ENZYMES

JEANNETTE BJERRE*, OLE SIMONSEN, JESPER VIND

*Corresponding author
Novozymes A/S
Krogshoejvej 36, 2880 Bagsvaerd, Denmark

Jeannette Bjerre

Detergent enzymes – from discovery

to product
The power of nature harvested in eco-friendly catalysts
for laundry applications

KEYWORDS: Detergent, Enzymes, Laundry, Expression, Screening, Formulation

Abstract Enzymes represent nature’s work of brilliance, affording efficient catalysis of reactions and acceleration of
biochemical processes, selectively and at a remarkable speed. The detergent industry is an important
application area where industrial enzymes are utilized to boost cleaning performance. Enzymes represent environmentally sound
alternatives to the use of toxic chemicals and pollutants, with reduced generation of waste and added performance benefits as the
result. The discovery, application screening, optimization, and production of industrial enzymes for detergents are presented in this
article, including examples of how formulation technology and protein engineering can enhance liquid stability. Proteases are used as
the main example as this was the first enzyme class to find wide-spread use in laundering, and proteases remain hallmark enzymes in
household care today.

INTRODUCTION in cold water. Added performance qualities by enzymes,

such as fabric care and maintenance of whiteness or
Detergent enzymes have been used for a century in colour clarity by cellulases, are also desirable traits. In
laundering, and continue to be of great benefit in automatic dishwash (ADW), proteases and amylases
household care today. In 1913 the first words in the story boost the cleaning of tableware (3), and enzymes are also
of laundry proteases were written, when pancreatic applied in the industrial and institutional cleaning sector.
trypsin was introduced as an additive in the European The workflow from discovery to product for industrial
pre-soak detergent product Burnus by Röhm and Haas enzymes involves several steps. It begins at
(1). In 1963, a major development in the protease field microorganism level with basic characterization of
took place when the first bacterial subtillisin protease secreted enzymes. Promising enzymes are tested for
from Bacillus licheniformis (subtilisin Carlsberg) was wash performance in detergent application testing,
introduced into commercial detergent. The importance concomitantly with the use of protein engineering to
of properly formulating technical enzymes became clear improve enzyme properties. In the last steps involving
during the early seventies, after the discovered risk of production, the fermentation process is scaled up,
allergic reactions from dusty proteases caused a major recovery and purification processes are optimized,
setback for the detergent industry (2). This issue was and a suitable enzyme formulation is developed. The
solved by proper formulation of the protease product novel detergent enzyme is then ready to be used within
into non-dusting granulates or prills, resulting in protease laundry or ADW applications.
enzyme products that are completely safe to use.
A common denominator for detergent enzymes is that
they target tough stains and hydrolyse the various
components in the soiling into smaller more water-
soluble fragments. This helps boost the mechanical
removal of the stain during wash, which is also assisted
by surfactants and builders in the detergent. The use of
laundry enzymes results in far more efficient stain removal
than that achieved by the detergent and mechanical
action alone. The growing trend towards lower washing
temperatures, with lower water solubility of many stains
Table 1. Commercially available detergent enzyme classes
as a consequence, calls for added cleaning power by
and their applications.
detergent enzymes to ensure efficient stain removal even

H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013 37
MICROBIAL SCREENING
Before the catalytic power of nature can be captured
in the form of a technical enzyme, one must be able to
express the enzyme in a microorganism that can yield
commercially relevant amounts. Countless microorganisms
including the bacteria genus Bacillus, secrete several enzyme
classes including proteases into their external environment.
Microorganisms do this to degrade surrounding biomolecules
into smaller entities which the microorganism can take in
and use. The challenging part is to identify and pinpoint a
microorganism which secretes a protease that can work in
a detergent solution. An initial small scale screening of many
different proteases from several microorganisms is used to
examine basic parameters related to the profile and activity
of the protease. In the case when the secreted protease
shows indications of being functional in detergent, the next
step is to ferment the microorganism to produce larger
amounts of protease for further testing in the laboratory.
Some decades ago this strain was used for the large scale
production of the enzyme, but new techniques have
changed this today. The production of proteases became
easier in the eighties with the introduction of recombinant
DNA technology.
Figure 1. A model of a subtilisin serine protease showing
the binding site and the amino acids of the catalytic triad
(Serine-221, Histidine-64, and Aspartate-32).
Once the microorganism expressing the desired proteases
was identified, a library was made containing the
gene pool from the organism. The gene encoding the
protease of interest is cloned from the genome of the
identified microorganism into another host such as Bacillus
licheniformis, which is easy to ferment, has GRAS status
(Generally Regarded As Safe) and can secrete enzymes at
commercially relevant levels (4). After the millennium, this field
took a further leap forward when numerous microorganisms
had their genome sequenced, and the price of artificially
synthesizing new genes (DNA Synthesis) has been drastically
lowered. Today many genomes from microorganisms
have been sequenced and the information put into large
databases which can be mined. Thus, when a new high-
38 H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013
performing protease is found, genes for homologous
proteases can be identified simply by comparing the DNA
or protein sequence of the high-performing protease with
sequences in the databases. These genes can be cheaply
synthesized, and the used codons can be chosen to fit the
preferences of the expression host. By optimizing the gene
and codon to suit the host, there is an increased chance
that the gene of interest will be well-expressed. Despite these
measures, there is no guarantee that a particular enzyme can
be expressed. Once expressed, the potential of the protease
is tested in application screening.
DETERGENT APPLICATION SCREENING
Factors influencing wash performance
Finding “a needle in a haystack” is a frequently used
metaphor describing the search for promising enzymes in an
application screening flow comprising thousands of different
molecules. To pick the right candidates, having the right and
relevant application screening assays is absolutely essential.
Plain enzyme activity measurements on simple substrates such
as the synthetic 4-nitroanilide peptides for proteases are useful
in the early screening stages before application testing. These
can be performed in high throughput using automated liquid
dispenser systems and microplate readers. However, these
simple activity measurements fall short in case of application
purposes. This is because there is a world of difference
between simple activity and the complex multi-factorial
situation, which a laundry full scale wash represents, with its
natural complex substrate types, mechanics, and textile load,
to name just a selected few. In fact, a vast range of factors
influence the observed wash performance by a laundry
enzyme, such as wash cycle duration, substrate specificity,
temperature, catalytic activity, water ion concentrations,
enzyme stability, ballast (unsoiled textile) load, inhibitors,
enzyme concentration, wash and soil load, interaction
with other enzymes, impact from detergent ingredients,
mechanical action in the wash machine, pH, adsorption
of enzyme onto substrates, etc. Most detergent enzymes,
including proteases work predominantly in-wash, while others
such as lipases are also very active post-wash (5).
Stain type
Detergent enzymes are typically evaluated based on their
ability to remove natural and artificially produced stains on
textile. Cotton and polyester are the most frequent fabric
types used. Choice of stain type is a critical factor since most
proteases are extremely effective and will often remove all
measurable amounts of protein substrate in a consumer-type
natural soiling. In application screening this effectiveness
induces the need for technically produced stain types that
are particularly resilient and very difficult to completely
remove. The technical stain types ensure that there is a
window left for response measurement and performance
ranking, also with high-performing protease variants.
Commercially produced textiles with common soiling types
(e.g. milk, grass) are available in many versions, containing
one or several substrates, sometimes with particulate
matter added, e.g. carbon black. Another benefit of using
commercially produced technical stains is that these often
are highly uniform. This elevates data quality when comparing
thousands of molecules in a screening flow. A wide selection
of protease-relevant stain types are used (e.g. blood, grass,
milk), since the substrate specificity profile can vary to some
extent between different proteases. Enzyme performance
is quantified by measuring the enzyme’s net contribution to
colour removal from the stain measured as reflectance of
light recorded in intensity or remission units. In special cases,
mechanistic studies can also be undertaken involving analysis
of stain components using e.g. fluorescence or infrared (IR)
spectroscopy, or other.
Figure 2. FT-IR spectra illustrating the protein content of home-
made skim milk stains (indented image), before wash (blue),
after wash without enzyme (red), and after wash with 80 nM
of the subtilisin serine protease Savinase® (green), with CN-11
cotton textile background subtracted (TOM wash at 20°C, 30
min., 120 rpm, 15°dH, pH 8 in liquid laundry detergent). The
catalysis by the protease results in a significant increase in
protein removal, seen by the reduced intensity in the peaks
from the amide groups in the milk protein.
Detergent
(6). In the AMSA, laundry enzyme and detergent solutions
are placed in a microtiter plate, soiled fabric is added,
and the system is sealed tightly by a lid. Mechanical
action is induced by rapid oscillating movements, and the
wash is conducted at a selected duration and controlled
temperature. For automatic dishwash enzymes, soiled hard
surface melamine plates are used in the AMSA.
Those enzymes that outshine their opponents and make
it through the AMSA screening have still to undergo more
trials; for laundry enzymes a range of small-to-medium
scale wash assays exist. One example is the Launder-O-
Meter (LOM) wash assay, which mimics European drum-
type machines. The LOM uses revolving sealed beakers
containing enzyme, detergent solution, soiled textile
swatches, ballast, and small stainless steel balls that add
mechanical action. The Terg-O-toMeter (TOM) is a medium-
sized wash assay, simulating a US top loader machine.
The TOM uses 1 L open metal beakers containing enzyme,
detergent solution, soiled textile swatches, ballast, and
mechanical action by an agitator (7). If an enzyme still
delivers promising results at this stage, the next and final test
in the application screening flow will usually be full scale
wash trials. Analogously, for automatic dishwash enzymes,
the final evaluation typically involves washing of soiled
tableware in a dishwasher. Alongside the main application
screening flow, smaller side-flows will often be incorporated,
which use custom designed assays to examine selected
features of enzymes, with the knowledge gained feeding
directly into the main screening flow. Information gathered
during application screening is used to improve the design
of promising enzyme variants in a joint learning loop with
protein engineering.

When assessing stain removal by a detergent enzyme,

the choice of detergent is of hallmark importance, since
enzymes perform differently in different detergents. Wash
solutions arising from liquid versus powder detergents differ
in several ways regarding e.g. alkalinity, inclusion of bleach
components etc, and these parameters all impact enzyme
performance. There are also differences in the composition
of powder detergents for use in laundry versus for automatic
dishwash, which contain, in the case of the latter, relatively
more builders, only little surfactant and a much higher
enzyme load. Also the compositions of detergents may
vary significantly across different regions of the world, as a
consequence of local differences in water hardness, pH,
washing traditions and procedures, etc.

Wash assays
When a protease enters the application screening process,
it engages in a series of elimination races, where the bar for
satisfactory assay performance is continuously raised. Only
the most promising enzymes that display desired features
are taken to the next screening step, and only a selected
few out of many thousands of candidates make it all the
way to final full scale wash trials. Application assays are to
different extents predictive of the performance seen in a full
scale wash. Throughout the screening flow the assays get
more advanced, complex, time- and enzyme-consuming, so
a balance of data quantity versus quality must be kept.
The first application assay an enzyme will be subjected to
Figure 3. Terg-O-toMeter (TOM) apparatus with capacity for
when it enters the detergent application screening process
12 metal beakers (1 L) with metal agitators (on tray).
will often be the AMSA (automated mechanical stress assay)

H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013 39
PROTEIN ENGINEERING
Protein engineering is used to improve properties of selected
enzymes that have shown the ability to be expressed in a host,
have shown promising results in the initial screening, and have
performed well in the first rounds of application testing. Protein
engineering can be used to change the type, number,
or position of some of the amino acids in the protein. This
technique is well-suited to solve issues related to detergent
enzyme stability, a challenge commonly encountered with
non-engineered proteases. It is often more tricky to improve
protease performance by protein engineering than it is to
increase stability, but this can also be done (8, 9). There are
several strategies to improve stability, and many papers
describe how stability has been increased. Improved stability
is a feature that is often easy to demonstrate and report,
using a simple screen in which the temperature is increased.
By contrast, it is far more challenging and costly to design and
conduct a performance screen which correlates with results
in real application.
Bleach stability
Proteases can be inactivated during storage by bleach
present in the detergent. The methionine in position 221
(Subtilisin Carlsberg numbering), which is close to the protease
active site serine, gets oxidized by the bleaching agents
which leads to inactivation of the protease. Simple site
directed mutation of this methionine to other non-oxidizable
amino acids leads to improved bleach stability (10), but
also causes reduced protease performance. It has been
shown that protease oxidation stability can be increased by
introducing a methionine in position 216 in subtilisin Carlsberg,
likely due to the methionine 216 acting as a “suicide antioxidant”
which safeguards the important methionine 221 (11).
Chelator sensitivity
Chelators are added to the detergent to lower the
concentrations of free ions naturally present in wash water,
especially divalent calcium and magnesium ions. This is
relevant since calcium ions can interact with surfactants
such as LAS (linear alkylbenzenesulphonate), producing LAS/
calcium complexes, which precipitate and lead to reduced
wash performance (12). Lowering the free ion concentrations
can also be problematic since many detergent enzymes, e.g.
amylases, depend on calcium. Protein engineering can be
used to improve the binding of the structural calcium ion to
the protease, thereby lowering the chelator-sensitivity of the
protease. A mutation such as N76D in the Bacillus protease
Savinase® seems to improve the binding of the calcium found
in the structure, and makes the protease more thermostable
(10). An alternative way to improve protease storage stability
towards chelators is to completely remove the calcium
binding site from the protease. One example is the BPN´
protease, which after multiple rounds of protein engineering was
evolved into a calcium free, stable and active subtilisin (13).
Proteolytic stability
Since enzymes are proteins, proteases can potentially be
their own target. Apart from auto-proteolysis, proteases
can hydrolyse other enzymes, e.g. lipases, which can
be degraded by the action of a protease included in a
detergent formulation. By means of protein engineering, it
is possible to mutate those sites which are highly prone to
proteolytic cleavage to less protease-sensitive sites, and
40 H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013
thereby increase the enzyme stability (14). An alternate
solution can be to add protease inhibitors that bind and
stabilize the protease when present in the concentrated
detergent mixture. Once diluted into the wash liquor, the
inhibitor is diluted too, freeing the protease to perform
catalysis and consequently stain removal. The binding of the
inhibitor can be optimized by either mutating the protease or
altering the molecular structure of the inhibitor (15, 16).
Thermostability and detergent stability
High temperatures and anionic surfactants such as LAS (linear
alkylbenzene sulphonate) can denature detergent proteases
during storage. Improving the thermostability of a protease
can in many cases lead to improved stability in a detergent
(17). A classic approach that has proven to work well in the
case of proteases consists in the addition of disulphide bridges
by introducing cysteine at the proper positions in the enzyme
tertiary structure (18). Another possibility is to replace an
amino acid with proline, which will increase the rigidity of the
protein due to the inclusion of the pyrrolidine ring structure in
the protein main chain. A third possibility is to use database
sequences of ancestral genes as inspiration for the build-up
of a more thermostable enzyme. This is based on a theory
that the earth was warmer a long time ago, and therefore
ancestral enzymes had to be more thermostable. This theory
is being highly debated. A different approach is molecular
dynamic analysis, in which computer modelling is used to
deduce the most mobile regions in the protein based on the
protein X-ray crystal structure. Amino acid substitutions can
then be made in these particular regions with the aim of
obtaining more rigidity, which in turn can increase enzyme
thermostability. An elegant feature of evolving a more
thermostable enzyme is that different mutations that confer
stability to the enzyme can be combined in the same enzyme.
This can boost enzyme stability further because the stabilizing
effects of the identified mutations are often additive.
PRODUCTION
Once the needle in the haystack has been found by
application screening and further optimized by protein
engineering, the expression of the enzyme is transferred into a
production-relevant microorganism (18).
Mastering microbial enzyme expression
The majority of industrial enzymes are produced in host
microorganisms such as filamentous fungi (e.g. Aspergillus
oryzae, Aspergillus niger or species of the Fusarium and
Trichoderma genus) or gram-positive bacteria (e.g. Bacillus
licheniformis and Bacillus subtilis), using simple and cheap
substrates. The host microorganisms may be genetically
manipulated in order to further improve their characteristics
for the industrial level fermentation, e.g. for removal of
undesired enzyme activities that could interfere with the
function or stability of the intended product. The fermentation
process is developed and optimized with regard to
fermentation time, temperature, pH, addition of substrates
etc., and subsequently scaled up (19).
Recovery and purification
In parallel to the expression the enzyme recovery/purification
process is developed, optimized, and scaled up too. The
main purpose of recovery is to remove non-enzymatic
components from the enzyme fermentation broth. Most
industrial enzymes produced today are extra-cellular
enzymes, i.e. the enzymes are secreted into the medium
by the producing microorganism and not confined within
the cell. The first recovery step is often the removal of whole
cells and other debris by centrifugation and/or filtration.
Ultrafiltration is increasingly used to remove water, salts, and
other low molecular weight impurities after the first recovery
step. Precipitation of the enzymes by use of salts, polymers, or
organic solvents is another simple method employed for the
recovery and purification of enzymes. Vacuum evaporators
are often used to remove water and concentrate the
enzyme. In some cases spray-drying may also be used if a
powdery intermediate product is desired. The concentrates
need to be completely free from genetically modified
organisms. The colour and odor of the concentrate should
not negatively affect the final detergent and enzyme
concentrates for liquid detergents need to be clear.
FORMULATION
In the final step of the process, the concentrated enzyme
formulations are developed, which are divided into solid
products for powder detergents and liquid products for
liquid detergents. The final product will have to live up to a
multitude of quality parameters (20).
Powder
For powder detergents, an enzyme granulate is developed
which is typically 0.3-1.2 mm in diameter. The main quality
parameters are particle size, dissolution rate, stability in
detergent, dust, colour, and flowability. The largest challenge
for enzyme stability in powder detergents is bleach, which
in combination with humidity can oxidize the enzyme (21).
This is typically handled by inclusion of antioxidant/reducing
agents and protective coatings, such as those used in the
Novozymes Evity® detergent enzyme range, developed to
deliver consistent performance after storage in detergent
even at harsh conditions.
Liquid
Stability is very challenging in liquid detergent products as
the enzyme can diffuse in the solution and is not separated
from other detergent components. This can lead the highly
efficient proteases to auto-proteolysis or to digest other
detergent enzymes. This issue can be pronounced for low
temperature proteases that are highly active below and at
room temperature which is a typical storage condition.
A neat solution is to mitigate the proteolytic attack using
reversible protease inhibitors. The inhibitor (I) works by
inhibiting the protease (P) when present in high concentration
in the detergent, thus forming an inactive inhibited protease
complex (PI):
Upon dilution with water in the washing machine the
chemical equilibrium is pushed towards free protease (P),
which then becomes active in the wash liquor. The effect of
an inhibitor is typically given by its inhibition constant KI, the
dissociation constant for the enzyme-inhibitor complex (PI).
Small KI values correspond to strong inhibitors.
H&PC Today - Household and Personal Care Today, Vol. 8 nr. 6 November/December 2013
Protease inhibitors
Traditionally Borax (Na 2 B 4 O 7 ·10 H 2 O, a sodium salt of
boric acid) has been used as a protease inhibitor in
liquid detergents. However, Borax is a weak inhibitor (K I
in the 10 mM range) such that large amounts, typically
around 2%, are needed in the detergent formulation
to provide a sufficient degree of inhibition. This high
percentage causes problems in complex detergent
solutions, such as reduced solubility of surfactants etc..
Moreover, Borax has also been found to be reprotoxic.
Therefore, a good incentive exists to develop and
explore new efficient inhibitors, which should be custom
designed for the specific protease enzyme to be most
effective. There is a delicate balance between the
use of too strong inhibitors, which may not release the
protease to a sufficient extent during the wash causing
lowered wash performance, and too weak inhibitors,
which may need a high dosing resulting in added
costs and cause problems related to solubility and
the physical stability of the detergent. One example
of a solution for liquid detergents is the inhibitor
4-formylphenyl boronic acid (4-FPBA) in the Ultra brand
by Novozymes, which is about 100 times more efficient
than Borax on a weight basis and is co-formulated into
concentrated enzyme products (22).
Figure 4. Docking of 4-formylphenyl boronic acid into the
binding pocket of a subtilisin serine protease.
In the future, non-boron based ingredients are
expected to play an increasingly important role in
enzyme formulations. The demand for non-boron based
inhibitors is predicted to rise accordingly, with one
recent example being the second generation inhibitor
under the Evity ® brand by Novozymes, which is far
more efficient than 4-FPBA, and not based on boron.
Improvements in liquid stability solutions and formulation
pave the way for further growth in the application of
industrial enzymes. The liquid Evity ® products are based
on an inhibitor which was specifically designed for
the Novozymes protease family. This delivers improved
stability in liquid detergent both of the protease and
41
other enzymes added to the detergent thus enabling a REFERENCES AND NOTES
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