests, endospores of Geobacillus stearothermophilus were used as bioindicators and five thermocouples were installed in the autoclave for temperature measurement throughout the chamber space. The healthcare waste was synthesized by characterizations such as gravimetric composition, granulometric distribution, apparent specific density, apparent specific mass and moisture content. A methodological proposal for the evaluation of waste treatment was introduced taking into consideration the difficulties encountered. Tests were performed at 116ºC, 125ºC and 134ºC, observing the fraction of inactivation at six different exposure times. Since the sterilization of materials at 121ºC is standardized in health establishments, even though no scientific studies were found to prove the sterilization efficiency in these conditions, tests were performed at 50 and 60 minutes of exposure to evaluate this condition. The concentration of 106 endospores were inoculated in the samples and the recovery was done with filtration after washing the waste, and the \"pour plate\" technique was used to count the colony forming units. The same procedure was done with the sample removed from the autoclave. The inactivation fraction of the endospores reached 100% in the time of 30 minutes of exposure to the maximum temperature of 134ºC and absolute pressure of 2.3 kgf/cm2, a result obtained by the ratio of the number of recovered microorganisms considered as inoculated and the number of microorganisms surviving the treatment. At 121ºC there was recovery of colony forming units after treatment. The occupation fraction was not a limiting factor for the inactivation of endospores, since the temperature remained the same regardless of the amount of waste submitted to autoclaving. Another objective was to experimentally evaluate the reproduction of the microorganisms taking into consideration the operational conditions of autoclaving and the amount of time that the waste remains under environmental conditions. This study proved that the inactivation under these optimized conditions sterilizes the waste, and that no microorganism would reproduce again after days exposed at room temperature, therefore, there would be no risk of contamination in a landfill when the waste is deposited. Having found these ideal conditions and evaluated the autoclaving process can mean a major advance in the treatment units themselves, which will have an established parameter for work.
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Proposta metodológica e avaliação da inativação de endósporos de Geobacillus stearothermophilus no tratamento de resíduos de serviços de saúde por autoclavagem / Methodological proposal and evaluation of the inactivation of endospores of Geobacillus stearothermophilus in the treatment of healthcare waste by autoclavingAmanda Borges Ribeiro de Oliveira 27 January 2017 (has links)
Resíduos de Serviços de Saúde (RSS), ainda que tratados e dispostos em aterros sanitários, podem ser causas de impactos ambientais por apresentarem algum indicador de periculosidade. Segundo o apêndice IV da Resolução da Agência Nacional de Vigilância Sanitária (Anvisa) RDC nº 306/2004, para as tecnologias de tratamento desses resíduos é necessário que se atinja pelo menos o Nível III de inativação microbiana. Perante a ausência de dados na literatura que revelassem o tempo de exposição, temperatura e pressão ideais para a inativação microbiana no tratamento de RSS por autoclavagem e se a fração ocupada dos resíduos na autoclave poderia interferir na eficiência da desinfecção definiu-se o objetivo do trabalho para otimização do processo. Para os testes foram utilizados endósporos de Geobacillus stearothermophilus como bioindicadores e instalados cinco termopares na autoclave para aferição da temperatura dentro de todo o espaço da câmara. O RSS foi sintetizado diante das caracterizações como composição gravimétrica, distribuição granulométrica, densidade específica aparente, massa específica aparente e teor de umidade. Apresentou-se uma proposta metodológica para a avaliação do tratamento de RSS frente às dificuldades encontradas. Realizou-se ensaios com 116ºC, 125ºC e 134ºC, observando a fração de inativação em seis tempos de exposição diferentes. Sabendo-se que é padronizada em estabelecimentos de saúde a temperatura ideal para esterilização de materiais de 121ºC, apesar de não terem sido encontrados trabalhos científicos que comprovem a eficiência da esterilização nessa circunstância, foram realizados testes a 50 e 60 minutos de exposição para avaliar essa condição. Inoculou-se 106 endósporos nas amostras e a recuperação foi feita com filtração após a lavagem dos resíduos, sendo realizada a técnica de pour plate para contagem das unidades formadoras de colônias (UFC). O mesmo procedimento foi feito com a amostra retirada da autoclave. A fração de inativação dos endósporos atingiu 100% no tempo de 30 minutos de exposição à temperatura máxima de 134ºC e pressão absoluta de 2,3 kgf/cm2, resultado obtido através da relação do número de micro-organismos recuperados considerados como inoculados e o número de micro-organismos sobreviventes ao tratamento. A 121ºC houve recuperação de UFC após tratamento. A fração de ocupação não foi um fator delimitante para a inativação de endósporos, pois a temperatura se manteve a mesma independentemente da quantidade de resíduo submetido à autoclavagem. Um outro objetivo era avaliar experimentalmente a reprodução dos micro-organismos frente às condições operacionais de autoclavagem e do tempo de permanência do rejeito em condições ambientais. Esse estudo comprovou que a inativação nessas condições otimizadas esteriliza o resíduo, sendo que nenhum micro-organismo voltou a se reproduzir após dias expostos em temperatura ambiente, ou seja, não haveria riscos de contaminação em aterro sanitário quando depositados os rejeitos. Ter encontrado essas condições ideais e avaliado o processo de autoclavagem pode significar um grande avanço nas próprias unidades de tratamento, que terão um parâmetro estabelecido para trabalho. / Healthcare waste, even when treated and disposed of in landfills, can be causes of environmental impacts because they present some hazard indicator. According to appendix IV of Resolution of the National Health Surveillance Agency of Brazil (Anvisa) RDC nº 306/2004, for technologies of healthcare waste treatment, it is necessary to achieve at least Level III of microbial inactivation. The purpose of this research to optimize the process was defined taking into account the absence of data in the literature that revealed the optimal exposure time, temperature and pressure for microbial inactivation through the healthcare treatment by autoclaving and whether the fraction occupied by waste in the autoclave could interfere with the effectiveness of the disinfection. For the tests, endospores of Geobacillus stearothermophilus were used as bioindicators and five thermocouples were installed in the autoclave for temperature measurement throughout the chamber space. The healthcare waste was synthesized by characterizations such as gravimetric composition, granulometric distribution, apparent specific density, apparent specific mass and moisture content. A methodological proposal for the evaluation of waste treatment was introduced taking into consideration the difficulties encountered. Tests were performed at 116ºC, 125ºC and 134ºC, observing the fraction of inactivation at six different exposure times. Since the sterilization of materials at 121ºC is standardized in health establishments, even though no scientific studies were found to prove the sterilization efficiency in these conditions, tests were performed at 50 and 60 minutes of exposure to evaluate this condition. The concentration of 106 endospores were inoculated in the samples and the recovery was done with filtration after washing the waste, and the \"pour plate\" technique was used to count the colony forming units. The same procedure was done with the sample removed from the autoclave. The inactivation fraction of the endospores reached 100% in the time of 30 minutes of exposure to the maximum temperature of 134ºC and absolute pressure of 2.3 kgf/cm2, a result obtained by the ratio of the number of recovered microorganisms considered as inoculated and the number of microorganisms surviving the treatment. At 121ºC there was recovery of colony forming units after treatment. The occupation fraction was not a limiting factor for the inactivation of endospores, since the temperature remained the same regardless of the amount of waste submitted to autoclaving. Another objective was to experimentally evaluate the reproduction of the microorganisms taking into consideration the operational conditions of autoclaving and the amount of time that the waste remains under environmental conditions. This study proved that the inactivation under these optimized conditions sterilizes the waste, and that no microorganism would reproduce again after days exposed at room temperature, therefore, there would be no risk of contamination in a landfill when the waste is deposited. Having found these ideal conditions and evaluated the autoclaving process can mean a major advance in the treatment units themselves, which will have an established parameter for work.
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Effects of Thermosonication on Microbial Population Reduction and Solubillity Index in Skim Milk PowderBeatty, Nicola F. 01 May 2016 (has links)
The effects of thermosonication (high intensity ultrasound coupled with thermal treatment), on the reduction of thermophilic spore-forming microorganisms and its effects on the solubility index in reconstituted skim milk powder (RSMP) were evaluated. Thermosonication was applied to RSMP at various solids concentrations, temperatures, and lengths of time based on commercial milk powder processing conditions. Microbial counts were determined prior to and after treatments to determine the log reduction of Geobacillus stearothermophilusvegetative cells and spores. Log reductions were recorded, and data were analyzed by response surface analysis. The log reductions induced by temperature and time without high intensity ultrasound (HIU) were compared to reductions observed with HIU. Thermosonication was also applied to RSMP to determine effects on solubility using a continuous flow cell system. Thermosonication yielded a significantly higher level of microbial destruction for both vegetative cells and spores than heat treatment alone. For experiments involving vegetative cells, the interaction of treatment time and temperature proved to have the greatest influence on microbial inactivation. In comparison, the interaction of total solids content and length of HIU treatment demonstrated the greatest effect on the increased log reductions for spores. The solubility of RSMP treated with HIU did not significantly differ from the solubility of RSMP not treated with HIU. Further data showed the implementation of HIU, or thermosonication, during milk powder processing would be most effective before and after the evaporation stage when the total solids content of product is 9.2% and 50% at 75°C and 60°C, respectively. Based on preliminary data, it is assumed HIU applied for 10 s at these two locations would produce an additive effect, thereby reducing overall microbial counts by 5.76 log and 0.51 log for G. stearothermophilus vegetative cells and spores, respectively, in the product prior to entering the drying stage. All research findings and observations suggest HIU, or thermosonication, to be a successful method for reducing microbial populations during milk powder processing without sacrificing skim milk powder solubility
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EVALUATION OF A RAPID BIOLOGICAL SPORE ASSURANCE TEST FOR DENTAL INSTRUMENT STERILIZATIONLee, Andie Hyunkyung January 2021 (has links)
Objectives: Dental instrument sterilization with steam autoclaves is critical to
maintaining infection control standards in dental practice, and preventing patient-to-patient
transmission of pathogenic bacteria and viruses. The American Dental
Association and the United States Centers for Disease Control and Prevention
recommend, and many state dental laws require, weekly use of biological spore tests to
verify dental instrument sterilization outcomes. However, the most widely used
biological spore test needs microbial culture incubation for 2 days after autoclave
exposure, which limits swift identification of sterilization failure.
To address this issue, this study evaluated the reliability of a new rapid biological
spore test for determining the sterilization efficacy of dental steam autoclaves within 20
minutes.
Methods: Two commercial biological spore tests were evaluated in Temple
University dental school steam autoclaves, 1.) the Steris Celerity 20 Steam Biologic
Indicator with a 20-minute outcome time requirement, and 2.) the 3M Attest 1262
Biological Indicator with a 48-hour outcome time requirement. Both biological spore
tests employed live thermoresistant Geobacillus stearothermophilus spores as an
indicator of whether sterilization conditions in steam autoclaves were met or not. To
compare their efficacy, a total of 157 pairs of the two biological spore tests were placed
into dental steam autoclaves with dental instrument cassettes, and subjected to
manufacturer-recommended steam autoclave temperature and air pressure operating
conditions for an adequate sterilization time of 15 minutes. Two additional groups of 10
pairs each of the two biological indicators were subjected to appropriate steam autoclave
temperature and air pressure settings, but only for aborted non-sterilizing time periods of
10 and 5 minutes, respectively. Subsequent aseptic processing and laboratory incubation
of both biological indicators was initiated within 2-24 hours, and followed manufacturer
recommendations.
After autoclave exposure, Steris Celerity 20 Steam Biologic Indicator test
ampoules were incubated in a specialized instrument for 20 minutes at 57 °C, which also
spectrophotometrically evaluated the microbial culture medium for fluorescent α-
glucosidase enzyme signal changes. No change in fluorescent intensity represented
successful sterilization, whereas increased fluorescence indicated survival of viable G.
stearothermophilus spores germinating into vegetative bacterial cells after failed
sterilization.
3M Attest 1262 Biological Indicator ampoules were incubated for 48 hours in a
laboratory heating block at 57 °C, after which a pH-based color change in the microbial
culture broth was visually assessed. No change in the color of the culture broth (purple
color remains) indicated successful sterilization, whereas development of a yellow color
in the culture broth, as a result of viable G. stearothermophilus spore germination into
vegetative bacterial cells, denoted failed sterilization.
Results: A total of 354 biological indicators were exposed to dental steam
autoclaves sterilization cycles, incubated for either 20 minutes or 48 hours, and evaluated
for G. stearothermophilus spore growth. The Steris Celerity and 3M Attest biological
spore tests both uniformly detected successful sterilization, with no G. stearothermophilus
spore growth, after 15 minutes of steam autoclave exposure at manufacturer recommended
steam autoclave temperature and air pressure operating conditions. This
provided 100% agreement, and no statistically significant difference in the prevalence of
successful sterilization outcomes, between 157 pairs of both biological indicator types
after 15 minutes of steam autoclave exposure.
Similarly, both biological spore test systems were also in complete agreement
after only 5 minutes of steam autoclave exposure, with 100% of both biological
indicators positive for G. stearothermophilus spore growth, indicating failed sterilization.
In contrast, after 10 minutes of steam autoclave exposure, there was a complete
lack of agreement between the two types of biological indicators. All 10 Steris Celerity
spore tests were positive, whereas all 10 3M Attest ampoules were negative, for G.
stearothermophilus spore growth after 10 minutes of steam autoclave exposure. Relative
to this disagreement, a non-biological chemical indicator strip that was part of the Steris
biological indicator test system failed to have a darkened bar develop and extend into the
“Accept (OK)” portion of the strip for all Steris Celerity spore tests exposed to either 5
minutes or 10 minutes of steam autoclave exposure, indicating that adequate autoclave
steam, temperature and/or time parameters had not been attained for sterilization.
Conclusions: The Steris Celerity biological spore test was successful in rapidly
determining the sterilization efficacy of dental steam autoclaves within only a 20-minute
incubation time period, as compared to 48 hours of incubation required by the widely-used
3M Attest biological spore test. As a result, this rapid assay offers earlier detection
of steam autoclave sterilization failure before potentially contaminated dental instruments
are used in clinical patient care.
The alarming failure of 3M Attest biological spores to grow after a non-sterilizing
10-minute steam autoclave exposure time warrants further product evaluation. / Oral Biology
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Characterization of the Group II Intron Gs. Int1 from the Thermophilic Bacterium <em>Geobacillus stearothermophilus</em>.Sun, Huijing 14 August 2007 (has links) (PDF)
Group II Introns are small segments of DNA that reside in the chromosome of bacteria or the organelles of primitive eukaryotes. These elements have some very interesting properties. First, they are retrotransposons that can move from one location to a new location in DNA via a reverse transcription mechanism. Second, they form a large ribozyme that mediates self-splicing of the intron from pre-mRNA. A Group II Intron type protein with similarity to reverse transcriptase was discovered in the thermophilic bacterium Geobacillus stearothermophilus strain 10 (Vellore et al., 2004, Appl. Environ. Microbiol. 70: 7140-7147). Numerous copies of the intron, designated Gs. Int1, are present in the chromosome of strain 10 but absent from a related strain ATCC 12980. Experiments to detect the in vivo splicing of intron Gs.Int1 from G. stearothermophilus cells did not work. Plasmids to that will over-express the Gs. Int1 intron to detest splicing in vivo in Escherichia coli have been constructed.
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Structural, Kinetic and Mutational Analysis of Two Bacterial CarboxylesterasesLiu, Ping 04 August 2007 (has links)
The crystal structures of two thermostable carboxylesterase Est30 and Est55 from Geobacillus stearothermophilus were determined to help understand their functions and applications in industry or medicine. The crystal structure of Est30 was determined at 1.63 Å resolution by the multiple anomalous dispersion method. The two-domain Est30 structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. A 100 Da tetrahedral ligand, propyl acetate, was observed to be covalently bound to the side chain of Ser94 in the catalytic triad. This ligand complex represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily. Est55 is a bacterial homologue of the mammalian carboxylesterases involved in hydrolysis and detoxification of numerous peptides and drugs and in prodrug activation. Est55 crystals were grown at pH 6.2 and pH 6.8 and the structures were determined at resolutions of 2.0 and 1.58 Å respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. This structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is pointing away from the active site. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the entry of substrate to its binding site. This structure suggested a self-inactivation mechanism, however, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains at this position were favorable, while polar serine was unfavorable for enzyme activity. Both Est30 and Est55 were shown to hydrolyze the prodrug CPT-11 into the active form SN-38. Therefore, Est30 and Est55 are potential candidates for use with irinotecan in cancer therapy. The catalytic efficiency (kcat/Km) of Est30 is about 10-fold lower than that of Est55. The effects of the Cys408 substitutions on Est55 activity differed for the two substrates, p-NP butyrate and CPT-11. Mutant C408V may provide a more stable form of Est55.
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<b>Microbial Inactivation and Validation of Aseptic Processing and Packaging System Using Vapor Peroxide</b>Manoj Ram Krishna Sawale (16840431) 23 June 2024 (has links)
<p dir="ltr">Liquid hydrogen peroxide (LHP) and vapor hydrogen peroxide (VHP) efficacy as a sterilant for <i>Bacillus atrophaeus</i> and <i>Geobacillus stearothermophilus</i> spores in aseptic packaging systems under commercial sterilization conditions were evaluated in this research. The work centers on quantifying and modeling the kinetic parameters that impact peroxide sterilization efficacy, including the D and z values, that relate to the change in concentration required for a 1-log reduction in spore population and a novel Z<sub>conc</sub> parameter This comprehensive study is divided into four key investigations, each contributing critical insights to the overall understanding of peroxide sterilization processes.</p><p dir="ltr">The first study examined the inactivation kinetics of <i>B. atrophaeus</i> spores in liquid hydrogen peroxide. By evaluating different concentrations (20%, 28%, and 33%) and temperatures (up to 82.2°C), the study revealed that higher concentrations and elevated temperatures significantly enhanced spore inactivation. The Weibull model provided a more accurate fit for the data, indicating a non-linear relationship between spore reduction and exposure time.</p><p dir="ltr">The second part of the research explored the use of VHP for sterilizing <i>B. atrophaeus</i> spores. With VHP concentrations of 2500 ppm and 4450 ppm at various temperatures, the study demonstrated that higher concentrations and temperatures are highly effective for spore inactivation. Both log-linear and Weibull models accurately described the inactivation kinetics, with the Weibull model showing a slightly better fit, emphasizing the potential of VHP in achieving commercial sterility.</p><p dir="ltr">The third investigation focused on developing predictive models for VHP concentration and its efficacy in spore inactivation. The study evaluated VHP concentrations ranging from 2.32 mg/L to 7.35 mg/L and their impact on spore inactivation rates. The Weibull model proved particularly effective in predicting the inactivation of <i>G. stearothermophilus</i> and <i>B. atrophaeus</i> spores, offering a robust tool for optimizing sterilization protocols in aseptic packaging.</p><p dir="ltr">The fourth and final study of the research investigated the influence of surface roughness on spore survival during VHP sterilization cycles on plastic packaging materials. Artificial roughness on high-density polyethylene (HDPE) coupons was created using sandpaper with different grits. <i>B. atrophaeus</i> spores were applied to both roughened and smooth HDPE surfaces. The study finds that rougher surfaces provide more shelter for spores, reducing sterilization efficacy. For example, surfaces roughened with P-36 grit showed a 2.75 log reduction in spore count, whereas smoother surfaces with P-220 grit achieved a 4.42 log reduction. Contact angle measurements indicated that increased roughness led to more hydrophilic surfaces, with water contact angles decreasing from 149.7° for the pristine sample to 105.4° for the P-36 sample. Scanning electron microscopy (SEM) images confirmed that spores were more likely to reside in the valleys of rough surfaces, highlighting the importance of surface characteristics in optimizing VHP sterilization protocols.</p><p dir="ltr">The findings of this dissertation underscore the significant impact of hydrogen peroxide concentration, application conditions, and packaging material surface properties on the efficacy of spore inactivation during sterilization. By providing a comprehensive understanding of these factors, the research contributes to the development of optimized aseptic sterilization protocols, enhancing the reliability and safety of aseptically packaged food and pharmaceutical products. This work will ensure compliance with regulatory standards and improve food safety in commercial manufacturing, laying a solid foundation for future research and practical applications in VHP sterilization technology.</p>
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