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PublicationPROCESS BIOCHEMISTRY
Year2015
Volume50
Pages918-927
International

EVALUATION OF DIVINYLSULFONE ACTIVATED AGAROSE TO IMMOBILIZE LIPASES AND TO TUNE THEIR CATALYTIC PROPERTIES

Authors:Roberto Fernandez-Lafuente, José Cleiton Sousa dos Santos
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Jose C. S. dos Santosa,b, Nazzoly Ruedaa,c,  Rodrigo Torresc, Oveimar Barbosad, Luciana R.B. Gonçalvesb and Roberto Fernandez-Lafuentea,*.
 
a: ICP-CSIC. Campus UAM-CSIC. Cantoblanco. 28049 Madrid. Spain.
b:   Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
c: Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
d: Departamento de Química, Grupo de investigación en productos naturales (GIPRONUT), Universidad del Tolima, Ibagué, Colombia.


Divinylsulfone (DVS) activated agarose beads have been used to immobilize several lipases, those from Pseudomonas flourescens, Rhizomucor miehei, and Thermomyces lanuginosus, (TLL) as well as the artificial chimeric phospholipase Lecitase Ultra. The best results in terms of activity recovery and immobilization yield were achieved using TLL. This enzyme could be immobilized at pH from 5 (with poor yield) to pH 10 (with 100% yield). The incubation of the immobilized enzymes for 72 h at pH 10 before the blocking step (using ethylenediamine) improved the enzyme stability whatever the immobilization pH value, but the stabilization achieved in each case depended on the immobilization pH value, and also on the inactivation conditions.
            The enzyme activities versus different substrates were very dependent on the immobilization protocol and the conditions of activity determination. That way, TLL immobilized at pH 5 on DVS activated support was the most active versus methyl mandelate, even more active than TLL immobilized on octyl-agarose (between 3 and 7 fold factors depending on the pH of measure). Using ethyl hexanoate the most active preparation was the octyl-TLL preparation. The most active among the enzymes immobilized in DVS-activated supports  was that just immobilized at pH 5 if the activity was determined at pH 7 or 8.5, while at pH 5 the most active was that enzyme but after incubation at pH 10.

            The results show that this support may be very useful for tuning lipase properties just by altering the first immobilization pH value, and that the further incubation at pH 10 improved enzyme stability, and in some cases, even increased  activity.
Keywords:Enzyme stabilization, Lipase immobilization, multipoint covalent attachment, divinylsulfone, lipase modulation, lipase from Thermomyces lanuginosus.
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