Q2. How can the immobilization rate be modulated?
The immobilization rate can be modulated using genetic engineering to add anamino acid on the enzyme surface that facilitates its immobilization on the support.
Q3. What is the effect of direct adsorption on the supports?
direct adsorption on very highly activated supports promotes a significant thermal stabilization of both enzymes, mainly under dissociation conditions, because the face of the protein that involves more enzyme subunits will be those that where it is easier to have a higher number of enzyme-support interactions.
Q4. What are the main uses of affinity domains?
The affinity domains, in order to yield a very high purification factors, require tobe properly designed to prevent unspecific adsorption of other proteins.
Q5. How many kDa can be purified by this technique?
The purification Purification depends on the distribution of charges and size of the target protein and the contaminant ones, having a protein of 200 KDa and contaminants not larger than 100 KDa purification may be almost total by this technique.
Q6. What is the way to immobilize lipases?
Interfacial activation of lipases on hydrophobic supports is a very useful reversible protocol to immobilize-purify lipases (see section 4.1 of this review) (Fernandez-Lafuente et al., 1998).
Q7. what is the irreversible inactivation of lipases on this kind?
Irreversible inactivation of the lipases adsorbed on this kind of supports usingchemical Ser-hydrolases inhibitors, that need to have access to the catalytic Ser and depend only in the exposition of this group to the medium, is much more rapid after the immobilization on hydrophobic supports than in the free enzyme or in other immobilized preparations (Carrasco-López et al., 2009, Manoel et al., 2015, Santos et al., 2014a, b).
Q8. What is the role of the support in the purification of enzymes?
There are not many heterofunctional supports in the literature, but most of the chromatographic supports described to purify enzymes (affinity ligands, dyes, etc.) may be compatible with the introduction of other moieties that may permit a covalent immobilization, changing from a support useful to purify enzymes (that is, a support that can easily release the adsorbed proteins), to a support useful to immobilize proteins.
Q9. What is the main problem of the protocol?
The protocol has as main problem the use of drastic conditions, which in certain cases may produce enzyme precipitation, perhaps by this reason the authors have been unable to find any actual industrial biocatalyst using this technology.
Q10. What is the criterion for lipases immobilized on hydrophobic supports?
The lipases thus immobilized may be used under a variety of conditions; even in relatively high concentrations of organic cosolvents the enzyme will remain attached to the support (Fernandez-Lafuente et al., 1998).
Q11. How does the pH of the support affect the enzyme?
the increase of the pH value to increase the reactivity of the nucleophilic groups on the enzyme surface is recommended to achieve some additional enzyme-support bounds (Pedroche et al., 2007).
Q12. What is the way to achieve the covalent attachment of lipases?
Immobilization was suggested to proceed via interfacial activation (activity improved after immobilization), and the final covalent attachments improve enzyme stability (Abaházi et al., 2014, Boros et al., 2013)This kind of heterofunctional hydrophobic supports may greatly improve theprospects of using interfacial activation of lipases as a tool to prepare biocatalysts useful under any experimental conditions, using the hydrophobic groups to have the a selective adsorption, and the other groups to transform this in an irreversible immobilization.
Q13. What is the main reason for the lipase to be adsorbed?
On the other hand, as lipase adsorption is related to interfacial affinity, somelipases become adsorbed only on certain hydrophobic supports and not in on others even permitting even the separation of different lipases contained in a sample in certain cases.