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Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.

BackgroundThis investigation comprises a contribution on the production of a new generation biofuel using the industrial liquid waste of bioethanol distilleries, known as vinasse. This study focuses on the exploitation of vinasse as an acidogenesis substrate for volatile fatty acids and simultaneous ethanol production. These products can be used for ester production, which is the new generation biofuel. Therefore, the aims of the present study are (i) to examine any promotional effect of γ-alumina on acidogenesis of a sucrose-raffinose mixture simulating vinasse, (ii) to study the operational stability of the continuous acidogenesis of sucrose and raffinose and subsequently of vinasse, and (iii) to determine the volatile fatty acid chemical composition and ethanol formation.ResultsBatch acidogenesis of sucrose and raffinose mixtures showed that γ-alumina promoted fermentation leading to an increase in the volatile fatty acid yield factor from 40% to 95% compared to free cells. The application of the system in continuous mode for more than 3 months showed that the continuous volatile fatty acid productivity obtained was higher than 7 g/L/day. Lactic acid was the predominant acid when sucrose and raffinose were used while butyric acid in the case of vinasse. The highest volatile fatty acid concentration reached was 19 g/L for vinasse.ConclusionsA promoting effect of γ-alumina in acidogenic fermentation of sucrose-raffinose and vinasse is reported. Continuous acidogenesis of sucrose-raffinose mixtures and vinasse using γ-alumina with immobilized cells showed high operational stability (more than 3 months). These findings result in easy scale up of the process that will produce a high annual added value of $11,000,000 in a daily bioethanol production plant of 50,000 L.

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