In this study, effects of different carbon sources and their feeding strategies on recombinant human growth hormone (rhGH) production by Pichia pastoris were investigated by means of cell growth, recombinant protein production and expression levels of hGH and alcohol oxidase (AOX) genes. In this content, firstly, the strain to be used for high level rhGH production was selected between the two phenotypes, i.e., P. pastoris hGH-Mut+ and P. pastoris hGH-MutS. In this selection both phenotypes were compared in two different media containing glycerol/methanol or sorbitol/methanol and P. pastoris-hGH-Mut+ strain grown on medium containing 30 g/L sorbitol with 1% (v/v) methanol was found to have the highest hGH expression level and rhGH production level, 9.84x109 copies/mg CDW and 120 mg/L, respectively. Thereafter, effects of sorbitol, mannitol, fructose, lactose, sucrose, citric acid, lactic acid and acetic acid were investigated by using P. pastoris hGH-Mut+ strain in laboratory scale bioreactors. Among them sorbitol and sucrose were selected to be compared for production in pilot scale bioreactors by adding them batch-wise at the beginning of induction phase with fed batch methanol feeding scheme at &amp / #956 / =0.03h-1. It was shown that sucrose does not support cell growth as sorbitol although it does not repress recombinant protein production. Then three different feeding strategies were applied to develop sorbitol/methanol mixed feeding i) single sorbitol addition at t=0, ii) besides at t=0, adding second batch-wise sorbitol at t=9 h, iii) giving pulse methanol at t=24 h to trigger AOX promoter. These three strategies were compared with a production without addition of co-substrate sorbitol. Substrate consumption, cell growth, recombinant protein production and expression levels of hGH and AOX were investigated for these different feeding strategies. The highest cell concentration was achieved in third strategy as 55 g/L where the highest extracellular rhGH production (301 mg/L) was achieved in the second strategy, with addition of two times of sorbitol. For this highest recombinant protein production case, overall cell and product yield on total substrate were found as 0.17 g/g and 1.71 mg/g, respectively. Moreover, the highest hGH and AOX expression levels were obtained in this strategy.

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No description available.

No description available.

High nitrogen discharge from the municipal wastewater is a major concern for the South Australian Government, primarily due to negative impacts on the marine environment. Therefore, under the South Australian Environmental Improvement Program, (SA EIP), all metropolitan wastewater treatment plants have been reconfigured to achieve enhanced nitrogen removal. Secondary treatment (denitrification process) at the metropolitan wastewater treatment plants must be optimised to meet the discharge guideline of 10 mg/L total nitrogen. However, secondary treatment at some plants is carbon limited (low C/N ratio), and external carbon supplementation is required to meet this discharge guideline. Molasses provides the current external carbon source at two plants. It is relatively inexpensive, but other carbon sources, particularly industrial waste streams, may be more attractive, due to the potentially lower material cost, as it is practically free, and environmentally friendly. Potato starch and sonicated return activated sludge (RAS) were considered. In this study, the bioavailability of the soluble carbon in potato starch and ultrasound treated RAS were assessed. The associated objective was to investigate the potential of both carbon sources as an external carbon donor for the denitrification zone of wastewater treatment plants to economically improve biological nitrogen removal. The economic analysis was performed using mainly United States dollars and the fixed capital investments and total capital costs were converted to Australian dollars. This was due to the United States dollars currency quotes obtained for the materials and unit operations required. SCOD from the three sources was quantified and preliminary results were presented. Molasses provided the highest SCOD release of 1.1285 x 10⁶ mg-SCOD/L, sonicated RAS produced 5.6 to 68.4 times the SCOD release of the untreated RAS (35.6 mg-SCOD/L) depending on the ultrasound intensity and treatment time, while the highest soluble carbon release obtained using potato starch was 809 mg-SCOD/L (using 20.9 g/100 mL potato starch concentration). Based on the experimental SCOD results, batch denitrification tests using the proposed carbon sources were carried out. The nitrogen removal efficiency at low dose (12.48 mg-SCOD/L) using molasses, potato starch and sonicated RAS were 77.54%, 57.24%, and 72.76% respectively, whilst at high dose (124.80 mg-SCOD/L) were 94.04%, 66.32%, and 92.10% correspondingly. In similar order of the proposed carbon sources, the nitrate removal rates for the first phase denitrification with low dose were 1.44, 1.16, and 1.18 mg-NO₃ − /h respectively, whilst the nitrate removal rate of the first phase denitrification with high dose improved to 2.01, 1.26, and 1.96 mg-NO₃ −/h correspondingly. From the denitrification test results, molasses proved to be the optimal carbon source in terms of nitrate removal. However sonicated RAS possesses similar denitrification performance and may be a suitable alternative. An economic analysis for sonicated RAS Option 2 confirmed it as the most viable substitute. The time to recover the initial investment (payback period) is approximately 6.5 years and the breakeven point is approximately 8 years. Both denitrification tests and economic analyses demonstrate that sonicated RAS may be a viable and attractive substitute for the molasses. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337059 / Thesis (M.Eng.Sc.) - University of Adelaide, School of Chemical Engineering, 2008

High nitrogen discharge from the municipal wastewater is a major concern for the South Australian Government, primarily due to negative impacts on the marine environment. Therefore, under the South Australian Environmental Improvement Program, (SA EIP), all metropolitan wastewater treatment plants have been reconfigured to achieve enhanced nitrogen removal. Secondary treatment (denitrification process) at the metropolitan wastewater treatment plants must be optimised to meet the discharge guideline of 10 mg/L total nitrogen. However, secondary treatment at some plants is carbon limited (low C/N ratio), and external carbon supplementation is required to meet this discharge guideline. Molasses provides the current external carbon source at two plants. It is relatively inexpensive, but other carbon sources, particularly industrial waste streams, may be more attractive, due to the potentially lower material cost, as it is practically free, and environmentally friendly. Potato starch and sonicated return activated sludge (RAS) were considered. In this study, the bioavailability of the soluble carbon in potato starch and ultrasound treated RAS were assessed. The associated objective was to investigate the potential of both carbon sources as an external carbon donor for the denitrification zone of wastewater treatment plants to economically improve biological nitrogen removal. The economic analysis was performed using mainly United States dollars and the fixed capital investments and total capital costs were converted to Australian dollars. This was due to the United States dollars currency quotes obtained for the materials and unit operations required. SCOD from the three sources was quantified and preliminary results were presented. Molasses provided the highest SCOD release of 1.1285 x 10⁶ mg-SCOD/L, sonicated RAS produced 5.6 to 68.4 times the SCOD release of the untreated RAS (35.6 mg-SCOD/L) depending on the ultrasound intensity and treatment time, while the highest soluble carbon release obtained using potato starch was 809 mg-SCOD/L (using 20.9 g/100 mL potato starch concentration). Based on the experimental SCOD results, batch denitrification tests using the proposed carbon sources were carried out. The nitrogen removal efficiency at low dose (12.48 mg-SCOD/L) using molasses, potato starch and sonicated RAS were 77.54%, 57.24%, and 72.76% respectively, whilst at high dose (124.80 mg-SCOD/L) were 94.04%, 66.32%, and 92.10% correspondingly. In similar order of the proposed carbon sources, the nitrate removal rates for the first phase denitrification with low dose were 1.44, 1.16, and 1.18 mg-NO₃ − /h respectively, whilst the nitrate removal rate of the first phase denitrification with high dose improved to 2.01, 1.26, and 1.96 mg-NO₃ −/h correspondingly. From the denitrification test results, molasses proved to be the optimal carbon source in terms of nitrate removal. However sonicated RAS possesses similar denitrification performance and may be a suitable alternative. An economic analysis for sonicated RAS Option 2 confirmed it as the most viable substitute. The time to recover the initial investment (payback period) is approximately 6.5 years and the breakeven point is approximately 8 years. Both denitrification tests and economic analyses demonstrate that sonicated RAS may be a viable and attractive substitute for the molasses. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337059 / Thesis (M.Eng.Sc.) - University of Adelaide, School of Chemical Engineering, 2008

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-10-19Bitstream added on 2014-06-13T19:03:38Z : No. of bitstreams: 1 barroso_cb_dr_jabo.pdf: 424873 bytes, checksum: c11fcf246d6ada6dcb329194e9814468 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Devido a baixa disponibilidade de P no solo e a alta capacidade do fungo Aspergillus niger F111 em solubilizar fosfatos inorgânicos, este trabalho teve por objetivo geral avaliar a possibilidade de inocular no solo esporos ou pellets imobilizados com vista a prolongar sua habilidade de solubilização e averiguar o mecanismo de solubilização de fosfatos inorgânicos de Ca, Al e Fe por este fungo. Os pellets inoculados em meio de cultura agitado proporcionaram maior solubilização dos fosfatos, principalmente o fosfato de Fe por ser de baixa solubilidade. No solo, os pellets livres e imobilizados promoveram as maiores solubilizações de fosfato de Fe e maior produção de CO2. Avaliando-se o efeito da fonte de N, as seguintes proporções foram obtidas na solubilização dos fosfatos de Ca, glicina > Al, nitrato de amônio > Fe, ácido l-glutâmico. Os açúcares que mais solubilizaram os fosfatos foram manitol, maltose e d-galactose. Dentre os metais somente o FeCl3.6H2O promoveu maior solubilização do fosfato de Fe e os metais FeSO4.7H2O e FeCl3.6H2O promoveram maiores solubilizações do fosfato de Ca. As concentrações de álcoois que mais favoreceram a solubilização do fosfato de Fe foram 3 e 4% de etanol e metanol, para o fosfato de Ca foi 3% de etanol. A combinação dos metais com o metanol, indicou que o metanol foi o principal responsável pela solubilização. Fatores como queda do pH, a maior produção de ácidos e o menor crescimento do fungo influíram neste trabalho, principalmente em relação a solubilização do fosfato de Fe. No solo, os pellets solubilizaram quantidades semelhantes de fosfato de Fe que os esporos imobilizados de A. niger, podendo ser utilizados com vantagem devido a sua facilidade de obtenção. / Considering the low P availability in the soil and the high capability of Aspergillus niger F111 in solubilizing inorganic phosphates, this work aimed to evaluate the possibility of inoculating spores or immobilized pellets in the soil to prolong the solubilization capability and study the solubilization mechanism of inorganic calcium phosphate, aluminum phosphate and iron phosphate by this fungus. Pellets inoculated in culture medium under agitation allowed higher phosphate solubilization, especially iron phosphate, which is low soluble. In the soil, free and immobilized pellets allowed the highest solubilization of iron phosphate and CO2 production. Evaluating the effect of N sources, the following proportions were obtained in the solubilization of calcium phosphates, glycine > Al, ammonium nitrate > Fe, l-glutamic acid. The sugars that most solubilized phosphates were mannitol, maltose and d-galactose. Among the metals, only FeCl3.6H2O promoted higher iron phosphate solubilization, and FeSO4.7H2O and FeCl3.6H2O promoted higher solubilization of calcium phosphate. The alcohol concentrations that most favored iron phosphate solubilization were 3 and 4% of ethanol and methanol, while the highest solubilization of calcium phosphate was reached with 3% ethanol. The combination of metals with methanol indicated this alcohol was mainly responsible for solubilization. Factors as pH decrease, higher acid production and lower A. niger growth influenced the results, especially in the solubilization of iron phosphate. In the soil, pellets and immobilized spores solubilized similar amounts of iron phosphate. Pellets are thus preferable because they are more easily obtained.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2017-09-21T20:27:37Z No. of bitstreams: 2 Tese - Fabiana Ribeiro da Mata - 2017.pdf: 5224228 bytes, checksum: 062f7a43994431350fa0acd55b4f076c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-22T11:44:01Z (GMT) No. of bitstreams: 2 Tese - Fabiana Ribeiro da Mata - 2017.pdf: 5224228 bytes, checksum: 062f7a43994431350fa0acd55b4f076c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-22T11:44:01Z (GMT). No. of bitstreams: 2 Tese - Fabiana Ribeiro da Mata - 2017.pdf: 5224228 bytes, checksum: 062f7a43994431350fa0acd55b4f076c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-13 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Members of the genus Paracoccidioides are known pathogens of humans that can be isolated from different infection sites. To investigate the expression rates of proteins expressed by different isolates, of the genus Paracoccidioides, including one isolate of P. lutzii (Pb01), and three isolates of P. brasiliensis (Pb03, Pb339 and PbEPM83) using sodium acetate, as carbon source, proteins quantities were determined using label-free and data independent LC-MSE. Statistical analysis provided the comparison of proteins profiles in the isolates. A total of 1160, 1211, 1280 and 1462 proteins were reproducibly identified, and relatively quantified in P. lutzii e P. brasiliensis isolates Pb03, Pb339 e PbEPM83, respectively. Notably, a total of 526, 435, 744 and 747 proteins were differentially expressed among P. lutzii and the P. brasiliensis isolates Pb03, Pb339 and PbEPM83, respectively with a fold change equal or higher than 1.5. The analysis revealed the reorganization of metabolism through the induction of proteins related to gluconeogenesis, stress response and amino acid degradation in the four isolates evaluated. The differences between the isolates were observed as follows: greater increases in the expression levels of proteins belonging to the cycle of glyoxalate, TCA and respiratory chain, ethanol production and β-oxidation were observed in the isolatesPPEPM83 and Pb01; Cyclic and cyclocyclic proteins of the methylcitrate were induced in Pb339. Proteomic profiles indicate that the four isolates reorganize the metabolism for the use of acetate as a carbon source. / Membros do gênero Paracoccidioides são patógenos conhecidos de seres humanos que podem ser isolados de diferentes locais de infecção. Para investigar as taxas de expressão das proteínas expressas por diferentes isolados, do gênero Paracoccidioides, incluindo um isolado de P. lutzii (Pb01) e três isolados de P. brasiliensis (Pb03, Pb339 e PbEPM83) usando acetato de sódio, como fonte de carbono, As quantidades de proteínas foram determinadas usando o uso de LC-MSE independente de etiquetas e de dados. A análise estatística proporcionou a comparação de perfis de proteínas nos isolados. Um total de 1160, 1211, 1280 e 1462 proteínas foram identificadas de forma reprodutiva, e relativamente quantificadas em isolados de P. lutzii e P. brasiliensis Pb03, Pb339 e PbEPM83, respectivamente. Nomeadamente, um total de 526, 435, 744 e 747 proteínas foram diferencialmente expressas entre P. lutzii e os isolados de P. brasiliensis Pb03, Pb339 e PbEPM83, respectivamente, com uma mudança de dobra igual ou superior a 1,5. A análise revelou a reorganização do metabolismo através da indução de proteínas relacionadas à gliconeogênese, resposta ao estresse e degradação de aminoácidos nos quatro isolados avaliados. As diferenças entre os isolados foram observadas como segue: maiores aumentos nos níveis de expressão de proteínas pertencentes ao ciclo do glioxalato, TCA e cadeia respiratória, produção de etanol e β-oxidação foram observadas nos isoladosnPbEPM83 e Pb01; As proteínas do ciclo da e do ciclo do metilcitrato apresentaram induzidas no Pb339. Os perfis proteômicos indicam que os quatro isolados reorganizam o metabolismo para o uso do acetato como fonte de carbono.

Dermatófitos são fungos filamentosos com a habilidade de invadir substratos queratinizados e causar dermatofitoses em humanos e animais, penetrando profundamente apenas em hospedeiros imunocomprometidos. Trichophyton rubrum é um fungo antropofílico e cosmopolita, o mais comum agente de micoses superficiais, que usa componentes celulares como proteínas e lipídeos após uma específica regulação de sua expressão gênica governada pelo pH ambiente e sensoriamento celular. A virulência de T. rubrum é relacionada com a secreção de enzimas proteolíticas, um importante fator determinante na invasão, utilização e subseqüente disseminação através do estrato córneo. O objetivo desse estudo foi identificar através de Hibridização Subtrativa Supressiva (SSH) genes de T. rubrum preferencialmente expressos durante o crescimento na presença de queratina e lipídeos, quando T. rubrum degrada fontes de carbono tipicamente encontradas em células epidérmicas. Inicialmente, nós avaliamos as mudanças no pH extracelular durante seu crescimento em queratina e lipídeo (depois de 6, 12, 24, 48, 72 h e 7 dias) em pH inicial 5,0, onde foi observado um gradual aumento do pH basal sob ambas as condições de teste, comparado com a condição glicose (controle). Também identificamos 576 transcritos de T. rubrum diferencialmente expressos por SSH usando conídios cultivados por 72 h em queratina como teste e em glicose como controle. Os genes de T. rubrum ativados codificam proteínas putativas que foram validadas por cDNA dot-blot e northern blot, mostrando similaridade com proteínas fúngicas envolvidas no metabolismo básico, crescimento e virulência, p. ex., transportadores ABC-MDR, MFS e ATPase de cobre, permease, NIMA interactive protein, poliproteína Gag-Pol, fatores de virulência subtilisinas serino-proteases (Sub 3 e 5) e metaloproteases (Mep 3 e 4) e Hsp30. Adicionalmente, entre os 762 clones obtidos na biblioteca da condição lipídeo (72 h), 80 transcritos superexpressos foram confirmados por cDNA dot-blot, revelando 14 unigenes similares à proteínas de vários organismos patogênicos, como glicoproteína 43 kD, transportador MDR, proteína G, quitina sintase e serino/treonina fosfatase. Transcritos do gene TruMDR2, codificador de um transportador ABC, foi isolado tanto na presença de queratina quanto em lipídeo, e a análise da linhagem mutante TruMDR2 de T. rubrum mostrou uma redução na atividade infectante, caracterizada pelo baixo crescimento em unhas humanas comparada com o tipo selvagem. A alta expressão de transportadores por T. rubrum em condições que mimetizam a infecção e a redução na virulência de TruMDR2 durante o modelo in vitro sugerem que transportadores estão envolvidos na patogenicidade de T. rubrum. Outro linhagem mutante (pacC-1) com um nocaute no gene pacC que codifica um fator de transcrição regulado pelo pH local, mostrou a expressão de proteases (Sub 3 e 5 e Mep 4) diminuída após o crescimento em queratina em comparação com o tipo selvagem em análises de northern blots. Essas proteases tem uma atividade ótima em pH alcalino, e nossos resultados indicam uma regulação defectiva do gene pacC de T. rubrum na ativação de proteases. Em conclusão, através do uso de SSH foi possível identificar genes de T. rubrum ativados após tratamentos específicos, o que sugere a importância dos mesmos na interação dermatófito-hospedeiro, instalação e manutenção da doença. Esses resultados disponibilizam novos dados sobre T. rubrum que levarão a um melhor entendimento dos mecanismos moleculares envolvidos no crescimento, metabolismo geral e patogenicidade, e também auxiliar na identificação de novos e efetivos alvos de drogas para dermatófitos. / Dermatophytes are a group of fungi filamentous that have the ability to invade keratinized substrates, causing dermatophytosis in humans and animals and only penetrate deeper if the host is immunocompromised. Trichophyton rubrum is an anthropophilic and cosmopolitan fungi, the most common agent of superficial mycoses, which uses cell components such as proteins and lipids after a specific regulation of its gene expression governed by pH environment and sensing cell. The virulence T. rubrum is related to secretion of proteolytic enzymes, an important factor determinant in the invasion, utilization and subsequently dissemination through the stratum corneum. The aim of this study was to indentify by Suppression Subtractive Hybridization (SSH) T. rubrum genes preferentially expressed during growth in the presence of keratin and lipids, upregulated when this fungus degrades carbon source typically found at epidemic cells. Initially, this work evaluated the changes in the extracellular pH during its growth in keratin and lipid (after 6, 12, 24, 48, 72 h and 7 days) at initial pH 5.0, where we observed a gradual increase of basal pH under both tests when compared to glucose condition (control). Also, we identified 576 T. rubrum transcripts differentially expressed by SSH using conidia cultivated for 72 h in keratin as tester, and in glucose as driver. The T. rubrum genes upregulated encode putative proteins that were validated by cDNA dot-blot and northern blot, showing similarity to fungi proteins involved in basic metabolism, growth and virulence, i.e., transporters ABC-MDR, MFS and ATPase of copper, permease, NIMA interactive protein, Gag-Pol polyprotein, virulence factors serine-protease subtilisins (Sub 3 and 5) and metalloproteases (Mep 3 and 4) and Hsp30. Additionally, among the 762 clones obtained in a library of lipid condition (72 h), 80 over-expressed transcripts were confirmed by cDNA dot-blot, revealing 14 unigenes similar to proteins of several pathogenic organisms, like glicoprotein 43 kD, MDR transporters, G protein, chitin synthase and serine/threonine-protein phosphatase. Transcripts of TruMDR2 gene, encoding an ABC transporter in T. rubrum, were isolated in the presence of keratin and lipid, and the examination of TruMDR2 mutant T. rubrum showed a reduction in infecting activity, characterized by low growth in human nails compared to wild-strain. The high expression of transporter by T. rubrum in conditions that mimetize the infection and the virulence reduction of TruMDR2 in an in vitro model suggests that transporters are involved in T. rubrum pathogenicity. Another mutant, pacC-1 with a knockout in PacC gene that encodes a transcription factor regulated by local pH, showed the expression of proteases (Sub 3, Sub 5 and Mep 4) decreased after growth in keratin (72 h) in comparison to wild-strain in northern blot analyzes. These proteases have an optimal activity in alkaline pH, and our results indicate a defective regulation of T. rubrum pacC gene in the activation of proteases. In conclusion, by means of SSH to identify genes upregulated in T. rubrum after specific treatments, their importance in the dermatophyte-host interaction, installation and maintenance in the disease is suggested. These results provide new insights about T. rubrum that will contribute to a better understanding of molecular mechanisms about the growth, metabolism and pathogenicity, and may also aid in the identification of novel effective drug targets for dermathophytes.

Philosophiae Doctor - PhD / In bacteria, nitrile hydratases and enzymes of nitrilase and signature amidase superfamilies hydrolyse nitriles and amides to their corresponding carboxylic acids releasing ammonia. Bacteria expressing these enzymes are typically isolated where a sole nitrogen and/or carbon source is used to support their growth. The majority of characterised enzymes of industrial potential have been identified for their stabilities at elevated temperatures. To date, no reports of such enzymes have been isolated from cold adapted bacteria.In this study, an extensive screening program of cold-active microbial isolates for enzymes of this group led to the selection and detailed characterisation of an aliphatic amidase from Nesterenkonia.Nesterenkonia AN1, a new psychrotrophic isolate of the genus, was isolated from soil samples collected from the Miers Valley, Antarctica. AN1 showed significant 16S rRNA sequence identity to known members of the genera, but this is the only strain that had optimal growth at approximately 21oC. AN1, similar to known members, is an obligately alkaliphilic (pH 9-10) and halotolerant (Na+ 0- 15% (w/v)) strain.The genome of Nesterenkonia AN1, sequenced in-house, revealed two ORFs encoding putative nitrilases, referred to as Nit1 and Nit2. Based on analysis of their deduced protein sequences, both belonged to the nitrilase superfamily. Both sequences showed conserved catalytic residues (EKEC), glycine residues and contained the characteristic áââá monomer fold. Homology modelling using known structures suggested that both genes could encode N-carbamoyl D-amino acid amidohydrolases, although neither showed conserved residues implicated in the hydrolysis of carbamoyls.Nit1 and Nit2 were expressed in Escherichia coli BL21 (DE3) pLysS as Cterminal and N-terminal hexahistidine tagged fusion proteins, and purified using Ni-chelation chromatography. Nit1 showed no activity towards nitrile, amide and carbamoyl substrates. This protein, unlike members of the multimeric enzymes of the nitrilase superfamily, was a monomer ~30 kDa protein. It is possible that the C-terminal hexahistidine tag might have prevented Nit1 from forming multimeric proteins.Nit2 showed substrate specificity similar to known aliphatic amidases with a preference for small amides. Nit2 had maximal activity at 30oC and between pH 6.5 and 7.5, properties compatible with its cold-adapted alkaliphilic origins. In addition, the enzyme was irreversibly inactivated at temperatures above 30oC and had a half-life of approximately 7 mins at 60oC. The crystal structure of Nit2 was solved to 1.66 Å. It revealed a ~45.5 kDa dimer, composed of two tightly bound ~30 kDa monomers. These monomers associated along the A surface forming a áââá-áââá sandwich architecture that is conserved in known structures of the nitrilase superfamily.Nit2 is distinct from known aliphatic amidases in both its structure and enzymic activity: the enzyme did not possess an extended C-terminal region; is active in dimeric form; has high affinity for 3C amides rather than 2C amides; and has a low overall catalytic rate. The short C-terminal region of Nit2 may have contributed to the low stability of the enzyme at elevated temperatures. A dendrogram composed of protein sequences of members of the nitrilase superfamily and Nit2 further supported evidence that this aliphatic amidase falls within a distinct group of enzymes.This is the first report of the enzymic characterisation and structural analysis of an aliphatic amidase from a psychrotolerant, alkaliphilic and halotolerant extremophile.