New Biotechnology  Volume 00, Number 00  August 2010 RESEARCH PAPER

Research Paper
Advances in protease engineering for
laundry detergents
Q1
Ljubica Vojcic1,5, Christian Pitzler1,5, Georgette Wirtz1,5, Felix Jakob2,5,
Ronny Martinez1,3, Karl-Heinz Maurer4 and Ulrich Schwaneberg1,2
1
RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany
2
DWI - Leibniz Institute für Interaktive Materialen, Forckenbeckstraße 50, D-52074 Aachen, Germany
3
EW-Nutrition GmbH, Enzyme Technology, Nattermannallee 1, D-50829 Köln, Germany
4
AB Enzymes GmbH, Feldbergstraße 78, D-64293 Darmstadt, Germany

Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin
proteases employed in the laundry detergent industry have been engineered by directed evolution and
rational design to tailor their properties towards industrial demands. This comprehensive review
discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for
laundry detergents comprise simultaneous improvement of thermal resistance and activity at low
temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase
promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The
three protease engineering campaigns presented provide in-depth analysis of protease properties and
have identified principles that can be applied to improve or generate enzyme variants for industrial
applications beyond laundry detergents.

Introduction Serine initiates a nucleophilic attack on the peptide bond in

Q2 Proteases (proteinases or peptidases) are enzymes that catalyze the
hydrolysis of peptide bonds. They are found in all organisms,
where they play an essential role in metabolic and physiological
processes. Substrate unspecific proteases participate in protein
recycling and digestion, whereas sequence specific proteases are
essential for zymogen activation, catalytic cascades, and other
physiological processes related to cell survival or death [1]. Pro-
teases are classified by their catalytic mechanism into aspartic,
glutamic, serine, cysteine or metalloproteases, by their ability to
cleave terminal amino acids as exo- or endopeptidases, or by pH
conditions for optimal activity (acid, neutral or alkaline proteases)
[2].
Serine proteases are the most abundant type of protease
containing a serine as essential catalytic amino acid residue.
Corresponding author: Schwaneberg, U. (u.schwaneberg@biotec.rwth-aachen.de)
5
Joint first authorship.
an electronic environment provided by a neighboring histidine
and aspartic acid [3]. Further classification of serine proteases is
dependent on substrate specificity and structural homology to well
established proteases [4]. The main subclasses of serine proteases
are subtilisin-like, chymotrypsin-like, wheat serine carboxy-
peptidase II-like, prolyligopeptidase-like, myxobacter a-lytic and
staphylococcal proteases [2].
In addition to their physiological importance, proteases are of
great use in industrial enzymatic applications such as laundry
detergents, automatic dishwashing, feed additives, food prepara-
tion, leather, diagnostics, therapeutics and pharmaceutical indus-
tries [2,5]. The major use of proteases in the food industry is the
enhancement of flavor in dairy, meat, and fish products. In the
leather and wool industry, proteases find their application for
soaking, de-hairing, and hydrolysis of overlapping scales on wool
fibers [6]. In different medical treatments, proteases are used as
active agents (treatment of osteoarthritis, removal of dead tissue,
wound healing) [7].

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Subtilisin-like proteases are serine proteases produced as extra-
cellular enzymes with a molecular weight ranging from 18 to
90 kDa and are mainly employed in industry due to their out-
standing properties such as high stability and broad substrate
specificity [5].
Proteases as additives in laundry detergent
applications
Proteases in the form of trypsin and chymotrypsin were intro-
duced for the first time as an active ingredient in laundry deter-
gents for degradation of proteinaceous stains in 1913 by the
Research Paper
German chemist Otto Röhm [8]. The first commercial detergent
containing bacterial proteases was produced by Gebrüder Schny-
der in 1959. By 1985, approximately 70% of the heavy-duty
laundry detergents in Europe contained enzymes. In the last
50 years, proteases and other enzymes in laundry detergents
switched from being minor additives to key ingredients. The
selection and evaluation of proteases to be used in detergents is
based on important parameters defined by laundry detergent
manufacturers. A detergent protease needs to have efficient wash-
ing performance at broad alkaline pH and over a wide range of
temperatures (from low temperatures for synthetic fibers, to high
temperatures for cotton). The performance of a good detergent
protease is defined by multiple parameters such as proteinaceous
stain degradation, compatibility with other detergent components
(e.g. nonionic and anionic surfactants, complexing agents, per-
fumes, and other enzymes), stability in the presence of oxidizing
agents as bleach, and shelf life in detergent formulations. The
leading enzyme suppliers and detergent manufacturers are actively
pursuing the development of new enzyme activities that address
consumer needs for improved cleaning, fabric care and antimicro-
bial properties. Hence, research on proteases has focused on dis-
covery and engineering enzymes that are more robust with respect
to pH, temperature, stability and substrate specificity by using
techniques of protein engineering and rational design.
In this review we will focus on three important properties for the
application of subtilisin proteases in the laundry detergent indus-
try that have been tackled by protein engineering; activity and
thermal resistance of Bacillus gibsonii alkaline protease (BgAP) was
simultaneously increased [9], promiscuous activity of subtilisin
Carlsberg was increased towards peroxycarboxylic acids produc-
tion [10], and the pH activity profile of BgAP was shifted towards
higher activity at lower pH (pH range 8.5–10) [11].
Hot and cold, the temperature challenge of modern
detergent proteases
Nowadays trends in energy efficiency raise the awareness in society
for washing at low temperature. Designing sustainable laundry
detergents with high performance at low temperatures requires
the development of enzymes with high efficiency at broad tem-
perature range especially at temperatures <208C [12]. Proteases
adapted to low temperatures can be isolated from naturally occur-
ring psychrophilic microorganisms, displaying high proteolytic
activity (158C) [13–15]. Unfortunately, such enzymes generally do
not meet industrial requirements due to inherent low stability at
temperatures above 208C and low product yields in large scale
production [5,12,16]. On the other hand, subtilisins isolated from
mesophilic organisms exhibit at the same time higher catalytic
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New Biotechnology  Volume 00, Number 00  August 2010
efficiency and temperature stability at temperatures from 308C to
458C. In order to adapt mesophilic subtilisins to the current trend
of washing at low temperature (208C) directed evolution and
rational design approaches have been employed. One directed
evolution campaign was performed with Bacillus sphaericus subtil-
isin (SSII) using random mutagenesis followed by recombination
of improved variants. A remarkable increase in the turnover num-
ber (kcat at 108C increased 6.6-fold) and increased catalytic
efficiency (9.6-fold higher than wild type) was achieved [17]. In
another approach a chimeric enzyme was generated by replacing
the highly flexible 12 amino acid region (MSLGSSGESSLI) in the
psychrophilic Antarctic Bacillus TA39 protease (S39) with the
corresponding amino acid sequence (LSLGSPSPSATL) of the meso-
philic subtilisin Savinase1 with a temperature optimum at 558C.
The resulting hybrid enzyme showed a higher specific activity for
synthetic substrates and a broadened substrate profile at room
temperature [18].
Stability of subtilisins is a property which has been studied
extensively by random mutagenesis and rational approaches.
Bryan et al. summarized, in an intensive analysis, amino acid
substitutions and their effects in over 50% of the 275 amino acids
of subtilisin [19]. Research on subtilisin stabilization focused
on calcium dependent and independent stability, as well as
stabilization by the introduction of disulfide bonds [19–25]. How-
ever, a general mechanism describing the stability of proteases has
to be elucidated. In most proteins, intramolecular interactions
such as salt bridges are essential for thermal stability. It has been
shown that thermal stability was dramatically reduced by removal
of salt bridge-networks in aqualisin I, a thermostable subtilisin
protease [26,27]. In another approach, the apparently opposite
properties of high thermal stability in combination with increased
activity at low temperatures were investigated in B. gibsonii alka-
line protease (BgAP) [9]. BgAP was of special interest for laundry
detergent applications as it exhibits superior activity and stability
in comparison to other subtilisins [28]. The mesophilic protease
BgAP with a temperature optimum of 45–508C shows a rapid
decrease in activity at temperatures lower than 458C, as well as
at temperatures higher than 508C. In order to broaden the tem-
perature range a directed evolution campaign using three iterative
rounds of Sequence Saturation Mutagenesis (SeSaM) was per-
formed [29]. In the first round of SeSaM, five variants were identi-
fied with increased activity at 158C for the artificial substrate
succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (suc-AAPF-pNA) ac-
companied by decreased thermal resistance at 588C. The amino
acid substitutions identified in these five variants were indepen-
dently saturated to elucidate the effect of each position on the
increased activity at 158C. Upon sequence analysis, key mutations
Ile21Val, Met122Leu and Asn253Asp were identified as essential
for increased activity at 158C. In parallel, screening for increased
thermal resistance at 588C resulted in a variant harboring amino
acid substitutions Ser39Glu, Asn74Asp and Asp87Glu. Both sets of
amino acid substitutions were combined in one hybrid variant
MF1 (Ile21Val, Ser39Glu, Asn74Asp, Asp87Glu, Met122Leu, and
Asn253Asp) in order to recover thermal resistance (see Fig. 1).
MF1 showed 1.5-fold improved kcat (35.3 s 1) and a 100-fold
increased half-life at 608C (224 min) in comparison to BgAP wild
type (kcat of 23.2 s 1 and half-life at 608C of 2 min). In order to gain
deeper insights into the location of the amino acid substitutions, a
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New Biotechnology  Volume 00, Number 00  August 2010
FIGURE 1
Summary of the directed evolution campaign for Bacillus gibsonii alkaline protease (BgAP) using sequence saturation mutagenesis (SeSaM) and a parallel screening
towards high activity at low temperatures (upper path) and thermal resistance (lower path). Variants with improved temperature-activity profiles were identified, and
beneficial amino acid substitutions were combined to generate one single enzyme variant with a wider temperature profile compared to the wild type [9].
homology model was constructed using the routine function from
YASARA based on the coordinates of an alkaline protease from
Bacillus lentus (78.4% sequence identity; PDB ID 1GCI; [30,31]).
Surprisingly, amino acid substitutions identified in variant MF1
were not located close to the catalytic triad. Substitutions Ile21Val
and Met122Leu were identified in four out of five variants with
increased proteolytic activity at 158C. The contribution of Ile21Val
and Met122Leu to increased proteolytic activity on a molecular
level was not elucidated and seems to be challenging. Amino acid
substitutions Ser39Glu, Asn74Asp, Asp87Glu and Asn253Asp
were identified in variants showing higher proteolytic activity
at elevated temperatures (608C) [9]. Through the generation of
a homology model of the most stable variant MF1, it could be
observed that all four mutations are located on the surface of
BgAP and comprise negatively charged amino acids (Glu or Asp).
Negatively charged residues on the surface were also observed in
thermophilic organisms supporting the increased thermal resis-
tance in variant MF1 [32]. In addition, it was reported that position
76 in subtilisin E leads to conformational changes in the Ca-1
binding loop, allowing an increase in thermal stability [33]. This
explanation of increased thermal resistance might be transferred
to position 74 in BgAP (serving as an equivalent of position 76 in
subtilisin E) where the negatively charged amino acid probably
stabilizes the Ca-1 binding pocket.
The MF1 variant, after a directed evolution campaign, showed
improved activity at 158C and more than 100-fold improved
thermal resistance at 608C. For the first time the contrary proper-
ties of high thermal resistance (requiring strong interactions) and
high activity (often requiring flexibility) were combined in one
hybrid enzyme variant optimized for washing at low temperatures
[9].
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RESEARCH PAPER
Research Paper
Engineering of pH-dependent activity
The success of subtilisins for their application in detergents
depends, as discussed before, on several factors such as com-
patibility with the detergent matrix, broad substrate specificity,
thermal resistance, activity at temperatures from 208C to 608C, as
well as high activity in a wide alkaline pH range. Commercially
relevant enzymes for application in detergent industry are subtil-
isin proteases originated from Bacillus sp., comprising the widely
used proteases of Bacillus amyloliquefaciens (subtilisin BPN’)
[34,35], B. gibsonii (BgAP) [9,11], B. lentus (subtilisin BL) [36],
and Bacillus licheniformis (subtilisin Carlsberg) [37,38]. The iden-
tification of new alkaline proteases is an ongoing challenge [39].
Activity at high alkaline pH is a prerequisite for protease applica-
tion in detergent formulations. So far the pH dependent activity
of subtilisins or hydrolases in general is not completely under-
stood. Several attempts to modulate pH dependent activity
focused on the engineering of charge distribution, since the pH
dependent activity is primarily determined by the p Ka values of
the active site residues. Therefore ‘large’ pH-activity profile shifts
are normally related to mutations located in close proximity to
the active site which unfortunately increase the chance to obtain
enzyme variants with reduced or no activity. Amino acid sub-
stitutions distant from the active site, such as surface exposed
residues, mostly result in enzyme variants maintaining the
wild type activity and exhibiting a ‘small’ shift in the pH profile
[40].
The influence of protease surface charges on the pH dependent
subtilisin activity was demonstrated already by amino acid sub-
stitutions in 1987 [41]. Recently a new amino acid selection
strategy to engineer the pH dependent activity of the B. gibsonii
alkaline protease (BgAP) (pH optimum 11), following the
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RESEARCH PAPER
deamidation principle, was reported. The posttranslational auto-
catalytic deamidation process converts asparagine and glutamine
residues into negatively charged aspartic acid and glutamic acid,
respectively. This process changes the net charge of the proteins
and thus their pH dependent activity. This deamidation principle
was mimicked by site-directed mutagenesis (Gln to Glu and Asn to
Asp). Three criteria for amino acid selection were defined by
(1) amino acids of the deamidation type (Asn or Gln), (2) non-
conserved residues and (3) surface exposed residues neighbored by
glycine. The protease BgAP consists of 270 amino acids, wherein
113 amino acids are surface exposed and 31 of the surface exposed
Research Paper
residues are Asn or Gln residues. For evaluation 18 individual
substitutions (11 Asn and 7 Gln) fulfilling one, two or all three
criteria were selected and generated. Site-directed mutagenesis
following the deamidation principle in five (Asn97, Asn253,
Gln37, Gln200, and Gln256) out of eight (Asn97, Asn154,
Asn250, Asn253, Gln37, Gln107, Gln200, and Gln256) amino
acids meeting all three criteria resulted in increased proteolytic
activity at pH 8.6. Variants Asn253Asp and Gln256Glu and the
combined variant Asn253Asp/Gln256Glu showed a pH optimum
at 10 (wild type pH optimum 11). The combined variant
Asn253Asp/Gln256Glu showed 2-fold increase in activity at
pH 8.5 compared to the wild type [11].
The criteria presented of the deamidation principle are in theory
independent of the protein/enzyme class together with the gen-
erality regarding the effect of surface charge changes on pH
dependent enzyme activity, it is likely that properties of enzymes
from different classes can be modified using the deamidation
approach.
Catalytic promiscuity in subtilisin proteases: switching
proteolysis towards perhydrolysis
One of the key components of modern powder laundry detergent
is the presence of oxygen-based bleaching agents. Commonly,
hydrogen peroxide is used as an oxidative agent produced by
spontaneous decomposition of perborate and percarbonate com-
bined with tetraacetylethylendiamin or nonanoyloxybenzensul-
phonate [42]. The concentrations of chemically produced
hydrogen peroxide are usually very high and harmful for textile
and surfaces as well as for the enzymes present in detergent
formulations. Due to these harsh local concentrations of hydrogen
peroxide it is extremely challenging to maintain constant level of
mild bleaching. Therefore, constant enzyme mediated production
of oxidative agents in low concentrations is much more attractive
for laundry and disinfectant applications [43]. The oxidizing
capacity of peroxycarboxylic acids shows a high potential to be
used as a bleaching component in powder detergent formulations
for soil removal. Peroxycarboxylic acids show superior perfor-
mance compared to hydrogen peroxide, but also a high level of
spontaneous hydrolysis [44,45]. In situ production of peroxycar-
boxylic acids is known for serine hydrolases such as lipases,
esterases, and proteases as a side catalytic reaction [42,46–48].
The ability of an enzyme to catalyze an additional chemical
reaction with different transition states is defined as catalytic
promiscuity [49]. Lipases and esterases have been engineered
towards increased perhydrolytic/hydrolytic ratio in several
reports. For both enzymes, strategies for engineering were based
on site-saturation mutagenesis at positions located within 12 Å
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New Biotechnology  Volume 00, Number 00  August 2010
to 15 Å from catalytic triad [50]. Based on this approach, the
perhydrolytic activity of Pseudomonas fluorescens esterase was
increased 28-fold [51].
Subtilisin Carlsberg is able to catalyze conversion of methyl-
butyrate into peroxybutyric acid and methanol in the presence of
hydrogen peroxide [42]. The level of perhydrolysis reported for
subtilisin Carlsberg is relatively low (2.1 s 1; 82-fold lower com-
pared to its proteolytic activity) therefore a directed evolution
campaign was used to improve the peroxybutyric acid production.
After screening an error-prone PCR library the identified subtilisin
Carlsberg variant (Thr58Ala/Leu216Trp) showed an increased level
of peroxybutyric acid production (3-fold higher than subtilisin
Carlsberg wild type) while the level of proteolysis was simulta-
neously reduced 8-fold [42]. The identified variant was analyzed by
molecular dynamic simulations to study the influence of benefi-
cial substitutions on increased peroxybutyric acid production [52].
The molecular dynamics analysis revealed a possible stabilization
effect of the second tetrahedral intermediate by Trp216 and
provided the first insights in the mechanism of perhydrolysis
observed in subtilisin protease.
Due to the high application relevance of perhydrolytic reaction,
subtilisin Carlsberg variant (Thr58Ala/Leu216Trp) was further
subjected to directed evolution. A semi-rational approach was
used to engineer subtilisin Carlsberg substrate binding pocket in
order to increase specificity for ester substrates [10]. Site-saturation
mutagenesis libraries were expressed in Bacillus subtilis DB104 host
[53] and comprised 9 positions (Thr33, Val67, Ser124, Ser155,
Gly165, Trp216, Asn217, Gly218, and Met221) located in the
vicinity of the active site or within the substrate binding pocket
(Fig. 2). Only position Gly165 was found to have an effect on
promoting promiscuous perhydrolytic activity in protease var-
iants. Three variants having amino substitutions at acid position
FIGURE 2
Amino acid positions of subtilisin Carlsberg located in the vicinity of the
active site or within the substrate binding pocket. The catalytic triad (Asp32,
His63 and Ser220) and the oxyanion hole (Asn154) are represented in
element color. The residues initially subjected to site-saturation mutagenesis
are colored blue.
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New Biotechnology  Volume 00, Number 00  August 2010
FIGURE 3
Hypothetical effect of mutations at position 165 in the S1 pocket for the catalysis of methylesters (methyl propionate). Docking studies revealed that the
modulation of the S1 pocket can change the configurations in which the methyl ester binds to the protease active site. In the wild type, there are two possible
modes of the substrate (A), of which only one is in the productive conformation (A2). When Gly165 is substituted by bulkier Ile, Leu or Tyr residues (B, C, D), the
non-productive binding mode does not occur, possibly increasing the reaction rate [10].
Gly165 (M1: Thr58Ala/Gly165Leu/Leu216Trp, M2: Thr58Ala/
Gly165Ile/Leu216Trp, and M3: Thr58Ala/Gly165Tyr/Leu216Trp)
showed increased production of peroxycarboxylic acids using
methyl-propionate, methyl-butyrate, and methyl-pentanoate as
substrates (up to 5.4-fold higher compared to subtilisin Carlsberg
wild type); simultaneously proteolytic activity towards standard
suc-AAPF-pNA substrate was decreased (up to 99.5-fold lower
for variant M2 compared to subtilisin Carlsberg). From the
obtained data analysis the preferred ester substrate for subtilisin
Carlsberg variants is methyl-butyrate (kcat values are significantly
increased compared to the subtilisin Carlsberg wild type) while the
KM values are decreasing with increase in the substrate chain
length. The latter, suggests the favoring of hydrophobic inter-
actions between residues in the S1 binding pocket and the ester
substrates.
The amino acid substitutions Gly165Leu, Gly165Ile, and
Gly165Tyr resulted in a size reduced S1 binding pocket and
subsequently influenced the binding orientation of the ester sub-
strates. Substitutions of Gly by more bulky amino acids (Leu, Ile,
and Tyr) resulted only in the productive binding mode of the
substrate compared to the subtlisin Carlsberg while type in which
non-productive and productive mode have been observed. The
productive binding mode (ester group of the substrate is located
close to the catalytic residue Ser220) observed in all three variants
provides a first hypothesis to explain increased level of perhydro-
lysis (Fig. 3) [10].
This work suggested that amino acids at position Gly165 are
crucial for substrate specificity in the peroxycarboxylic acid pro-
duction. Three substitutions at position Gly165 converted the
protease subtilisin Carlsberg into a perhydrolase with comparable
absolute activities of natural perhydrolases [54]. Computational
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Please cite this article in press as: Vojcic, L. et al., Advances in protease engineering for laundry detergents, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2014.12.010
RESEARCH PAPER
Research Paper
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Summary
Subtilisins are important industrial enzymes and essential addi-
tives in modern laundry detergents to boost washing performance
by removing efficiently proteinaceous stains. Reported investiga-
tions focus on the engineering of the subtilisin BgAP towards
increased activity at low temperatures (158C) and simultaneous
improvement of thermal resistance; two contrary properties
requiring strong molecular interactions and flexibility in one
enzyme. Additionally, a general applicable rational protein engi-
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adjust pH dependent activity was presented. Finally, the substitu-
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a perhydrolase. The three protein engineering examples demon-
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well-established role as model systems in the field of protein
engineering.
Acknowledgments
This work was supported through funds from German
Government through the Bundesministerium für Bildung und Q3
Forschung (BMBF), Bioindustrie-2021 Programme, FKZ0315250
and FKZ0315035A. US thanks the Chinese Academy of Sciences
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