期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 160, 期 -, 页码 28-35出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2019.03.016
关键词
beta-glucosidase; Endoglucanase; Immobilization; Co-immobilization; Nanoparticles
类别
资金
- Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)
- Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)
- Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2016/17582-0, 2017/13734-3]
Nanoparticles can act as support materials for enzymatic immobilization, introducing a balance of characteristics that modulate the efficiency of biocatalysts, such as specific surface area, resistance to mass transfer and effective enzymatic loading. Magnetic nanoparticles can be easily separated using an external magnetic field, and in this work two recombinant enzymes, the beta-glucosidase from Humicola insolens (Bglhi) and the endoglucanase from Scytalidium thermophilwn (Egst) were immobilized on synthesized Fe3O4 nanoparticles derivatized with chitosan/glutaraldehyde/N-(5-amino-1-carboxy-pentyl) iminodiacetic acid and functionalized with NiCl2. The immobilization yields were about 20% for Bglhi and Egst with efficiencies of 132% and 115%, respectively. The two enzymes were also co-immobilized with yield was about 49%. The optimal temperatures of the immobilized enzymes were 70 degrees C and 55 degrees C for Egst and Bglhi, respectively. Egst hydrolyzed CMC in the presence of 4 mM MnCl2 with V-max = 625.0 +/- 6.7 U mg(-1) and K-M = 6.4 +/- 0.5 mg mL(-1) resulting in a catalytic efficiency (k(cat)/K-M) of 107.4 +/- 5.4 mg(-1) s(-1) mL. Bglhi hydrolyzed pNP-Glc with V-max = 52.7 +/- 2.7 U mg(-1) and K-M = 0.23 0.01 mM resulting in a catalytic efficiency (k(cat)/K-M) of 214.3 +/- 10.25(-1) mM(-1). The individually immobilized enzymes when combined showed a synergistic effect on the substrates tested and a very similar action when compared to the co-immobilized enzymes, suggesting excellent potential for application in biotechnological processes.
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