Proteases, Production

O P Ward, University of Waterloo, Waterloo, ON, Canada

M B Rao, National Chemical Laboratory, Pune, India
A Kulkarni, National Chemical Laboratory, Pune, India
ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Industrial Production

Introduction Industrial Application
Proteases and their Mechanisms of Action Proteases in Organic Synthesis
Physiological Functions Further Reading
Protease Engineering

Glossary pepstatin A natural peptide, produced by various

aspergillopepsin Pepsin-like aspartic endoprotease
from Aspergillus species.
chymosin The predominant milk-clotting enzyme from
the true stomach or abomasum of the suckling calf.
clostripain Papain-like cysteine endoprotease from
Clostridium histolyticum.
directed evolution Method used in protein engineering
to harness the power of Darwinian selection to evolve
proteins or RNA with desirable properties.
endothiapepsin Pepsin-like aspartic endoprotease
from Endothia species.
necrosis Cell death in which cells swell and break
open, release their contents, and may damage
neighboring cells.
penicillopepsin Pepsin-like aspartic endoprotease
from Penicillium species.
Actinomyces species, which inhibits aspartic proteases.
protease engineering Techniques for creation of
proteases with new or artificial amino acid sequences.
proteasome A large multicomponent protease
complex that can digest a variety of proteins into short
polypeptides and amino acids.
streptokinase The extracellular hemolytic protease of
Streptomyces species, which dissolves fibrin clots.
streptopain Papain-like cysteine endoprotease from
Streptococcus spp.
subtilisin A serine endopeptidase initially obtained
from Bacillus subtilis, but secreted in large amounts
from many Bacillus species.
thrombolytic enzymes Enzymes that break down or
lyse blood clots.

Abbreviations HslV proteasome subunit

CHO Chinese hampster ovary TLCK tosyl-L-lysine-chloromethylketone
DAN diazoacetyl-DL-norleucine methyl ester TPCK tosyl-L-phenylalanine-chloromethyl ketone
DFP diisopropylfluorophosphate PCMB p-chloromercuribenzoate
EDTA ethylenediaminetetraacetic acid SA chymotrypsins
EPNP 1,2-epoxy-3-( p-nitrophenoxy)propane SB subtilisins
FPC fermentation produced chymosin SC carboxyprotease C
HCN hydrogen cyanide SE D-Ala–D-Ala protease A

HslU ATP-binding subunit

Defining Statement regulation, and in microbial pathogenesis. Natural

and modified commercial proteases are produced with
Microbial proteases are classified based on their mode of applications in detergents, leather, food processing, and
action and biocatalytic mechanisms. Proteases participate medicine. They are also used as biocatalysts in organic
in most aspects of cell nutrition, physiology, and synthesis.

495
496 Applied Microbiology: Industrial | Proteases, Production
Introduction
The topic microbial proteases deals with a very large
group of enzymes from the complete diversity of micro-
organisms. Microbial proteases are ubiquitous in all
microorganisms where they have a variety of biochemical,
physiological, and regulatory functions. Extracellular
proteases of eukaryotic pathogenic microorganisms play
important roles in infectious diseases by attacking the
protein component of mammalian tissues. Over the cen-
turies, microbial proteases also had key roles in the
production of traditional fermented foods, and now a
large and vibrant industrial enzyme industry, dominated
by microbial protease products, supplies the world with
biocatalysts for use in food processing, for detergents, for
use as therapeutics, and for organic chemical synthesis.
Proteases have been classified by the Enzyme
Nomenclature Committee as EC 3.4.-.-, namely subgroup
4 of group 3 (the hydrolases). Depending on their site of
action, proteases are categorized into two major groups,
that is, exoproteases and endoproteases, and further clas-
sification relies on the distinct arrangement of functional
groups present at the active site of the enzyme. There is
continuing interest in investigating the mechanisms of
actions of enzymes in general and of proteases in parti-
cular as a means to understanding the structural and
mechanistic factors that affect biocatalytic properties,
including substrate affinity, reaction rates, and substrate
specificity. Modern molecular techniques enhance our
knowledge of enzyme structure and functional relation-
ships, which can be exploited to engineer new enzymes
with desired new biocatalytic activities and perhaps also
to create totally synthetic organic molecules that can
simulate biocatalytic systems. Developments in our
knowledge of the crystalline structure of industrial pro-
teases have been achieved through a great variety of
research strategies, including use of inhibitors and avail-
ability of genomic information and of cloned genes. Use of
random and site-directed mutagenesis has facilitated the
development of proteases with altered pH-activity pro-
files and substrate specificities, enhanced thermostability,
greater metal binding capacity, and greater resistance to
oxidizing agents with a view to understanding reaction
mechanisms or tailoring better enzymes for industrial use.
Proteases and their Mechanisms of Action
In general, the active sites of protease are flanked on one
or both sides by one or more subsites capable of accom-
modating the side chain of specific single amino acid
residue from the substrate. The degree of amino acid
specificity delineated by the active site and the subsites
ultimately defines the general biocatalytic properties and
especially the specificity of the proteolytic reaction.
Exoproteases act only near the ends of the N- or
C-termini of the polypeptide chains and are classified
accordingly as amino- and carboxyproteases, respectively
(Table 1). Peptide cleavage by aminoproteases liberates a
single amino acid residue, a dipeptide, or a tripeptide by
acting on a free N-terminus of the polypeptide chain.
The N-terminal Met, present in many heterologously
expressed proteins but not in many naturally occurring
proteins is also cleaved off by these enzymes. While
intracellular aminoproteases are produced by a wide vari-
ety of bacterial and fungal species, Aspergillus oryzae is
known to produce an extracellular aminoprotease.
Bacterial and fungal enzymes exhibit distinctly contrast-
ing substrate specificities such that substrate preferences
may be used to differentiate the host organisms. These
enzymes have been reported from Escherichia coli, Bacillus.
licheniformis, and Bacillus stearothermophilus. The carboxy-
proteases correspondingly cleave off a single amino acid
or a dipeptide from the C-terminal ends of the polypep-
tide chain. Carboxyproteases are characteristically
differentiated based on amino acid substituents at the active
site into three major groups: serine carboxyproteases, metal-
locarboxyproteases, and cysteine carboxyproteases.
These enzymes have been reported from a number of
fungal species like Penicillium, Saccharomyces, and Aspergillus.
Endoproteases attack peptide bonds in more central
locations of the polypeptide chain more remote from the
N- and C-termini and indeed free amino or carboxyl
groups that are known to inhibit or retard enzyme action.
They are divided into six subgroups based on their cata-
lytic mechanism in the following sections.
Serine Endoproteases (EC 3.4.21)
As the name implies, serine proteases contain a serine
group in their active site, which is essential for substrate
binding and cleavage. Generally, serine proteases are
characterized by their broad substrate specificity, and
their activity extends beyond purely peptidase to include
esterase and amidase activities. Serine proteases exist
among exoprotease, endoprotease, oligoprotease, and
omega protease groups. Important representative enzyme
groups include the chymotrypsins (SA), subtilisins (SB),
carboxyprotease C (SC), and Escherichia D-Ala-D-Ala pro-
tease A (SE), and these have primary structures that are
totally unrelated. Serine proteases are characterized by
having a conserved glycine-containing peptide, Gly-
Xaa-Ser-Yaa-Gly, associated with the catalytic serine.
A common reaction mechanism in the form of a catalytic
center containing serine as a nucleophile, aspartate as an
electrophile, and histidine as a base, is exhibited by groups
SA, SB, and SC, respectively. Interestingly, distinctive
 Applied Microbiology: Industrial | Proteases, Production
Table 1 Proteases and their mechanisms of action
Proteases Mode of action
Exopeptidases
3.4.11 Aminopeptidases
3.4.14 Pipeptidyl peptidases
3.4.14 Tripeptidyl peptidases
3.4.16–3.4.18 Carboxypeptidase
3.4.16 Serine type protease
3.4.17 Metalloprotease
3.4.18 Cysteine-type protease
3.4.15 Peptidyl dipeptidase
3.4.13 Dipeptidases
3.4.19 Omega peptidases
3.4.21–3.4.34 Endopeptidases
3.4.21 Serine protease
3.4.22 Cysteine
3.4.23 Aspartic protease
3.4.23.19 Glutamic acid protease
3.4.24 Metalloproteases
3.4.25 Threonine proteases
Open circles represent the amino acid residues in the polypeptide chain. Solid circles indicate the terminal amino acids, and stars signify the blocked
termini. Arrows show the sites of action of the enzyme. This table expands upon and updates information provided in Rao et al. (1998).
protein folding strategies among these groups accomplish
similar geometric orientations of these residues, suggest-
ing a convergent evolutionary background. Some groups
may be differentiated from the latter groups in that they
lack the serine-aspartate-histidine catalytic center.
Serine proteases generally exhibit pH optima in the
range 7–11 and manifest isoelectric pH values in the range
4–6. Serine alkaline proteases, produced by certain bacteria
including Arthrobacter, Streptomyces, and Flavobacterium species,
filamentous fungi, including Conidiobolus, Aspergillus, and
Neurospora species, and some yeasts represent the largest
subgroup of serine proteases, and are active at the higher
end of the above pH range (pH optimum 10) with an
unusually high isoelectic pH around 9. These enzymes are
inhibited by diisopropylfluorophosphate (DFP) or a potato
protease inhibitor, but not by tosyl-L-phenylalanine-
chloromethyl ketone (TPCK) or tosyl-L-lysine-chloro-
methylketone (TLCK), which inhibit other serine
proteases. These enzymes are characterized as having sub-
strate specificities similar to but broader than that of
chymotrypsin, where the carboxyl side of the peptide bond
being attacked contains a tyrosine, phenylalanine, or leucine
497
Common active site substituents/metal
Free N-terminus
Not applicable
Not applicable
Free C-terminus
Xaa-Ser-Yaa
HEXXH-H/HEXXH-E, zinc or cobalt ions
Cys-His/His-Cys
Not applicable
Not applicable
Blocked N-terminus
Blocked C-terminus
Xaa-Ser-Yaa
Cis-His/His-Cys
Asp-Xaa-Gly
Glu-Gln
HEXXH-H/ HEXXH-E
Thr
residue. The second largest family of serine proteases con-
tains the subtilisins, which are best represented by subtilisin
Carlsberg and subtilisin BPN, produced by B. licheniformis
and Bacillus amyloliquefaciens, respectively. The active site
conformations of both the enzymes are similar to trypsin
and chymotrypsin despite their contrasting molecular struc-
tures. While the subtilisin from Conidiobolus coronatus exhibits
catalytic similarities with subtilisin Carlsberg, its protein
structure is distinct.
The serine protease reaction mechanism involves the
formation of a covalently-linked enzyme-substrate inter-
mediate through acylation, resulting in loss of the
corresponding amino acid or peptide fragment, and
nucleophilic attack on the intermediate by water results
in deacylation, thereby completing hydrolysis of the pep-
tide. In the endoproteases, the amino acids directly
supplying the amino and carboxyl substituents to the
peptide bond exclusively determine the candidate site
or sites for peptide bond attack whereas the subsites
may affect reaction rate. These enzymes were classified
as trypsin-, chymotrypsin-, or elastase-like if they had
positively charged, large hydrophobic, or small
498 Applied Microbiology: Industrial | Proteases, Production
hydrophobic primary substrates, respectively. Some of
the serine proteases from Achromobacter spp. are lysine-
specific, while clostripains from Clostridium spp. are argi-
nine –specific, and some B. licheniformis and Staphylococcus
aureus endoproteases are glutamic acid-and aspartic acid-
specific. A general substrate-binding mechanism for pro-
teases exhibiting specificity to glutamic acid consists of a
histidine residue, and a hydroxyl function is suggested.
Some of the subtilisins have an active site triad con-
taining the same amino acid residues as are found in the
chymotrypsin family, with the exception that the the
order is changed (Asp-His-Ser). Also Cys 173 adjacent
to the active site histidine in some subtilisins from the
yeasts Tritirachium and Metarhizium spp. has rendered
these enzymes thiol-dependent for their activity. A glu-
tamate residue preceding the catalytic Ser in serine-
dependent carboxyproteases is thought to be responsible
for the acidic pH optimum of these enzymes.
Aspartic Endoproteases (EC 3.4.23)
The microbial aspartic acid proteases have a catalytic pair
of these amino acids present at their active sites. Because of
their pH optima (3–4) they are called acid proteases. They
have low isoelectric points (pH 3–4.5). They are typically
sorted into two groups: (1) pepsin-like enzymes produced
by Aspergillus, Penicillium, Rhizopus, and Neurospora; and (2)
rennin-like enzymes produced by Endothia and Mucor spp.
Pepstatin, a natural peptide produced by various
Actinomyces species, is a well-known inhibitor of aspartic
proteases and may be applied to inhibit these proteases
where they cause undesirable degradation of other pro-
teins, for example, when heterologous proteins are
produced by a host, which also produces aspartic proteases.
The enzymes are also inhibited by diazoketone com-
pounds, such as diazoacetyl-DL-norleucine methyl ester
and 1,2-epoxy-3-(p-nitrophenoxy)propane, in the presence
of copper ions. The specificity of the microbial acid pro-
teases is broader than that of pepsin, and it is analogous to
pepsin in that the enzymes attack peptide bonds where
both contributing amino acids contain aromatic groups or
bulky side chains.
Penicillopepsin and endothiapepsin biocatalysis is
mediated by a general base catalytic mechanism with a
lytic water molecule participating in the reaction. The
pepsin generally has bilobal structures surrounding the
active site with each homologous lobe contributing a pair
of aspartic acid residues to the catalytic process. In both
N- and C-terminal lobes in most pepsins, catalytic aspar-
tate residues consist of a Asp-Thr-Gly-Xaa motif
(Xaa¼Ser or Thr, with side chains hydrogen bonding to
Asp). In general, pepsins and other aspartic proteases
exhibit broad-based specificity toward cleavage in pep-
tides consisting of at least six hydrophobic amino acids at
specific substrate positions.
Cysteine/Thiol Endoproteases (EC 3.4.22)
Cysteine proteases generally may be assigned to one of
the following four groups according to their side chain
specificities: (1) papain-like (includes clostripain and
streptopain), (2) trypsin-like with preference for cleavage
at the arginine residue, (3) specific to glutamic acid, and
(4) others. Most have neutral pH optima. All cysteine
proteases have cysteine/histidine catalytic dyad, although
the order of these residues, Cys-His or His-Cys, may
vary. They generally need reducing agents such as
sodium bisulfite, hydrogen cyanide, or cysteine for
activity retention. Sulfydryl agents such as p-
chloromercuribenzoate are inhibitor or denaturants,
whereas DFP and metal-chelating agents are not.
Clostripain (Clostridium histolyticum), which shows high
specificity for arginyl residues contributing the carboxyl
group to the peptide bond contrasts with papain in that it
requires calcium for activity. Streptopain (Streptococcus
spp.) manifests broad specificity toward synthetic sub-
strates and oxidized insulin B chain.
Biocatalysis is mediated by a double-displacement
pathway involving general acid-base formation and
hydrolysis of an acyl-thiol intermediate. These enzymes
have broad specificity and also attack amide ester, thiol
ester, and thiono ester bonds. The enzyme initially binds
noncovalently to the substrate, after which acylation of
the enzyme occurs together with release of the first pro-
duct. Water reacts with the acyl enzyme releasing the
second product through deacylation.
Metalloendoproteases (EC 3.4.24)
The divalent metal-requiring metalloproteases are a very
diverse group of proteases, which include both endopro-
teases and exoproteases. They are inhibited by chelating
agents such as ethylenediaminetetraacetic acid, but not by
sulfhydryl agents or DFP. Thermolysin, a neutral zinc
protease produced by B. stearothermophilus, is one of the
most thoroughly characterized metalloproteases where a
histidine and glutamine participate in the active site,
providing a ligand for zinc and a catalytic function,
respectively. As its name suggests, thermolysin exhibits
high thermostability and has a half-life of 1h at 80  C.
Four calcium atoms enhance the thermostability of the
protein. The metalloprotease collagenase is produced by
a variety of microorganisms, including Achromobacter
iophagus and Clostridium hystolyticum. Pseudomonas aeruginosa
produces the neutral metalloprotease elastase as well as
alkaline metalloproteases. The alkaline protease I from
Myxobacter lyses the cell walls of Arthrobacter crystellopoites,
whereas Myxobacter protease II cannot lyse the bacterial
cells. Generally, the metal-binding site motif includes the
motif His-Glu-Xaa-Xaa-His.
 Applied Microbiology: Industrial | Proteases, Production
Biocatalysis requires bound divalent cations, and
removal of the metal through dialysis or with chelating
agents causes inactivation. In thermolysin, Glu143 parti-
cipates in the nucleophilic attack of a water molecule on
the carbonyl carbon of peptide bond being cleaved, which
has been polarized by the Zn ion.
Glutamic Acid Proteases (EC 3.4.23.19)
This newly established family of glutamic proteases
(family G1) was derived from the former pepstatin-
insensitive carboxyl proteases. There were several enzymes
from bacteria and fungi, which were found to be resistant
to pepstatin. The unusual active site proved difficult to
characterize, but it was soon established that the bacterial
enzymes were acid-acting serine proteases, having a dif-
ferent catalytic mechanism from the family of aspartic
proteases. These enzymes have been discovered from
Stylidium lignicola and Aspergillus niger var. macrosporus.
The catalytic mechanism is based on the two enzymes
from the aspergilloglutamic and scytalidoglutamic pro-
teases. The active site diad glutamic acid and glutamine
play a critical role in substrate binding and catalysis.
These amino acids along with their associated water
molecules act as nucleophiles to exhibit an acid-base
mechanism distinct from that of the aspartic proteases.
The glutamic acid acts as a general acid in the first phase
of catalysis, donating a proton to the carbonyl oxygen of
the scissile peptide bond of the substrate. Simultaneously,
an OH– is donated by water associated with the active site
of the enzyme to the carbonyl oxygen of the peptide bond
of the substrate. Sometimes, two water molecules are
involved in the reaction. The transition state of the sub-
strate is thought to be stabilized by hydrogen bonding
with the two catalytic residues. Then, glutamic acid
donates a proton to the amide nitrogen atom of the scissile
peptide bond triggering the breakdown of the tetrahedral
intermediate and thus effecting peptide bond cleavage.
The glutamine residue is then responsible for recovering
the original state of the glutamic acid residue.
Threonine Endoproteases (EC 3.4.25)
This new class of proteases was discovered in 1995 as part
of proteasome complex. The discovery of a fifth protease
catalytic type came when the crystallographic structure
of the eukaryotic proteasome (XT01-001) from
Saccharomyces cerevisiae was determined, showing an
N-terminal threonine at the active site. The catalytic
type of the proteasome had been a puzzle because of its
resistance to inhibition by most standard protease inhibi-
tors. The structure also revealed the number of subunits
in the protease complex as 14, and the N-terminal threo-
nine residues of some of the subunits as catalytic residues.
Prokaryote homologues have a simpler structure: the
499
bacterial proteasome (T01.005) has four different kinds
of subunits, the archaean (T01.002) two, and the bacterial
HslVU (T01.006) protease consists of a proteasome sub-
unit (HslV) and an ATP-binding subunit (HslU).
These enzymes are part of a multicomponent protea-
some complex in microbial cells. The archaebacterial
proteasome has 14 active sites in the inner channel,
one on each subunit. The hydrolytic sites are spatially
separated from the intracellular components. Nonspecific
proteolysis of proteins is prevented by the barrier of
ring, which allows only unfolded proteins to enter the
small opening of the central channel. This entry point is
the first regulation site, which is under the control of
associated factors like the 19S complex. The exact
mechanism of hydrolysis has been a mystery for quite
some time now. All prokaryotic and eukaryotic and
subunits are homologous. They do not show any resem-
blances to other proteases. Recent reports have indicated
that the active site nucleophile is the hydroxyl group on
the threonine at the N-terminus of the subunit.
Archaebacterial proteasome replacements of the terminal
threonine by serine allows full proteolytic activity.
Therefore, the conservation of the threonines in the
active sites of all threonine proteases from bacteria to
eukaryotes is unclear. Looking at the diverse functions
of the threonine proteases in bacteria and mammals, it is
evident that the phylogenetically ancient proteasome has
undergone adaptations that favor different functions in
different physiological situations.
Physiological Functions
Proteases participate in a variety of metabolic and regu-
latory functions in all organisms. Perhaps, the simplest
and most obvious role of microbial proteases is in nutri-
tion where extracellular proteases, following their
secretion by the cell, degrade insoluble proteins and
soluble large polypeptides into smaller peptides and
amino acids, which are accessible to the cell. However,
the roles played by proteases, which participate in many
essential general processes of cells and their regulation,
are far more complex. Examples of some of the cellular
functions mediated by proteases are set out below:
Regulation of Gene Expression
Proteases participate in modulation of gene expression
through enzyme modification of repressor proteins, RNA
polymerase, ribosomal proteins, or regulatory proteins
associated with other physiological events such as heat
shock or programmed cell death. ATP-requiring pro-
tease-mediated modification of repressor proteins can be
involved in gene derepression. Protease-mediated hydro-
lysis of the B subunit of Bacillus thuringiensis RNA
500 Applied Microbiology: Industrial | Proteases, Production
polymerase can result in an alteration in its specificity
of transcription. Proteases may participate in regulation
of translation processes by proteolytic modification of
ribosomal proteins. ATP-dependent proteases may also
participate as chaperones, promoting insertion of proteins
into membranes and the disassembly or oligomerization
of protein complexes. In addition, regulatory proteins are
involved in control of physiological processes such as the
heat shock response, the SOS response to DNA damage,
and programmed bacterial cell death; hence these regu-
latory proteins represent targets for protease-mediated
interventions of these processes.
Enzyme Modification
Covalent modification of enzyme and protein precursors
by specific proteases is an important step in the physio-
logical regulation of many rate-controlling processes and
enzyme cascades. Conversion of zymogenic forms of
chitin synthase into the active enzyme by limited proteo-
lysis has been observed in organisms such as Candida
albicans, Mucor rouxii, and Aspergillus nidulans. In eukar-
yotes, the Kex-2 protease (kexin; EC 3.4.21.61), originally
discovered in yeast, is an example of a eukaryotic
precursor processing enzyme. Kex-2 participates in
hydrolysis of membrane-associated proteins of the secre-
tory pathway by specific cleavage at the carboxyl side of
pairs of basic residues such as Lys-Arg or Arg-Arg.
Systems such as this are now being widely applied in
biotechnology to engineer secretory pathways for pro-
duction of extracellular recombinant heterologous
proteins. Proteolytic modification of enzymes may alter
enzyme specificity and hence physiological function. In
this manner, removal of a small peptide converts E. coli
leucyl-L-RNA synthetase into an enzyme catalyzing
leucine-dependent pyrophosphate exchange. Yeast pro-
teinases A and B participate in covalent modification and
inactivation of certain yeast enzymes. Ultimately, more
intensive protease hydrolysis of specific enzymes are part
of the more general cellular protein turnover process, and
results in reductions or losses of particular biocatalytic
activities with the associated physiological outcomes as
indicated below.
Protein Turnover
Only a fraction of a cell’s genetic potential is expressed at
any given time and hence the proportions of the total
number of proteins encoded by the cell that are expressed
and their amounts present in the cell are highly variable.
Indeed, the cell and its proteins and metabolites are in a
completely dynamic state whereby the concentration of
each protein in the cell is represented by the difference
between its rate of synthesis and its rate of breakdown.
Especially in the case of microbial cells, which have the
capacity to adjust their physiology and metabolism to
adjust to a wide range of external environmental factors,
this dynamic characteristic is of paramount importance.
Cells need to synthesize new enzymes and other func-
tional proteins and indeed restrict the actions of other
enzymes and other functional proteins to make the
requisite physiological and biochemical adjustments.
Intracellular proteases facilitate the breakdown of unde-
sired proteins and generate in this way at least part of the
amino acid pool required to support synthesis of new
enzymes or other functional proteins and thus play an
essential role in mediating protein turnover in the cell.
Deficiencies in protein turnover may be demonstrated by
generating protease minus mutants. In many microbial
cells, ATP-dependent proteases often participate in pro-
tein turnover mechanisms.
The proteasomes identified as being an essential part
of the ubiquitin signaling pathway in prokaryotes and
eukaryotes are known to mediate the rapid elimination
of highly abnormal proteins that arise by mutation or
postsynthetic damage. A large number of critical regula-
tory proteins, which have to be eliminated for normal
growth and metabolism of the cells, are targeted by
the proteasome. Formation of tumors and stoppages in
cell cycle are a prominent feature of blockage of the
ubiquitin-mediated pathway and of the proteasome. The
proteasome also precisely regulates the slow degradation
of most cell proteins. Generally, proteasomes degrade
proteins to small peptides, most of which are rapidly
hydrolyzed to amino acids by cytosolic exoproteases.
However, some of the oligopeptides generated by protea-
somes, especially in response to interferon, are presented
to the immune system as major histocompatibility com-
plex class I molecules.
Sporulation, Conidial Discharge, and
Germination
It is obvious that substantial protein turnover occurs dur-
ing the major cellular physiological adjustments
associated with such events as production of microbial
spores, ascospores, and fruiting bodies, and in the
development and discharge of fungal conidia and in ger-
mination events. Many sporulating organisms are known
to synthesize large amounts of protease during sporula-
tion and indeed sporulation processes rely on protease
activity. An increase in protease A activity in yeast
diploids occurs during ascospore formation. Slime mold
fruiting body development and onward differentiation
involve high rates of protein degradation. In C. coronatus,
an alkaline serine protease participates in the conidial
discharge process.
The amino acids necessary for spore germination are
not available in dormant spores. Serine endoproteinases,
specific only toward spore storage proteins in dormant
 Applied Microbiology: Industrial | Proteases, Production
spores, cause their hydrolysis to generate the amino acids
and nitrogen for biosynthesis. A specific alkaline serine
protease also mediates microconidal germination and
hyphal fusion events. Germination-associated disruption
of the cell wall polypeptide linkages of Dictyostelium dis-
coideum spores and Polysphondylium pallidum microcysts are
mediated by extracellular acid proteases.
Microbial Pathogenesis
Proteases play key roles in many pathophysiological con-
ditions caused by microorganisms such as AIDS, malaria,
candidiasis, staphylococcal infections, and cancer, and this
has led to major emphasis on development of protease
inhibitors as therapeutic agents targeting these proteases.
Of all the proteases, aspartic, cysteine, and metallo pro-
teases are the major classes involved in disease states.
A few interesting reports implicating proteases in
apoptosis, especially cysteine proteases, have recently
emerged, more so in eukaryotes than prokaryotes.
The role of microbial proteases in human pathogenesis
is well illustrated with reference to just one specific
enzyme type, collagenase. Collagen amounts to 25–33%
of total protein in mammalian organisms; in skin, tendons,
and cartilage, it is the dominant constituent. It is also the
major protein component of bone, teeth, and the cornea.
Not surprisingly, pathogenic bacteria that produce col-
lagenases are responsible for many mammalian diseases.
These diseases are associated with skin, the oral cavity,
amniotic membranes, the lungs, the eye, muscles and soft
tissues, and the central nervous system. Collagenase from
Actinomadura madurae is associated with mycetoma.
Collagenases from Clostridium spp. are involved in necro-
tizing diseases. Collagenase produced by Bifidibacterium
sp., Eubacterium alactolyticus, and Peptostreptococcus spp., are
associated with necrotic pulp chambers. Collagenases
from Peptostreptococcus spp. are also associated with breast
abscess and diabtic foot ulcer. Collagenase from Vibrio
vulnificus is associated with scepticemia while the enzyme
from Streptococcus agalactiae (group B strepcocci) partici-
pates in neonatal septicemia. Collagenase from
P. aeruginosa is associated with emphysema and ecthyma
gangrenosum. Collagenases from Brucella melitensis,
Bacteroides spp., Clostridium spp., E. coli, Fusobacterium nucle-
atum, Peptococcus sp., Peptiostreptoccus spp., Proteus mirabilis,
Staphylococcus spp., and S. agalactiae have been
associated with pregnancy complications. Collagenases
from Actinobacillus actinomycetemcomitans, Bacillus cereus,
Porphyromonas (Bacteroides) spp., Provotella (Bacteroides)
spp., Streptococcus mutans, and Treponema species have
been reported in periodontal disease. Collagenases from
Enterococcus faecalis and S. mutans are associated with den-
tal caries.
501
Protease Engineering
Advances in molecular and cell biology, and genetic
engineering, combined with developments in computing,
X-ray crystallography, and structural biology have facili-
tated our ability to determine rapidly the amino acid
sequences and three-dimensional structures of enzymes.
The field of protein engineering allows us to predict
three-dimensional structures based on altering amino
acid sequences to modify the function of a particular
protein. Thus, in the context of proteases, protein engi-
neering may be used to determine enzyme mechanisms,
modify enzyme specificity and stability, and to determine
the contributory roles of one or more amino acids to these
properties.
Since subtilisin has been the subject of some of the
most detailed protein engineering studies, this section
will be confined to addressing this enzyme. The compo-
nent amino acids of the active site of subtilisin BPN
include the well-known serine protease catalytic triad,
Ser221, His64, and Asp32; the oxyanion binding site,
Asn155; and the amide chain of Ser221. Mutation of any
of Ser221, His64, and Asp32 reduces kcat by four to six
orders of magnitude, whereas Km is little affected, indicat-
ing the observed reductions in catalytic activity are not
due to alterations in substrate binding. With 86 of 125
amino acids being identical, subtilisin BPN and subtilisin
Carlsberg have quite contrasting sequences and structures
and catalytic efficiencies (kcat/Km) toward the synthetic
substrate tetra-amino acid-p-nitroanilide, with the
Carlsberg enzyme exhibiting a catalytic preference for
the low molecular weight substrate. When subtilisin
BPN residues Tyr217, Glu156, and Gly169 involved in
substrate binding were replaced by the corresponding
amino acids in the Carlsberg enzyme, specificities were
very similar, suggesting these amino acids are responsible
for the differences. Deprotonation of active site His64 of
subtilisin BPN increases the enzyme’s pH optimum. This
can be effected by making specific substitutions to surface
amino acids on the enzyme. The catalytic function of
subtilisin BPN may be shifted from peptide hydrolysis
to peptide synthesis, through improved stabilization of
the enzyme in organic solvents, by substituting Ser221
and Pro225 with cysteine and alanine, respectively.
Storage stability of most native alkaline proteases in
bleach-containing detergents was problematic due to the
presence of a bleach-sensitive methionine near the pro-
tease’s active site. To address this, protein-engineered
proteases were developed where the bleach-sensitive
methionine was replaced by other amino acids, which
were insensitive to oxidation.
The more recent approaches to protease engineering
involve site-directed mutagenesis, directed evolution, and
DNA shuffling. In the future, rational enzyme design
502 Applied Microbiology: Industrial | Proteases, Production
approaches combined with advanced computational tech-
niques may be expected to result in generation of novel
proteases for the diversity of microbial protease
applications.
Protease Engineering Strategies for Organic
Synthesis
Protease engineering approaches, mediated by deliberate
chemical modification of application of genetic techni-
ques to remodel wild-type enzymes, have also been
applied to improve the applicability of proteases to
organic synthesis. The goals may be (1) to make the
protease more stable to the organic solvent; (2) to increase
synthesis efficiency of the protease by reducing the rate of
competitive hydrolysis; (3) reduce unwanted proteolytic
activity to prevent competitive hydrolysis of peptides
during synthesis, and (4) to expand or change the native
enzyme’s substrate specificity or enantioselectivity to
meet the desired synthesis needs. An example of an
enzyme engineering approach is the preparation of
thiol-subtilisin, which was the first enzyme engineering
approach tested, resulting in the conversion of active site
serine 221 into cysteine. It has been found useful in pep-
tide synthesis and transesterification reactions, for
example, in sugar regioselective acylation. With respect
to genetic modifications to proteases, mutations to more
than 50% of the 275 amino acid residues of subtilisin have
been described and some other microbial proteases have
been similarly investigated. As indicated above, these
studies related of course not just to investigating use of
proteases in organic synthesis, but were also aimed at
deriving an understanding of general characteristics of
proteases, including stability, specificity, activity, and
catalytic mechanisms.
Industrial Production
Enzymes have been used as biocatalysts since ancient
times. Proteolytic enzymes were used to produce cheese
in ancient Greece where milk in calf stomachs was found
to clot (due to the activity of calf rennet, i.e., chymosin).
Christian Hansen started commercialization of chymosin
by extraction from calves’ stomachs with saline, in
Denmark. The original industrial microbial enzyme pro-
duction processes were derived from traditional
food fermentations where molds such as Aspergillus and
Rhizopus species produced amylases and proteases to con-
vert semisolid starch and protein-rich media, including
soya and rice, which were further converted by lactic acid
and/or ethanol-producing strains into edible and nutri-
tious foods. Well-known examples of these foods include
soy sauce, miso, and tempeh. Proteases and amylases from
these molds continue to be produced commercially to this
day. There have also been age old fermentations invol-
ving Bacillus species. In the traditional Natto production
process, B. subtilis (natto) was used in Japan to produce the
fermented food, natto, and other versions of this process
were implemented in Thailand and China to produce
ithiki-natto. The most important enzymes involved in
the production of natto are proteases, responsible for
development of the main flavor through hydrolysis of
soybean protein, having pH optima at 8.5 and 10.8.
Recently, acidic, neutral, and alkaline proteases have
seen an enhanced industrial use in their respective indus-
tries in the natural as well as the recombinant form. With
better knowledge and purification of enzymes from
diverse sources, the number of applications has increased
manifold. Microbial proteases have been among the most
significant hydrolytic enzymes studied extensively for
their industrial applications. Ever since their inception
in 1914, they have seen applications in diverse industries
such as food industry, leather industry, and textile, and
detergent industries. While some commercial fungal
microbial enzymes are still produced in Asia by semi-
solid surface culture, industrial microbial enzymes of both
fungal and bacterial origin are predominantly produced
by large-scale submerged fermentation processes using a
limited number of high-producing bacterial and fungal
hosts producers.
These fermenters have typical volumes of 50 000–
200 000l, which are designed to be sterilized at 15 psi
and 121  C, prior to inoculation with the production
strains. Fermenter design also facilitates supply of sterile
air to the culture as a source of oxygen for the aerobic
fermentation and as a form of mixing via the air sparging
arrangement or using stirring impellers and baffles. The
temperature and pH of the culture are typically con-
trolled during the fermentation and in many cases
nutrients added prior to inoculation may be supplemen-
ted by feeding carbon, nitrogen, or inducer compounds
at particular points during fermentation. All of the pre-
dominant industrial microbial enzymes and indeed all of
the major microbial proteases are secreted by their pro-
ducers into the extracellular medium. Durations of the
fermentation are about 2–4 days for bacterial processes
and 3–5 days for fungal processes. Extracellular enzyme
product concentrations of up to 30 gl–1 may be achieved
in these industrial enzyme fermentations. In the case of
bacteria, these enzymes are recovered in the supernatant
following a continuous centrifugation process to remove
cells and other particulates. In the case of fungi, the
fungal mycelium and other particulates are removed on
a rotary vacuum filter producing the enzyme contained
in the filtrate. The resulting supernatants or filtrates are
usually further concentrated by reverse osmosis. With a
notable exception, most microbial enzymes are marketed
as liquid concentrates as powdered enzyme products
are known to cause allergies and enhaled powdered
 Applied Microbiology: Industrial | Proteases, Production
enzymes, especially proteases can cause serious damage
to lung tissues. However, it is necessary to incorporate
dry alkaline proteases into detergent powders and these
enzymes are typically prepared as wax-containing gran-
ules, so they are not in powder form, in a process known
as prilling.
As indicated above, major protease producing organ-
isms having industrial potential became evident from
traditional food fermentations involving high protein-
containing ingredients, such as soybean. Industrial work-
horse strains developed among species of Bacillus produce
the two very important commercial proteases, the alka-
line serine protease used predominantly in detergents, but
also in food processing, and a neutral protease used in
brewing and other food processing products. Perhaps the
most important industrial protease now produced by a
species of Aspergillus is not a native enzyme, but rather
recombinant heterologous calf rennet. However,
Aspergillus species also produce several important fungal
proteases used in the food industry. Hence, we will focus
on Bacillus and Aspergillus hosts for protease production.
Bacillus Protease Production
Enzymes produced by Bacillus species represent about
50% of the total enzyme market with the Bacillus alkaline
serine proteases (subtilisins) being the single most domi-
nant commercial enzyme because of its application in
household detergents. The most important commercial
serine alkaline protease, subtilisin Carlsberg, produced by
B. licheniformis, consists of a single peptide chain with 274
amino acid residues, contains no cysteine amino acids,
and consequently has no disulfide bonds. This enzyme is
also produced by Bacillus pumilus. Another Bacillus serine
alkaline protease, subtilisin BPN, produced by B. amyloli-
quefaciens, B. subtilis, and B. stearothermophilus has little
commercial significance compared with subtilisin
Carlsberg. While the enzyme structures have significant
similarities, the Carlsberg enzyme has a broader pH-
activity curve, and it retains a much greater percentage
of its optimum pH activity, when the pH is reduced to 7.0.
The three largest enzyme manufacturers, Novo
Nordisk, Genencor International, and DSM N.V., have
reported market shares of about 41–44, 21, and 8%,
respectively, with other producers in North America,
Europe, and Asia accounting for most of the remaining
27–30%. Commercial proteases from some of the major
manufacturers are listed in Table 2. The subtilisin
Carlsberg-type alkaline serine protease is produced by
B. licheniformis, B. pumilus, and B. subtilis. Highly alkalophi-
lic Bacillus species, such as Bacillus clausii and Bacillus
halodurans, produce more highly alkaline stable enzymes
used in heavy-duty detergent formulations, such as
Esperase produced by Novo Nordisk. Commercial neu-
tral proteases produced by Bacillus species have activity
503
optima in the region of pH 7 and these are zinc metallo-
proteases. They have applications in milk and soy protein
modification, in brewing for control of amino nitrogen,
and in optimization of cereal mash extraction and chill-
haze removal. Commercially important proteases of
Bacillus species are typically produced constitutively,
and the production is associated with the exponential
and postexponential phases of growth.
Bacillus species are attractive hosts for production of
proteases for a variety of reasons. They exhibit high
growth rates, which result in short fermentation cycles.
Several strains of Bacillus, including B. subtilis and B. liche-
niformis, have GRAS status from the United States Food
and Drug Administration, which means these organisms
and their products are generally regarded as safe. The
complete sequence of B. subtilis and a number of other
Bacillus species have now been published, which is facil-
itating the further development and engineering of these
strains and their enzyme products. Bacillus species, includ-
ing B. subtilis, B. licheniformis, and B. amyloliquefaciens, have a
high capacity to secrete the proteases of interest into the
extracellular medium. In this regard, the identification of
several genes in the B. subtilis genome, which encode
proteins of the major secretion pathway, including five
type-I signal peptidase genes and one type-II signal pep-
tidase, was of special significance. Genomic analysis of the
alkaliphilic B. halodurans has also been especially interest-
ing from the perspective of producing commercial
alkaline proteases. Through protein engineering techni-
ques, it has been possible to develop commercial protease
variants of the B. clausii alkaline protease to generate
enzymes with improved performance for use in low-
temperature washes and in bleach containing detergents.
A variety of molecular strategies are used for develop-
ment of recombinant Bacillus strains for commercial
production. Engineered versions of natural plasmids
from the same organism or a closely related species are
introduced into hosts using transformation, cell fusion,
and electroporation approaches. Native promoters, such
as the promoter of the highly efficient B. subtilis
-amylase, are often exploited. It was found that more
stable clones could be developed by direct insertion of
DNA into the chromosome. Antibiotic resistance strate-
gies cannot be used in commercial enzyme production
fermentations for stable plasmid maintenance, and other
strategies such as incorporating the only copy of another
gene required for microbial growth into the plasmid have
been used. Amplification of gene copy number has gen-
erated high enzyme-producing strains. It has been
suggested that the ideal production strain is a genetically
stable and chromosomally integrated gene, supported by
high levels of expression and secretion.
In a different context, the ability of Bacillus species to
produce both a large variety and high quantities of extra-
cellular proteases has presented a barrier to the use of
504 Applied Microbiology: Industrial | Proteases, Production

Table 2 Examples of commercial proteases’ sources, applications, and their industrial suppliers

Product trade
Supplier name Microbial source Application

Novo Nordisk, Alcalase Bacillus licheniformis Detergent (high alkaline, high temperature), silk degumming,
Denmark protein hydrolysates, meat processing, fuel alcohol
fermentation enhancement
Biofeed pro B. licheniformis Feed
Durazym Bacillus sp. Detergent
Esperase B. lentus Detergent, food, silk degumming
Everlase Bacillus sp Detergent (bleach stable)
Flavorzyme Aspergillus oryzae Protein hydrolysates (mixture of exo-, endo-proteases)
Kannase Bacillus sp. Detergent (cold wash)
Kojizyme Not specified Protein hydrolysates
Neutrase B. subtilis Brewing and baking
NovoBate WB Bacillus sp. Leather
Novozyme 243 B. licheniformis Denture cleaners
Novozyme Genetically modified Very broad specificity, hydrolyses amide, and ester bonds,
539HP F Bacillus sp. enantioselective catalyst in synthesis of optically active
amines, alcohols, carboxylic acids, and amines; peptide
synthesis
NUE Bacillus sp. Leather liming (active at pH 12–13)
Ovozyme Bacillus sp. Dishwasher detergent (egg inhibitor-resistant)
Protamex Bacillus protease Production of protein hydrolysates, flavor development, and
complex meat processing.
Savinase Bacillus sp. Detergent (higher pH, medium temperature), textile.
Subtilisin A Genetically modified Very broad specificity, hydrolyses amide and ester bonds,
Bacillus sp. enantioselective catalyst in synthesis of optically active
amines, alcohols, carboxylic acids, and amines; peptide
synthesis
Genencor Acid fungal Aspergillus sp. Protein hydrolysis
International, protease
USA Fermgen Bacillus sp. Ethanol fermentations
Fungal protease Aspergillus sp. Baking, dairy-selective protein hydrolysis
HT proteolytic Bacillus sp. Protein hydrolysis (high temperature)
Multifect neutral Bacillus sp. Protein hydrolysis (neutral pH), wheat ethanol fermentation
Primatan Bacterial source Leather (bating)
Properase Bacillus sp. Detergent (high alkaline, low temperature)
Protex Bacillus sp. Protein hydrolysis (alkaline)
Purafect B. lentus Detergent (high alkaline), leather (alkaline soak)
DSM, NL Accelerezyme Non GMO Cheesemaking – flavor development
BakeZyme Bacillus sp. Baking
B500BG
BakeZyme Aspergillus sp. Baking
B500BG
Brewers Bacillus sp. Brewing (neutral protease)
protease
Fromase Rhizomucor miehei. Cheesemaking:fungal coagulant
Maxiren Recombinant Cheesemaking: fermentation produced chymosin
Kluyveromyces lactis
Suparen/ Cryphonectria Cheesemaking fungal coagulant
Surecurd parasitica
Amano Acid protease A A. niger Protein hydrolysis (pH 3.0)
Pharmaceuticals Acid protease A. niger Dietary supplement
JP DS
Collagenase Clostridium sp. Tissue culture
Newlase F Rhizopus niveus Protein hydrolysis (pH 3.0)
Proleather Bacillus sp. Protein hydrolysis (pH 10)
Protease A 2 A. oryzae Protein hydrolysis (pH 7.0)
Protease A-DS A. oryzae Dietary supplement
Protease DS A. mellius Dietary supplement
Protease M A. oryzae Protein hydrolysis (pH 4.5)
Protease N B. subtilis Biotransformations, protein hydrolysis

(Continued )
 Applied Microbiology: Industrial | Proteases, Production 505

Table 2 (Continued)

Product trade
Supplier name Microbial source Application

Protease S B. stearothermophilus Biotransformations, protein hydrolysis

Protin Bacillus sp. Leather bating, dehairing, and silver recovery (pH 10–11)
Prozyme Aspergillus mellius Digestive aid
Streptokinase/ Streptococcus sp. Anti-inflammatory pharmaceutical
streptodornase
Thermoase Bacillus sp. Leather bating, dehairing, and silver recovery (pH 7–8.5)
Enzyme Enzeco alkaline B. licheniformis Industrial (pH 9.5)
Development, protease
USA
Enzeco alkaline B. licheniformis Food
protease-L FG
Enzeco high Bacillus sp. Industrial
alkaline
protease
Enzeco neutral B. subtilis Food, meat tenderizing
bacterial
protease
Enzeco neutral B. licheniformis Vegetable protein hydrolysis (pH 6–9.5)
bacterial
protease

these hosts for production of cloned foreign proteins
because the proteases tend to degrade the secreted cloned
proteins. As a result, strategies to use protease-deficient
mutants have been employed and successfully used to
produce a number of intact heterologous proteins.
Aspergillus Protease Production
The long-time acceptance of Aspergillus species as hosts
for production of enzymes for use in food processing
derive from the traditional use of these fungi in fermented
foods. Indeed, Aspergillus species are also used to produce
other food additives including citric and gluconic acids.
A. niger and A. oryzae are GRAS listed organisms. In addi-
tion, hyperproducing Aspergillus species are capable of
producing upto 25–30 g l-1 of commercial extracellular
enzyme. Because of these valuable properties, the genes
and genomes of Aspergillus species have been extensively
studied to understand the characteristic mechanisms
involved and to facilitate further developments. Detailed
genomic information is available on all of the Aspergillus
strains of relevance to protease production, namely, A.
oryzae, A. niger, and A. nidulans; this information and the
associated methodologies, combined with other classical
and molecular methods, is supporting the ongoing devel-
opment of these strains as fermentation hosts.
Pepsin-like acid protease is one of the important com-
mercial proteases produced by Aspergillus species, with a
major application in the hydrolysis of soybean protein in
soy sauce manufacture. One of the most important
sources of this enzyme is Aspergillus saitoi (A. phoenicis).
Aspergillopeptidase A, produced by various Aspergillus
species, has a low pH optimum of 23.5–3.0, and its main
application reflecting this pH optimum is as a digestive
aid. The main commercial acid protease of A. oryzae has a
pH optimum of 4–4.5 and is very much an endo-acting
protease, which only liberates small amounts of free
amino acids. Commercial preparations of this enzyme
used in soy sauce preparation need to be augmented
with high carboxypeptidase activity to generate the high
amino acid concentrations, which are characteristic of soy
sauce preparations and essential to their flavor. Proteases
predominantly produced by Aspergillus species in sub-
merged fermentation solid state systems are also
still used. Interesting observations were made in some
solid-state fermentation experiments with recombinant
Aspergillus strains producing chymosin. Much higher
yields of chymosin were observed in solid state media
containing 2% wheat bran as compared to submerged
culture. There are indications that fungal extracellular
enzyme secretory mechanism are altered when the strains
are grown in solid-state culture as compared with sub-
merged liquid cultures.
Production of heterologous extracellular proteins by
Aspergillus species may be limited at the level of transcrip-
tion, translation, secretion, or by extracellular proteolytic
degradation. In addition to secretion, limitations which
occur at the posttranslational level have been cited to
include inefficient translocation, folding, transport, and
processing. Consequently, a range of strategies have had
to be implemented to overcome these bottlenecks. These
have included: gene fusion strategies, development of
506 Applied Microbiology: Industrial | Proteases, Production
protease-deficient mutants, control of morphology, over-
production of foldases and chaperones, and other
molecular and physiological approaches.
Chalf chymosin, a key proteolytic enzyme used in chee-
semaking was produced in a recombinant Aspergillus species.
Indeed, this was the first recombinant heterologous product
to receive approval from the United States Food and Drug
Administration. It is synthesized as preprochymosin.
During the secretion process, the 16-amino acid prese-
quence is cleaved off. The 42-amino acid prosequence is
removed automatically during further processing by expo-
sure of the protein to low pH. Yield improvements of the
product were obtained through gene fusion technology and
classical mutation and selection strategies. Indeed, novel
mutants of A. nidulans, which were deficient in the aspartic
protease gene encoding aspergillopepsin were useful in
production of recombinant chymosin.
In Aspergillus species, proteases degrading heterologous
proteins may be localized in intracellular compartments,
associated with the cell wall or be secreted. A variety of
strategies have been employed to reduce this proteolysis
including use of protease-deficient strains, physiological
and process engineering, cell immobilization, and pro-
tease inhibition. Aspergillus hosts with inactivated key
protease-producing genes, areA, pepC, and/or pepE, were
found to be useful in heterologous protein production.
Manipulation of fungal morphology of Aspergillus species
also has a profound effect on production of proteases
during heterologous protein production, with pelleted
growth resulting in greater proteolytic activity, probably
due to intrapellet cell lysis. Manipulation of media com-
position with removal of peptide nitrogen and shifting pH
away from the optima for proteolytic activity are other
strategies that may be used to reduce the impact of
Aspergillus proteases on degradation of recombinant pro-
teins produced by these hosts.
Industrial Application
Currently, the enzyme industry is thriving worldwide,
with the US market responsible for about one third of
the total market. The estimated value of the worldwide
sales of industrial enzymes in 2003 was $2.3 billion per
year. Approximate distribution between sectors are food
enzymes (29%), feed enzymes (15%), and general tech-
nical enzymes (56%). Proteases account for about 60% of
the market. The detergent industry represents the single
most important industry, accounting for about 35% of
total enzyme sales.
Detergents
In 1961 Novo Industri introduced the microbial alkaline
serine protease from B. licheniformis, alcalase, having high
stability and activity in the pH 8–10 range to the deter-
gent industry. Since then laundry enzymes have become
accepted with market penetration of 75–95% in the
United States, Europe, and Japan. The main laundry
products may be divided into three product categories:
low pH (7.5–9.0), low ionic strength liquid detergents
containing no bleach; European regular detergents pow-
ders having a high pH (9.5–10.5), high ionic strength with
an activated bleach system (also contain sodium sulfate
and sodium triphosphate and zeolite); and General North
American and European/Japan compact powders with
high pH (9.5–10.5) detergents without sodium sulfate.
All proteases used in these detergents are serine alkaline
proteases from Bacillus species. Alcalase or similar pro-
tease products from B. licheniformis are used in the low pH
detergents described above, whereas highly alkaline pro-
teases derived from alkalophilic Bacillus species, such as
B. clausii and B. halodurans, are used in the highly alkaline
detergents. These latter enzymes have higher pH optima
than those derived from B. licheniformis and the enzymes
have also been engineered to be more resistant to bleach
components.
Some of the enzyme detergent preparations presently
marketed by different suppliers are included in Table 2.
Food Industry
The ancient discoveries about naturally occurring
enzymes and their use in enhancing food quality have
led to the development of various food applications for a
wide range of available proteases from many sources,
usually microbial. Proteases may be used at various pH
values, and they may be highly specific in their choice of
cleavable peptide links or quite nonspecific. Examples of
many of the proteases used in the food industry are
provided in Table 2.
Enzymes were first used in the food (dairy) industry as
milk clotting agents with rennin being the enzyme of
choice. Industrially, acidic proteases (aspartic proteases)
have been in demand for their ability to coagulate pro-
teins especially milk proteins to form curds from which
cheese is prepared after the removal of whey. The major
application of proteases in the dairy industry is in the
manufacture of cheese. There are three categories of
milk-coagulating enzymes: animal rennets, microbial
milk coagulants, and genetically engineered chymosin.
Animal and microbial milk-coagulating proteases are
aspartic proteases. Rennet extracted from the fourth sto-
mach of unweaned calves contains a mixture of the milk
coagulating enzyme, chymosin (EC 3.4.23.4) together
with pepsin, but with a high ratio of rennet to pepsin
activity. A world shortage of calf rennet developed as a
result in an increased demand for cheese production and
this triggered a search for alternative microbial milk coa-
gulants. In cheesemaking, the unique property of rennet is
 Applied Microbiology: Industrial | Proteases, Production
its ability to hydrolyse a single specific peptide bond (the
Phe105-Met106 bond) in the k-casein fraction of milk to
generate para-k-casein and macropeptides without cata-
lyzing hydrolysis of other caseins. This k-casein is
responsible for stabilization of the milk micelles in sus-
pension and its hydrolysis by rennet results in
destabilization of the milk micelles leading to the coagu-
lation associated with curd formation. Thus the key
characteristic sought in the search for microbial rennet
substitutes was that they should exhibit proteolytic char-
acteristics similar to rennet, namely that they should have
a very high ratio of milk clotting activity to general
proteolytic activity. Another desired characteristic was
that the microbial candidates would have a similar low
thermal stability, such that activity could be eliminated by
low thermal treatment such that proteolysis would not
continue in storage. Many of the potential microbial
rennet substitutes found did not have as high a ratio of
milk clotting activity to general proteolytic activity or
were more thermostable than rennet. The effect of the
greater or more prolonged proteolytic activity was that it
produced weaker curds and/or the cheese produced
became more liquid or softer than desired during storage.
Technically and commercially, the most successful native
microbial cheesemaking enzyme was produced by the
mold Rhizomucor miehei. In the late 1980s, calf chymosin,
synthesized through recombinant DNA technology, was
first produced on a large scale for use in cheesemaking by
Genencor International using Aspergillus niger var. awamori
as the microbial host. Maxiren is the commercial fermen-
tation recombinant chymosin product, produced in
Kluyveromyces lactis by DSM.
Proteases are also used in the baking industry. Where
appropriate, dough may be prepared more quickly if its
gluten is partially hydrolysed. A heat-labile fungal pro-
tease is used so that it is inactivated early in the
subsequent baking. Weak-gluten flour is required for
biscuits in order that the dough can be spread thinly and
retains decorative impressions. In the past, this has been
obtained from European domestic wheat but this is being
replaced by high gluten varieties of wheat. The gluten in
the flour derived from these high gluten-containing
wheats must be extensively degraded if such flour is to
be used efficiently for making biscuits or for preventing
shrinkage of commercial pie pastry away from their alu-
minium dishes. The bacterial proteases are used for
improving the functional, nutritional, and flavor proper-
ties of proteins. Neutrase is a bacterial protease, which is
used in alcohol production for improving yeast growth. In
baking, it is used to degrade proteins in flour for biscuits,
crackers, and cookies. In brewing, it is used for extracting
sufficient proteins from malt and barley and for obtaining
the desired level of nitrogen nutrients. Acid protease from
A. saitoi, aspergillopepsin I, is useful for the production of
seasoning materials from foods containing various
507
proteins, the degradation of the turbidity complex result-
ing from protein in fruit juices and alcoholic liquors, and
the improvement of quality of protein-rich foods.
Flavourzyme is a fungal complex of exoproteases and
endoproteases derived from A. oryzae is used for extensive
hydrolysis of proteins. Kojizyme is a similar complex,
which finds application in the fermentation of soy sauce.
These enzymes are also products of Novozymes,
Denmark (Table 2).
Traditionally, microbial proteases, especially alkaline
proteases (serine proteases), have been used in the pre-
paration of protein hydrolysate of high nutritional value.
The protein hydrolysates play an important role in blood
pressure regulation and are used in infant food formula-
tions, specific therapeutic dietary products, and the
fortification of fruit juices and soft drinks. Commercial
protein hydrolysates are derived from casein (Miprodan
MD Foods, Viby, Germany), whey (Lacprodan,
MD Foods), and soy protein (Proup, Novo Nordisk,
Bagsvaerd, Denmark). Other applications, which exploit
the hydrolytic property of proteases, include soy protein
hydrolysis, soy sauce production, gelatin hydrolysis,
casein and whey protein hydrolysis, meat protein recov-
ery, fish protein hydrolysis, and meat tenderization.
Proteases from B. subtilis have been used to deproteinize
crustacean waste to produce chitin and also in the pro-
duction of fish hydrolysates of high nutritional value.
A novel application of neutral protease is in combination
with the physical method of ultrasonication for the
extraction of rice starch. Another very interesting appli-
cation is the production of proteinaceous fodder from
waste feathers or keratin-containing materials wherein
the keratinolytic activity of alkaline proteases from
Bacillus and Streptomyces species are utilized for the
hydrolysis to obtain a protein concentrate for fodder
production. The limited proteolysis of inexpensive mate-
rials such as soya protein can increase the range and value
of their usage as it occurs naturally in the production of
soy sauce. Hydrolysed proteins may develop properties
that contribute to the elusive, but valuable, phenomenon
of ‘mouth feel’ in soft drinks. Proteases are used to recover
protein from parts of animals and are used in canned
meats and soups. Dehaeming is done using subtilisin and
the purified hydrolysate is used in cured meats, sausages,
and luncheon meats.
Protein hydrolysates have a variety of applications as
nutritional supplements and flavor agents in infant for-
mulae and as functional food additive supplements. For
example, soya hydrolysate products having different
functional or taste properties may be produced by selec-
tion of specific hydrolytic conditions where alkaline
protease from Bacillus species are used as catalysts. The
development of bitter tasting protein hydrolysates is a
recognized problem, caused by a significant presence of
hydrophobic amino acid residues as well as the presence
508 Applied Microbiology: Industrial | Proteases, Production

of proline residues within the hydrolyzed peptides, and product in terms of hardness. Examples of proteases used
proteolytic conditions must be manipulated to minimize in the various aspects of leather processing are provided
these effects. A protease from Pseudomonas fluorescens R098 in Table 2.
has been reported to hydrolyse the peptides found in
cheese, which are responsible for the bitter taste. The
action of endogenous proteases in meat after slaughter is Medical Applications
complex, but ‘hanging’ meat allows flavor to develop, in
There are three main areas where microbial enzymes are
addition to tenderising it. It has been found that peptides
used in medicine, as thrombolytic enzymes, in certain
with terminal acidic amino acid residues give meaty,
cancer treatments and as digestive aids.
appetising flavors akin to that of monosodium glutamate.
Streptokinaseis an extracellular protease produced by
Nonterminal hydrophobic amino acid residues in
hemolytic Streptococcus species, which rapidly dissolves
medium-sized oligopeptides also confer a bitter flavor,
fibrin clots. It is used to treat acute blocking of arteries,
whereas this is not observed in larger peptides.
deep vein thrombosis, and pulmonary embolism. It is
Application of this knowledge allows the tailoring of the
typically the preferred therapy in coronary thrombosis
flavor of protein hydrolysates. The presence of proteases
caused by myocardial infarction. It is particularly effec-
during the ripening of cheeses is not totally undesirable
tive in the first couple of hours of thrombosis, indeed
and a protease from B. amyloliquefaciens may be used to
thrombolytic efficacy of the enzyme decreases signifi-
promote flavor production in cheddar cheese.
cantly when it is administered more than 3 h after
Meat tenderisation by the endogenous proteases in the
occlusion. Streptokinase has been tested in combination
muscle after slaughter is a complex process, which varies
with aspirin for treatment of acute myocardial infarction
with the nutritional and physiological state of the
and it has been found that while each exhibited positive
animal at the time of slaughter. These properties are
effects, the combination showed an effect which was
now mediated by use of proteases derived from
additive. Administration of streptokinase is not clot selec-
microorganisms.
tive and it causes extensive activation of plasmin, which in
addition to attacking thrombus can degrade blood factors
Leather Industry including factor V, factor VIII, and fibrinogen, events
which may contribute to hemorrhaging. Streptokinase
The major components of skin and hair, and hence leather,
can also induce immune responses or indeed be neutra-
contain predominantly proteins, including elastin and ker-
lized by antibodies present in the patients’ blood
atin. The major steps in leather processing are soaking,
produced as a result of a previous treatment with strepto-
dehairing, bating, and tanning. The purpose of soaking is
kinase. While efforts have been made to synthesize the
to swell the hide and this objective may be achieved using
human thrombolytic enzyme, tissue plasminogen activa-
alkali. Chemical methods for dehairing and dewooling
tor in microorganisms, this has not yet been achieved and
exploit extremely alkaline condition followed by treat-
the FDA-approved recombinant product is currently pro-
ment with hydrogen sulfide, which attacks, solubilizes,
duced in culture by Genentech using Chinese hampster
and removes the proteins of the hair root. These conven-
ovary (CHO) cells.
tional approaches used in leather processing involve
Carboxypeptidase G1 from Pseudomonas sp. hydrolyses
addition of harsh chemicals, which create safety, effluent
the terminal L-glutamic acid substituents of folic acid that
disposal, and pollution problems. Thus, use of enzymes in
can cause a state of folic acid deficiency, which negatively
general and proteases in particular wherever possible as
impacts certain tumor cells. Carboxypeptidase G1 also
substitutes to toxic chemicals has the potential to reduce
attacks an analogue of folic acid in a manner that enables
environmental problems and may also improve leather
one of the reaction products, methothrexate, to exert a
quality. Proteases may be exploited to achieve more selec-
selective antiproliferative action against a brain tumor
tive hydrolysis of noncollagenous skin constituents and for
while protecting the normal tissues from drug side
removal of nonfibrillar proteins such as albumins and
effects. Microinjection of solid tumors with proteases
globulins.
from Serratia marcescens results in tumor necrosis and
It has been found that time for absorption of water
solubilisation.
during leather soaking is reduced by application of micro-
bial alkaline proteases. Application of alkaline proteases
with hydrated lime and sodium chloride for dehairing
Other Biotechnological Applications
significantly reduces volumes of wastewater produced.
Different combinations of Bacillus and Aspergillus proteases Proteases find significant uses in many more industries,
with trypsin can be used in leather bating depending on including the textile and waste water management indus-
the nature of the leather matrix proteins being targeted, tries. In addition to their dominant role in detergents and
while enzyme dosage relates to the desired leather type their uses in food processing, alkaline proteases are used
 Applied Microbiology: Industrial | Proteases, Production
for diverse applications, including silk processing, silver
recovery, and in some nutritional applications.
Recently, proteases have found novel applications in
the silk industry wherein they find use in the process of
degumming of silk. Sericin is the protein that imparts
rough texture to the raw silk fibre. This is conventionally
removed from the inner fibroin protein by shrink proofing
and twist setting using starch, an expensive process. A few
patents describing the use of proteases in degumming of
silk prior to dyeing have been filed. Besides these uses,
other novel applications for proteases have been found in
the management of industrial and household waste.
Proteases in Organic Synthesis
Conventional applications of proteases involve hydrolysis
reactions in aqueous media, where water itself is a reagent
in the reaction, and its presence in high concentrations
drives the reaction equilibrium in favor of hydrolysis.
Nevertheless, while historically proteases have been asso-
ciated with digestion processes, reverse reactions
involving peptide synthesis have been known for about
a century, in the phenomenon known as plastein forma-
tion where very high concentrations of low molecular
weight amino acids or peptides were used to drive for-
mation of peptide bonds. In the past two decades,
strategies by which the water phase in the reaction is
wholly or partially replaced by introduction of an organic
solvent phase, thereby substantially reducing the concen-
tration of water as a reactant, has made it possible to shift
the equilibrium of the reaction in favor of synthesis. In
addition to the benefit of being able to reverse hydrolytic
reactions, nonaqueous-phase enzymatic reactions may be
applied to the array of substrates that are more soluble in
nonpolar solvents. There is now a great deal of research
ongoing on applications of enzymes in organic synthesis
and among published papers in this area, application of
proteases ranks second only to lipases and esterases. Thus,
proteases are viewed in chemical synthesis as highly
regiospecific and stereospecific catalysts, having moder-
ate pH optima, that are easy to handle and do not require
cofactors. They are also advantageous in that many pro-
teases, including the range of industrial microbial
proteases, are readily available at low cost and offer syn-
thetic flexibility. Protease engineering strategies to
improve biocatalysts have been discussed above. Several
commercial enzymes are marketed for this purpose by
Novo Nordisk and Amano Pharmaceuticals (Table 2).
Medium Engineering Strategies for Organic
Synthesis
Two main medium engineering strategies are used in the
application of proteases in organic synthesis, namely use
509
of aqueous-organic biphasic mixtures and use of neat
organic solvents. While these systems have many draw-
backs, especially related to undesired hydrolysis and
potential substrate insolubility in the aqueous phase,
they have been successfully applied with many proteases
including thermolysin. However, many reactions, includ-
ing esterifications and transesterifications and reactions
involving water-sensitive moieties, are not possible in
these two-phase systems and need to proceed only in an
organic phase. The presumption has been that a dramatic
decrease in protease stability occurs due to fast enzyme
unfolding in neat organic media, but in recent years this
assumption has been proven not to be always correct. For
example, the microbial Thermus strain Rt4A2 protease is
truly organic solvent stable. However, despite the fact
that proteases may adopt the correct conformation in
neat organic solvents their catalytic activities are far
lower than those observed in water or aqueous-organic
mixtures and this has lead to intensification of research to
improve the catalytic activities of proteases in nonaqu-
eous organic solvents. Improvements in activity and
solubility of subtilisin in organic solvents were observed
by covalent chemical modifications of subtilisin with cer-
tain polymers and other strategies have involved
noncovalent physical modifications using polymers such
as lipids, surfactants, and polyethyleneglycol. Covalent
and noncovalent binding of proteases, including subtilisin,
to immobilization supports have also been successfully
utilized, although the well- known disadvantages of sup-
ports where access of substrate to the enzyme active site
may still prevail. However, the most exciting results
relate to finding proteases, which are truly solvent stable
and can still attain remarkably high reaction rates and
produce high product yields, and P. aeruginosa PST-01
protease complies with this requirement. P. aeruginosa
PST-01 is highly homologous to thermolysin except
that the Pseudomonas protease contains two disulfide
bonds, one of which has been linked to its solvent stability.
The improved solvent stability is not accompanied by
improved thermostability. Another approach to using
organic solvent reaction media involves using supercriti-
cal fluids, and indeed, among many example applications,
subtilisin and proteases of Aspergillus species have been used
to catalyse the transesterification reaction of N-acetyl-(L or
D)-phenylalanine ethyl ester with methanol.
Organic Synthesis Applications
The most obvious application of proteases is in peptide
synthesis, especially synthesis of short peptides. For
example, a new thermophilic protease from Clostridium
thermohydrosulfuricum and pronase from Streptomyces griseus
have both been used for synthesis of various dipeptides,
while the prolyl-endopeptidase of Flavobacterium meningo-
septum was used in model peptide synthesis studies. In an
510 Applied Microbiology: Industrial | Proteases, Production
example of the application of microbial proteases to
stepwise synthesis of oligopeptides, subtilisin and thermo-
lysin, along with chymotrypsin and papain, were used for
preparative scale synthesis of Leu-enkephalin and Met-
enkephalin.
Because of the flexibility afforded by some proteases to
acylate not only nucleophiles generated from natural
amino acids, but also these enzymes can generate novel
products through acylation from nonnatural amino acids
and nonamino acid-derived amines. In addition, in theory,
all proteases may participate in transesterification and
esterification reactions. However, because esterases and
lipases generally exhibit broader substrate specificities
than proteases, these enzymes are typically more efficient
than proteases in catalyzing the esterification reaction.
Proteases also participate in the synthesis of glycocon-
jugates where subtilisin has been the dominant enzyme
used. Subtilisin may act in glycoconjugate synthesis
through either catalysis of a new peptide bond linking a
glycopeptode to a peptide or through direct acylation of
carbohydrates. The latter approach is complicated by the
fact that carbohydrates present multiple hydroxyl groups
for acylation. Other microbial enzymes, including clostri-
pain from C. histolyticum have also been applied to
glycoconjugate synthesis.
Proteases may also be exploited in organic synthesis to
resolve a pair of enantiomeric forms in racemic mixtures
through the generalized process of kinetic resolution,
whereby one enantiomer in the mixture is more rapidly
transformed than the other. While protease catalysts
could be exploited to resolve enantiomers through a
variety of reactions they catalyze, including hydrolysis
of esters or amides of carboxylic acid, esterification or
transesterification reactions or amide bond formation, the
most widely used approach is through enantioselective
ester hydrolysis. A. oryzae protease is one of the most
widely used proteases in kinetic resolution, while other
prominently cited proteases are subtilisin and the serine
alkaline protease from Thermoactinomyces vulgaris.
Aspartame, a dipeptide composed of L-aspartic acid
and the methyl ester of L-phenylalanine, is used as a
food additive and in soft drinks as a noncalorific artificial
sweetener. Maintenance of the L-configurations of the
two amino acids is a perquisite for achievement of the
required sweet taste of aspartame and this is most effec-
tively achieved commercially by enzymatic synthesis of
aspartame. Implementation of reverse proteolytic
reactions in low aqueous media facilitates synthesis of
aspartame from precursor amino acids. In this reaction,
the L-phenylalanine complete with amino group blocking
agent is reacted with L-aspartyl-methyl ester to form the
dipeptide. This reaction may be catalyzed by the immo-
bilized thermolysin from Bacillus thermoproteolyticus at
about 50  C. Later, the phenylalanyl-amino blocking
agent is chemically removed to produce aspartame or
L-phenylalanyl-L-aspartyl-methyl ester.
Human insulin differs only from porcine insulin in the
carboxyl amino acid number 30 of the heavy B insulin
chain. In the case of porcine insulin, this amino acid is
alanine whereas the corresponding amino acid in human
insulin is threonine. The penultimate carboxyl amino acid
in both cases is lysine. Thus, human insulin can be synthe-
sized from porcine insulin by making the amino acid
substitution in protease-mediated sequential peptide
hydrolysis and peptide synthesis steps. In the first step, a
lysine-specific protease clips off the alanine amino
acid from porcine insulin, producind des-B30-insulin.
Threoninyl-tert-butyl ester is then reacted with des-
B30-insulin in 60% ethanol/dimethylformamide solvent
in a reaction catalyzed by lysine-specific protease from
Achromobacter sp., the tert butyl substituent is then
removed from Thr30 with trifluoroacetic acid/anisole to
produce the human insulin product.
The aminopeptidase from Pseudomonas putida ATCC
12633 has been used in the resolution of a wide range of
amino acid amides using different reaction strategies
resulting in the production of optically pure L- and
D-amino acid derivatives. The enzyme has been cloned
and expressed in E. coli K-12 and is characterized as
belonging to the group of leucine aminopeptidases. This
approach has been applied to produce optically pure
enantiomers with more than 100 natural and synthetic
amino acids. This so called P. putida ATCC 12633 ami-
noamidase process has been commercialized by DSM for
production of several L- and D-amino acids.
See also: Enzymes, Industrial (overview); Fermented
Foods; Industrial Biotechnology, (overview); Industrial
Fermentation Processes; Quinolones
Further Reading
Barett AJ (1994) Proteolytic enzymes: Serine and cysteine peptidases.
Methods in Enzymology, vol. 244, pp. 1–15. New York: Academic
Press.
Barett AJ (1995) Proteolytic enzymes: Aspartic and metallopeptidases.
Methods in Enzymology, vol. 248, pp. 183–198. New York:
Academic Press.
Fujinaga M, Cherney MM, Oyama H, Oda K, and James MNG (2004)
The molecular structure and catalytic mechanism of a novel carboxyl
peptidase from Scytalidium lignicolum. Proceedings of the National
Academy of Sciences 101: 3364–3369.
Gupta R, Beg QK, and Lorenz P (2002) Bacterial alkaline proteases:
Molecular approaches and industrial applications. Applied
Microbiology and Biotechnology 59: 15–32.
Harrington DJ (1996) Bacterial collagenases and collagen-degrading
enzymes and their potential in human disease. Infection and
Immunity 64: 1885–1891.
Kumar CG and Takagi H (1999) Microbial alkaline proteases: From a
bioindustrial viewpoint. Biotechnology Advances 17: 561–594.
Löwe J, Stock D, Jap B, Zwickl P, Baumeister W, and Huber R (1995)
Crystal structure of the 20S proteasome from the archaeon T.
Acidophilum at 3.4Å resolution. Science 268: 533–539.
 Applied Microbiology: Industrial | Proteases, Production 511

Rao MB, Tanksale AM, Ghatge MS, and Deshpande VV (1998)
Molecular and biotechnological aspects of microbial
proteases. Microbiology and Molecular Biology Reviews
62: 597–635.
Schallmey M, Singh A, and Ward OP (2004) Developments in the use of
Bacillus species in industrial production. Canadian Journal of
Microbiology 50: 1–17.
Sumantha A, Larroche C, and Pandey A (2006) Microbiology and
industrial biotechnology of food-grade proteases: A perspective of
food-grade proteases. Food Technology and Biotechnology
44: 211–220.
Ward OP, Qin WM, Dhanjoon J, Ye J, and Singh A (2006) Physiology
and biotechnology of Aspergillus. Advances in Applied Microbiology
58: 1–75.