Download G5. Strategies for Stabilization of Enzymes in Organic

Aalto University
School of Chemical Technology
CHEM-E3140 Bioprocess Technology II
Summary: Strategies for Stabilization of
Enzymes in Organic Solvents
Group Members:
Evgen Multia
Jimi Leivo
Karl Mihhels
1
1
Introduction
Enzymes are used in the industrial biocatalysis in wide variety of applications. These
vary from synthesis of pharmaceuticals to large-scale production of biofuels. Most
important biocatalysts are hydrolases, particularly lipases, since they are able to
convert wide variety of substances. They have many favourable properties; stability
at extreme temperatures, high chemo-, regio-, and enantioselectivity, and no need
for cofactors. Biocatalysts as such have evolved to work in cellular environments,
which makes them intolerant to harsh industrial conditions.
The solvent in cellular environment is water, which is not the best solvent for most
synthetic reactions. This raises a problem, even though organic solvents make synthesis more favourable, it affects the enzyme activity, and majority of enzymes display lower catalytic efficiency under organic solvents. This can be solved by either
optimizing the process conditions for existing enzymes, or prepare new biocatalysts
that can work under optimal process conditions. The latter is the strategy most
often employed strategy today. With help of modern technology biocatalysts have
been engineered to work even in neat organic solvents, which makes enzyme stability
engineering both of commercial and scientific interest.
2
Biocatalyst stability
The main factor that affects biocatalysis in nonconventional media is water content.
Biocatalysts that work efficiently in neat organic solvents are lipases, proteases and
many others, but even these biocatalysts have few water molecules bound to protein molecule. This leads to a general belief that waterless proteins are inactive.
While water promotes the conformational mobility required for optimal catalysis,
organic solvents lead to stronger intermolecular interactions. Thus water content
of an organic solvent promotes conformational mobility which leads to tendency
of an enzyme to denature. This results in decrease of enzyme activity when watermiscible cosolvent is added to water. For most enzymes the limit of organic cosolvent
2
concentration is 60-70 % (v/v), after which they become inactive.
Hydrophilic solvents have a tendency to remove protein-bound water, which is essential for proteins structure and function. Hydrophobic organic solvents are thus
much better for maintaining the protein structure. Because enzymes are not soluble
to hydrophobic organic solvents, they are added to hydrophobic solvents as powders
prepared by lyophilization. Lyophilization dehydrates the proteins, thus altering the
structure, which makes the enzyme activity lower also in hydrophobic solvents. The
denaturation of lyophilized protein is reversible upon addition of some water to the
protein, however, not unproblematic when protein is in hydrophobic solvent, since
it reduces the structural mobility of the protein.
3
Assessing biocatalyst stability
Protein stability can be defined as energetics of unfolding reactions, which occur
via reversible and irreversible unfolding. Reversible unfolding is characterized by
an equilibrium between native and unfolded states, and is called thermodynamic
stability or conformational protein stability. It reflects protein’s stability to refold
after different extreme conditions. Irreversible unfolding can be due to protein aggregation, misfolding, chemical modification, or a lack of chaperons. Irreversible
unfolding is kinetically driven, it is thus termed the kinetic stability.
Thermodynamic stability alone doesn’t guarantee that protein will remain active
during the process, since typical conditions in bioprocesses often result in irreversible
denaturation. This makes kinetic control of operational stability important for successful biocatalytic process.
4
Assessing biocatalyst stability
Three major strategies for obtaining stable biocatalysts in organic solvents can be
employed: (i) isolation of new enzymes that can work under extreme conditions,
3
(ii) modification of enzyme structure to make it more resistant to organic solvents,
(iii) modification of solvent environment so that it has less denaturing effect on the
enzyme.
Figure 1: Strategies for stabilization of enzymes toward organic solvents. [1]
4.1
(i) Isolation of stable biocatalysts
Biodiversity prospecting can be utilized to isolate the enzymes that are staying functional under harsh conditions from living organisms. These so-called extremozymes
are collected from microorganisms that can grow under extreme conditions. In these
cases nature employs many different structural strategies to obtain highly stable enzymes. The most important characteristic of these extremozymes is probably their
improved hydration capabilities, since loss of water molecules under extreme conditions is a main factor affecting the functionality of an enzyme. This makes it possible
to use extremozymes in nonaqueous environments. Important sources of these extremozymes are for example various strains of Pseudomonas aeruginosa, also some
halophilic and alkaliphilic organisms. Since there is certain difficulties related to
isolation of extremophilic organisms, it is more convenient to clone genes of these
organisms into suitable mesophilic hosts.
4
4.2
(ii) Modification of biocatalysts
Modification of biocatalysts can be divided into five parts: enzyme immobilization,
chemical modification, propanol-rinsed enzyme preparation, ionic liquid coating and
genetic modification. The most effective method to enhance enzyme stability is replacing free enzymes to immobilized forms. This immobilization creates stiffer structure of biocatalyst preventing them to and unfold enzymes and form malformations
of its active sites. Furthermore, the method increases the recovery of enzymes and
lowers the industrial costs by improving usability in continuous processes. Basically,
immobilization can be occurred onto or within enzymes or form water-insoluble particles. The most effective way for increasing stability is physical adsorption of an
enzyme.
Chemical modification of amino acid residues was one of the most common way
to improve chemical stability before broader understanding of molecular biology.
Even though the use of other methods have been increased, chemically modifying
has kept its popularity as a decent alternative. Usually polyethylene glycol (PEG)
and its derivatives have been used to process the properties of organic solvents.
Surface modification of proteins and covalent modification of subtilisin Carlsberg
are examples PEG can be used.
Other methods for modifying enzymes have not yet been able to achieve same popularity than previously mentioned methods. The basics of propanol-rinsed enzyme
preparation (PREP) are based on reactions with dry n-propanol. The goal is to
achieve low-water media by rinsing enzymes with n-propanol. Apart from immobilized ones, studies has shown that it is also possible to run PREP with free form
enzymes.
Another less-studied method is ionic liquid coating which utilizes ionic liquids (IL)
as cosolvents and enzyme-coating agents. IL are organic salts in a liquid form in
room temperature. They consist of only different cations and anions, and their properties bring new innovative ways to process enzymes e.g. polar substrates. Some
of those properties are low volatility and melting point (usually under 100 C), non-
5
flammability and thermal stability. IL have been examined in an organic solvent by
coating Burkholderia cepacia enzyme in [PPMIM][PF6]. In spite of increased stability, the activity of enzyme wasn’t improved, however compared to that, activation
improved during lyophilization.
Genetic modification manipulates directly the genome of organisms using biotechnology. It is technology to improve the genetic characterization of different cells.
This kind of modification has three different categories: directed molecular evolution, rational design and semirational design. The difference between methods are
how mutations are generated (randomly, predicted, with degenerate oligonucleotide,
etc.).
4.3
(iii) Modification of solvent environment
Solvent environment can be modified with additives or surfactants. Modification is
relatively easy to perform, as additives, such as inorganic salts, polyols and sugars,
can be added straight to the reaction solution or before lyophilization. Currently, the
most of the market enzymes have been produced using additives. The method needs
still more studying, since often estimations are based on presumptions and empirical
values. Several studies have been shown that simple salts can affect to enzymes by
activating them in organic media by lyophilization. Furthermore, other proofs about
the salt activation have been obtained using spectroscopic methods. Instead when
adding polyols or sugars in aqueous solution, they have been reported to strengthen
the hydrophobic interactions among nonpolar amino acid residues. Thus, proteins
stiffen. Polyols and sugars also affect water activity and microbial contamination
by lowering it. When stabilizing and activating proteins in nonaqueous conditions,
crown ether is one of the alternatives to consider.
Surfactants lower the surface tensions between two liquids or liquid and solid. When
adding into organic solvents, enzymes can form reverse micelles or water-in-oil microemulsions. Especially microemulsions have several benefits what comes to stabilization. They are clear, thermodynamically stable and isotropic liquid mixtures of
6
water, surfactants and organic compounds which are in different phase than water.
The enzyme stands in the aqueous phase while reacting hydrophobic substrate can
be dissolved in the organic phase. The encapsulation of enzymes to microdroplets
are the main reason for chemically activation and stabilization. New studies have
given new promising results, when conventional organic solvent has been replaced
by ionic liquid.
5
Conclusions
Enzymes play a significant role in today’s chemical technology and wide range of
reactions. Water can be used as a solvent for many different reactions, however, it
is relatively ineffective in most synthesis where organic solvents are needed. Using
organic solvents at the same time with the enzymes require modification and optimizing. One way is to produce new biocatalysts. Lipases and proteases are good
and effective examples of enzymes that are stable in organic liquids. The main factor
for the biocatalysts in nonconventional media is water content.
Stabilization is needed to improve reaction economy. For organic solvents, there are
three main methods to produce stable biocatalysts: Isolation of stable catalysts,
modification of biocatalysts and modification of solvent environment. Isolation is
done by separation of biocatalysts from extremozymes. Modification of biocatalysts
can be performed in several ways such as enzyme immobilization, chemical and
genetic modification, or with the presence of ionic liquids. Solvent environment is
basically modified with additives or surfactants, of which additives are predominant.
6
References
[1] V Stepankova, S Bidmanova, T Koudelakova. . . - Acs . . . , 2013 - ACS Publications