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Extracorporeal membrane oxygenator (ECMO) is a well-established therapy for respiratory failure. Refractory hypoxemia, despite the use of ECMO, remains a challenging problem. The ECMO circuit may not provide enough oxygenation support in the presence of high cardiac output, increased physiologic demand, and impaired gas exchange. Adding a second ECMO oxygenator using the same pump (sometimes needing a second drainage cannula) can improve oxygenation and facilitate lung-protective ventilation in selected patients. We describe a 3-patient series with severe ARDS secondary to SARS-CoV-2 infection and refractory hypoxemia during ECMO support successfully treated with this approach.

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Ionic additives affect the structure, activity and stability of lipases, which allow for solving common application challenges, such as preventing the formation of protein aggregates or strengthening enzyme-support binding, preventing their desorption in organic media. This work aimed to design a biocatalyst, based on lipase improved by the addition of ionic additives, applicable in the production of ethyl esters of fatty acids (EE). Industrial enzymes from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida antárctica B (CALB) and Lecitase®, immobilized in commercial supports like Lewatit®, Purolite® and Q-Sepharose®, were tested. The best combination was achieved by immobilizing lipase TLL onto Q-Sepharose® as it surpassed, in terms of %EE (70.1%), the commercial biocatalyst Novozyme® 435 (52.7%) and was similar to that of Lipozyme TL IM (71.3%). Hence, the impact of ionic additives like polymers and surfactants on both free and immobilized TLL on Q-Sepharose® was assessed. It was observed that, when immobilized, in the presence of sodium dodecyl sulfate (SDS), the TLL derivative exhibited a significantly higher activity, with a 93-fold increase (1.02 IU), compared to the free enzyme under identical conditions (0.011 IU). In fatty acids ethyl esters synthesis, Q-SDS-TLL novel derivatives achieved results similar to commercial biocatalysts using up to ~82 times less enzyme (1 mg/g). This creates an opportunity to develop biocatalysts with reduced enzyme consumption, a factor often associated with higher production costs. Such advancements would ease their integration into the biodiesel industry, fostering a greener production approach compared to conventional methods.

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Processes involving lipases in obtaining active pharmaceutical ingredients (APIs) are crucial to increase the sustainability of the industry. Despite their lower production cost, microbial lipases are striking for their versatile catalyzing reactions beyond their physiological role. In the context of taking advantage of microbial lipases in reactions for the synthesis of API building blocks, this review focuses on: (i) the structural origins of the catalytic properties of microbial lipases, including the results of techniques such as single particle monitoring (SPT) and the description of its selectivity beyond the Kazlauskas rule as the "Mirror-Image Packing" or the "Key Region(s) rule influencing enantioselectivity" (KRIE); (ii) immobilization methods given the conferred operative advantages in industrial applications and their modulating capacity of lipase properties; and (iii) a comprehensive description of microbial lipases use as a conventional or promiscuous catalyst in key reactions in the organic synthesis (Knoevenagel condensation, Morita-Baylis-Hillman (MBH) reactions, Markovnikov additions, Baeyer-Villiger oxidation, racemization, among others). Finally, this review will also focus on a research perspective necessary to increase microbial lipases application development towards a greener industry.

Assuntos

RESUMO

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg-1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg-1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.

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RESUMO

Immobilization on Glyoxyl-agarose support (Gx) is one of the best strategies to stabilize enzymes. However, the strategy is difficult to apply at neutral pH when most enzymes are stable and, even when possible, produces labile derivatives. This work contributes to overcoming this hurdle through a strategy that combines solid-phase amination, presence of key additives, and derivative basification. To this end, aminated industrial lipases from Candida artarctica (CAL), Thermomyces lunuginosus (TLL), and the recombinant Geobacillus thermocatenulatus (BTL2) were immobilized on Gx for the first time at neutral pH using anthranilic acid (AA) or DTT as additives (immobilization yields >70%; recovered activities 37.5-76.7%). The spectroscopic evidence suggests nucleophilic catalysis and/or adsorption as the initial lipase immobilization events. Subsequent basification drastically increases the stability of BTL2-glyoxyl derivatives under harsh conditions (t1/2, from 2.1-54.5 h at 70 °C; from 10.2 h-140 h in 80% dioxane). The novel BTL2-derivatives were active and selective in fish oil hydrolysis (1.0-1.8 µmol of polyunsaturated fatty acids (PUFAs) min-1·g-1) whereas the selected TLL-derivative was as active and stable in biodiesel production (fatty ethyl esters, EE) as the commercial Novozyme®-435 after ten reaction cycles (~70% EE). Therefore, the potential of the proposed strategy in producing suitable biocatalysts for industrial processes was demonstrated.

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Early intercellular signaling in Coffea arabica L.-Hemileia vastatrix host-pathogen interaction was studied, using inside-out plasma membrane from two varieties of coffee leaf and a fungal fraction to determine the plant's biochemical responses. Microsomal pellets (100,000 x g) from the susceptible (Caturra) and resistant (Colombia) coffee leaf varieties were purified by partitioning in two-polymer DEX (6.3% w/w) and PEG (6.3% w/w) system aqueous phase. Fungal material was obtained from orange rust Hemileia vastatrix Berk and Br. race II urediospore germ tubes. Plasma membrane vesicles were preferentially localized to PEG phase, as indicated by its enzyme marker distribution. Both H(+)-ATPase activities displayed similar kinetic and biochemical characteristics, comparable to those described for P-type ATPases. Several enzymes may play pivotal roles in plants regarding early interaction with fungal elicitors. Studies of fungal fractions' effects on H(+)-ATPase and both varieties' proton pumping activities were thus carried out. Concentration as low as 0.1 Gluc eq. ml(-1) fungal fraction induced specific inhibition of H(+)-ATPase and the resistant variety's proton pumping activities. The present work describes characterizing the H(+)-ATPase plasma membrane from two Coffea arabica L. varieties (Caturra and Colombia) for the first time and the race specific inhibitory effect of a crude fungal fraction on both H(+)-ATPase and the resistant variety's proton pumping activities.

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