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CHAPTER 13
ASPARTIC PROTEASES USED IN CHEESE MAKING
FÉLIX CLAVERIE-MARTÍN
∗
AND MARÍA C. VEGA-HERNÁNDEZ
Unidad de Investigación, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de
Tenerife, Spain
∗
fclamar@gobiernodecanarias.org
1. INTRODUCTION
The use of aspartic proteases (APs) in cheese manufacture is among the earliest
applications of enzymes in food processing, dating back to approximately 6000 B.C.
(Fox and McSweeney, 1999). Enzymatic milk coagulation is a two-phase process.
In the first phase, APs hydrolyse the Phe
105
-Met
106
bond of bovine -casein splitting
the protein molecule in two, yielding hydrophobic para--casein and a hydrophilic
part known as the macropeptide. The second phase consists of the coagulation of
the casein micelles that have been destabilized by the proteolytic attack.
Milk-clotting enzymes are obtained from mammals, plants and fungi. They can
also be produced using recombinant DNA technology. Enzymes extracted from
the fourth stomach (abomasum) of suckling calves (rennet) have traditionally been
used as milk coagulants for cheese production. In addition to its chymosin content,
conventional rennet also contains lower levels of pepsin A, the most representative
peptidase of Family A1, characterized by its general proteolytic activity that makes
it unsuitable for milk clotting (Harboe and Budtz, 1999). Plant and fungal milk
coagulants present high levels of non-specific, heat-stable proteases the prolonged
action of which cause bitterness in the cheese after a period of storage (Harboe and
Budtz, 1999; Roserio et al., 2003). A world shortage of bovine rennet, due to the
increased demand for cheese, encouraged the search for alternative milk coagulants.
Research on fungal APs resulted in the production of enzymes that are inactivated at
normal pasteurisation temperatures and contain low levels of non-specific proteases
(Branner-Jorgensen et al., 1982; Yamashita et al., 1994; Aikawa et al., 2001).
In 1988, chymosin produced by recombinant DNA technology was first intro-
duced to the dairy industry for evaluation. A few years later, scientists at Genencor
207
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 207–219.
© 2007 Springer.
208 CLAVERIE-MARTÍN AND VEGA-HERNÁNDEZ
International were able to increase the production of chymosin in Aspergillus niger
var. awamori to commercial levels (Dunn-Coleman et al., 1991). Presently, several
recombinant chymosins such as Maxiren® produced by DSM, and Chymogen ®
produced by Christian Hansen, are available on the market. Recombinant chymosin
preparations are very pure and have high milk-clotting activity.
Chymosins from other mammalian species including lamb, kid goat, camel and
buffalo calves are being considered as alternatives for milk clotting in the production
of certain types of cheese (Mohanty et al., 1999; Elagamy, 2000; Rogelj et al., 2001;
Vega-Hernández et al., 2004).
2. STRUCTURE OF ASPARTIC PROTEASES
Aspartic proteases (E.C. 3.4.23) are peptidases and exhibit a wide range of activ-
ities and specificities. They are present in animals, plants, fungi and viruses
(Davies, 1990). APs have been linked to a variety of physiological functions
including mammalian digestion of nutrients (e.g. chymosin, pepsin A), defence
against pathogens, yeast virulence (e.g. candidapepsins), metastasis of breast cancer
(e.g. cathepsin D), pollen-pistil interactions (e.g. cardosin A), control of blood
pressure (e.g. renin), haemoglobin degradation by parasites (e.g. plasmepsins) and
maturation of HIV proteins (retropepsin).
Structurally APs belong to the A1 pepsin family (Rawlings et al., 2004). Like
other pepsin-like enzymes, APs are synthesized as preproenzymes (Fig. 1). After
cleavage of the signal peptide the proenzyme is secreted and autocatalytically
activated. In general the active enzymes consist of a single peptide chain of about
320-360 amino acid residues having molecular masses of 32-36 kDa. X-ray crystal-
lographic analyses of various APs show that they are composed mostly of -strand
secondary structures arranged in a bilobal conformation (Fig. 2) (Cooper et al., 1990;
Davies, 1990; Gilliland et al., 1990; Newman et al., 1991; Aguilar et al., 1997;
Yang et al., 1997). The two lobes are homologous to each other and have evolved
by gene duplication (Tang, 2004). The catalytic centre is located between the two
lobes and contains a pair of aspartate residues, one in each lobe, that are essential for
the catalytic activity. In most pepsin family enzymes, the catalytic Asp residues are
contained in an Asp-Thr-X motif, where X is Ser or Thr. These Asp residues activate
a water molecule that mediates the nucleophilic attack on the substrate peptide bond
Figure 1. Schematic representation of the primary structure of bovine chymosin. SP, signal peptide; P,
prosegment; M, mature enzyme. Arrows indicate processing sites
ASPARTIC PROTEASES USED IN CHEESE MAKING 209
Figure 2. Tertiary structure of chymosin showing the bilobal fold. The arrow points to the flap
(Gilliland et al., 1990)
(James, 2004). Andreeva and Rumsh (2001) have found another water molecule
that plays an essential role in the formation of a chain of hydrogen bonds that
determine substrate binding. The catalytic centre is large enough to accommodate at
least seven residues of the polypeptidic substrate. A flexible structure (flap) located
at the entrance of the catalytic site controls specificity (Hong and Tang, 2004;
James, 2004). APs are active at acidic pH (Chitpinitoyl and Crabbe, 1998). It has
been proposed that the optimum pH of each aspartic protease is determined by the
electrostatic potential at the active site, which in turn is determined by the position
and orientation of all residues near the active site (Yang et al., 1997).
210 CLAVERIE-MARTÍN AND VEGA-HERNÁNDEZ
Family A peptidases are strongly inhibited by pepstatin, a pentapeptide produced
by Streptomyces (Marciniszyn et al., 1976) which contains two residues of an
unusual amino acid, statine. Pepstatin binds to the flap in the catalytic site. The
hydroxyl oxygen of the first statine forms hydrogen bonds with both of the catalytic
aspartate residues (Davies, 1990; Yang and Quail, 1999). Pepstatin is effective
against APs in general but its affinity varies between enzymes. Pepsin A is inhibited
completely in the presence of equimolar amounts of pepstatin, while chymosin and
gastricsin are less susceptible, 10- and 100-fold molar excesses being required for
complete inhibition, respectively (Kageyama, 2002). Other inhibitors are the pepsin
A inhibitor from Ascaris, a parasitic nematode, potato cathespin D inhibitor, and
the highly selective saccaropepsin inhibitor (Kageyama, 2002).
3. CHYMOSIN
3.1. Bovine Chymosin
Chymosin (EC 3.4.23.4) is a gastric digestive aspartic peptidase that is respon-
sible for the coagulation of milk in the abomasum of unweaned calves (Fox and
McSweeney, 1999). In mammals, chymosin is expressed mostly in the foetus and the
newborn, and decreases gradually during postnatal development, becoming insignif-
icant in adults (Foltmann, 1992). The natural function of chymosin is the hydrolysis of
-casein once the milk is in the calf’s stomach, leading to the formation of a coagulum
that can be easily digested. The first step in the biosynthesis of chymosin (323 amino
acids, 35.6 kDa) by the cells of the gastric mucosa is the synthesis of preprochymosin,
a polypeptide of 381 amino acids and 42.1 kDa (Fig. 1). Preprochymosin is secreted
as an inactive precursor, known as prochymosin, with 365 amino acids and 40.8 kDa,
produced by cleavage of the N-terminal signal peptide (Foltmann, 1992). In the acidic
environment of the gastric lumen, prochymosin is activated by autocatalytic removal
of the 42-amino acid prosegment (Pedersen et al., 1979). There are two allelic forms
of calf chymosin, A and B, both of which are active and differ by a single amino acid
substitution, Asp/Gly, at position 243 (Foltmann et al., 1977). The three-dimensional
structure of bovine chymosin has been determined (Fig. 2) (Gilliland et al., 1990;
Newmanetal.,1991).Surprisingly,thenativecrystalstructureshowsthattheflapatthe
catalyticsite adoptsadifferent conformationto thatof other closelyrelated APssuch as
pepsin and renin. This conformation could prevent the binding of substrate-inhibitors
explaining the reduced susceptibility of chymosin to pepstatin. In addition, it could
determine the specificity of chymosin to -casein (Gustchina et al., 1996). However,
X-ray analysis of chymosin complexed with an inhibitor shows close resemblance to
other AP-inhibitor complexes (Groves et al., 1998).
Bovine chymosin is used in cheese production as a milk-clotting agent because
it cleaves -casein in a specific manner at the Phe
105
-Met
106
bond, and has low
proteolytic activity (Fox and McSweeney, 1999; Mohanty et al., 1999). The yeasts
Saccharomyces cerevisiae and Kluyveromyces lactis, and the filamentous fungi
Aspergillus niger var awamori and Trichoderma reesei have been successfully used
ASPARTIC PROTEASES USED IN CHEESE MAKING 211
as hosts for the expression of recombinant calf chymosin (Mohanty et al., 1999).
Recombinant chymosin has also been produced in Escherichia coli but the use of this
product in cheese manufacture is not accepted. Several biotechnology companies
are producing the recombinant enzyme for commercial application, and different
types of conventional cheeses have been produced using these preparations at
experimental or pilot scales. No major differences have been detected between
cheeses made with recombinant chymosin or natural enzymes regarding cheese
yield, texture, smell, flavour and ripening. The absence of bovine pepsin in the
recombinant preparations improves cheese yield and cheese flavour development.
3.2. Other Chymosins
Chymosin is also produced by other mammalian species such as sheep, goat,
buffalo, pig, camel, humans, monkeys and rats. The characterization of chymosins
from these species has been the subject of several reports (Houen et al., 1996;
Elagamy, 2000; Rogelj et al., 2001; Mohanty et al., 2003; Vega-Hernández
et al., 2004). Recently, our group has cloned the cDNA for goat prochymosin and
expressed it in yeast (Vega-Hernández et al., 2004). The cDNA encodes a protein
of 381 amino acids with an N-terminal leader sequence and a proenzyme region of
16 and 42 amino acids, respectively. The deduced sequence shows high similarity
to other preprochymosins (99, 94, and 94% amino acid identity with lamb, calf and
buffalo sequences, respectively). The two catalytic aspartate residues of APs are
conserved in the caprine sequence and the presence of six cysteine residues suggests
the presence of three disulfide bridges similar to those reported for the bovine
enzyme (Foltmann et al., 1979; Vega-Hernández et al., 2004). In caprine prochy-
mosin, glutamate occupies position 36 in the propeptide. This non-conservative
replacement in aspartic protease zymogens has also been observed in lamb, sheep
and mouflon prochymosins (Pungercar et al., 1990; Francky et al., 2001). The
recombinant caprine chymosin shows high specificity towards -casein and has
been used experimentally to produce cheese from goat’s milk (Vega-Hernández
et al., 2004; Vega-Hernández and Claverie-Martín, unpublished). This proteolytic
capability is in agreement with the observation by Francky et al. (2001) that a basic
residue at position 36 of prochymosin is not essential for its autocatalytic activation.
Buffalo (Bubalos bubalis) milk is the major milk source in India, and it has
a different composition from that of cow. Mohanty et al. (2003) have purified
chymosin, (molecular weight of 35.6 kDa) from the stomach of buffalo calves.
Slight differences in stability and relative proteolytic activity are found compared
to bovine chymosin. This indicates that buffalo chymosin could be the best choice
for cheese production from buffalo milk.
Studies on the characteristics of rennet extracted from camel stomach
(Camelus dromedaries) have been reported (Elagamy, 2000). Camel chymosin
has a specific -casein hydrolysis activity superior to that of bovine chymosin
(Kappeler et al., 2004). Consequently, in cheese made with camel chymosin the
loss of protein due to non-specific degradation is decreased, yield is improved and
212 CLAVERIE-MARTÍN AND VEGA-HERNÁNDEZ
the development of bitter taste is reduced. Were camel chymosin to be commercially
available, more efficient clotting of camel’s milk could be achieved at the industrial
level. Furthermore, camel chymosin is very suitable for the coagulation of bovine
milk.
Lamb preprochymosin cDNA has been cloned and expressed in E. coli and
the recombinant lamb chymosin has been tested for its potential use in cheese
production (Rogelj et al., 2001). The coagulation properties of recombinant lamb
chymosin and the overall quality of the cheese made with this enzyme are similar
to those of recombinant bovine chymosin. A characteristic of recombinant lamb
chymosin is its instability at temperatures above 45
C (Rogelj et al., 2001).
This could be an advantage in the production of hard cheeses where relatively
high incubation temperatures are used. The production of cheeses made from
ovine milk is also a potential area for the application of recombinant lamb
chymosin.
4. PLANT ASPARTIC PROTEASES
Similarly to other aspartic proteases, plant APs are synthesized as single-chain
zymogens. Subsequent maturation is a crucial step in the regulation of their activity.
The primary structure of plant APs comprises a signal peptide, responsible for
translocation to the endoplasmic reticulum; a prosegment of 46-50 amino acids
involved in correct folding, stability and sorting of the enzyme (Simöes and
Faro, 2004); and the mature enzyme which possesses two catalytic sequence motifs
(Fig. 3). In contrast to other APs, the two catalytic Asp residues are contained
within Asp-Thr-Gly and Asp-Ser-Gly motifs (Simöes and Faro, 2004). Plant APs
contain an extra region of approximately 100 amino acids named the plant-specific
insert (PSI) that presents no homology to any other aspartic protease sequence.
The PSI is usually removed during the maturation process and resembles saposin-
like proteins (SALIPS). The function of PSI is still unclear but a possible role in
vacuolar targeting has been proposed (Egas et al., 2000).
Most plant APs are located in seeds (suggesting a role in storage-protein
cleavage), in leaves (indicating a role in mechanisms of defence against pathogens),
Figure 3. Schematic representation of the primary structure of cardosin A. SP, signal peptide; P,
prosegment; H, heavy chain of the mature enzyme; L, light chain of mature enzyme; PSI, plant-specific
insert. Arrows indicate processing sites
ASPARTIC PROTEASES USED IN CHEESE MAKING 213
or in flowers (implying a role in sexual reproduction) (Simöes and Faro, 2004).
Plant APs are also involved in defence mechanisms and cell death events associated
with plant senescence and response to stress.
Plant extracts have been used as coagulants in cheese making for many centuries
(Roserio et al., 2003). In contrast to chymosin which is specific for -casein, the APs
present in plant extracts cleave -, - and -caseins. This causes excessive acidity,
bitterness and texture defects in cheese, thereby limiting their use. However, these
characteristics are responsible for the special flavour, smell and consistency of the
cheese varieties produced using plant enzymes. Cheeses made with plant coagulants
are found mainly in Southern European and West African countries. Flower extracts
of cardoon and red star thistle (Cynara sp. and Centaurea calcitrapa) are used in
Portugal and Spain for the manufacture of traditional cheeses (Roserio et al., 2003).
The main milk-clotting APs present in these extracts are known as cardosins,
cyprosins and cenprosins (Ramalho-Santos et al., 1997; White et al., 1999;
Domingos et al., 2000). There are other APs isolated from Cynara sp., referred to as
cynarases, but they have not been well characterized (Roserio et al., 2003; Sidrach
et al., 2004). In Nigeria, extracts from the Sodom apple (Calotropis procera) are
used in the production of traditional cheese. Plant recombinant APs, including
cardosins and cyprosins, have been expressed in yeast but these enzymes are not
yet commercially available for industrial application (Soares Pais et al., 2000;
Castanheira et al., 2005).
4.1. Cardosins
Cardosin A, the most abundant of the cardosins, accumulates in the protein storage
vacuoles of the stigmatic epidermal papillae and in the vacuoles of the epidermal
cells in the stylus (Ramalho-Santos et al., 1997). Preprocardosin A is encoded by
the CARDA gene and consists of 504 amino acid residues (Fig. 3). The mature
enzyme is formed by two peptides of 31 and 15 kDa and has low proteolytic
activity (Roserio et al., 2003). The conversion to the active enzyme probably
takes place inside the vacuoles, during which process the PSI is removed prior to
cleavage of the prosegment (Ramalho-Santos et al., 1998). The catalytic residues
are located in positions 32 and 215 of the heavy chain (Frazao et al., 1999).
The unique feature of cardosin A among plant APs is the presence of the RGD
cell attachment motif Arg
176
-Gly
177
-Asp
178
(Frazao et al., 1999) which is a well-
known integrin-binding sequence. It has been suggested that this enzyme may
participate in an RGD-dependent proteolytic mechanism in pollen-pistil inter-
actions (Simöes and Faro, 2004). The C-terminus of cardosin A contains the
hydrophobic sequence Val
320
-Gly
321
-Phe
322
-Ala
323
-Glu
324
-Ala
325
-Ala
326
which is
conserved among plant APs and has been proposed to play a role in vacuolar
targeting (Frazao et al., 1999; Ramalho-Santos et al., 1998). The crystal structure
of cardosin A has been determined and shows the bilobal structure observed for
other APs (Frazao et al., 1999). The two independent polypeptides are held together
by hydrophobic interactions and hydrogen bonds.
214 CLAVERIE-MARTÍN AND VEGA-HERNÁNDEZ
In contrast to cardosin A, cardosin B accumulates in the cell wall and in the
extracellular matrix of the transmitting tissue, suggesting that the two cardosins
may play different roles in the pistil of Cynara cardunculus (Vieira et al., 2001).
Although less abundant than Cardosin A, cardosin B has more proteolytic activity
and may take part in general protein digestion (Faro et al., 1995). This enzyme
displays 73% similarity to cardosin A (Vieira et al., 2001). The cardosin B
precursor (506 amino acids) is encoded by the CARDB gene. The mature enzyme
is formed by two peptides of 34 kDa and 14 kDa, the catalytic residues being
located in the former (Vieira et al., 2001). Cardosin B lacks the RGD motif and
an additional putative N-glycosylation site is created by the replacement of an Asp
by Asn.
4.2. Cyprosins
Cyprosins are another type of AP isolated from the flowers of C cardunculus
(Ramalho-Santos et al., 1997). Preprocyprosin (509 amino acid residues) is encoded
by the CYPRO1 gene. The precursor of recombinant cyprosin is processed to
different isoforms by excision of the prosegment and most of the PSI (White
et al., 1999). The mature enzyme shares 52% identity with animal cathepsin D, the
closest AP of non-plant origin (White et al., 1999).
5. FUNGAL ASPARTIC PROTEASES
Due to the worldwide shortage of calf chymosin, fungal APs have been being
used as milk-clotting enzymes in the dairy industry for about 30 years. The
enzymes produced by Mucor miehei, Mucor pusillus and Cryphonectria (Endothia)
parasitica, marketed under the trade names Rennilase®, Fromase®, Novoren®,
Marzyme®, Hannilase®, Marzyme® and Suparen®, are widely used for the
production of different kinds of cheese. The proteolytic specificities of the fungal
coagulants are different from those of calf chymosin, resulting in the production of
some bitter peptides during the process of cheese ripening (Harboe and Budtz, 1999).
The crystal structures of several fungal APs including those of R. miehei,
R. pusillus, C. parasitica and Irpex lacteus, alone and complexed with inhibitors,
have been determined (Blundell et al., 1990; Newman et al., 1993; Yang et al., 1997;
Yang and Quail, 1999; Coates et al., 2002; Fujimoto et al., 2004). The tertiary
structures of these enzymes are very similar to those of other APs.
5.1. Mucorpepsin
The APs produced extracellularly by the two closely related species of zygomycetes,
M. pusillus and M. miehei, possess relatively high milk-clotting activities due to
their selective cleavage of -casein, along with relatively low proteolytic activities
(Harboe and Budtz, 1999; Awad et al., 1999). These enzymes, referred to as
mucorpepsins or rennins, exhibit the highest levels of thermal stability among
ASPARTIC PROTEASES USED IN CHEESE MAKING 215
the APs and therefore are of limited use as milk coagulants. This high thermal
stability results in the persistence of enzyme activity after cooking of the curd and
thus causing off-flavours in the cheese during long maturation periods (Yamashita
et al., 1994).
The aspartic protease from M. miehei (MAP, EC 3.4.23.23) is the most glycosy-
lated of the AP enzymes (Rickert and McBride-Warren, 1974). The carbohydrate
moieties may stabilize the conformation of MAP, conferring the enzyme a high level
of thermal stability and protecting it from proteolytic attack (Yang et al., 1997).
MAP was first crystallized by Jia et al. (1995), and Yang et al. (1997) refined
the native enzyme structure. Based on this structure, useful modifications of the
enzyme resulting in reduced thermal stability and higher milk-clotting activity have
been designed.
MAP preparations used in the cheese industryhavebeen produced both in M. miehei
and in A. oryzae (Budtz and Heldt-Hansen, 1998; Harboe and Kristensen, 2000).
When these preparations are deglycosylated by treatment with endo--N-
acetylglucosaminidase H a significant increase in clotting activity is observed
(Harboe and Kristensen, 2000). Two procedures have been described to reduce the
thermal stability of MAP; one by treatment in aqueous solution with oxidizing agents
containing active chlorine, and the other by acylation with an active derivative of a
carboxylic acid (Branner-Jorgensen, 1981; Branner-Jorgensen et al., 1982).
As expected, M. miehei MAP is almost identical to that of M. pusillus AP (MPAP)
(Tonouchi et al., 1986; Yang et al., 1997). While they share a common antigenic
structure and almost identical enzymatic properties, there are some differences
between these two enzymes with respect to their peptide cleavage patterns and
glycosylation. The structural gene for the M. pusillus aspartic protease, mpr, has
been expressed in S. cerevisiae (Aikawa et al., 1990). The product secreted to the
culture medium is the proenzyme: The 44 amino acid prosegment is removed by
autocatalytic processing at acid pH (Hiramatsu et al., 1989).
Deglycosylation studies have shown that removal of the N-linked carbohydrate
groups from MPAP increases its milk-clotting activity whilst decreasing both prote-
olytic activity and thermal stability (Aikawa et al., 1990). Site-directed mutage-
nesis at several positions has been carried out in order to improve its practical
properties in cheese production. Yamashita et al. (1994) have obtained mutant
forms of this aspartic protease having decreased thermal stability. Mutant mpr
genes carrying Gly186Asp or Ala101Thr have been expressed in S. cerevisiae. Both
mutations cause a marked decrease in thermal stability of the enzyme. The double
mutant shows the lowest thermal stability without affecting the enzymatic activity
(Yamashita et al., 1994). By contrast, replacement of Tyr
75
in the flap by Asn has
been shown to reduce the non-specific proteolytic activity of this enzyme, leading
to a considerable enhancement of the specific clotting activity (Park et al., 1996).
In addition, mutant Glu13Ala shows a 5-fold increase in the ratio of clotting versus
proteolytic activity without significant loss of clotting activity (Aikawa et al., 2001).
Residue Glu
13
seems to play a critical role in forming the correct hydrogen bond
network around the active centre.