Problem Set 3.

2
(No. 3)
Pagara, Krishna Faith A.
Palaganas, Patricia Ann O.
 SK 3.12.
You are working for company A and you join a
research group working on immobilized enzymes.
Harry, the head of the lab, claims that
immobilization improves the stability of the
enzyme. His proof is that the enzyme has a half-life
of 10 days in free solution, but under identical
conditions of temperature, pH, and medium
composition, the measured half-life of a packed
column is 30 days. The enzyme is immobilized in a
porous sphere 5 mm in diameter. Is Harry’s
reasoning right? Do you agree with him? Why or
why not?
 Aggregation is often a problem with proteins in solution; the
higher the concentration of enzyme, the quicker the aggregation, and
usually, the faster the enzyme dies. This can be further increased if
redox sites are involved, at least in part due to cysteine reactivity and
divalent bonds forming between enzymes leading to inactive sludge
(often why DTT or other reducing agents are included in enzyme
storage buffers).
Additionally, enzymes which undergo conformational
changes during their catalysis can also become more prone to
denaturation in a purified state; denatured proteins also tend to glom
up more readily, rendering dead enzyme quite quickly. Certain
enzymes (those designed to chew up other molecules) will also exhibit
some activity against themselves (even if low, this adds up quickly in
the high concentration, low other-substrate type environment of
storage). Immobilization solves several of these problems - enzymes
are at a relatively low concentration for aggregation and inter-enzyme
reactions with each other, while they can still be at a high relative
concentration for reaction with substrate flowed through the beads.
Supporting Arguments
O “Insolubilization of the enzyme by
attachment to a matrix also imparts several
added benefits such as (1) rapid arrest of
the reaction by removal of the enzyme from
the reaction solution and (2) improvement
of enzyme stability against temperature,
solvents, pH, contaminants and impurities”.

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434042/
O “Insolubilization of the enzyme by
attachment to a matrix also imparts several
added benefits such as (1) rapid arrest of
the reaction by removal of the enzyme from
the reaction solution and (2) improvement
of enzyme stability against temperature,
solvents, pH, contaminants and impurities”.

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434042/
O The stability of the immobilized enzymes can
increase or decrease depending on the type of
matrix as well as on the interaction between the
enzyme and the matrix. The following are three
important parameters that are the determinants
of enzyme stability during immobilization.

O Immobilizing matrix
O Immobilization conditions
O Rigidification by multipoint attachment

Source: A. Dwevedi, Enzyme Immobilization, DOI 10.1007/978-3-

319-41418-8_2
O Increase in half-life and thermal stability of enzymes
has been achieved by covalent coupling with
different supports like mesoporous silica, chitosan,
etc. (Hsieh et al. 2000; Ispas et al. 2009). Cross-
linking of enzymes to electrospun nanofibers has
shown greater residual activity due to increased
surface area and porosity. Use of such
nanodiametric supports have brought a turning point
in the field of biocatalyst immobilization (Wu et
al. 2005; Kim et al. 2006; Ren et al. 2006; Li et
al. 2007; Huang et al. 2008; Sakai et al. 2010).

O Source:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563746/