Geobacillus thermoglucosidasius (GT) is a thermophilic, ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. One strategy to improve fermentation yields would be to engineer GT strains to secrete hydrolases to increase the amount of available sugars from various feedstocks. Therefore, optimised protein secretion would be vital to improve feedstock utilisation. Secretion in the related mesophile Bacillus subtilis (BS) has been well studied, and several strategies have been developed to improve secretion of heterologous proteins in BS, one such strategy being the manipulation or changing of the signal peptide. One aim is to identify any differences in the secretion machinery and signal sequences between GT and BS. Another aim is to analyse any effects of overproduction of hydrolases and to identify any bottlenecks in protein secretion in GT. Using bio-informatics tools we find that although GT is a thermophile, the signal peptides in this organism do not differ significantly from those in BS. From a shotgun mass spectrometry approach it was also observed that unlike BS, GT undergoes significant cell lysis during growth releasing cytoplasmic proteins into the extracellular milieu, which could have implications on the levels of secreted hydrolases. A model enzyme was selected and over-produced at high levels in order to stress the secretion system in GT so as to identify any bottlenecks in secretion. The results thus far indicate that the rate limiting step in secretion could be post-translocation where the enzyme is degraded by proteases in the cell wall and extracellular milieu. The addition of protease inhibitor to growth media, increases the activity and abundance of the enzyme, suggesting that proteolysis may be a major factor when over-producing secreted enzymes at high levels.

<p>Nitrile hydratases (NHases) are enzymes that catalyse the conversion of organocyanides to amides via a non-hydrolytic hydration reaction. They are industrially relevant enzymes, currently used in the manufacture of nicotinamide and acrylamide. The target of this study belongs to the thermophilic bacteria Geobacillus pallidus.</p>

Fifty two cellulolytic thermophilic microorganisms were analyzed for their physiological characterization and phylogenetic systematics. Based on 16S rDNA sequence analysis, 3 strains from DCB and 4 novel isolates from southern Taiwan are close related to the genera of Bacillus and Geobacillus respectively. Among 4 new Geobacillus strains, strain T4, a Gram negative, motile, aerobically growing sporulating rod, can secrete thermostable endoglucanase. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70¢XC. Based on 16S rDNA sequence analysis, DNA G+C content, phenotypic and physiological characteristics, as well as DNA-DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). Furthermore, a phylogenetic tree of 20 related microorganisms was also constructed based on their thermostable cellulase amino acid sequences. Our sequence analysis shows that cellulases belonging to the large family of glycoside hydrolases (GHs) can be divided into four subfamilies: TC-1 (GH family 12 group), TC-2 (bacterial group £L in which fungal species Thermoascus aurantiacus fits), TC-3 (bacterial group £L£L), and TC-4 (GH family 1 group). Together with the 16S rDNA sequence analysis of strain T4 and 10 related microorganisms, strain T4 has a close phylogenetic relationship with subfamily TC-4 but far from subfamily TC-1.

<p>Nitrile hydratases (NHases) are enzymes that catalyse the conversion of organocyanides to amides via a non-hydrolytic hydration reaction. They are industrially relevant enzymes, currently used in the manufacture of nicotinamide and acrylamide. The target of this study belongs to the thermophilic bacteria Geobacillus pallidus.</p>

Tesis de Magíster en Bioquímica área de Especialización en Bioquímica de proteínas y biotecnología y Memoria para optar al Título de Bioquímico / Las lacasas son enzimas de la familia de las multi-cobre oxidasas que han suscitado mucho interés debido a su amplia especificidad de sustrato, la variabilidad de sus propiedades entre cada especie, y su todavía elusivo mecanismo de reacción. La presencia de cuatro centros de cobre en su sitio activo la convierte en un modelo interesante para el estudio de la relación entre la estructura y la función de proteínas. Estos átomos de cobre son además responsables de las propiedades catalíticas de las lacasas y exhiben propiedades redox y espectroscópicas que son propias de estas enzimas. Es posible encontrarlas en hongos, plantas y en procariontes. Las lacasas bacterianas poseen muchas ventajas desde el punto de vista aplicado por sobre las lacasas de hongos, que hasta hoy se encuentran mejor caracterizadas. En general poseen una mayor termoestabilidad, y exhiben un perfil de pH distinto, lo que permite ampliar el número de aplicaciones de estas enzimas. Sus propiedades las hacen buenas candidatas para diversas aplicaciones biotecnológicas, que van desde la industria textil hasta la industria de alimentos, e incluyen su aplicación como biosensores, su uso en bioremediación de suelos y aguas entre otros. En este trabajo se caracteriza una lacasa recientemente detectada en el microorganismo termófilo antártico Geobacillus sp. ID17. Esta es la primera lacasa caracterizada de una bacteria antártica, y la primera lacasa intracelular descrita de este género bacteriano. La purificación de la enzima se logró utilizando cromatografía de intercambio aniónico y exclusión molecular. Luego de obtener una muestra pura se determinaron sus propiedades bioquímicas y su rango de sustratos. La lacasa purificada demostró poseer una alta estabilidad térmica a pH neutro y 55 ºC. Se detectó la mayor actividad enzimática en el rango entre 55 y 60 ºC, a pH 7,5. Además la enzima posee una mayor selectividad de sustrato que otras lacasas descritas a la fecha. Adicionalmente, se describe la purificación parcial de una segunda enzima presente en el extracto crudo del Geobacillus sp. ID17, que demostró poseer una actividad peroxidasa dependiente de cobre / Laccases are enzymes from the Multi-Copper Oxidase family that have gathered high attention due to their wide range of substrates, their interspecies variability and their still elusive mechanism of action. The presence of four copper atoms in their active site makes them an interesting model for the study of the relationship between the structure and function of proteins. These copper atoms are responsible for the catalytic properties of laccases and exhibit redox and spectroscopic features unique to this class of enzymes. It is possible to find them in fungi, plants, and prokaryotes. Bacterial laccases display many advantages over fungal laccases for their application. They have, in general, a higher thermal stability and a different pH profile, which contributes to widen the possible application of laccases. In this thesis a new laccase from an Antarctic microorganism, Geobacillus sp. ID17, is purified and characterized. This is the first Antarctic bacterial laccase to be functionally described, and the first intracellular laccase from this bacterial genus. The pH profile, catalytic constants, and thermal behavior of the enzyme is described. A screening of different laccase substrates reveals that this new laccase has different substrate specificity than the commercial laccase from Trametes versicolor. The enzyme was purified using anionic exchange and size-exclusion chromatography. After obtaining a pure enzyme, its biochemical parameters and substrate specificity were determined. The purified laccase showed high thermal stability at neutral pH and 55 ºC. The highest activity was detected between 55 ºC and 60 ºC, at pH 7,5. Additionally, this enzyme shows higher substrate selectivity than other previously described laccases. Additionally, the partial purification of a second enzyme present in the crude extract of Geobacillus sp. ID17, which seems to display a copper dependent peroxidase activity, is described

Magister Scientiae - MSc / Nitrile hydratases (NHases) are enzymes that catalyse the conversion of organocyanides to amides via a non-hydrolytic hydration reaction. They are industrially relevant enzymes, currently used in the manufacture of nicotinamide and acrylamide. The target of this study belongs to the thermophilic bacteria Geobacillus pallidus. The G. pallidus RAPc8 NHase is a heterotetramer that has a 28 kDa α subunit and a 29 kDa β subunit, with a α2β2 configured functional unit. The G. pallidus RAPc8 NHase operon has been cloned, sequenced and expressed at high levels in E. coli, with subsequent crystallisation for X-ray diffraction analysis. Current work is based on the study of the reaction mechanism of NHase as proposed by Mitra and Holz (2006). This mechanism implicated βY72 and βW76 in the hydration of nitriles. The study involved the site directed mutation of the β subunit of G. pallidus RAPc8 NHase, resulting in the generation of a βY72FβW76V mutant. This was followed by sequencing, expression and purification of the recombinant protein. The pH-activity and temperature- activity profiles of the mutant showed the enzyme to be optimally active at pH 6.4 and 55oC, respectively. Further kinetic analysis of the mutant found optimal kcat/KM values of 94.7 + 7.5 at 50oC and pH 5.8. The determination of the kinetic parameters under differing environmental conditions such as changes in temperature and pH revealed the ionisation constants of the enzymesubstrate complex (pKES1 and pKES2) to be 5.9 and 7.7, respectively. These results suggest that the active site sulphinic acid as opposed to the active site tyrosine may be responsible for catalysis. The active site tyrosine probably plays a substrate-stabilising role. The fluctuation of the kcat, KM and kcat/KM values demonstrated the need to use an alternative measure of enzyme efficiency. A more appropriate term could be the enzyme efficiency function, Ef, which considers the dependence of the enzyme efficiency on substrate concentration. / South Africa

The aim of this master's thesis is to study the biosorption of heavy metals copper, lead and zinc on thermophilic bacteria of the genus Geobacillus – G. thermodenitrificans CCM 2566 and G. thermocatenulatus CCM 2809. Biosorption was carried out in a batch stirred system for each metal separately. The influence of pH, biomass concentration and initial metal concentration on sorption capacity of bacteria was investigated. For both bacteria the optimum biosorption pH values for copper, lead and zinc were 5; 4 and 5, respectively. Sorption capacity of both bacteria was higher at lower concentrations of biomass and increased with increasing initial metal concentration. The confrontation of sorption capacities showed that the bacterium G. thermocatenulatus has greater sorption potential than G. thermodenitrificans.

Three thermophilic aerobic bacterial strain, Geobacillus thermoleovorans T4, Bacillus subtilis b5 and Bacillus licheniformis b6 were isolated from the wastewater of a Taiwan Sugar Company sugar refinery in Kaohsiung. All strains were capable of growth in cellulose containing medium. We found out that G. thermoleovorans T4 possessed both of the activity of cellulses and xylanase. This was the first report ever in this bacterial spices. Its endoglucanase was active at temperatures ranging from 30¢J to 100¢J, and pH ranging from 4.0 and 8.0. Its £]-glucosidase activities could be measured at 40¢J - 90¢J and pH 5.0 - 9.0. Its xylanase was active from 30¢J to 90¢J and pH from 3.0 to 10.0, while the exoglucanase was active at temperatures from 30¢J to 90¢J. The pH values of xylasnase was better than G. thermoleovorans K-3d. This enzyme was worth of possible in industry application. Both of B. subtilis b5 and B. licheniformis b6 contained a thermoactive endoglucanase. The endoglucanase of B. subtilis b5 was active at pH values between 4.0 and 7.5, and was thermostable at 100¢J for at least 1 hour ( retaining 60% of the original activity ). The stability of this enzyme was better than all known enzymes extracted from Bacillus spices. The endoglucanase of B. licheniformis b6 was active at pH values between 4.0 and 9.0, and thermostable at 60¢J for least 1 hour.

>Magister Scientiae - MSc / Due to economic and environmental concerns associated with use of fossil fuels, humanity is seeking alternative fuels. Ethanol is one of the alternative fuels produced commercially. Current ethanol production technologies using first generation ethanol processes is criticised for depleting the food supply and escalating food prices. Biomass is a target feedstock for use in bioethanol production and would resolve the criticism associated with the current bioethanol industry. Bacterial strains such as Geobacillus thermoglucosidasius NCIMB 11955 can be used to produce ethanol from biomass because they assimilate hexose and pentose sugars, a property that is lacking in first generation ethanol producing microbes (Saccharomyces cerevisiae and Zymomonas mobilis) (Riyanti and Rogers, 2009). Due to the low ethanol tolerance (4 % v/v (maximum)) of G. thermoglucosidasius, use of this species for bioethanol production is not economically viable. GroES and GroEL genes are involved in stress tolerance in bacteria: activation of these genes has been observed in stress induced bacteria (Rasouly and Ron, 2009). In this study the ethanol tolerance of G. thermoglucosidasius NCIMB 11955 was characterised by culturing at 45 ºC and 55 ºC in the presence of ethanol. A greater ethanol tolerance was observed at the sub-optimal growth temperature of 45 ºC. Escherichia coli metabolic systems are well understood. Aiming to improve the ethanol tolerance G. thermoglucosidasius NCIMB 11955, the GroES and GroEL genes of the organism were cloned in an expression vector and expressed in E.coli before testing their ability to confer an increased tolerance to ethanol. Proteomic analysis of the recombinant E. coli strain showed that GroES was over-expressed while GroEL was not. After over expression of GroES, the optical density of cultures was periodically measured. Over-expression of the G. thermoglucosidasius NCIMB 11955 GroES gene improved the ethanol tolerance of E. coli Rosetta pLySs growing in 4% (v/v) ethanol.

Bendrąja prasme kolagenolizė - dalinė arba visiška konkretaus kolageno tipo molekulės proteolizė, vykstanti in vivo (fiziologinių ar patologinių procesų metu) ar in vitro. Vandens molekulė, kaip įprasta hidrolizės reakcijoms, būtinas antrasis kolagenolizės proceso substratas. Kolagenolizę katalizuoja kolagenoliziniu specifiškumu pasižyminčios peptidazės. Kolagenai -vieni svarbiausių baltymų tiek atliekamų funkcijų, tiek biotechnologinio panaudojimo atžvilgiais. Jų panaudojimas neįmanomas be kolagenolizės proceso, taigi -kolagenolizinių peptidazių. Tik nedaugeliui peptidazių būdingas kolagenolizinis specifiškumas. Eukariotų ir prokariotų skirtumai kolageno kaip kolagenolizės substrato produkavimo galimybės atžvilgiu, taip pat netapačios šio fibrilinio baltymo biologinės funkcijos šiuose gyvybės domenuose lėmė skirtumus tarp prokariotų ir eukariotų kolagenolizinių peptidazių. Nors prokariotinės kolagenolizinės peptidazės tiriamos vis intensyviau, daugiau dėmesio skiriama patogeninių kamienų (Clostridium, Porphyromonas genčių) produkuojamoms kolagenazėms. Tuo tarpu nepatogeninių prokariotų kolagenazių tyrimai vykdomi rečiau. Ilgainiui tai gali lemti nevisavertį prokariotų kolagenazių biotechnologinio potencialo išnaudojimą bei iš dalies riboti pačio kolageno taikymą, sprendžiant konkrečias problemas. Biotechnologinio panaudojimo poreikiams tenkinti būtina klonuoti kolagenolizinių peptidazių genus, pritaikant efektyvias ekspresijos sistemas. Darbo tikslas: Atlikti termofilinių... [toliau žr. visą tekstą] / Collagens are one of the most important proteins including their functions and biotechnological application. The biotechnological application of collagens wouldn‘t be possible without the process of collagenolysis which is catalysed by collagenolytic peptidases. The aim of this work was to perform transcriptional analysis of U32.002 (Helicobacter-type) collagenolytic peptidase gene and its cloning as well as basic analysis. The analysis of U32.002 peptidase gene transcription was performed after the extraction of total RNA from G. lituanicus DSM 15325T cells that were in two different stages of growth. Reverse transcription assays were performed after total RNA extraction. After the process of reverse transcription samples of cDNA were used for the diagnostic PGR that was carried out by using five different primers. This gene was cloned with and without putative signal/pro sequence in order to produce the preparation of U32.002 peptidase. Full sequence of U32.002 peptidase gene was cloned into pTZ57R/T and then into the expression vector pET28c(+). U32.002 gene without putative signal/pro sequence was cloned into pJET1.2, and then into pET28c(+). SDS-PAGE and MALDI-TOF analyses were performed in order to determine the fact of expression in E. coli BL21 (DE3). Full scale expression optimisation was performed, as well. The influence of calcium and zinc ions to the structural stability of U32.002 peptidase with putative signal/pro sequence was analysed. The collagenolytic... [to full text]