Abstract
Eversa is an enzyme recently launched by Novozymes to be used in a free form as biocatalyst in biodiesel production. This paper shows for first time the immobilization of Eversa (a commercial lipase) on octyl and aminated agarose beads and the comparison of the enzyme properties to those of the most used lipase, the isoform B from Candida antarctica (CALB) immobilized on octyl agarose beads. Immobilization on octyl and aminated supports of Eversa has not had a significant effect on enzyme activity versus p-nitrophenyl butyrate (pNPB) under standard conditions (pH 7), but immobilization on octyl agarose beads greatly enhanced the stability of the enzyme under all studied conditions, much more than immobilization on aminated support. Octyl-Eversa was much more stable than octyl-CALB at pH 9, but it was less stable at pH 5. In the presence of 90% acetonitrile or dioxane, octyl-Eversa maintained the activity (even increased the activity) after 45 days of incubation in a similar way to octyl-CALB, but in 90% of methanol, results are much worse, and octyl-CALB became much more stable than Eversa. Coating with PEI has not a clear effect on octyl-Eversa stability, although it affected enzyme specificity and activity response to the changes in the pH. Eversa immobilized octyl supports was more active than CALB versus triacetin or pNPB, but much less active versus methyl mandelate esters. On the other hand, Eversa specificity and response to changes in the medium were greatly modulated by the immobilization protocol or by the coating of the immobilized enzyme with PEI. Thus, Eversa may be a promising biocatalyst for many processes different to the biodiesel production and its properties may be greatly improved following a suitable immobilization protocol, and in some cases is more stable and active than CALB.
Highlights
Lipases are very interesting enzymes in biocatalysis due to their broad substrate specificity, their selectivity and high stability, combined in many instances with a very high enantio or regio selectivity and specificity [1,2,3,4,5,6,7,8,9,10]
CALBremained slightly almost intact, being immobilization marginally more rapid,while usingthe both enzymes immobilization decreased its activity after immobilization on octyl agarose, activity of Eversa remained yield is over maintains full activity, but immobilization yield is around almost intact, being immobilization marginally more rapid, using both enzymes immobilization yield is80%
Retention times were 2.4 min for acid and 4.2 min for ester. Eversa properties such as stability or activity may be enhanced after a proper immobilization, the supplier recommends use in free form
Summary
Lipases are very interesting enzymes in biocatalysis due to their broad substrate specificity, their selectivity and high stability, combined in many instances with a very high enantio or regio selectivity and specificity [1,2,3,4,5,6,7,8,9,10]. They can perform their function in a wide diversity of reaction media [11,12,13]. In the closed form, a polypeptide (named lid) blocks the active center, making the Catalysts 2018, 8, 511; doi:10.3390/catal8110511 www.mdpi.com/journal/catalysts
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