RESUMO
d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses. ONE-SENTENCE SUMMARY: This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.
Assuntos
Etanol , Fermentação , Oxigênio , Xilose , Xilose/metabolismo , Etanol/metabolismo , Oxigênio/metabolismo , Leveduras/metabolismo , Leveduras/crescimento & desenvolvimento , Cinética , Saccharomycetales/metabolismo , Saccharomycetales/crescimento & desenvolvimento , AerobioseRESUMO
Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in REDOX reactions and oxidative stress defense systems. Furthermore, NAD is used as substrate by proteins that regulate essential cellular functions as DNA repair, genetic, and signal transduction, among many others. NAD biosynthesis can be completed through the de novo and salvage pathways, which converge at the common step catalyzed by the nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT EC: 2.7.7.1/18). Here, we report the kinetic characterization of the NMNAT of Leishmania braziliensis (LbNMNAT), one of the etiological agents of leishmaniasis, a relevant parasitic disease. The expression and homogeneous purification of the recombinant 6xHis-LbNMNAT protein was carried out and its kinetic study, which included analysis of K m , V max , K cat and the equilibrium constant (K D ) for both the forward and reverse reactions, was completed. The oligomeric state of the recombinant 6xHis-LbNMNAT protein was studied through size exclusion chromatography. Our results indicated the highest and lowest K m values for ATP and NAD, respectively. According to the calculated K D , the pyrophosphorolytic cleavage of NAD is favored in vitro. Moreover, the recombinant 6xHis-LbNMNAT protein showed a monomeric state, although it exhibits a structural element involved in potential subunits interaction. Altogether, our results denote notable differences of the LbNMNAT protein in relation to the human orthologs HsNMNAT1-3. These differences constitute initial findings that have to be continued to finally propose the NMNAT as a promissory pharmacological target in L. braziliensis.
RESUMO
The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.
Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Leucil Aminopeptidase/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/microbiologia , Concentração de Íons de Hidrogênio , Cinética , Leucina/análogos & derivados , Leucina/química , Leucil Aminopeptidase/antagonistas & inibidores , Leucil Aminopeptidase/química , Leucil Aminopeptidase/isolamento & purificação , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , TemperaturaRESUMO
Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pKa of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants Km for NADH was 23.4 ± 1.8 µM, and for pyruvate was 203 ± 25 µM, while Vmax was 7.45 µmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia.
Assuntos
Proteínas de Artrópodes , Expressão Gênica , L-Lactato Desidrogenase , Penaeidae/genética , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , L-Lactato Desidrogenase/biossíntese , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/isolamento & purificação , Penaeidae/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
In this work we communicate the heterologous expression of a laccase from Coriolopsis gallica in Pichia pastoris. This enzyme has been reported to efficiently degrade a variety of pollutants such as industrial dyes. The expression strategy included using a previously reported modified α-factor preproleader for enhanced secretion and pAOX1, a methanol-responsive promoter. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in this heterologous system. A volumetric activity of 250 U/L was achieved after 12-day culture in Fernbach flasks. The protein was recovered from the supernatant and purified, obtaining a preparation with 90% electrophoretic purity. The catalytic constants of the recombinant enzyme are almost identical to the fungal enzyme, thus rendering this system a useful tool for protein engineering of laccase from C. gallica.
Assuntos
Coriolaceae/genética , Proteínas Fúngicas , Expressão Gênica , Lacase , Coriolaceae/enzimologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Lacase/biossíntese , Lacase/química , Lacase/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70 percent) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20 percent of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.
A enzima glucanase de Moniliophthora perniciosa foi produzida em meio líquido e purificada a partir do sobrenadante da cultura. A metodologia de superfície de resposta (MSR) foi usada para avaliar os efeitos das variáveis, incluindo indutor (extrato de levedura) e tempo de fermentação, na atividade da glucanase de M. perniciosa detectada no meio de cultura. A enzima presente no sobrenadante foi purificada em duas etapas: precipitação com sulfato de amônio (70 por cento) e cromatografia de filtração em gel em Sephacryl S-200. A produção da enzima glucanase foi maior na concentração de 5,9 g L-1 de extrato de levedura e 13 dias de fermentação. Os resultados mostraram três diferentes isoformas (GLUI, GLUII e GLUIII) com fatores de purificação de 4,33, 1,86 e 3,03, respectivamente. O extrato enzimático parcialmente purificado mostrou um pH ótimo de 5,0 e uma temperatura ótima de 40°C. A atividade enzimática aumentou na presença de KCl em todas as concentrações estudadas. A atividade da glucanase foi maior na presença de NaCl 0,2 M. A enzima apresentou alta estabilidade térmica, perdendo apenas 10,20 por cento de sua atividade específica após 40 minutos de incubação a 90°C. Os resultados de termoestabilidade e a atividade em baixo pH mostraram que a enzima glucanase de M. perniciosa tem características promissoras para futuras aplicações industriais.