O1. Improvement of stability and activity under drastic conditions through different immobilization protocols for Polygalacturonase from A. niger
Lucas Dal Magro1,2, Jakub F. Kornecki1,4, Manuela P. Klein3, Rafael C. Rodrigues2, Roberto Fernandez-Lafuente1
1Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, ZC 28049, Madrid, Spain; 2Biotechnology, 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; 3Department of Nutrition, Federal University of Health Sciences of Porto Alegre (UFCSPA), ZC 90050-170, Porto Alegre, RS, Brazil; 4Orion High Technologies Calle Turín 15, Nave 3, 28983 Parla, Madrid
Correspondence: Jakub F. Kornecki (yakokornecki@hotmail.es)
Polygalacturonases (PGs) are enzymes that act over the pectic acid eliminating the α-1,4 bond between two non-esterified galacturonic acid residues. PGs from fungi present a high rate of activity and they perform best at slightly acidic pH values and an optimum temperature between 30°C and 50°C. The addition of PGs during juices production is focused on the degradation of pectin and other big molecules, reducing its viscosity and achieving a clear aspect for the juice, besides increasing the juice production and halving the required filtration time. PG from Aspergillus niger was immobilized using three different supports: glyoxyl, vinylsulfone and glutaraldehyde-activated amino support. The use of supports pre-activated with glutaraldehyde had the best results. PG immobilization was carried for 24h at pH 5, and at pH 5, 6.5 and 8 for 3h, and passed this time they were switched to pH 8 to complete the 24h. Another protocol used pH 8 adding 300 mM NaCl to prevent ionic exchange between the enzyme and the support. The immobilization under all conditions produced a significant increase in thermal stability during stress inactivation experiments at pHs from 4, up to 10. This permitted that at temperatures over 70°C or pH values that went over 7, the biocatalyst maintained significant levels of activity while the free enzyme was completely inactive. The immobilization conditions were key over enzyme activity, thermostability and operational stability, making us think that the different conditions applied, allowed PG to have different orientations while being immobilized. The interest on the performance of each biocatalyst depends on the parameter of most value (activity or stability) and the conditions used during the reaction. Optimal PG immobilized biocatalysts could be reused up to ten times without significant losses in enzyme activity and offered a very linear reaction courses.
Funding: This work was supported by grants and scholarships (L. Dal Magro) from Capes, CNPq (process 403505/2013-5) and FAPERGS (process 17/2551-0000939-8). We also gratefully recognize the economic support from the Comunidad Autónoma de Madrid (project Ref. IND2017/IND-7640) and the MICIU from Spanish Government, (project number CTQ2017-86170-R). The authors wish to thank Amazon group and LNF Latinoamericana for kindly supplying the enzymes used in this research.