Effects of site-directed PEGylation on L-asparaginase thermostability / Efeitos da PEGuilação sítio-dirigida na termoestabilidade da L-asparaginase

The enzyme L-asparaginase (ASNase) is broadly applied as a drug to treat acute lymphoblastic leukemia, as well as in the food industry to avoid acrylamide formation in baked and fried food. In the present work, ASNase was covalently attached to polyethylene glycol (PEG) of different molecular weights (ASNase-PEG-5, ASNase-PEG-10, ASNase-PEG-20, and ASNase-PEG-40) at the N-terminal portion (monoPEGylation). Native and PEGylated forms were analyzed regarding thermodynamics and thermostability based on enzyme activity measurements. ASNase (native and PEGylated) presented maximum activity at 40 °C and denaturation followed a first-order kinetics. Based on these results, the activation energy for denaturation (E*d) was estimated and higher values were observed for PEGylated forms compared to the native ASNase, highlighting the ASNase-PEG10 with a 4.24-fold increase (48.85 kJ.mol-1) in comparison to the native form (11.52 kJ.mol-1). The enzymes were evaluated by residual activity over time (21 days) under different storage temperatures (4 and 37 °C) and the PEGylated conjugates remained stable after the 21 days. Thermodynamic parameters like enthalpy (ΔH‡), entropy (ΔS‡) and Gibbs free energy (ΔG‡) of ASNase (native and PEGylated) irreversible denaturation were also investigated. Higher - and positive - values of Gibbs free energy were found for the PEGylated conjugates (61.21 a 63.45 kJ.mol-1), indicating that the process of denaturation was not spontaneous. Enthalpy also was higher for PEGylated conjugates (18.84 a 46.08 kJ.mol-1), demonstrating the protective role of PEGylation. As for entropy, the negative values were more elevated for native ASNase (-0.149 J/mol.K), pointing out that the denaturation process enhanced the randomness and aggregation of the system, which was observed by circular dichroism. Thus, PEGylation proved its potential to increase ASNase thermostability

A enzima L-asparaginase (ASNase) é amplamente usada como medicamento para tratamento da leucemia linfoblástica aguda, bem como na indústria de alimentos para evitar a formação de acrilamida em alimentos cozidos e fritos. No presente trabalho, ASNase foi covalentemente ligada ao polímero poli(etilenoglicol) (PEG) de diferentes massas moleculares (ASNase-PEG-5, ASNase-PEG- 10, ASNase-PEG-20, and ASNase-PEG-40) na região N-terminal (monoPEGuilação) a fim de se estudar os efeitos da PEGuilação na termoestabilidade da enzima. As formas PEGuiladas e nativa foram analisadas em relação à termodinâmica e termoestabilidade a partir de atividade enzimática. A ASNase (nativa e PEGuilada) apresentou atividade máxima a 40 °C e a desnaturação ocorreu por cinética de primeira ordem. Com base nesses resultados, a energia de ativação para desnaturação (E*d) foi estimada e maiores valores foram observados para as formas PEGuiladas em comparação à enzima nativa, destacando-se a ASNase-PEG10 com aumento de 4.24 vezes (48.85 kJ.mol-1) em comparação com a forma nativa in (11.52 kJ.mol mol-1). As enzimas foram avaliadas por sua atividade residual ao longo do tempo em diferentes temperaturas de armazenamento (4 e 37 °C) e os conjugados PEGuilados mostraram-se mais estáveis após os 21 dias de ensaio. Parâmetros termodinâmicos como entalpia (ΔH‡) de desnaturação irreversível foram analisados. Valores maiores - e ), entropia (ΔS‡) de desnaturação irreversível foram analisados. Valores maiores - e ) e energia livre de Gibbs (ΔG‡) de desnaturação irreversível foram analisados. Valores maiores - e positivos - da energia livre de Gibbs foram encontrados para os conjugados PEGuilados (61.21 a 63.45 kJ.mol-1), indicando que o processo de desnaturação não ocorreu de forma espontânea. A entalpia também foi maior para os conjugados PEGuilados (18.84 a 46.08 kJ.mol-1), demonstrando o efeito protetivo da PEGuilação. Já para a entropia, os valores negativos foram mais elevados para a ASNase nativa (-0.149 J/mol.K), apontando que o processo de desnaturação aumentou a aleatoriedade e agregação do sistema, o que foi confirmado pelo dicroísmo circular. Dessa forma, a PEGuilação revelou o seu potencial de aumento de termoestabilidade para a ASNase

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase.

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185-192 and 382-392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60°C and 65°C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55°C or 60°C for 1h. Interestingly, H192P was also 2-times more active than wild-type TK at 25°C. Optimal activity was achieved at 60°C for H192P compared to 55°C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.