Detergent protease

Article (PDF Available)inCurrent Opinion in Biotechnology 15(4):330-4 · September 2004with802 Reads
DOI: 10.1016/j.copbio.2004.06.005 · Source: PubMed
Abstract
Over the past 20 years, the development of subtilisins as typical detergent proteases has employed all the tools of enzyme technology, resulting in a constant flow of new and improved enzymes. The number of molecules identified and characterized, however, is in clear opposition to the number of molecules that are entering the market. Will the next-generation detergent proteases be based on new backbones different from subtilisins, or will the use of all available technologies (rational design, directed evolution and exploitation of natural diversity) yield improved subtilisins, ending the current era dominated by high alkaline subtilisins? These questions will have to be answered not only by the performance of the molecules themselves, but also by their yield in fermentation and their compatibility with existing production technologies.

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Available from: Karl-Heinz Maurer
Detergent proteases
Karl-Heinz Maurer
Over the past 20 years, the development of subtilisins as typical
detergent proteases has employed all the tools of enzyme
technology, resulting in a constant flow of new and improved
enzymes. The number of molecules identified and characterized,
however, is in clear opposition to the number of molecules that
are entering the market. Will the next-generation detergent
proteases be based on new backbones different from subtilisins,
or will the use of all available technologies (rational design,
directed evolution and exploitation of natural diversity) yield
improved subtilisins, ending the current era dominated by high
alkaline subtilisins? These questions will have to be answered
not only by the performance of the molecules themselves, but
also by their yield in fermentation and their compatibility with
existing production technologies.
Addresses
Henkel, Enzyme Technology, Henkelstrasse 67 40191, Duesseldorf,
Germany
e-mail: karl-heinz.maurer@henkel.com
Current Opinion in Biotechnology 2004, 15:330–334
This review comes from a themed issue on
Protein technologies and commercial enzymes
Edited by Karl-Erich Jaeger
Available online 1st July 2004
0958-1669/$ see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2004.06.005
Introduction
Apart from their importance in physiology (e.g. in the
activation of zymogenic preforms of enzymes, blood clot-
ting, the lysis of blood clots, the processing and transport
of secretory proteins across membranes and as pathogenic
factors) proteases are highly relevant in technical enzyme
applications. Of these, their use in detergents is the most
prominent with respect to market volume and tonnage.
The idea of using proteases in industry — and specifically
in detergents goes back to the use of pancreatic extracts
by Roehm in 1913. Only with the availability of enzymes
from bacteria in the 1960s, however, did their use become
efficient in the technical as well as in the economic sense.
From the beginning these enzymes were produced using
Bacillus species, starting with Bacillus amyloliquefaciens
and Bacillus licheniformis. The alkaline proteases from
these species represent the lead molecules for the sub-
tilisins. Members of the subtilisin superfamily of pro-
teases have now been identified, with different functions,
in practically all living organisms [1]. Nevertheless, the
subtilisins from Bacillus species still provide all the
proteases used in the detergent industry. In the 1980s
the high-alkaline subtilisins were identified, and within a
short time they had replaced the ‘standard’ subtilisins in
all but liquid detergents. In the mid-1980s the subtilisins
were shown to be an excellent model for testing genetic
engineering approaches, and by the end of the decade
they were amongst the first technical enzymes to be
manufactured using recombinant strains. Protein-engi-
neered enzymes entered the market at the beginning
of the 1990s and established themselves as benchmarks
in several applications. Their importance is illustrated
by the fact that the amount of subtilisin produced and
used in the European Union in 2002 was 900 tons of
pure enzyme (Human Health and Environmental Risk
Assessment ‘risk assessment of detergent raw materials’;
http://www.hera-project.org).
Here, we review recent attempts to develop new and
improved proteases for use in detergents either through
the protein engineering of traditional subtilisins or by
searching the metagenome for new enzymes. We also
discuss aspects of production and formulation and consider
how these factors will effect future developments in the
field.
Subtilisins
Subtilisins are defined by their catalytic mechanism as
serine proteases. Their amino acid sequence and three-
dimensional structure can be clearly differentiated from
the other serine proteases, such as chymotrypsin, carbox-
ypeptidase and Peptidase A from Escherichia coli. The
catalytic triad of subtilisins consists of aspartic acid,
histidine and serine. Although the size of subtilisins
varies from 18 kDa to 90 kDa, all the subtilisins used in
detergents have a size of approximately 27 kDa.
The success of subtilisins is based on several factors,
including their high stability and relatively low substrate
specificity features common in extracellular proteases.
Their production as extracellular enzymes is of course an
important factor in itself, as this greatly simplifies the
separation of the enzyme from the biomass and facilitates
other downstream processing steps. Another important
point is the ability of Bacillus strains to secrete enzymes
over a very short period of time into the fermentation broth.
Subtilisins are used in all types of laundry detergents and
in automatic dishwashing detergents. Their function is
to degrade proteinaceous stains [2,3

,4]; typical stains
include blood, milk, egg, grass and sauces. For testing
purposes, such stains are commercially available from test
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institutes. For automatic dishwashing tests the prepara-
tion of stains has been described in great detail. An aspect
that has to be considered when screening candidate
enzymes for better performance is that they are not acting
on soluble substrates in solution, but on substrates bound
to the surface of a solid, water-insoluble substrate.
In contrast to more biochemical environments, where the
denaturation of protein substrates normally leads to
improved enzyme activity, the denaturation of proteinac-
eous stains by aging, heating and oxidization makes them
less accessible to enzymic degradation. The effect of
oxygen bleach on heat-denatured blood or milk stains
is an excellent example: the presence of oxygen bleach
transforms an otherwise easy protease target into an
extremely difcult one. Therefore, test results depend
critically on the type of stain, the composition of the
detergent and the nature and status of the textiles used in
the washing test as ller material (ballast). The same
holds true for automatic dishwashing detergents, where
the nature, composition and amount of ballast stain are
crucial aspects in the evaluation of enzyme performance.
The identification and optimisation of
detergent proteases
At present, less than 15 different enzyme molecules are
used in detergents worldwide. These enzymes originate
from B. amyloliquefaciens, B. licheniformis, Bacillus clausii,
Bacillus lentus, Bacillus alkaloophilus, and Bacillus halodur-
ans [2]. Some of these species assignments have in recent
years been changed; for example, Savinase
1
and Esper-
asee
1
were assigned for several years to Bacillus subtilis or
B. lentus (see Table 1) [5
,6].
Protein engineering methods
Since protein engineering with subtilisins began in 1984,
all the amino acid positions have been modied either by
site-directed mutagenesis based on rational design or,
later, by various methods of random mutagenesis. Most
of this work is published in patents and not in the
scientic literature. The example of replacing Met222
(adjacent to the active Ser221) with amino acid residues
that are stable towards hydrogen peroxide has become a
textbook example of a rational approach to site-directed
mutagenesis [7]. Hydrogen peroxide and peroxo acids are
typical bleaching agents generated in the cleaning process
of bleach-containing products. The oxidation of certain
methionine residues to sulfoxides was known for more
than a decade before the rst approaches to site-directed
mutagenesis were realized. In 1991 the rst proteases
modied in this way for hydrogen peroxide stability were
marketed, even though the performance of these variants
did not full their promise. By 1996 substitutions at nearly
every position in the mature 275 amino acid BPN
0
sub-
tilisin (Bacillus Protease Novo type, subtilisin from B.
amyloliqefaciens) had been claimed in patents. The BPN
0
subtilisin is generally considered to be the lead molecule
for subtilisin modications, and mutations in other sub-
tilisins often refer to the homologous position in this lead
molecule. There are some excellent general reviews on
the protein engineering of subtilisin, as well as articles on
more specic detergent applications [8

,9].
Since 1997, several gene shufing approaches have been
performed with subtilisins. Interesting results from the
shufing of 26 protease genes have been described for
properties such as activity in organic solvents, tempera-
ture stability, and activity at high or low pH [1012].
Little, however, has been published on stain removal.
Owing to the large number of variant molecules gener-
ated by shufing and other random techniques, screening
methods with high-throughput and increased relevance
have had to be developed. Unfortunately, these methods
are still not entirely satisfactory, which might explain why
Table 1
Subtilisin variants used in detergents.
Trade mark Producer Origin WT/PE
c
Production strain Synonym
Alcalase
1
Novozymes B. licheniformis WT B. licheniformis Subtilisin Carlsberg
FNA
a
Genencor B. amyloliquefaciens PE B. subtilis
Savinase
1
Novozymes B. clausii WT B. clausii Subtilisin 309
Purafect
TM
Genencor B. lentus WT B. subtilis
KAP
b
Kao B. alkalophilus WT B. alkalophilus
Everlase
TM
Novozymes B. clausii PE B. clausii
Purafect OxP
TM
Genencor B. lentus PE B. subtilis
FN4
a
Genencor B. lentus PE B. subtilis
BLAP S
b
Henkel B. lentus PE B. licheniformis
BLAP X
b
Henkel B. lentus PE B. licheniformis
Esperase
1
Novozymes B. halodurans WT B. halodurans Subtilisin 147
Kannase
TM
Novozymes B. clausii PE B. clausii
Properase
TM
Genencor B. alkalophilus PB92 PE B. alkaliphilus
a
Exclusive molecules for specific customer.
b
Exclusive molecules for captive use. The names of captive use products are often based on technical terms or acronyms.
c
PE, protein engineered; WT, wild type.
Detergent proteases Maurer 331
www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:330334
no outstanding new subtilisin variant created by one
of the gene shufing technologies is yet present in
detergents.
Phage display, because of the close link between the
polypeptide and its encoding gene, is considered to be
an excellent method for the selection of enzymes with
desired properties. Legendre et al. [13] used subtilisin 309
to illustrate the potential of this method, modifying its
substrate specicity with respect to the amino acid at
the P4 site of the substrate. Soumillion and Fastrez [14

]
give further examples of phage-display applications with
subtilisins.
As all mutations affecting the specicity of subtilisins may
also inuence the autoproteolytic processing of the proen-
zyme to the mature form, the engineering of the pro-
region or its uncoupling from this biosynthesis step has
become relevant [15,16]. So far, no further information on
the success of such approaches has become publicly
available.
The search for new proteases
New interest in properties such as low-temperature
performance, as well as the complexity of the patent
situation, has led to renewed interest in screening for
novel enzymes in nature. The search for new proteases is,
of course, not limited to subtilisins, but is also directed at
nding completely new protease backbones. Some inter-
esting molecules have been identied, but none of them
has as yet made it into a detergent product [17

]. Every
year approximately ten new wild-type subtilisins are now
being described in the scientic or patent literature.
Interesting enzymes are still being found by classical
microbiological screening methods; for example, the
oxidation-stable subtilisin found by Seiki and colleagues
[18,19]. In addition to microbiological screening methods
based on the cultivation of protease-producing microor-
ganisms, the exploitation of genome programs and meta-
genomic screening methods have been established and
have enlarged the screening pool [20,21
].
Chemical modications
The chemical modication of amino acids in subtilisins
has a long tradition, but owing to the high costs of such
processes no application in detergents has yet been put
into practice. The value of chemically modifying amino
acids currently lies in testing various aspects of enzyme
action, such as the effect of net charge on substrate
specicity [22].
Production aspects
Production strains
All major subtilisins for detergents are produced in
Bacillus, because these species are able to secrete large
amounts of extracellular enzymes [23
]. The control
mechanisms involved in the production of proteases in
Bacillus are extremely complex and still not fully under-
stood. An example is the two-component regulatory sys-
tem that acts as a quorum sensing mechanism in B. subtilis
and which has been found to control the expression of the
alkaline protease [24]. This regulatory system is encoded
on the chromosome and on endogenous plasmids.
Industrial strain improvement programs using classical
microbiological methods have been carried out over many
years and have resulted in the development of several
highly productive strains. These have often been used as
hosts for the expression of recombinant genes; however,
these industrial production strains have frequently been
described as resistant to transformation. As plasmid-based
production strains commonly display instability pro-
blems, it is now standard practice to generate strains in
which the recombinant gene is integrated into the chro-
mosome in multiple copies [25,26].
Fermentation considerations
The fermentation of subtilisins is subject to a large
number of variables. Recent publications by C¸alik and
colleagues [2729] give detailed descriptions of investi-
gations into the fermentation parameters for SAP
(B. licheniformis alkaline protease; subtilisin Carlsberg).
The parameters range from the media composition, pH
and oxygen-transfer rate to the different Bacillus species
used as hosts for recombinant production.
Industrial production processes are normally run as large-
scale, fed-batch fermentations at high cell density. Con-
tinuous fermentations are used to analyse critical produc-
tion parameters, but are not used for production [30].
The media used in industrial fermentations have, above
all, to fulll economic requirements; therefore, these
media are often based on complex, inexpensive nitrogen
sources. The composition of fermentation media and the
details of the fermentation processes and yields are nor-
mally considered company secrets and thus almost no
reliable information is available in the public domain.
The general principles on fermentation and downstream
processing have been published, however [31]. Recently,
publications on the production of industrial Bacillus
strains have described yields in the range of 2025 g/L
enzyme in fermentation broth [32].
Granulation processes
Subtilisin preparations are marketed either as a stabilized
enzyme solution or as encapsulated and coated granu-
lates. The liquid preparations normally have a reduced
water content and contain signicant amounts of 1,2-
propane diol. The original granulate type (prill), which
was based on a mixture of enzyme and polyethylene
glycol, has now practically disappeared from the market.
Granulation processes include the use of extrusion, high
shear mixing and uidised beds [33]. The equipment
332 Protein technologies and commercial enzymes
Current Opinion in Biotechnology 2004, 15:330334 www.sciencedirect.com
used for granulation also determines the type of granulate
generated in the process with respect to shape, chemical
composition and structure. In all cases, one or several
coating layers ensure that granulates have low dusting
properties. This requirement results from the recognition
in 1970 that enzyme dust, generated during the detergent
manufacturing process, can lead to the sensitisation of
exposed workers. Granulation technology was developed
to avoid the release of this enzyme-containing dust. This
technology has now been further improved and additional
steps in the production process (e.g. encapsulation and
ventilation) have been introduced to eliminate the pro-
blem. Training and various control mechanisms, as part of
occupational safety programs, have also been established
in the detergent industry.
Formulation in detergents
Granulated enzymes place few restrictions on the for-
mulation of powder detergents and tablets. Intensied
contact with bleaching components, encountered for
example in the pressing of tablets, necessitates higher
levels of storage stability. This in turn has promoted
the development and use of oxidation-stable enzyme
variants.
The stabilization of proteases in liquid preparations is still
a eld for research [34,35]. The major problem in aqueous
environments is autoproteolysis. Some general principles
in formulating liquid detergents include the reduction of
the free water concentration and the use of reversible
inhibitors like borate or phenyl boronic acid derivatives.
In addition, the composition and nature of the surfactants
in the liquid detergent greatly inuence the storage
stability of the enzyme. Liquid detergents have to be
formulated around the needs of the enzymes they con-
tain, optimising ways to stabilize and inhibit them
reversibly.
Conclusions
Enormous efforts over the past 20 years to nd improved
detergent proteases and to improve known ones have not
succeeded in bringing forth a single new enzyme as
versatile and universally applicable as the high-alkaline
subtilisins. The promise offered by gene-shufing tech-
niques has not resulted in successful products. One reason
for this might be that the conditions in detergents are by
no means extreme when compared with those where the
shufing technologies have given their best results. In
general, it might be that so much work has already been
done on subtilisins that intellectual property considera-
tions make it extremely difcult to combine various
positive attributes and to transfer results quickly into
production strains. Nevertheless, there is an expectation
that signicantly improved proteases can still be made by
combining shufing technology with rational design or by
using metagenome screening. In both cases, the applica-
tion of improved screening methods represents a crucial
step [36

]. With time running out for some major sub-
tilisin protein engineering patents, the opportunities for
the production and application of improved molecules
will increase. If this expectation is not fullled protease
development will continue incrementally. In any case, it
is probable that we will see an increasing number of more
specialized proteases designed to perform well in niche
applications.
Future achievements will include a much greater appre-
ciation of structurefunction relationships in proteases,
leading to a deeper understanding of the factors inuen-
cing the cleaning performance of detergent proteases, and
an improved knowledge of functional genomics relevant
to production. The future will also bring an increasing
number of enzymes suitable for use in more specic
environments (e.g. low-temperature applications and
applications in automatic dishwashing detergents). As
improved performance must be based on both enzyme
development and on screening using detergent composi-
tions and relevant stains, new and specialized enzymes
will most probably emerge from the joint efforts of
enzyme producers and detergent formulators.
Acknowledgements
I want to thank Nicholas Kennedy for his help and careful proofreading
of the manuscript.
References and recommended reading
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review, have been highlighted as:
of special interest

of outstanding interest
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334 Protein technologies and commercial enzymes
Current Opinion in Biotechnology 2004, 15:330334 www.sciencedirect.com
    • "Alkaline and high-alkaline proteases are the most prominent detergent enzymes and contribute – alone or in combination with α-amylases – to the basic performance of modern detergents. Such proteases already reached in 2002 an annual tonnage of about 900 metric tons equivalent of pure enzyme for the European market, tendency increasing [3]. "
    [Show abstract] [Hide abstract] ABSTRACT: Since volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis. To develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel's industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an alpha-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer. In this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the basis for a next generation production host, since the strain has still a large potential for further genetic engineering. The final amylase titer of 65% in reference to B. licheniformis protease titer suggests that the developed B. pumilus expression platform is also suitable for an efficient production of non-proteolytic enzymes reaching a final titer of several grams per liter without complex process modifications.
    Full-text · Article · Mar 2014
    Tobias KüppersTobias KüppersVictoria SteffenHendrik HellmuthHendrik Hellmuth+3 more authors ...Wolfgang WiechertWolfgang Wiechert
    • "Gram-positive bacteria of the genus Bacillus are widely appreciated as industrial workhorses for the production of various enzymes owing to their ability of secreting proteins into the extracellular medium [1]. Among the huge number of commercial enzymes, proteases are of great importance for their extensive applications in detergent , tanning, food processing, silk degumming, medical diagnosis, bioconversion, waste treatment, and peptide synthesis [2] [3] [4] [5]. Currently, proteases have accounted for up to 60% of the total enzyme sales in the global market, in which alkaline proteases represent the largest portion [6]. "
    Data · Dec 2013 · Microbial Cell Factories
    • "Gram-positive bacteria of the genus Bacillus are widely appreciated as industrial workhorses for the production of various enzymes owing to their ability of secreting proteins into the extracellular medium [1]. Among the huge number of commercial enzymes, proteases are of great importance for their extensive applications in detergent , tanning, food processing, silk degumming, medical diagnosis, bioconversion, waste treatment, and peptide synthesis [2] [3] [4] [5]. Currently, proteases have accounted for up to 60% of the total enzyme sales in the global market, in which alkaline proteases represent the largest portion [6]. "
    Data · Dec 2013 · Microbial Cell Factories
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