BLBS102-c09 BLBS102-Simpson March 21, 2012 11:15 Trim: 276mm X 219mm Printer Name: Yet to Come
188 Part 2: Biotechnology and EnzymologyAcid ProteasesThe acid proteases (also known as aspartate proteases) are so
called because the enzymes in this group tend to have one or
more side chain carboxyl groups from aspartic acid as essential
component(s) of their active sites, and they are optimally active
within the acid pH range. Examples include pepsins, chymosins,
rennets, gastricins (from animal sources), cyprosin, and cardosin
(from plant sources), and several other aspartic acid proteases
from microorganisms such asRhizomucor miehei,R. pusillus,
R. racemosus,Cryphonectria parasitica, andA. niger(Mistry
2006). Most of the known acid proteases are inhibited by the
hexapeptide pepstatin. The major food industry use of acid pro-
teases is in dairy processing, such as for the milk coagulation
step in cheese making. In cheese making, the acid proteases
added (e.g., rennet of chymosin) act to cleave critical peptide
bonds between phenylalanine and leucine residues inκ-casein
of the milk to form micelles that aggregate and precipitate out as
the curd. The characteristic specificity of these proteases in these
dairy products is important to restrict hydrolysis to the peptide
bonds between the critical phenylalanine and leucine residues in
κ-casein; otherwise, excessive proteolysis could elicit undue tex-
ture softening, bitterness, and/or “off-odors” in the product(s).Serine ProteasesThe serine proteases are the most abundant group of the known
proteases (Hedstrom 2002). The characteristic features of this
group of enzymes include the presence of a seryl residue in their
active sites, as well as a general susceptibility to inhibition by ser-
pins, organophosphates (e.g., di-isopropylphosphofluoridate),
aprotinin (also known as trasylol), and phenyl methyl sul-
fonyl fluoride. Examples of the animal serine proteases are
chymotrypsin, trypsin, thrombin, and elastase; examples of
plant serine proteases are cucumisin (from melon; Kaneda and
Tominaga 1975), macluralisin, and taraxilisin (from the osage
orange fruit and dandelion, respectively; Rudenskaya et al.
1995); and examples of microbial serine proteases are pro-
teinase K and subtilisin A (Couto et al. 1993). Several of the
serine proteases function optimally within neutral to alkaline
pH range (pH 7–11), and they find extensive use in the food
and animal feed industries. They are used to produce highly
nutritive protein hydrolysates from protein substrates such as
whey, casein, soy, keratinous materials, and scraps from meat
processing, as well as from fish processing discards; for the
development of flavor during ripening of dairy products; and
for the production of animal fodder from keratinous waste
materials generated from meat and fish processing (Dalev 1994,
Wilkinson and Kilcawley 2005). Other industrial applications
of serine proteases include their use for the regioselective
esterification of sugars, resolution of racemic mixtures of
amino acids, and production of synthetic peptides to serve as
pharmaceutical drugs and vaccines (Chen et al. 1999, Guzman ́
et al. 2007, Barros et al. 2009). Serine proteases are also used
commercially for the treatment and bating of leather to remove
undesirable pigments and hair from leather (Valera et al. 1997,
Adıguzel et al. 2009). They are also used for the treatment of ̈industrial effluents and household waste, to lower the biological
oxygen demand, and viscosity, to increase the flow properties
of solid proteinaceous waste (e.g., feather, hair, hooves, skin,
and bones), as well as wastewater from animal slaughterhouses
and fish processing plants (Dalev and Simeonova 1992). Serine
proteases also find some use in the degumming and the detergent
industries (Godfrey and West 1996), and in medicine for the
treatment of burns, wounds, and abscesses (Lund et al. 1999).Sulfhydryl ProteasesThe sulfhydryl (also known as thiol or cysteine) proteases are
so called because they contain intact sulfhydryl groups in their
active sites and are inhibited by thiol reagents such as alkylat-
ing agents and heavy metal ions. Examples include plant types
like actinidain, papain, bromelain, chymopapain, and ficin; ani-
mal types like cathepsins B and C, calpains, and caspases; and
clostripain, protease 1 and others from various microorganisms
such asStreptococcussp (Whitaker 1994), thermophilicBacillus
sp (Almeida do Nascimento and Martins 2004), andPyrococ-
cussp (Morikawa et al. 1994). The activities of this group of
enzymes are enhanced by dithiothreitol and its epimer dithio-
erythritol, cysteine, and 2-mercaptoethanol, and they are inhib-
ited by compounds such as iodoacetate,p-chloromercuribenzoic
acid, cystatin, leupeptin, andN-ethylmaleimide. The enzymes
in this group tend to be optimally active around neutral pH (i.e.,
pH 6–7.5) and are relatively heat stable, which accounts for their
use in meat tenderizers. Thiol proteases act to degrade myofibril
protein and collagen fibers to make meats tender. Meat tender-
ness is a very important factor in meat quality evaluation. Ficin
tends to have the greatest effect on myofibril protein hydrolysis
due to its relatively higher thermal stability and broader speci-
ficity, followed by bromelain and papain in that order, although
bromelain tends to degrade collagen fibers more extensively
(Miyada and Tappel 1956). In commercial application, papain
and bromelain are more frequently used because the higher hy-
drolytic capacity of ficin often results in excessive softening and
mushiness in the treated meat (Wells 1966). These proteases
may be applied in various ways to achieve meat tenderization,
as by blending, dipping, dusting, soaking, spraying, injection,
and vascular pumping (Etherington and Bardsley 1991). A com-
mon drawback with most of these approaches is the unequal
distribution of the enzyme that could result in excessive ten-
derization or inadequate tenderization of different parts of the
meat. The problem of uneven distribution of the enzyme may be
minimized by using an additional tumbling step after enzyme
treatment to increase distribution or by pre-slaughter injections
of the animal with the enzyme (Beuk et al. 1962, Etherington
and Bardsley 1991).
Sulfhydryl proteases are also used in the brewing industry to
control haziness and improve clarity of the beverage. Haziness
may form in the product during the aging process via aggrega-
tion of larger peptide and polyphenol molecules to form high
molecular weight complexes. This is particularly enhanced dur-
ing chilling of the beer, where solubility is minimal, and this
group of enzymes is also used in the baking industry to im-
prove the stretchability and firmness of the dough, through the