Legionaminic acid (Leg5,7Ac2) is a nonulosonic acid similar to sialic acid (Neu5Ac), which can be found in the extracellular glycoconjugates of several bacterial pathogens. Due to the similarity in stereochemistry of the two compounds, legionaminic acid has great potential in the production of pharmaceutical drugs. A novel biosynthetic pathway to produce legionaminic acid was created to overcome the limitations of organic synthesis. This is the first study involving the scale-up of legionaminic acid production by high cell density fermentation processes. In this work, fed-batch cultivations of recombinant Escherichia coli BRL04 were carried out in shake flasks and 5-L bioreactors. The final process was optimized by determining the effects of different carbon sources, induction temperatures, pH, dissolved oxygen (DO) content, induction optical density and N-acetylglucosamine (GlcNAc) feed rate on the production of legionaminic acid. Overall, results showed that the titer, yield and productivity for legionaminic acid production achieved relatively high levels, which were 5.53 g/L, 73.29% and 0.092 g/(Lh), respectively. It is hoped that this study accelerates research into the production of legionaminic acid for therapeutic treatments as well as for further study in glycobiology.

La bio-transformation naturelle des médicaments peut produire des métabolites toxiques; l’identification de ces métabolites est essentielle dans la stratégie de choix de molécules thérapeutiques. En appliquant les technologies de fermentation en bioréacteur des cellules hétérologues (souches d’E. coli recombinantes exprimant une iso-enzyme de cytochrome P450 humain avec la réductase humain), la bioconversion du substrat (principe actif) en ses métabolites de dégradation, a été réalisée à grande échelle (g-g). Notre choix s’est porté sur le complexe hCYP3A4/HR fonctionnel produit par un hôte E. coli. Les cellules intactes ou les membranes cellulaires peuvent être exploitées comme biocatalyseur dans un système bioréacteur. Cependant, la faible solubilité des principes actifs dans des milieux de bioconversion aqueuse limitent le rendement. Un bioréacteur biphasique a été étudié. En solution, plusieurs combinaisons eau/solvants organiques conciliant la viabilité des cellules, la solubilité des principes actifs et produits de réaction et la catalyse des complexes enzymatiques ont conduit à l’établissement d’un mélange approprié. Cependant, ces combinaisons présentent toujours une inhibition importante du pouvoir catalytique des complexes enzymatiques. Pour minimiser un effet dénaturant possible des solvants sur le système enzymatique, ce dernier a été maintenu dans un environnement aqueux en immobilisant les cellules et/ou les membranes cellulaires dans une matrice hydrophile. L’alginate de calcium apparaît être une matrice d’immobilisation idéale pour les membranes assurant la fonctionnalité du complexe CYP/HR et permettant en outre un stockage à long terme des préparations. Par contre, l’immobilisation des cellules dans diverses matrices, si elle permet une viabilité et une conservation à long terme des souches recombinantes, ne permet aucune expression de l’activité enzymatique présente dans les cellules. La combinaison d’une localisation du complexe hCYP/HR fonctionnel dans la membrane interne et d’une perméabilité réduite des cellules d’E. coli (immobilisées) en est une explication possible mais non-démontrée. Entre-temps, cette technologie de bioréacteur homogène biphasique ou par immobilisation des membranes cellulaires a été utilisée plusieurs reprises pour produire des métabolites humains à partir de divers principes actifs. Ces métabolites ont été purifiés avec succès, démontrant que cette approche technologique est compétitive comparée aux procédures conventionnelles. Néanmoins, de nouvelles pistes de recherche seraient extrêmement intéressantes. La localisation des complexes enzymatiques recombinants en surface des cellules permettrait de concilier les propriétés hydrophobes des principes actifs et l’environnement hydrophile nécessaire aux enzymes. D’autre part une investigation de complexes enzymatiques résistant aux solvants pourrait remplacer avantageusement l’immobilisation.

In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxygen transfer rate, volumetric and specific oxygen uptake rates, mass transfer and biochemical reaction resistances / moreover, the variation in product and by-product distribution, specific substrate uptake rates, yield and maintenance coefficient were investigated in the pilot scale batch bioreactor at QO/VR = 0.5 vvm and agitation rates of N= 250, 500, 625, and 750 min-1, and dissolved oxygen levels DO= 20%, 40% conditions, while medium components were CGlucose= 8.0 kg m-3, C(NH4)2HPO4= 5.0 kg m-3 and salt solution at controlled pHc=7.2. The highest cell concentration and benzaldehyde lyase activity were obtained at DO=40% condition as 3.0 kg m-3 and A=1095 Ucm-3, respectively. v Then a mathematical model was proposed to estimate benzaldehyde lyase activity as function of time, agitation rate, cell concentration, dissolved oxygen concentration, and by-product concentration with reasonable accuracy.

This study focuses on the molasses based complex medium design for benzaldehyde lyase production by recombinant E. coli BL21 and development of a feeding strategy based on the designed complex medium. For this purpose, firstly, the effects of molasses were investigated in laboratory scale bioreactors. As E. coli BL21 was not able to utilize sucrose, molasses was pretreated and hydrolyzed to fructose and glucose. Thereafter, effect of pretreated molasses concentration was investigated in the range of 16 to 56 kg m-3 by batch-bioreactor experiments / and the highest cell concentration and benzaldehyde lyase activity were obtained as CX=5.3 kg m-3 and A=1617 U cm-3, respectively, in the medium containing 7.5 kg m-3 glucose and 7.5 kg m-3 fructose. Then, different feeding strategies were developed to produce efficient cells with high concentration and BAL activity. In the first strategy, after 10 hours of batch-cultivation with molasses based medium having 7.5 kg m-3 glucose and 7.5 kg m-3 fructose concentration, based on the airflow rate, pretreated molasses was fed to the system. When air flow rate decreased considerably, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3. At the end of the process, the highest cell concentration obtained was CX=7.4 kg m-3. The maximum activity was reached at 20th hour as A=2360 U cm-3. On the other hand, as air flow variation only demonstrated the absence of glucose not fructose, a second strategy, based on the detection of the fructose and glucose concentrations during the process, was applied. In this strategy when glucose and fructose were depleted, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3 / and the highest BAL activity was obtained as 2370 U cm-3 at t= 26 h where the cell concentration was 7.5 kg m-3. At the last strategy, when glucose and fructose were depleted, fed was given to the system that results in increase in CGlucose=1.5 kg m-3 and CFructose=1.5 kg m-3 in the production medium to decrease the accumulation of acetic acid. By this strategy highest cell concentration was obtained as 8.04 kg m-3 at t=24 h and the highest BAL activity was 2315 U cm-3. These strategies could be accepted having the same BAL activity with little distinctions. However, cell concentration of the last one was higher than others and also the lowest amount of carbon source was used. Thus, last one could be chosen as the most favorable strategy.

In this study, the effects of bioprocess operation parameters on benzaldehyde lyase production were systematically investigated. For this purpose, the research program was carried out in mainly four parts. In the first part of the study, Escherichia coli K12 (ATCC 10798), having the highest benzaldehyde lyase production capacity, was selected as the host microorganism. Next, using the selected microorganism, the production medium was designed in terms of its carbon and nitrogen sources. Among the investigated media, the highest cell concentration and benzaldehyde lyase activity were obtained as 1.8 kg m-3 and 745 U cm-3, respectively, in the medium containing 8.0 kg m-3 glucose, 5.0 kg m-3 (NH4)2HPO4 and the salt solution. Thereafter, by using the designed medium, the effects of bioreactor operation parameters, i.e., oxygen transfer and pH, were investigated in pilot scale bioreactor. Oxygen transfer effects on benzaldehyde lyase production were investigated at QO/VR=0.5 vvm / N=250, 375, 500, 625, 750 min-1 and at QO/VR=0.7 vvm, N=750 min-1 conditions. The highest cell concentration and benzaldehyde lyase activity were obtained at 0.5 vvm, 500 min-1 condition as 2.3 kg m-3 and 860 U cm-3, respectively. Finally, the effect of pH was investigated for benzaldehyde lyase production process at Qo/VR=0.5 vvm, N=500 min-1 condition, at pHC=5.0, 6.4, 6.7, 7.0, 7.2 and 7.8 values. Among the investigated pH values, the highest cell concentration and enzyme activity were obtained at pHC=7.0 condition as 2.1 kg m-3 / 775 U cm-3. However, the values obtained at this condition, were lower than the values obtained at pHUC=7.2 uncontrolled pH operation. Hence, medium oxygen transfer condition and uncontrolled pH operation are found to be favorable for benzaldehyde lyase production.

In this study, the benzaldehyde lyase (BAL, EC 4.1.2.38) production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3) pLySs cells, the effect of bioprocess parameters was systematically investigated. Among the investigated media, the highest cell concentration and benzaldehyde lyase activity were obtained as 2.0 kg m-3 and 1060 U cm-3, respectively, in the medium containing 20.0 kg m-3 glucose, 11.8 kg m-3 (NH4)2HPO4 and the salt solution. Thereafter, oxygen transfer effects on benzaldehyde lyase production were investigated at air inlet v rate of QO/VR = 0.5 vvm, and agitation rates of N=500 and 750 min-1 and at QO/VR = 0.7 vvm, N=750 min-1 in pilot scale bioreactor and the highest cell concentration and volumetric BAL activity were found as 1.7 kg m-3 and 990 U cm-3, respectively, at 0.5 vvm, 750 min-1 condition. Next, the signal DNA sequence of serine alkaline protease (SAP) from B. licheniformis DSM 1969 chromosomal DNA (pre-subC) was fused in front of the bal by using PCR-based gene splicing by overlap extension (SOE) method. The fusion product of hybrid gene first cloned into pUC19 plasmid, thereafter sub-cloned into pBR374 shuttle vector and recombinant plasmid was transferred into various Bacillus species. However, no extracellular production of benzaldehyde lyase was observed in none of the developed recombinant Bacillus species, probably because of ineffective secretion system, inefficient folding of heterologous protein, degradation of enzyme due to proteolytic activity or high inactivation rate of the enzyme.

Made available in DSpace on 2014-12-17T15:01:53Z (GMT). No. of bitstreams: 1 MichelleRFV_TESE.pdf: 2132732 bytes, checksum: 80b95f923491eeb118d74b0376a64ba9 (MD5) Previous issue date: 2011-12-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Escherichia coli has been one of the most widely used hosts in recombinant protein production, in both laboratory and industrial scale since the advent of recombinant DNA technology. Despite the substantial progress of studies on the molecular biology and immunology of infections, there is currently no medication-based prophylaxis capable of preventing leishmaniasis. As such, there is a great need to identify specific antigens for the development of vaccines and diagnostic kits against visceral leishmaniasis. Thus, the primary goal of the present study is to assess the influence of cultivation conditions on the production of Leishmania chagasi antigens, carried out in a rotating incubator and bioreactor. To that end, several assays were conducted to evaluate the kinetic behavior of antigens (648, 503) of Leishmania. i. chagasi in two different compositions of media (2xTY, TB), with and without an inducer. In order to improve expression, assays were performed in a benchtop bioreactor using the best conditions obtained in a rotating incubator, in addition to assessing the influence of stirring speed. Results show that high complexity of the cultivation medium favored kinetic growth of clones (648, 503). However, in assays submitted to induction by IPTG, this elevated complexity did not promote the expression of recombinant proteins. Expression of antigens 648 and 503 exhibited behavior associated with growth and, in terms of location, proteins 648 and 503 are intracellularly stored. Lactose may be the most adequate inducer in protein expression, when considering factors, cost, toxicity and stability. Elevated stirring may increase cell growth in clone 53, although it may not result in high concentrations for the protein of interest. On the other hand, positive results were obtained for all recombinant clones (648, 503) tested, confirmed by the electrophoretic profile / A Escherichia coli ? um dos hospedeiros mais utilizados para produ??o de prote?nas recombinantes tanto em escala de laborat?rio como em escala industrial desde o advento da tecnologia do DNA recombinante. Apesar do avan?o expressivo dos estudos da biologia molecular e da imunologia das infec??es, n?o existe, atualmente, nenhuma droga profil?tica capaz de prevenir o calazar. Desta forma, existe uma grande necessidade de identifica??o de ant?genos espec?ficos para o desenvolvimento de vacinas e kits para diagn?sticos contra a Leishmaniose visceral. Com base no exposto, o presente trabalho tem como foco principal avaliar a influ?ncia das condi??es de cultivo na produ??o dos ant?genos de Leishmania i. chagasi em cultivos realizados em incubador rotativo e biorreator. Para atingir o objetivo proposto, v?rios ensaios foram realizados a fim de se avaliar o comportamento cin?tico dos clones (648, 503) de Leishmania i. chagasi em duas diferentes composi??es de meio (2xTY, TB), com e sem adi??o de indutor em incubador rotativo. Para melhorar a express?o, ensaios foram conduzidos em biorreator de bancada com as melhores condi??es obtidas em incubador rotativo, al?m da avalia??o da influ?ncia da velocidade de agita??o. Com base nos resultados obtidos, pode-se observar que a elevada complexidade do meio de cultivo favoreceu a cin?tica de crescimento dos clones (648, 503), no entanto, ao se tratar dos ensaios submetidos ao procedimento de indu??o por IPTG, a elevada complexidade do meio de cultivo n?o favoreceu a express?o das prote?nas recombinantes. Pode-se observar que a express?o dos ant?genos 648 e 503 apresentam um comportamento associado ao crescimento e que em termos de localiza??o, as prote?nas 648 e 503, s?o armazenadas intracelularmente. A lactose pode ser o indutor mais adequado na express?o das prote?nas tendo em vista os fatores, custo, toxicidade e estabilidade. A elevada agita??o pode aumentar o crescimento celular do clone 503, entretanto, pode n?o acarretar em altas concentra??es da prote?na de interesse. Por outro lado, foram obtidos resultados positivos para todos os clones recombinantes (648, 503) testados, confirmada atrav?s do perfil eletrofor?tico

Polyhydroxyalkanoates (PHAs) are polyesters, produced by many bacteria and some archaea. The most commonly characterised is polyhydroxybutyrate (PHB). Produced when nutrients are growth limiting and carbon available in excess, PHA serves as a carbon-energy storage material and forms generally spherical insoluble inclusions between 50-500nm in diameter in the cytoplasm. The key enzyme for PHA synthesis is the PHA synthase and this enzyme catalyses the polymerisation of (R)-3-hydroxy fatty acids into PHA. PHA synthase remains covalently attached to the growing polyester chain and is displayed on the surface of the PHA granule. Other proteins associated with PHA granules include depolymerases for mobilisation or degradation of granules, regulatory proteins and phasins, proteins that aid in PHA granule stability. PHA bio-beads displaying an IgG binding protein were produced and used to purify IgG from serum demonstrating that the PHA synthase can be engineered to display functional synthase fusion proteins at the PHA granule surface. Correctly folded eukaryotic proteins were also produced and displayed at the PHA granule surface as phasin fusion proteins. Multiple-functionality was also achievable by co-expression of various hybrid genes suggesting that this biotechnological bead production strategy might represent a versatile platform technology. The production of functional eukaryotic proteins at the PHA bead surface represents a novel in vivo matrix-assisted protein folding system. Protein engineering of PHA granule surface proteins provides a novel molecular tool for the display of antigens for FACS based analysis and offers promising possibilities in the development of future biotechnological production processes. Overall, the results obtained in this study strongly enhance the applied potential of these polyester beads in biotechnology and medicine.

The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5&rsquo / end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through PCR. The resulting hybrid gene pre(subC)::skc was cloned into the pUC19 plasmid. Then, the hybrid gene was sub-cloned to pMK4 plasmid which is an E. coli-Bacillus shuttle vector with high copy number and high stability. Recombinant plasmid pMK4::pre(subC)::skc was finally transferred into B. subtilis (npr- apr-) and B. licheniformis 749/C (ATCC 25972) species. Streptokinase production capacities of these two recombinant Bacillus species were compared. The highest production was observed in recombinant B. lichenifomis 749/C (ATCC 25972) strain in a defined medium which was optimized in terms of carbon and nitrogen sources by a statistical approach, namely Response Surface Methodology (RSM). RSM evaluated the streptokinase concentration as the response and the medium components as the independent variables. The highest recombinant streptokinase concentration was found as 0.0237 kgm-3 at glucose and (NH4)2HPO4 concentrations of 4.530 and 4.838 kgm-3 respectively. The fermentation and oxygen transfer characteristics of the streptokinase production were investigated in a 3 dm3 pilot scale batch bioreactor (Braun CT2-2) equipped with temperature, pH, foam, air inlet and agitation rate controls having a working volume of VR=1.65 dm3 using the production medium optimized for the recombinant B. lichenifomis 749/C (ATCC 25972) strain. Streptokinase and &amp / #946 / -lactamase activities, cell, glucose and organic acid concentrations, dissolved oxygen, pH, oxygen uptake rate, overall liquid phase mass transfer coefficient for oxygen, maintenance coefficient for oxygen, specific cell growth rate and yield coefficients were determined through the bioprocess. The bioprocess of recombinant streptokinase production was performed at uncontrolled pH of these bioreactor operation conditions: air inlet rate of Q0/VR=0.5 vvm, and the agitation rate of N=400min-1. The resulting streptokinase volumetric activity reached its maximum as 1.16 PUml-1 (0.0026 g/l streptokinase) at t=20 h.

Made available in DSpace on 2016-06-02T19:56:33Z (GMT). No. of bitstreams: 1 2531.pdf: 1826513 bytes, checksum: 1b4aeefe4e23520d603b495bbd0b8832 (MD5) Previous issue date: 2009-04-03 / Universidade Federal de Sao Carlos / Using recombinant DNA techniques, the lipase BTL2 gene of Bacillus thermocatenulatus was cloned in E. coli BL321 under control of the strong temperature-inducible λPL promoter. It was investigated, initially, in this work, the influence of different variables in cell growth and expression of lipase BTL2 by recombinant E. coli, through experiments performed in agitated flasks with LB medium. First, it was studied the influence of temperature of growth (between 27 ° C and 34.2 °C) and heat shock temperature (between 37.8 °C and 46.2 °C) in the expression of lipase BTL2 by E. coli recombinant, using statistical design of experiments. The results of this study, where the shock was performed in the early exponential phase, indicated as the best Tcresc = 27 °C and Tchoque = 45 °C, yielding cellular concentration of 0.6 g dry weight / L and enzyme activity of 121.000 U/g.wet.cel. Then, it was investigated the influence of the growth phase of the microorganism at the shock, through cultures with Tcresc = 27 °C and Tchoque = 45 °C, the results of these experiments showed how heat shock condition at the end of exponential phase, resulting in activity of 258.000 U/g.wet.cel It investigated the influence of different initial concentrations of glucose in the medium on cell growth and expression of the enzyme. The best results were obtained from cell mass with 10 g/L glucose and μmax = 0,16 h-1, Yx/s = 0,19 g/g, resulting in 1.2 g / L dry cel, enzymatic activity of about 250.000 U / g.wet.cel. Lower concentrations of glucose and higher concentrations led to smaller cell, but did not affect enzyme activity in the final. Based on previous cultures of E.coli were conducted simulations to calculate the best condition for feed-batch. The experimental test was performed with 10 g/L glucose at the beginning of cultivation, Tcresc = 30 °C, Tchoque = 45 °C. In this test, we were able to achieve 15 g/L dry.cel μmax = 0,38 h-1 with enzyme activity of 231.000 U/g.wet.cel, with resulting in 100 times more enzyme in this test than in cultivation in shaker in the best condition. The results of the simulation, obtained using model of Monod, predicted quite well those obtained experimentally. There was no significant accumulation of organic acids and all aminoacids consumed by the moment of shock. From the heat shock, those who were not exhausted with concentration remained constant. The enzyme produced in the test batch was recovered by breaking the cells in a French press to obtain with this methodology 272.000 U/g wet cells, whereas the results of the samples, which were disrupted by sonication resulted in much lower value. Was then investigated the efficiency of the protocol of sonication that was being used, by subjecting the cells to successive rounds of sonication. The results showed that actually the first cycle only 50% of the enzyme was released, indicating that the maximum production achieved was actually around 462.000 U/gcél.úmida. Study characterization of enzyme kinetics showed that the temperature of maximum activity is 65 °C with activation energy equal to 142.3 kJ/mol. Study of stability in solvent showed that the enzyme retains activity in the presence of 2- propanol. / Utilizando técnicas de DNA recombinante, o gene da lípase BTL2 de Bacillus thermocatenulatus foi clonado em E. coli BL321 sob controle do promotor λPL, com o qual a indução na produção da enzima é obtida através de choque térmico. Investigou-se, inicialmente, neste trabalho, a influência de diferentes variáveis no crescimento celular e na expressão da lípase BTL2 por E.coli recombinante, através de experimentos realizados em frascos agitados, com meio LB. Primeiramente, estudou-se a influência da temperatura de crescimento (entre 27°C e 34,2°C) e da temperatura de choque térmico (entre 37,8°C e 46,2°C) na expressão de BTL2 por E.coli recombinante, usando planejamento estatístico de experimentos. Os resultados desse estudo, onde o choque era realizado no início da fase exponencial, indicaram como as melhores temperaturas Tcresc=27°C e Tchoque=45°C, obtendose concentração celular de 0,6 g massa seca/L e atividade enzimática de 121.000U/gcél.úmida. A seguir, foi investigada a influência da fase de crescimento do microrganismo no momento do choque, através de cultivos com Tcresc=27°C e Tchoque =45°C, os resultados desses experimentos indicaram como melhor condição choque térmico no final da fase exponencial, obtendo-se nessa condição atividade de BTL2 de 258.000U/gcél. Úmida. Investigou-se assim a influência de diferentes concentrações iniciais de glicose no meio de cultivo no crescimento celular e expressão da enzima. Os melhores resultados de massa celular foram obtidos com 10g/L de glicose, obtendo-se 1,2 g/L de massa seca, μmax = 0,16 h- 1, Yx/s=0,19 g/g e atividade enzimática em torno de 250.000 U/gcél, úmida. Concentrações de glicose menores e maiores conduziram a menores concentrações celulares, mas não influenciaram na atividade enzimática final. Baseando-se em cultivos anteriores de E.coli foram realizadas simulações para cálculo de alimentação de meio em ensaio em batelada alimentada. O ensaio experimental foi realizado com 10 g/L de glicose no início do cultivo, Tcresc=30°C, Tchoque=45°C. Nesse ensaio, conseguiu-se atingir 15 g/L de massa seca, com μmax =0,38 h-1 e com atividade enzimática de 231.000 U/gcel.úmida, obtendo-se 100 vezes mais enzima nesse ensaio do que no cultivo em frasco agitado na melhor condição. Os resultados da simulação, obtidos usando modelo de Monod, previram bastante bem os obtidos experimentalmente. Não se observou acúmulo significativo de ácidos orgânicos e todos os aminoácidos.eram consumidos até o momento do choque. A partir do choque térmico, aqueles que não estavam esgotados permaneceram com concentração constante. A enzima produzida no ensaio em batelada foi recuperada rompendo-se as células em uma prensa francesa, obtendo-se com essa metodologia 272.000 U/g cel úmida, enquanto que os resultados das amostras , que eram rompidas por sonicação resultaram em valor muito menor. Investigou-se então a eficiência do protocolo de sonicação que vinha sendo utilizado, submetendo-se as células a sucessivos ciclos de sonicação. Os resultados mostraram que realmente no primeiro ciclo apenas 50% da enzima era liberada, o que indica que a máxima produção obtida estava na verdade em torno de 462.000U/gcél.úmida.Estudo de caracterização cinética da enzima mostrou que a temperatura de máxima atividade é 65°C, com energia de ativação igual a 142,3 kJ/mol. Estudo de estabilidade em solvente mostrou que a enzima mantém atividade na presença de 2-propanol.