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Invertase from Aspergillus niger C28B25 was produced by solid-state fermentation (SSF). Fermented solids were used directly as a biocatalyst for batch and continuous hydrolysis of sucrose in a packed-bed reactor under different operational conditions with various temperatures, sucrose concentrations, and feed flow rates. The SSF allowed obtaining a biocatalyst with an invertase activity of 82.2 U/g db. The biocatalyst maintained its activity in the range of 40 to 70 °C for at least 70 h of continuous operation. In a 20-mL packed bed reactor, the highest hydrolysis rate (12.3 g/g db h) was obtained at 40 °C with 2 M sucrose. Continuous hydrolysis in 20-mL and 200-mL reactors at 60 °C led to sucrose hydrolysis above 60% (8.5 residence times) and above 55% (4.5 residence times), respectively. The auto-immobilised biocatalyst produced by SSF without recovery, purification, and immobilisation stages offers an economical alternative for developing accessible biocatalysts that can be applied in batch or continuous sucrose hydrolysis processes. This study shows the potential of biocatalyst production by SSF for other enzymatic systems.

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E3 ubiquitin ligases mediate the last step of the ubiquitination pathway in the ubiquitin-proteasome system (UPS). By targeting transcriptional regulators for their turnover, E3s play a crucial role in every aspect of plant biology. In plants, SKP1/CULLIN1/F-BOX PROTEIN (SCF)-type E3 ubiquitin ligases are essential for the perception and signaling of several key hormones including auxins and jasmonates (JAs). F-box proteins, TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and CORONATINE INSENSITIVE 1 (COI1), bind directly transcriptional repressors AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) and JASMONATE ZIM-DOMAIN (JAZ) in auxin- and JAs-depending manner, respectively, which permits the perception of the hormones and transcriptional activation of signaling pathways. Redox modification of proteins mainly by S-nitrosation of cysteines (Cys) residues via nitric oxide (NO) has emerged as a valued regulatory mechanism in physiological processes requiring its rapid and versatile integration. Previously, we demonstrated that TIR1 and Arabidopsis thaliana SKP1 (ASK1) are targets of S-nitrosation, and these NO-dependent posttranslational modifications enhance protein-protein interactions and positively regulate SCFTIR1 complex assembly and expression of auxin response genes. In this work, we confirmed S-nitrosation of Cys140 in TIR1, which was associated in planta to auxin-dependent developmental and stress-associated responses. In addition, we provide evidence on the modulation of the SCFCOI1 complex by different S-nitrosation events. We demonstrated that S-nitrosation of ASK1 Cys118 enhanced ASK1-COI1 protein-protein interaction. Overexpression of non-nitrosable ask1 mutant protein impaired the activation of JA-responsive genes mediated by SCFCOI1 illustrating the functional relevance of this redox-mediated regulation in planta. In silico analysis positions COI1 as a promising S-nitrosation target, and demonstrated that plants treated with methyl JA (MeJA) or S-nitrosocysteine (NO-Cys, S-nitrosation agent) develop shared responses at a genome-wide level. The regulation of SCF components involved in hormonal perception by S-nitrosation may represent a key strategy to determine the precise time and site-dependent activation of each hormonal signaling pathway and highlights NO as a pivotal molecular player in these scenarios.

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The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1-cullin-F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.

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Lipases produced by solid-state fermentation were used directly as biocatalysts for continuous synthesis of ethyl oleate in a continuously stirred tank reactor. The effect of biocatalyst reutilisation, molar ratio of substrates, agitation rate and feed rate on the esterification of oleic acid with ethanol were investigated. The catalyst maintained 90% conversion for four batch cycles with a 1:2 molar ratio (oleic acid:ethanol). Mechanical agitation at 200 and 300 rpm during 12 h of continuous reaction did not affect the biocatalytic conversion, allowing substrate conversions greater than 90% that were obtained with 50 mM oleic acid at a molar ratio of 1:2 during 14 h reaction. In contrast, substrate conversion was 70% with 100 mM oleic acid at a flow rate of 2 mL/min during 25 h of reaction. These results are promising and offer a technical alternative for the development of accessible biocatalysts that can be used in continuous operations.

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This paper reports a study of the enzymatic esterification of oleic acid and ethanol. The reaction was catalyzed by lipases produced by solid-state fermentation with Rhizopus sp. Olive oil and perlite were used as an inducer and inert support, respectively. Synthesis of ethyl oleate was carried out in a 10-mL batch reactor with magnetic stirring. The effects of substrate ratios, biocatalyst concentration, and temperature on the reaction rate and conversion efficiency were evaluated. The highest reaction rate (1.64 mmol/L min) was reached with an oleic acid/ethanol mol ratio of 1:5 (oleic acid 50 mM:ethanol 250 mM) and 1 g of biocatalyst. Conversions approaching 100% were obtained after 60 min of reaction at 45 degrees C with n-hexane as a solvent. The initial reaction rate increased proportionally with respect to biocatalyst concentration, which suggests that the reaction rate was not controlled by mass transfer. The biocatalyst retained more than 80% of its catalytic activity after 7 months of storage at 4 degrees C. The results demonstrate that the biocatalyst produced by Rhizopus sp. in solid-state fermentation can be successfully used for ethyl oleate synthesis over short reaction periods under conditions when ethanol is in excess.

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