Saccharification of hemicellulose and cellulose with immobilized enzymes

• Autor(a) principal: Murilhas, Sara Sofia Adriano
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/141379
• topic: Enzyme immobilization; Cellulase; Hemicellulase; Magnetic nanoparticles; Chitosan microparticles; Lignocellulose saccharification; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Wine is a very appreciated beverage worldwide, and, in Portugal, the winemaking sector is of major socio-economic significance, substantially contributing to the national economy. However, it results in a lot of residual material called pomace, which is rich in cellulose, hemicellulose, and lignin. Cellulose and hemicellulose represent a significant portion of vegetal biomass, usually discarded as feed. However, both sugar polymers can be hydrolyzed into C5 and C6 monomers by a saccharification process and then fermented into multiple added-value products, like bioethanol. The greener and more specific option for saccharification is through enzymes. However, after the reaction is over, the enzymes are inactivated and remain in the final product, as there is no way to retrieve them from the solution. Nonetheless, these enzymes are expensive and still functional after a single use. So, this work’s goal is to develop immobilization protocols for cellulases and hemicellulases. That will allow for their reuse, making the process economically more viable. The immobilization protocols consisted of covalent attachment of cellulase to magnetic nanoparticles (mNPs), specifically superparamagnetic iron oxide nanoparticles (SPIONs), and of hemicellulase to chitosan microparticles (CMPs). SPIONs were used with multiple coatings – dimercaptosuccinic acid (DMSA), (3-aminopropyl)triethoxysilane (APTES), and chitosan. With this approach, the enzymes attached to the supports can be recovered by magnetic separation (for mNPs) or by filtration (for CMPs). This work aims at the saccharification of subcritical water (SBW) pretreated Red Wine Grape Pomace (RWGP). SBW results in a solid residue rich in lignin and cellulose, target of the cellulase complexes, and a liquor rich in hemicellulose oligomers, target of the hemicellulase complexes. The target characteristics for the complexes are reusability, high enzyme loading, and enzymatic activity, as well as operational and storage stability. Therefore, the complexes were assessed by their retained activity when comparing with the enzyme in its free form. The monomer yield was determined by High-Performance Liquid Chromatography (HPLC), while enzyme leaching was monitored through protein detection assays. A preliminary assessment of the complexes’ storage stability was also made. To develop the immobilization protocols, the project started with the characterization of three available commercial carbohydrase formulations and the analysis of their activity in their free form to establish a control to compare to the immobilization complexes. Two cellulases, CelluClast 1.5L and Cellic cTec2, and a hemicellulase, Viscozyme L, were tested. Protein content was determined by the Bradford assay using Bovine Serum Albumin (BSA) as standard, resulting in 54.8 mg/mL for CelluClast 1.5L, 99.1 mg/mL for Cellic cTec2, and 20.6 mg/mL for Viscozyme L. Total saccharification activities of 51 units/mL in CClast and 137 units/mL in cTec2 were determined for the cellulases. An equivalent activity measurement was determined for the hemicellulase Viscozyme L using two standard substrates, achieving an activity of 2 units/mL in xylan and 0.4 units/mL in arabinogalactan. Fifteen complexes were performed for the immobilization of cellulase onto mNPs, and two for hemicellulase onto CMPs (HM). From the produced cellulase complexes, the CAS protocols (cellulase immobilized onto APTES-coated SPIONs) were deemed less promising due to the loss of integrity of the matrix. The most promising protocols for cellulase are CDS-g 0, a version of cellulase immobilized onto DMSA-coated SPIONs, and CMm (cellulase immobilized onto CMPs with magnetic cores), while for hemicellulase the most promising protocol is HM 2 (the 2nd protocol of hemicellulase immobilization onto CMPs). However, they all must be reproduced in larger quantities and re-tested in multiple saccharification cycles.