Bioresource Technology 64 (1998) 175-183

1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain
0960-8524/98 $19.00
ELSEVIER

PII:SO960-8524(97)OOI82-X

ALKAI.INE PROTEASES: A REVIEW

Adil Anwar* & Mohammed Saleemuddin
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India
(Received 22 September 1997; revised version received 1 November 1997; accepted 10 November 1997)

useful biomass, protein concentrate or amino acids

using proteases derived from cerain microorganisms.
Other successful commercial applications (Table
1) of alkaline proteases include the possibility of
using them to catalyse peptide synthesis and to
resolve racemic mixtures of amino acids (Sutar et al.,
1991; Chen et al., 1991; Chen et al., 1995). The
detergent industry has now emerged as the single
major consumer of several hydrolytic enzymes acting
at highly alkaline pH. The major use of detergentcompatible proteases is in laundry detergent formulations. Alkaline proteases of bacterial (including
those from alkalophiles, organisms whose optimal
rate of growth is observed at least two pH units
above neutrality), fungal and insect origin can be, or
are exploited commercially and are reviewed in
detail here.

Abstract

In recent years there has been a phenomenal increase

in the use of alkaline proteases as industrial catalysts.
These enzymes offer advantages over the use of
conventional chemical catalysts for numerous reasons,
for example they exhibit high catalytic activity, a high
degree of substrate specificity, can be produced in large
amounts and are economically viable. Although many
other classes of enzymes are currently in industrial use,
the focus of this review is on alkaline proteases from
various sources. This is because the organisms
producing enzymes capable of catalysing the reactions
at the extremes of p H above neutrality, having been
considered as oddities thereby receiving little attention
until the last decade. A fresh look at some aspects of
stabilization of alkaline proteases, their limitations and
future strategies is also included. 1998 Elsevier
Science Ltd. All rights reserved
Key words: Alkaline proteases, industrial catalysts,
stabilization, natural and engineered stabilized
enzymes.

Production of alkaline proteases

Alkaline proteases for industrial use can be obtained

from different sources such as bacteria, fungi, or
certain insects. The feasibility of the use of proteases
for industrial applications depends on certain
factors. Jonsson & Martin (1965) studied many
fungal cultures for protease production and reported
that the amount of protease produced varies greatly
with strain and the media used. In order to obtain
high and commercially viable yields of protease it is
essential to optimize fermentation media for the
growth and production of protease. Banerjee &
Bhattacharya (1992) optimized the culture conditions for the production of an industrially important
alkaline protease from the fungal isolate Rhizopus
oryzae. The effects of organic and inorganic nitrogen
sources, metal ions, surfactants, fungicides and
phenolic compounds on protease production were
investigated. Organic nitrogen was found to give
better yields of protease. Economically, however,
inorganic salts are preferred for industrial production of proteases, because of the low costs involved.

INTRODUCTION
Proteases constitute one of the most important
groups of industrial enzymes, accounting for at least
a quarter of the total global enzyme production sales
(Layman, 1986). Proteases are by far the most
important group of enzymes produced commercially
and are used in the detergent, protein, brewing,
meat, photographic, leather and dairy industries
(Kalisz, 1988). Fibrous proteins, such as horn,
feather, nails and hair, are abundantly available in
nature as wastes, but these can be converted to
*Author to whom correspondence should be addressed at:
Department of Microbiology B172, University of
Colorado Medical School, 4200 E 9th Avenue, Denver,
Co 80262, USA.
175

176

A. Anwar, M. Saleemuddin

Among the inorganic nitrogen sources, 0-2% sodium

nitrate was found to be the best. Metal ions (Cu 2,
Zn 2, Ca 2, Ag 3, Pb 2, Hg 2) and surfactants
(Tween 80, SDS, Triton X-100) acted as inducers for
protease production, while fungicides and phenolics
were found to be inhibitory.
Maximum protease production was obtained from
the fungus Conidiobolus coronatus when the basal
medium contained 1% sucrose, 0"3% sodium nitrate,
0.1% dipotassium phosphate, 0.05% magnesium
sulphate, 0.1% potassium chloride and 0.001%
ferrous sulphate at pH 7.0 (Phadatare et al., 1993).
Cell growth and the production of the desired
metabolites can be continued over a prolonged
period in continuous cultures. Feasibility of steadystate operation makes continuous cultures very
attractive for industrial production of cell mass and
non-biomass products, using freely suspended or
immobilized cells.
Studies on protease production via continuous
bioreactor operations have included suspension
cultures of Bacillus subtilis (Heineken & O'Connor,

1972), B. firmus (Helmo et al., 1985), immobilized

cultures of B. lichenformis (Okita & Kirwan, 1988)
and Serrata marcescens (Vuillemard et aL, 1988).
Proteases produced by Bacillus sp. are by far the
most important group of enzymes being exploited
commercially. Some observations on protease
production in continuous suspension-cultures of B.
firmus NRS 783 have been documented (Moon &
Parulekar, 1993). These investigators have reported
a strategy providing useful guidelines in the
designing of bioreactors for the mass production of
alkaline protease by B. firmus. Continuous-culture
experiments were conducted by varying a single
parameter at a time. It was found that an increase in
dilution rate led to decreased utilization of principal
carbon, nitrogen and phosphorus sources (glucose,
ammonium chloride, potassium dibasic phosphate)
resulting in an increase in the specific production
rate of protease. Moon & Parulekar (1993) felt that
the approach would be of significant benefit to
industrial production on a large scale and that the
use of bioreactors could also lead to a substantial

Table 1. Some industrially important alkaline proteases

Species

Source

pH Optimum/ Industrial application(s)

stability

Streptococcus sp.
Bacillus stearothermophilus

Bacterial
Bacterial

8.0
9.5

Tritirachium album

Fungal

9.0-12.0

Fungal

7"0-10"0

Fungal

9"7

(proteinase T)
Tritirachium album

(proteinase R)
Conidiobolus coronatus

(alkaline proteinase B)
Bacillus sp. Y. (BYA)
Bacillus lichenformis

Bacterial
Bacterial

10.0-12.5
8.2

Bacterial
Fungal

12.0-13-0
3.0-11.0

(Alcalase)
Bacillus sp. (AH-101)
Rhizopus oryzae (RO, liT,

KGP)

Conidiobolus coronatus (NCI

Dairy/cheese production
Detergents and heavy duty
laundry powders
Laundry detergents
formulations
Laundry detergent
formulations
Resolution of racemic
mixtures of D,L-phenyl
alanine and glycine
Detergent formulations
Catalyst for N-protected
amino acids
Dehairing/leather industry
?

Reference
Van Boven et al. (1988)
Sato et al. (1990)
Samal et al. (1990)
Samal et al. (1990)
Sutar et al. (1991)
Shimogaki et al. (1991)
Chen et al. (1991)
Takami et al. (1992)
Banerjee & Bhattacharya
(1992)
Phadatare et al. (1993)

Fungal

8.5

Commercial detergents

Bacillus firmus
Bacillus sp. (P-001A)

Bacterial
Bacterial

8.0
9.5

Moon & Parulekar (1993)

Atalo & Gashe (1993)

Bacillus sp. (B 18)

Bacillus sp.
Bacillus sp.
Thermus Rt 41A
Bacillus sp. (Savinase/

Bacterial
Bacterial
Bacterial
Bacterial
Bacterial

Detergent industry
Production of biomass from
natural waste
?
?
Dehairing/leather industry
?
Detergent formulations
Synthesis of biologically
active peptides
Bating agent in leather
industry
?
Contact lens cleansing agent
Cheese and detergents

Chen etaL (1995)

86.8.20)

12.0
12.0
8.5
11"0
9.0-11-0

Fujiwara et al. (1993)

Masui et al. (1994)
Loperena et al. (1994)
Wilson et al. (1994)
Bossi et al. (1994)

Durazym)
Bacterial

8"2

Bacillus subtilis

Bacterial

8-5

Bacillus sp.
Bacillus subtilis
Amycolata/Amycolatopsis

Bacterial
Bacterial

Spilosoma obliqua

Insect larvae

Bacillus licheniformis

(Alcalase)

(Lepidoptera)

8.5
8-11.0
11-0

Commercial detergents and

stain remover
formulations

H a m e e d e t a L (1996)

Sinha et al. (1996)

Sanyo et al. (1996)
Denmark patent no. 04, 082
(1997)
Anwar & Saleemuddin
(1997)

Alkaline proteases: A review

improvement in protease production in a continuous-culture operation.

USES OF ALKALINE PROTEASES
Alkaline proteases in the dairy industry
Lactic acid bacteria constitute a very important
group in the dairy industry for the production of
fermented milk products such as cheese (Thomas &
Mills, 1981). For their supply of amino acids in milk,
these bacteria are solely dependent on their proteolytic system for the degradation of casein, which also
meets their demand for a supply of peptides. These
processes contribute significantly to the development
of flavour during cheese ripening (Schmidt et al.,
1976). In the cheese industry, Lactobacillus sp. and
Streptococcus cremoris are the two most important
bacteria (De Man et al., 1960; Van Boven et al.,
1988). Lactic acid bacteria have two main functions
as starter bacteria in the manufacturing of cheese:
(i) the acidification of milk; and (ii) the development of flavour during ripening. In both the
processes proteolytic activity plays a central role
(Hugenholtz et al., 1987). Owing to the importance
of proteases in cheese production, in recent years
much attention has been focused on these enzymes
in the study of starter bacteria (Law, 1979; Doi et al.,
1981; Thomas & Mills, 1981; Hugenholtz et aL,
1984; Geis et al., 1985).
The number of proteases among the strains has
been found to vary between one (Law, 1979; Geis et
al., 1985) and four (De Man et aL, 1960; Hugenholtz
et aL, 1984) and the localization of proteases in the
cell has been found to be either extracellular (Exterkate, 1976), cell-wall associated (Schmidt et al., 1976;
Law, 1978; Exterkate, 1981; Hugenholtz et aL, 1984;
Geis et al., 1985), membrane bound (Otto et al.,
1982), or cytoplasmic (Cliffe & Law, 1979; Cliffe &
Law, 1985; Law, 1979). However, most of the investigators agree that Ca 2+ ions are important for the
activity of these proteases (Exterkate, 1981; Gels et
aL, 1985), although in one case Mn 2+ and Co 2+ ions
were found to be necessary as co-factors (Doi et aL,
1981).
The protease from Streptococcus cremoris used in
cheese production was found to be optimally active
at pH 8"0, and at pH values lower than pH 5"0 no
hydrolysing activity could be detected whereas at pH
10 only 50% of the activity was found (Van Boven et
al., 1988), which implies the importance of the
alkaline nature of ,proteases in the dairy industry.
The role of lactic acid bacteria in the fermentation
of different foods, the composition of the fermentation, flora, metabolic activities and their use as
starters in cheese production have been reviewed
recently (Luecke et al., 1996). It is noteworthy to
mention that proteolytic enzymes from Amycolata
sp. and Amycolatopsis sp., having alkaline pH optima

177

(8-11), are also currently in use for the production

of cheese (Denmark patent No. 1997, 04,082).
Degradation of proteinaceous waste into useful
biomass by alkaline proteases
Fibrous proteins such as horn, feather, nail and hair
are abundantly available in nature as waste. This
waste can be converted into useful biomass, protein
concentrate or amino acids using proteases from
certain micro-organisms. Venugopal et al. (1989)
immobilized cells from B. megaterium in calcium
alginate and the extra-cellular proteases secreted by
these cells were useful to solubilize fish meat. Dalev
& Simeonova (1992) employed alkaline protease
from B. subtilis for the complete utilization of the
main waste of the leather industry for the production of useful products Such as animal glue, which
could be used as a high quality glue in carpentry,
and protein concentrate for fodder and animal
tallow. An alkaline protease from a thermophilic
Bacillus sp. (P 001A) has been isolated (Atalo &
Gashe, 1993). The protease from this bacterium was
found to be stable over a pH range of 4"5-11.5 with
a pH optimum of 9.5. It was maximally active at
55C and could hydrolyse 90, 60, 50% of skin,
feather and horn. This protease appears to have all
the properties of a good industrial catalyst for the
production of amino acids from protein concentrate.
More recently Dalev (1994) reported the use of
alkaline protease from B. subtilis in processing waste
feathers from poultry slaughter houses. Feathers
constitute about 5% of the body weight of poultry
and can be considered as a high protein source for
food and feed provided their rigid keratin is
destroyed completely. The solubilization of feathers
was achieved by pretreatment with NaOH, mechanical disintegration and enzymic hydrolysis, resulting
in a useful end product with a very high protein
content which could be used mainly as a feed
constituent.
Alkaline proteases in the leather industry
Another industrial process which has received attention is the enzyme assisted de-hairing of animal
hides and skin in the leather industry. Traditionally,
this process is carried out by treating animal hides
with a saturated solution of lime and sodium
sulphide. Besides being expensive and particularly
unpleasant to carry out, a strongly polluting effluent
is produced. The alternative to this process is
enzyme-assisted
de-hairing.
Enzyme-assisted
de-hairing is preferentially possible if proteolytic
enzymes can be found that are stable and active
under the alkaline conditions (pH 12) of tanning.
Early attempts using a wide variety of enzymes
were largely unsuccessful, but proteases from certain
bacteria which are alkalophilic in nature have been
shown to be effective in assisting the hair-removal
process (Horikoshi & Akiba, 1982; Sharpe &
Munster, 1986). Several alkaline proteases from

178

A. Anwar, M. Saleemuddin

alkalophilic actinomycetes have also been investigated for this purpose. Some of these have been
shown to be particularly active against keratinous
proteins such as hair, feather, wool, etc. at alkaline
pH and may have commercial applications (Sharpe
& Munster, 1986).
The substitution of chemical depilatory agents in
the leather industry by proteolytic enzymes
produced by Bacillus sp., could have important
economical and environmental impacts. A Bacillus
sp. exhibiting promising depilatory activity has been
isolated and optimal conditions for the maximum
protease production have been worked out in
laboratory bioreactors (Loperena et al., 1994). The
alkalophilic Bacillus sp. No. AH-101 produces an
extremely thermostable alkaline serine protease that
has a high pH optimum (12-13) and possesses
keratinolytic activity (Takami et al., 1992). Using a
different approach, these workers have cloned the
gene encoding the protease in Escherchia coli and
expressed it in B. subtilis. The cloned protease was
identical to AH-101 protease in its optimum thermostability in highly alkaline environments and suitable
for use in tanneries. In a recent study, Masui et al.
(1994) investigated the rational shift of the optimum
pH and the enhancement of thermostability of
Bacillus sp. B18'. The protease gene was cloned and
an even more thermostable mutant enzyme was
created by introducing a point mutation when the
position in j~-turn Thr-203 was replaced by proline.
The enzyme was optimally active in alkaline conditions (pH 12-13) hence the protease has the potential of replacing chemical depilatory agents in the
leather industry. In a more recent study, Hameed et
al. (1996) employed an alkaline protease from a B.
subtilis isolate as bating agent for the production of
high quality leather. Increase in the tensile, bursting
and tear strengths, along with elongation at breaking
of leathers is indicated with the increasing amounts
of alkaline protease used in the bating process.
Alkaline proteases in detergent formulations
The detergent industry has now emerged as the
single major consumer of several hydrolytic enzymes
acting in the alkaline pH range. Detergents
containing different enzymes; proteases, amylases
and lipases are available in the international markets
under several brand names. The use of different
enzymes as detergent additives arises from the fact
that proteases can hydrolyse proteinaceous stains,
amylases are effective against starch and other
carbohydrate stains while lipases are effective
against oily or fat stains. F o r an enzyme to be used
as a detergent additive it should have two qualities:
it should have an alkaline pH and it should also be
compatible with detergents. The major use of detergent-compatible proteases is in laundry detergent
formulations (Anstrup & Andersen, 1974; Van
T//burg, 1984). Detergents available in the international market such as Dynamo , Era plus

(Procter & Gamble), Tide (Colgate Palmolive)

contain proteolytic enzymes the majority of which
are produced by members of the genus Bacillus.
Although subtilisins have been the enzymes of
choice in detergent formulations (US patent Nos
1240058, 370482, 374971, 4266031 and UK patent
No. 13155937), these are not ideal detergent
enzymes due to their low stability in presence of
detergents (Samal et al., 1990).
To overcome the problem of low stability of subtilisins in detergent formulations Von der Osten et al.
(1993) successfully employed site-directed mutagenesis in the construction of subtilisin variants with
improved storage and oxidation stabilities. Sato et al.
(1990) investigated the effects of various poly(styrene sulfonate methacrylate) copolymers, polyacrylate and tripolyphosphate, anionic builders, as
well as poly-(vinyl alcohol vinyl acetate) non-ionic
co-polymers, on the proteolytic activity of B. stearothermophilus (Toyozyme NP), which is a detergent
additive. It was found that the addition of anionic
builders lowered the activity, while the non-ionic
co-polymeric builders enhanced the activity. In an
interesting study, Samal et al. (1990) separated two
novel proteases from the fungus Tritirachium album
and the proteases, termed as 'R' and 'T', were
checked for their thermal and detergent stabilities.
The pH optimum of the protease R was between 7.0
and 10.0, while the protease T was maximally active
between pH 9.0 and 12.0. The optimal temperature
for both proteases R and T was 55 to 60C. Stability
of these two proteases in the presence of commercial detergents was investigated by incubating the
enzyme at 50C in the presence of laundry detergents of different compositions. The stability was
determined in three laundry detergents containing
proteases (Eraplus ~, Tide , Dynamo ) and in
detergent (Wisk ) that does not contain any
protease. The stability of protease T was excellent in
all the detergent formulations in which the
endogenous enzyme had been destroyed by heat
prior to the addition of protease T. Protease R was
also found to be extremely stable, retaining 90% of
its original activity after 1 h incubation at 50C in
Eraplus or Dynamo ~. The stability of protease R
was, however, found to be less in detergent Tide ,
where it lost 50% of its activity after 1 h. The activity
of protease T tested in the presence of laundry
detergent Wisk, which does not contain any
endogenous enzyme, was found to be 90% after 1 h
at 50C, while in similar conditions protease R lost
up to 50% of its activity after 10 min. The use of
protease T in all the cases and that of R in most of
the cases would lead to use of less enzyme in detergent formulations. Because of the thermal stability
and detergent compatibility, these two novel
proteases, especially protease T, could have diverse
industrial applications in addition to use in detergent formulations (Samal et al., 1990).

Alkaline proteases: A review

In the course of a search for an alkaline-stable

protease for industrial use, an alkaline protease was
isolated from the Bacillus sp. Y (Shimogaki et al.,
1991). The protease from the Bacillus sp. Y was
found to have an optimum pH of 10.0-12.5. In
addition, it was also very stable towards surfaceactive agents, such as sodium dodecyl sulfate (SDS)
and sodium linear alkyl benzene sulfonate, implying
its potential for use in detergent formulations.
Although bacterial proteases have long been used
in detergents, the main drawback in their use is that
they require cost-intensive filtration methodologies
to obtain a microbe-free enzyme preparation.
However, proteases from fungal origin offer an
advantage that mycelium can be easily removed by
filtration techniques. Phadatare et al. (1993) investigated the properties of an alkaline protease from
Conidiobolus coronatus (NCL 86.8.20) related to
enzyme production and compatibility with commercial detergents. The optimized fermentation conditions for maximum protease production in order to
make its industrial application feasible economically,
particularly in detergent formulations have also been
reported (Phadatare et al., 1993).
An ideal detergent enzyme should be stable at
high pH and temperatures up to 40C, withstand
oxidizing and chelating agents, and be effective at
low enzyme levels in detergent solutions. Moreover,
it should also have broad substrate specificity. The
alkaline protease from C. coronams showed a high
level of stability up to pH 8.5, no loss in activity was
detected up to 40C and it was also compatible with
most of the detergents tested. The protease was also
able to hydrolyse various protein substrates tested.
The data obtained in the study (Phadatare et al.,
1993) implies that the protease of C. coronatus has
all the potential to be used as a detergent enzyme.
An alternative source of detergent compatible
enzymes could be insect guts. The advantages being
that cost-intensive filtrations are not involved as in
the case of bacterial and fungal sources. Taking
these things into consideration, in a more recent
study from our laboratory (Anwar & Saleemuddin,
1997) we reported on the alkaline pH-acting
digestive enzymes from the polyphagus insect pest
Spilosoma obliqua. Their stability and potential as
detergent additives have also been discussed. The
protease from the insect source was found to be
active at pH 11.0 and had a temperature optimum of
50C. An ammonium sulfate (60% saturation)
fraction from the larval guts of S. obliqua was found
to be highly effective in facilitating the removal of
old blood stains from cotton cloth both in the
presence and absence of detergents.
Anwar (1996) also showed that the purified
protease from the S. obliqua larval guts was compatible with most of the commercial detergents tested,
albeit to differing extents, while its broad substrate
specificity, as demonstrated by the cleavage of

179

different proteins, implied its effectiveness against a

wide variety of stains. The data obtained from both
studies imply that the larval protease has all the
potential to be used as a detergent additive and as a
stain remover. Although the economics of using an
enzyme from the insect source may not appear
feasible there is always a possibility of cloning the
enzyme (Von der Osten et al., 1993). A low-foaming
liquid detergent containing proteolytic enzymes with
longer shelf-life has been reported (Okamoto et al.,
1997). This liquid detergent is currently in use as a
dish-washing detergent and is available commercially
in the international market.
Studies on the possible use of enzymes in detergents should also be directed towards their stability
when formulated into detergent liquids or powders
in concentrated form as sold in the market.
However, caution must also be exercised in the
source of enzymes to be used as detergent additives
as some of these might cause allergic reactions in
humans.
Alkaline proteases in the synthesis and resolution of
o,L-amino acids
Amino acids are of increasing importance as dietary
supplements for both humans and domestic animals.
Only the L-amino acids can be assimilated by living
organisms and since the chemical synthesis of amino
acids produces a racemic mixture it is all the more
necessary to separate the isomers before commercial
use. Resolution is one of the best ways to produce
optically pure unnatural amino acids. An extracellular low molecular weight protease (6800) has
been purified to homogeneity from the culture
filtrate of C. coronatus (NCIM 1328) by Sutar et al.
(1991). The protease was found to have a pH
optimum of 9"7 and exhibited esterolytic acitivity on
N-benzoyl-L-tyrosine ethyl ester (BTEE) and it was
successfully employed to resolve the racemic mixture
of D,L-phenyl alanine and D,L- phenyl glycine and
can thus potentially replace subtilisin carlsberg in
resolving the racemic mixture of amino acids (Sutar
et al., 1991).
Kinetic resolution of N-protected amino acid
esters in organic solvents catalysed by a stable industrial alkaline protease 'Alcalase' has been reported
(Chen et al., 1991). Alcalase is a proteolytic enzyme
isolated from a selected strain of B. licheniformis, its
major component being subtilisin carlsberg. It was
found that Alcalase was stable in organic solvents
and capable of use as a catalyst in the resolution of
N-protected amino acids having unusual side chains.
Two resolution procedures, hydrolysis in aqueous
solution with dioxan as co-solvent and hydrolysis in
t-butanol containing 5% phosphate buffer have been
discussed in detail elsewhere (Chen et al., 1991).
In a recent study, Tsuchiya et al. (1993) found
that a serine alkaline protease from Cephalosporum
sp. KM 388 was specific against esters of aromatic

180

A. Anwar, M. Saleemuddin

and hydrophobic amino acids. The cleavage specificity of KM 388 protease D was broader than those
of other alkaline proteases and the site of Arg-Gly
(22-23) was cleaved, which is a specific site for
trypsin-like proteases. In a more recent study Chen
et al. (1995) reported a useful procedure for the
synthesis of biologically active peptides using (subtilisin carlsberg) Alcalase.
LIMITATIONS
The overall potential of alkaline proteases in industrial process is yet to be exploited fully. The inherent
disadvantages in the use of proteases, in particular,
are related to thermal, operational and storage
problems as they are easily prone to inactivation by
self-digestion (autolysis), whereas a good industrial
catalyst should be stable under the toughest
operating conditions and for long durations. To
overcome such limitations great attention has been
devoted to the problem of enzyme stabilization.
STABILIZATION OF ALKALINE PROTEASES
Stabilization of enzymes by immobilization can be
categorized according to whether the protein
becomes immobilized by chemical binding or by
physical retention. The most widely used immobilization techniques are based on the binding of
enzyme molecules to carriers by covalent bonds, or
by adsorptive interactions, entrapment into gels/
beads/fibres, cross-linking or co-cross-linking with
bifunctional reagents, encapsulation in microcapsules or membranes.

Based on these, numerous methods have been

employed to immobilize proteolytic enzymes and
stabilize them against pH, thermal, chemical
denaturants and storage inactivation (Martinek et
al., 1977; Church et al., 1992; Kumakura et al., 1984;
Dua et al., 1984, 1985; Kovalenko & Sokolovsky,
1987; Blanko & Guisan, 1988). Information related
to the stabilization of proteases such as carboxypeptidase (Dua et al., 1985), chymotrypsin
(Matveeva et al., 1985), pronase (Church et al.,
1992), trypsin (Reddy et al., 1986) and other
proteases (Johnson et al., 1990; Gusek & Kinsella,
1990) is available in the literature.
Certain factors such as cost, reuse and stability
profoundly influence the applications of immobilized
enzymes as industrial catalysts by giving an edge
over their soluble counterparts, as immobilization
confers additional stability to an enzyme and it can
also be reused. It is possible to recover the
immobilized enzymes by centrifugation or filtration.
This procedure is especially recommended when the
enzyme used is expensive as its reuse would reduce
operational cost. A process involving the use of
immobilized papain to eliminate the chill haze
caused by the proteins present in beer is in vogue.
An industrially important alkaline protease from
Trichoderma koningii immobilized by cross-linking
with glutaraldehyde could be stored in distilled
water at 4C for 60 days or recycled eight times in
enzymic reaction with practically no loss in activity
(Manonmani & Joseph, 1993). However, the data
available on the stabilization of alkaline proteases is
scanty (Table 2). One possible reason which can be
attributed to this discrimination is that alkaline
proteases are industrially important and are preferably used in soluble forms.

Table 2. Stabilization of some alkaline proteases by different modes of immobilization/modification

Enzyme

Stabilizing support/
modification

Alkaline protease (EC. 3.4. (a) Acylation with succinic

group)
and maleic anhydrides
(b) Aluminium oxide/silica
gel
Hydroxyl-ethyl-acrylate
Thermolysin (EC. 3.4.24.4.
tetradeca-ethylene glycol
group)
Site-directed mutagenesis
Subtilisin BPN'
Subtilisin (EC 3.4. group)
Porous chitosan beads
Subtilisin BPN'
Porous chitosan beads
Subtilisin carlsberg
Alkaline protease (Thermus Controlled pore glass beads
Rt 41A)
Alkaline protease
Glutaraldehyde
( Trichoderma koningii)

Subtilisin

Site-directed mutagenesis

Bacillus sp. B19'

Site-directed mutagenesis
Cellulose co-polymer
Polyurethane
Glutaraldehyde

Alkaline protease
Alkaline protease
Alkaline protease

Stability/improvement
against

Reference

Autolysis

Temperature

Maneepun & Klibanov

(1982)
Kovalenko & Sokolovsky
(1987)
Kumakura et al. (1984)

Temperature
Temperature/storage
Stability in organic solvents
Stability in organic solvents
pH/Temperature

Pantoliano et al. (1987)

Zaks & Klibanov (1988)
Kise & Hayakawa (1991)
Kise & Hayakawa (1991)
Wilson et al. (1993)

pH/Temperature

Manonmani & Joseph

(1993)
Von der Osten et al. (1993)

Temperature

Storage and oxidation

stabilities
Thermal stability
Improved shelf-life
?
Temperature

Masui et al. (1994)

Virnik et al. (1996)
Grajek et al. (1996)
Anwar & Saleemuddin
(1997)

Alkaline proteases: A review

Detrimental aspects of stabilization

Although enzyme stabilization is now a pressing
problem in biotechnological applications, stabilization of enzymes in soluble form has always been an
important desirable goal. Increased attention has
been focussed on the stabilization of enzymes in
soluble forms, and many efforts have been made in
search of new and different methods to obtain
soluble, but stabilized, enzymes (Schmidt, 1979;
Gray, 1988), as it is impossible to use insoluble
enzymes in several biotechnological applications
including detergent, food, cosmetic and textile
industries.
FUTURE STRATEGIES
The future strategies in the identification of industrially important, stable enzymes, particularly
proteases could be to search for naturally occurring
enzymes with intrinsic stability or to produce stable
enzymes by means of protein engineering. These
strategies are based upon two different but correlated approaches.

Naturally occurring, stable enzymes

The simplest approach to obtain a stable enzyme is
to look for the desired enzyme in a readily available
organism. During the last few years many enzymes
have been isolated from alkalophilic/thermophilic
organisms which are capable of surviving in
extremes of pH and temperature and are also stable
against a wide variety of denaturants such as urea
and detergents (Cowan & Daniel, 1982; Wojtczak,
1987; Takii et al., 1987). Hence great interest has
been generated in the search for new thermophilic
and alkalophilic strains for industrial applications.
Production of stable enzymes by enzyme engineering
There seems litle doubt that new organisms capable
of secreting industrially important enzymes will
continue to be discovered and those which are
already known have to be exploited practically.
Subtle alterations in the amino acid sequences, using
techniques such as site-directed mutagenesis, could
be beneficial in producing enzymes for industrial
applications. Indeed site-directed mutagenesis
experiments have produced subtilisins (Wells &
Powers, 1986; Pantoliano et al., 1987; Von der Osten
et al., 1993) and other enzymes (Liao et al., 1986;
Matsumura et al., 1986; Bae et al., 1987; Heslot et
al., 1987; Scrutton et aL, 1988; Masui et al., 1994)
with enhanced stability in soluble form.
CONCLUSIONS
In the present review we have tried to present a
general view of the wide and complex matter of the
industrial applications of alkaline proteases. It is
hoped that such a review will be an introduction to

181

the problem of alkaline proteases in industry for

newcomers to the field and will also encourage those
who have already thought about the problem to
consider the
new
experimental
approaches
discussed. Our apologies if just a part of the existing
ocean of literature has been mentioned and for
other unintentional omissions.
ACKNOWLEDGEMENTS
Financial assistance provided by the University
Grants Commission, Government of India, in the
form of a fellowship to one of us (A.A.) is thankfully
acknowledged.

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