Produção e aplicação de agregados enzimáticos reticulados e magnéticos (CLEAs e mCLEAs) usando coquetéis celulolíticos comerciais para hidrólise de fibra de coco verde

Abstract

Enzymes are responsible for catalyzing important reactions in biotechnological processes. However, its industrial viability requires easy handling, reuse and operational stability, which can be achieved through immobilization techniques. Among them, the strategy of cross-linked enzyme aggregates (CLEAs) stands out, as it allows direct and efficient contact between the enzyme and the substrate, as it eliminates the need for conventional solid supports. Furthermore, the use of non-porous supports, such as magnetic nanoparticles, in the formation of magnetic CLEAs (mCLEAs) can optimize the immobilization process. This is particularly advantageous when applied to industrial enzymes, such as cellulolytic cocktails, which consist of cellulases capable of hydrolyzing cellulose to produce glucose, a valuable precursor for the manufacture of ethanol. In this framework, green coconut fiber is considered, a lignocellulosic residue generated on a large scale after the consumption of coconut water, which represents around 70% of the total fruit. The use of CLEAs and mCLEAs could efficiently transform this biomass into glucose, demonstrating a significant practical application of this technology in the use of waste and the production of biofuels. This work aimed to produce CLEAs and mCLEAs from commercial cellulolytic cocktails (Celluclast 1.5L and Cellic CTec 2) for the hydrolysis of pretreated green coconut fiber, transforming a common waste into glucose, a value-added product. Initially, different precipitation solutions were evaluated, such as ethanol, acetone and ammonium sulfate, using glutaraldehyde and magnetic nanoparticles covered with aminopropyltriethoxysilane for the production of biocatalysts. The addition of bovine serum albumin (BSA) protein was also tested to improve immobilization. The biocatalysts showed better performance with ethanol and acetone, especially those with acetone and BSA, which achieved the best enzymatic activities (287.27 ± 42.59 U/g for CMCase and 0.00 ± 0.00 U/g for FPase with Celluclast 1.5L) and (425.37 ± 48.11 U/g for CMCase and 1.54 ± 1.10 U/g for FPase with Cellic CTec 2). To expand industrial applicability, magnetic nanoparticles were incorporated in the precipitation stage, producing mCLEAs with excellent thermal stability and high catalytic activities. Notably, the 5% mCLEA-Celluclast-AC-BSA-GA biocatalyst showed activity of 149.65 ± 2.65 U/g for CMCase and 5.30 ± 1.75 U/g for FPase, and maintained 58% of its activity after 72 hours at 70 ºC. The mCLEA-Celluclast-AC-BSA-GA 2.5% biocatalyst proved to be effective in the hydrolysis of coconut fiber and isolated cellulose, producing up to 9.60 ± 0.57 g/L of glucose. Based on the data presented, CLEAs appear to be an alternative for the efficient use of cellulases in the 2G ethanol chain.