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PublicationRSC Advances
Pages 27242 - 27251

Preparation and characterization of a Combi-CLEAs from pectinases and cellulases: a potential biocatalyst for grape juice clarification

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Lucas Dal Magro1, Plinho F. Hertz1, Roberto Fernandez-Lafuente2, Manuela P. Klein1,3,
Rafael C. Rodrigues1,*
1Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and
Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O.
Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil.
2Departamento de Biocatalisis. ICP-CSIC. Campus UAM-CSIC. Cantoblanco. 28049
Madrid. Spain
3Department of Bioprocesses, State University of Rio Grande do Sul, Porto Alegre, RS,
Combined cross-linked enzyme aggregates (combi-CLEAs) are a novel
prospective for immobilization of mixture of enzymes and the present study addresses
the preparation, characterization and application of pectinases-cellulases combi-CLEAs
for grape juice clarification. Initially, 8 enzymatic preparations were tested for turbidity
reduction in grape juice and Rohapect 10L provided the best results (around 50 % in 1
h) being selected for CLEA preparation. The optimization of combi-CLEAs, was
performed using a central composite design (CCD) and response surface methodology
(RSM) varying the glutaraldehyde concentration and reaction time, using iso-propanol
as precipitant agent. The best conditions for Rohapect 10L CLEAs preparation was 110
mM of glutaraldehyde and 2 h. Bovine serum albumin (BSA) was used as feeder and
improved the volumetric activity, recovered activity and thermal stability. Combi-
CLEAs-BSA prepared using 0.4 mg.mL-1 of enzyme mixture and 2.4 mg.mL-1 of BSA
presented an activity of 14 U.mL-1, 18 % of recovered activity and 3-times more thermal
stability compared to soluble enzymes. The combi-CLEAs and combi-CLEAs-BSA
were tested in repeated batches, being reused for 4 and 6 cycles, respectively, keeping
100 % of the initial activity. The combi-CLEAs and combi-CLEAs-BSA appear to be
suitable alternatives of immobilized biocatalyst for the clarification of grape juices.

Keywords:pectinase; cellulase; cross-linked enzyme aggregates; bovine serum albumin; grape juice clarification
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