Published in SOFW Journal (Feb 2019)

Title: Improving Protease Stability in Liquid Detergents by Protein Engineering

By: A.J. Hoekstra*, M.R. Stoner

Abstract:

Enzymes have been used to improve the cleaning efficiency and fabric care benefits of
detergents for several years, and are now well accepted as ingredients in a wide range of
product formats. The main challenge with enzymes in laundry detergents is that they can lose
activity over time. With the global rise of liquid laundry detergents, formulators need to
balance the shelf stability of the enzymes against destabilizing effects of anionic surfactants and
other chemicals. Recently, much effort has been invested to improve enzyme stability by means
of protein engineering. This article reports on the development of a structurally stable protease
that opens up avenues for the formulator to improve the aged cleaning performance of the
liquid detergent or use alternative chemical stabilizers.

Introduction

Formulating detergents has become more challenging due to pressures to reduce energy and
water consumption, as well as to eliminate ingredients that have a negative impact on health
and the environment. Functional ingredients like enzymes have become a vital part of
detergent formulations because they deliver significant performance benefits. Enzymes are
biocatalysts that target specific macromolecular substrates. Familiar examples include
proteases breaking down protein-based stains and amylases breaking down starch-based
stains. During the past decade, many other enzyme functionalities have been introduced to
improve both stain removal and fabric care properties of detergents. Enzymes contribute to
more sustainable detergents as a result of their efficacy at low inclusion levels and their ability
to (partially) replace chemical ingredients.

The main challenge with enzymes in laundry detergents is that they can lose activity over time.
Despite detergents being characterized as fast-moving consumer goods, it can take several
months before a detergent arrives at a consumer’s home. During its journey, the detergent can
experience high temperatures, especially when it is exposed to direct sunlight or stored under
ambient conditions in a hot climate. Retaining sufficient enzyme activity during these
conditions is essential to deliver enzyme performance at point of use. It is by delivering
robust enzyme performance time and again that the detergent or retailer brand delivers on its
promise, making consumers more inclined to repeat their purchases.

Enzyme Degradation in Liquid Detergent Formulations

Several factors can lead to enzyme activity loss in liquid laundry detergents. Enzymes are
proteins with a complex, three-dimensional structure. Figure 1 shows a structural model of an
enzyme-substrate complex. The active site of the enzyme is the locus that binds to the
substrate, breaks it down, and releases the reaction products. The active site catalyzes this
reaction as long as the enzyme retains its structure. However, the laundry liquid detergent
environment can be hostile resulting in a loss of this structure.

Figure 1 Space-filling model of an enzyme-substrate complex

Since enzymes are proteins, they can serve as substrates for protease enzymes. Typically, two
modes of hydrolysis are distinguished: autolysis and proteolysis. Autolysis occurs when a
protease behaves like a substrate and hydrolyzes itself into smaller peptides. Once the protease
has been broken apart, it loses its functionality. In contrast, proteolysis occurs when a protease
treats other enzymes in the detergent as the substrate and hydrolyzes them into smaller
peptides. Protein hydrolysis can happen in parallel with other mechanisms of inactivation such
as protein unfolding due to interaction with surfactants and increasing temperature. The
presence of water amplifies the detrimental effect of surfactants and enhances the rate of
undesirable reactions.

Stabilization by Chemical Additives

Considerable effort has been devoted to overcoming the challenge of formulating enzymes into
liquid laundry detergent products. Traditionally, various enzyme-stabilization strategies have
been developed based on chemical additives [2]. Boron compounds (boric acid, borate salts),
especially in conjunction with polyols (propylene glycol, glycerol) have been shown
to stabilize enzymes in liquid detergent formulations. Boronic acid-based derivatives have also
been promoted as a more weight-effective inhibitor. Carboxylic acid salts such as sodium
formate are also known for their inhibiting effect, but tend to be less weight-effective than
borate. The protein stability of enzymes like subtilisin is affected by calcium binding sites, which
is why some formulators add a low level of calcium chloride to the detergent. The effectiveness
of calcium salt as a stabilizer depends on its compatibility with anionic surfactants and chelants
in the formulation.

The Protein Engineering Approach

A drawback of using chemical additives to stabilize proteins in detergent formulations is that

the stabilizers themselves occupy valuable formulation space and can be expensive. Modern
biotechnology enables us to address this challenge by developing inherently more stable
enzymes through protein engineering. By using protein engineering, a large sequence space can
be systematically mapped, and proteins can be tailored for specific needs. The initial stage is to
find at least one protein sequence that meets the minimum performance requirement for the
desired properties. Once such a starting sequence is identified, additional sequence diversity is
generated based on a selected enzyme scaffold. High-throughput methods are used to screen
the variants, and consecutive multi-property optimization helps to identify the best set of
mutations. Then, combinatorial variants are created using computational strategies which are
guided by structural biology and bioinformatics. Finally, performance applications testing is
required to identify the engineered variants that exhibit superior properties.

Materials and Methods

The protease activity was measured by means of a biochemical assay at pH 8.7 and 30 °C. The
assay is colorimetric and monitors the rate of degradation of N-succinyl-ala-ala-pro-phe-p-
nitroanalide substrate from Sigma Aldrich. The release of the substrate's p-nitroanalide is
measured at 405nm on a Konelab auto-analyzer.
The cleaning performance of the heavy duty liquid (HDL) detergent was measured by using an
enzyme responsive stain set that is commercially available from CFT BV (The Netherlands).
Experiments were carried out in Miele W1935 WPS Ecoline (Cotton Short cycle) at 30 °C and
water hardness of 25 °FH using a cotton wash load and 4 strips SBL2004 soil ballast. The
detergent dosage was 55 ml/wash. Stain removal measurements were carried out using a
Mach5 multi area spectral imaging device (CFT BV/Colour Consult), averaged using 2 internal
and 4 external replicates, and expressed in a Stain Removal Index (SRI) % based on Delta E color
differences.

Improving the Structural Stability of Protease

The protein engineering approach has previously been demonstrated for improving the stability
of cellulase, and in particular its resistance to the degrading effects of protease [2]. Recently,
this approach has been applied to improve the stability of protease itself. Protease stability is
critical, because protease is a workhorse enzyme that cleans many common consumer relevant
stains such as food stuffs comprising milk, meat, and egg, outdoor stains like grass, and body
fluids comprising blood. As a result, proteases are present in virtually every enzymatic liquid
detergent. If the protease loses catalytic activity during storage, the laundry detergent will not
be able to deliver consistent cleaning performance on a wide range of stains.
The effect of targeted protein engineering for protease stability is shown in Figure 2. In this
experiment the thermostability of three different protease molecules was studied. Each
molecule was subjected to increasing temperature for 20 minutes after dissolution in a 10
w/w% aqueous solution of harsh detergent formula. The figure shows the residual protease
activity (measured by using a biochemical assay) for each molecule as a function of
temperature.
An existing commercially available protease demonstrates poor thermostability, while some
improvement could be achieved by selecting a different wild-type protease (isolated from a
natural Bacillus species). Subsequent protein engineering of this molecule resulted in a
significant improvement of its thermostability; a recently commercialized protease retained
nearly all of its activity, even at 65°C.

Figure 2 Residual protease activity curves as a function of temperature for an existing protease vs. an alternative wild type
protease and a protein engineered variant of this “parent” molecule (commercialized as PREFERENZ® P 300).

The higher thermostability of PREFERENZ® P 300 protease results in a step change in-detergent
stability improvement versus conventional proteases. The extent of stability improvement,
however, is not independent of the composition of a liquid detergent. This is demonstrated in
Figure 3 for a range of representative commercial liquid detergents that were sampled from
supermarkets in The Netherlands.
Four detergents could be characterized as heavy-duty liquid (HDL) and two detergents are
liquid unit dose sachets packed in a water-soluble flowwrap. The liquid detergents were
incubated at high temperature to inactivate any enzymes present in the formulation. A
protease engineered for improved stability was added to each formulation. Subsequently, the
residual protease activity was measured by means of a biochemical assay during an accelerated
storage study for 8 weeks at 37°C. The figure shows the measured protease activity (relative to
the initial activity) for both protease technologies after four and eight weeks of storage.
The data show that excellent protease stability can be achieved. In most of the detergents the
protease remains more than 50% activity after 8 weeks storage at these conditions. In the liquid
unit dose products the residual protease activity is even more than 90%. As expected, the
detergent formulation still impacts the protease stability profile which is likely related to the
presence of stabilizing chemical additives.

Figure 3 Residual protease activity in several commercially available liquid detergents during accelerated storage.

Alternative Stabilizer Approach for Enzymatic Liquid Detergents

The improved structural stability of PREFERENZ® P 300 protease enables two different avenues
for the detergent formulator. Firstly, improved protease stability results in a better aged
performance of the detergent at point of use, i.e. a more consistent cleaning performance can
be delivered during the shelf life of the detergent, even under stressed storage conditions.
Moreover, using a protease engineered for stability allows the formulator to revisit his or her
enzyme stabilization strategy if no protease stability upgrade is required. Less chemical
additives are required to maintain protease stability, or alternative stabilization systems can be
considered.
An example of using an alternative stabilizer approach is shown in Figure 4. In this study, a
representative European heavy duty liquid detergent was used to establish fresh and aged
cleaning performance on a wide range of enzymatic stains. Both detergent formulations
comprised a protease engineered for stability combined with several secondary enzyme
functionalities. A separate screening design was carried out to determine the optimal stabilizer
system based on boric acid or sodium formate combined with propylene glycol. A commercially
available HDL was used as the performance benchmark. The stain removal index was measured
for “fresh” detergent as well as detergent submitted to an accelerated storage test. The results
show that both stabilizer systems achieve equivalent cleaning performance across the stain set,
and a performance loss of less than 10 points SRI% after storage.

Figure 4 Fresh and “aged” cleaning performance of a multi-enzyme European HDL detergent using different stabilizer systems.
The enzyme system comprises PREFERENZ® P 300 protease, PREFERENZ® S 210 amylase, PREFERENZ® M 100 mannanase,
PREFERENZ® L 100 lipase, and REVITALENZ® 200 cellulase.

Conclusion and Outlook

In conclusion, protein engineering enables exciting opportunities to deliver more robust
enzyme performance in liquid detergents. There are numerous other developments in the
pipeline as DuPont builds a portfolio of enzymes that give new levels of flexibility to
formulators.
Acknowledgments:
Dr. Sina Pricelius (DuPont Industrial Biosciences) is greatly acknowledged for a critical review of
the article and correction of the German translation.
References:

[1] Stoner, M.R., Dale, D.A., Gualfetti, P.J., Becker, T., Manning, M.C., Carpenter, J.F., and
Randolph, T.W., Protease autolysis in heavy-duty liquid detergent formulations: effects of
thermodynamic stabilizers and protease inhibitors, Enzyme and Microbial Technology, 2004, 34,
114- 125.
[2] Krouwer, A.J.J., Adams, C.D., Engineering an Improved Cellulase for Fabric Care in Liquid
Detergents, SOFW Journal, 2016, 142, 64-67.

*Contact:
Dr. Arjen J. Hoekstra
DuPont Industrial Biosciences
Archimedesweg 30, 2333 CN Leiden
The Netherlands
Email: [email protected]