Abstract
Carrier-free immobilization is a key process to develop efficient biocatalysts able to catalyze the cell wall degradation in microalgae where the traditional solid supports cannot penetrate. Thus, the insolubilization of commercial Celluclast®, Alcalase®, and Viscozyme® enzymes by carrier-free immobilization and their application in microalgae pretreatment was investigated. In this study, different precipitants at different ratios (ethanol, acetone, and polyethylene glycol 4000) were tested in the first part of the method, to establish the precipitation conditions. The screening of the best precipitant is needed as it depends on the nature of the enzyme. The best results were studied in terms of immobilization yield, thermal stability, and residual activity and were analyzed using scanning electron microscopy. Moreover, a novel strategy was intended including the three enzymes (combi-CLEAs) to catalyze the enzymatic degradation of Nannochloropsis gaditana microalgal cell wall in one pot. The carrier-free immobilized derivatives were 10 times more stable compared to soluble enzymes under the same. At the best conditions showed its usefulness in the pretreatment of microalgae combined with ultrasounds, facilitating the cell disruption and lipid recovery. The results obtained suggested the powerful application of these robust biocatalysts with great catalytic properties on novel and sustainable biomass such as microalgae to achieve cost-effective and green process to extract valuable bioactive compounds.
Highlights
Nowadays, microalgae have gained attention as an alternative biomass due to a wide range of characteristics that make them an interesting biomass source
The use of enzymes has demonstrated to facilitate the hydrolysis of microalgae cell walls, which could be increased if the enzymes are combined between each other and with other physical disruption methods, obtaining increased oil yields
The optimum cross-linked enzyme aggregates (CLEAs) and combi-CLEAs obtained were applied to microalgae using the pretreatment described below and compared to soluble enzymes in terms of oil yield extracted [using the Folch method (Folch et al, 1957)] and microalgal cell wall disruption rate studied by scanning electron microscopy (SEM)
Summary
Microalgae have gained attention as an alternative biomass due to a wide range of characteristics that make them an interesting biomass source. The use of enzymes has demonstrated to facilitate the hydrolysis of microalgae cell walls, which could be increased if the enzymes are combined between each other and with other physical disruption methods, obtaining increased oil yields As it has been proved in previous studies on N. gaditana dry biomass, the combination of different enzymes, such as. When GA is the crosslinker, enzyme aggregates are irreversibly bonded through covalent bonds between its free amino groups (mainly from lysine residues) and both aldehyde moieties of GA, yielding an insoluble biocatalyst with high stability and activity It must be emphasized the importance of the cross-linking agent because it prevents the leaching of the enzyme and can cause steric hindrance. ® ® ® Viscozyme , Alcalase , and Celluclast have been developed and compared with soluble free enzymes regarding thermal and pH stability, to be applied in microalgae aqueous pretreatment combined with USs to enhance lipid recovery from biomass
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