Journal of Scientific & Industrial Research

Vol. 61. September

2002. pp 6<)0-70-1

A Review on Microbial Alkaline Proteases

P Ellaiah''',
Ph.umuccuticul

Alkaline
protcascs

prorcasex

isol.uion methods which enable

various
prorcuscs

nutritional

Hiotcchnolog

are of considcruhlc

that can resist extreme

alkaline

y Division,

and K Adinarayana'

University,

interest

in view of their activity

environments
and selection

parameters

affecting

by free and immobilized whole cells is discussed.

of Phannuceuticut

Department

Visakhap.nnam

Audhra

the screening

and cnvironmcntul

13 Srinivasulu

produced

530

Scicncc,.

om

and stability

at alkaline

pll. This review

by a wide range of ulk alophilic

of promising
the production

organisms
of alkaline

The purification.

1'01'

industrial

protcuscs

properties.

describes

microorg anisms.

production

are dclinctucd.

and applications

the

Different

are discussed.
The production

of these pretenses

The
of
are

also discussed.

Introduction
Alkaline
proteases
are a physiologically
and
commercially
important
group of enzymes used primarily as detergent additives. They playa specific catalytic
role in the hydrolysis
of proteins. In 1994 the total market for industrial
enzymes accounted
for approximately
$4(}O mill ion, of wh ich. cnzy mes worth $112 million were
used for detergent
purposesl.
In Japan, 1094 alkaline
protease
sales were estimated
at IS,OOO million yen
(equivalent
to Silo million)'.
This enzyme accounts for
4() per cent of the total worldwide
enzyme sales. There
is expected to he an upward trend in the use of alkaline
pro teases in the future.
Proicascs catalyze the cleavage of pcpt iele bonds
in proteins.
They arc enzymes
of class the hydroluscs
and subclass the peptide hydrolascs or pcptidascs. Proteases
may be either cxopeptidscs,
whose actionx are
directed by the amino or carboxyl terminus of proteins.
or endopeptidascs.
which cleave internal peptide bonds.
Endopcoridases
arc also termed as protcinascs. t\ more
rational system of prorcascs classification
is based Oil a
comparison
of active sites, mechanism
of action and
3-~ structure:'.
Protcases can also be classified Oil the basis or:
(a) pH
(b) Substrate
specificity
"' Corresponding
author
. E-Illail: adikunamncni Cr.!- rcdiurnail.com

(c) Similarity
in action to well characterized
enzymes
like trypsin, chymotrypsin
& elastase, and
(d) Active site amino acid residue & catalytic mechanism.
More conventionally,
proteases are classified
into four important groups like serine, cysteine, aspartic
and metallo proreases.

Serine Proteases
Serine proreases arc the most widely

distributed
group of proteolytic
enzymes of both microbial and animal origin". The enzymes have a reactive serine residue
in the active
site and arc generally
inhibited
by
diisopropyl fluorophosph.ue
(DlP) and phenyl methyl
sulphonyl
fluoride (PMSF).
Most or the protcascs
are
also inhibited
by some thiol reagents,
such as !Jch lororncrcuric
benzoate (pCM 13). prohabl y due to the
presence
cysteine residue near the active sill'. which
probably docs not participate
in the catalytic mechanism
of the enzyme. These arc genet'ally active at neutral and
alkaline pl-l, with an optimum
pH between 7-1/. They
have broad substrate
specificities,
including
considerable cstcrcolytic activity towards many ester substrates,
and are generally of low molecular weight (18.5-35 k Da),

or

Cvsteine

Proteases
Cysteine
prorcascs
arc sensitive
to sulphydryl
reagents, such aspCM13, Na-tosyl-L-Iysine
chlorornethyl
ketone (TLCK), iodoacetic
acid, iodoacetamidc,
heavy

ELLAIAH et al.: MICROBIAL ALKALINE PROTEASES

metals, and are activated by reducing agents such as

potassium cyanide or cysteine, dithiothreitol, and ethylene diaminetetraacetic acid (EDTA). The occurrence of
cysteine proteases has been reported in only a few fungi".
Intracellular enzymes with properties similar to cysteine
proteinases have been reported in Trichosporon species,
Oidiodendron kalrai, and Nannirzia fulva. Extracellular
cysteine proteases have been observed in Microsporium
species, Aspergillus
oryzae, and Sporotrichum
pulverulentum', Most of these enzymes are active at pH
5-8. Some are stimulated by reducing agents'.
Aspartic Pro teases
Aspartic proteases are characterized by maximum activity at low pH (3-4) and insensitivity to inhibitors of the other three groups of enzymes". They are
widely distributed in fungi, but are rarely found in bacteria or protozoa. Most aspartic proteases are sensitive
to epoxy and diazo-ketone compounds in the presence
of copper cations. They are also inhibited by pepstatin
or streptomyces pepsin inhibitor.
Most aspartic proteases have molecular weights
in the range 30-45 kDa, and their isoelectric points are
usually in the range pH 3.4-4.6. These enzymes are specific against aromatic or bulky amino acid residues on
both sides of the cleavage point. Catalytic activities involve two aspartic acid residues. The catalytic mechanism of the aspartic proteases requires the initial binding of a water molecule at the active site before nucleophilic attack on the substrate peptide bond. Most of the
fungal aspartic pro teases are unstable above neutral pH
and are not found in cultures growing at neutral or alkaline pH.
M etalloproteases
All these enzymes have pH optima between pH
5-9 and are sensitive to metal-chelating reagents, such
as EDTA, but are unaffected by serine protease inhibitors or sulphydryl agents". Many of the EDTA- inhibited
enzymes can be reactivated by ions, such as zinc, calcium, and cobalt. These are widespread, but only a few
have been reported in fungi. Most of the bacterial and
fungal metalloproteases are zinc-containing enzymes,
with one atom of zinc per molecule of enzyme. The zinc
atom is essential for enzyme activity. Calcium is required
to stabilize the protein structure.

691

Alkalophilic Microorganisms
All microorganisms follow a normal distribution pattern based on the pH dependence for their optimal growth, and the majority of these microorganisms
are known to proliferate well at near neutral pH values.
As the pH moves away from this neutral range the number of microorganisms
decreases. The number of
alkalophilic bacteria found in the soil is about 1/10 to 11
100 of that of neutrophilic bacteria. However, some neutrophilic organisms are capable of growth even at extreme pH conditions. This is primarily due to the special
physiological and metabolic systems, which they have
adopted by altering the bioenergitic membrane properties and transport mechanisms, enabling their survival
and multiplication under such adverse conditions. Such
microorganisms may also be refereed to as pH dependent extremophiles.
Alkalophilic microorganisms constitute a diverse
group that thrives in highly alkaline environments. They
have been further categorized into two broad groups,
namely alkalophiles and alkalotolerants.
The term
alkalophiles is used for those organisms that were capable of growth above pH 10, with an optimal growth
around pH 9, and are unable to grow at pH 7 or less. On
the other hand, alkalotolerant organisms are capable of
growing at pH values 10, but have an optimal growth
rate at pH 7 (ref. I). The extreme alkalophiles have been
further subdivided into two groups, namely facultative
and obligate alkalophiles. Facultative alkalophiles have
optimal growth at pH 10 or above but can grow
well at neutrality, while obligate alkalophiles fail to
grow at pH 7.
Isolation and Screening
Vedder" has reported the isolation of obligate
alkalophilic organisms from human and animal feces in
1934. He briefly described these organisms and proposed
the name Bacillus alcalophilus for his strains and also
stated that he had been able to prove that life exists that
not only tolerates, but also depends on, a highly alkaline
pH. Today, many of these alkalophilic Bacillus strains
are of considerable industrial importance, particularly
for use of proteases in laundry detergent. Normal garden soil was reported to be a preferred source for isolation, presumably because of the various biological activities that generate transient alkaline conditions in such
environment? These organisms were also isolated from

----~-- - -~ ..,.----.~-.--------,....."."'.~-~..........,.
.. .

692

.....

...

_._--- .......--------

-.~--.-,..-.
.

J SCI IND RES VOL 61 SEPTEMBER 2002

nonalkaline habitats, such as neutral and acidic soils,

and thus appear to be fairly widespread.
One of the most important and noteworthy features of many alkalophiles is their ability to modulate
their environment. They can convert neutral medium to
alkaline or acidify high alkaline medium to optimize
external pH for growth. However, their internal pH is
between pH 7 and 9, always lower than the external
medium. Thus, alkalophilicity is maintained by these
organisms through bioenergetic membrane properties and
transport mechanisms, and does not necessarily rely on
alkali resistant intracellular enzyme.
In natural environments, sodium carbonate is
generally the major source of alkalinity. Its addition to
the isolation media enhance the growth of alkalophilic
microorganisms. The addition of sodium carbonate to
the medium for the isolation of alkalophilic thermopiles
results in brown colour and cracking of the medium. At
temperatures of> 70C, agar based media usually lose
their gel strength and exhibit water of syneresis, making
them useless for isolation of thermopiles. As a result,
the need for gelling agents with good thermal stability
led to the discovery of agents, such as Gelrite" 8 and an
optimized concentration (3 per cent w/v) of bacteriological grade agar".
Enrichment and Selection
The primary stage in the development of an industrial fermentation process is to isolate strain(s) capable of producing the target product in commercial
yields. This approach results in intensive screening programs to test a large number of strains to identify high
producers having novel properties. The conventional
practice with many extracellular microbial products is
to grow a large number of organisms on agar plate media and to relate each organism's production capability
to the radius of the product's zone of diffusion around
the colony. In the course of designing a medium, for
screening proteases, it is essential that the medium should
contain likely inducers of the product and be devoid of
constituents that may repress enzyme synthesis. It has
been reported that B.licheniformis produces very narrow zones of hydrolysis on casein agar despite being
very good protease producer in submerged culture. Normally, alkalophilic organisms are isolated by surface plating on a high alkaline medium and subsequent screening for the desired characteristics. The organisms are
further grown on specific media for estimating proteolytic activities using appropriate substrates such skim

milk or casein. The isolates, exhibiting desired level of

activity are chosen and maintained on slants for further
use. The most commonly used general medium for the
isolation of alkalophiles
has been described by
Horikoshi 10. Several types of defined media have also
been used for their isolation which include nutrient agar,
glucose-yeast extract-asparagine agar (GYA), MYGP
agar, peptone-yeast extract-glucose (PPYG) medium, and
other undefined media, such as wheat meal agar. The
medium composition was varied by several workers to
isolate microorganisms of choice, such as those with high
proteolytic activity or those that were thermostable. For
any type of medium, a high pH value is essential to isolate the obligate alkalophiles.
Alkalophilic
Microorganisms Exhibiting
Protease
Activity
Of all the alkalophilic microorganisms that have
been screened for use in various industrial applications,
members of the genus Bacillus were found to be predominant and a prolific source of alkaline proteases. The
different alkaline protease-producing Bacillus species
and strains are summarized in Table 1. Several fungi have
also been reported to produce extracellular alkaline proteases. The different alkaline proteases producing fungal species are summarized in Table 2.
Alkaline proteases are also produced by some
rare actinomycetes. Kurthia spiroforme, a spiral shaped
Gram-positive bacterium possessing a distant relationship to genus Baillus, was reported to produce alkaline
proteases. Further, a bacterial isolate capable of producing alkaline pro teases and showing a symbiotic relationship with a marine shipworm, Psiloteredo healdi, was
also reported by Greene et al. II .
Table 1- Alkaline protease producing Bacillus species
Ref.
Bfirmus

86

B. alcalophilus

87

B. amyloliquefaciens

88

B. proteolyticus

40

Bacillus alcalophilus ATCC 21522

(Bacillus sp. No. 221)

10

B. subtilis

89

B. thuringiensis

55

Bacillus sp. Y

90

Bacillus sp. KSM-K 16

50

ELLAIAH et al.: MICROBIAL ALKALINE PROTEASES

Table 2 - Alkaline protease producing fungal species

Table 3 -

Ref.
Ac flavus

91

A. fumigatus

Microorganisms producing thermostable alkaline

proteases
Ref.

Bacillus licheniformis

27

A. stearothermophilus

13

Bacillus thermoruber BT2T

52

Bacillus sp. strain B 189

61

101

92,93

A. melle us

94

A. niger

95

Chrysosporium

keratinophilum

96

Fusarium graminearum

97

Thermomonospora

Penicillium griseofulvim

98

Thermoactinomycetes

Fusarium sp.

99

Staphylothermus

P. lilac inus
Scedosporium

693

100
apiosermum

fusca

sp.

24

102

marinus

Malbranchea pulchella var. sulfurea

47

48
Table 4 - Commercial producers of alkaline proteases

Bacillus licheniformis

Alcalase

Novo Nordisk, Denmark

Protein engineered
variant of Savinase

Durazym

Novo Nordisk, Denmark

Protein engineered
variant of alkalophilic
Bacillus sp.

Maxapem

Solvay Enzymes GmbH, German

Alkulophilic Bacillus sp.

Savinase, esperase

Novo Nordisk, Denmark

Alkalophilic Bacillus sp.

Maxacal, maxatase

Gist-brocades, The Netherlands

Alkalophilic Bacillus sp.

Opticiean, optimase

Solvay Enzymes GmbH, Germany

Alkalophilic Bacillus sp.

Proleather

Amano Pharmaceuticals

Ltd, Japan

Aspergillus sp.

Protease P

Amano Pharmaceuticals

Ltd. Japan

Halophiles that were described to produce alkaline proteases

included
Halobacterium
sp.,
Halobacterium ATCC 43214, and Halomonas sp. ES10. Alkalopsychrotropic and alkalopsychrophilic bacteria represent a new potential source for alkaline proteases.
These organisms are characterized by their adaptation
to both cold temperatures and alkaline conditions. An
alkalopsychrotropic Bacillus sp. capable of producing
alkaline proteases of high activity at low temperatures
was isolated by Margesin et al. 12.
Despite many published reports on alkaline proteases from alkalophilic Bacillus sp., very few reports
exist on thermostable
alkaline proteases
from
alkalophiles. Many of the thermophilic alkalophiles grow

at 60C (ref.13), with a few exceptions". Thermostable

alkaline pro teases from various thermophilic alkalophiles
are listed in Table 3. Because alkaline proteases are of
great commercial importance, considerable information
has been compiled on various industrially important producing organisms (Table 4).
Production of Alkaline Proteases
Most alkalophilic microorganisms produce alkaline proteases, though interest is limited only to those
that yield substantial amounts. It is essential that these
organisms be provided with optimal growth conditions
to increase enzyme production. The culture conditions
that promote protease production were found to be sig-

J SC I IND RES VO l. 6 1 SEPT EM BER 2002

nificantly different from the cu lture condi ti ons promo ting ce ll grow th. In th e industrial production o f alkaline
proteases, techni cal media w ere u.- ually employed that
contained very hi gh concentrations ( I 00 - I 50 g dry wt
I L ) of co mpl ex carbohyd rates, protein s, and oth er media co mponents. With a view to develop an economica ll y feasi bl e techno logy, efforts arc mainl y foc used on:
(i) Im provement in th e y ield s of alkaline proteases and
(ii ) Optimi zati on o f th e ferm entati on medium and product ion conditi ons.
flllp rol'e ll /enl ol Yield

Strain improvement plays a key role in the co mmerci al deve lop ment o r microb ial fer me ntati on processes . As a rul e th e w ild strain s usuall y produ ce lim i ted quantiti es o f th e des ired enzy me to be use ful for
comme rcial appli catio n 15 Howc ,er, in most cases , by
adopting simpl e se lecti on method s, such as spreading o f
the culture on specifi c media, it is possible to pick co lonies that show a substantial increase in y ield . Conventio nal ph ys ical and chemical muta ge ns arc used for
sc reenin g o f hi gh y ielding strain s.
Shah e1 a /. 1r' have developed a cyste in e
aux otropi c mutant of /J. Licl1 enUrnmis w ith improved
protease producti on. A n ad vantage imparted by cys teine
auxotrophy is that th e strain can be readil y rciso latcd in
the case of contamination wi th w ild ty pe 11ocil/us mutant s that were res istant to antibiotic s such as van co mycin and ri stocctin. Asporoge nous mut ant strain s o f Bacillus sp. are used industrially. A five-fold increase in
th e y ield o f enzy me was observed by the usc of alkalin e
protease pos iti ve asporogenic mut ants.
Th e adven t of protein eng inee ri ng and sophi sti cat ed mol ec ular technologies ha ve opened poss ibiliti es
fo r screen in g hi gh y ielding variant s of enzy mes and tai lor made protein s from alkalophilic microorgani sms w ith
en hanced prod ucti on in y ield s, w hi ch may be of int erest
for spec ifi c co mm ercial applicati ons. 1 ew co nstru cti ons
ha ve been made by th e trans fer of ge nes be tween organisms to produce hi gh yie ldin g va riant s 17 Further the prote in eng in ee ri ng approach ca n be exploited for th e improvement of alkaline proteascs and I or subtil is in s beyo nd it s current limit ati o ns. C urrentl y. t wo
concc pti onall y di ffe rent strat egies arc ava ilabl e for generation o f protein enginee red va ri an ts: rand om and sitedirected mutagenesis. With random mutagenes is, a large
number o f va ri ant s arc produced , but th e success o f thi s
approach largely depends on th e avai labilit y o f efficient

properti es. Site-di rec ted mut agenesis depend s on th e

access to structural or bi oche'11 ical data to reduce the
number o f variant s to be cons tru c:ecl . as every protein
var iant i s purifi ed and indi vidually tes ted for i mprovements . Promi sin g variants generated and identified by
random mutagenes is, o rten can be improved by further
si te- di reeled introducti on o f know n ad va ntagcous mutati ons.
Opti111i::,o!ion ol F enn enlalion M edi 11 111

A I kal i ne prot eascs are general! y prod uced by

submerged fermentati on. In addition , so lid state fermentation processes ha ve also been ex pl oited to a lesse r ex ten t for producti on o f th ese enzy mes 1x. Efforts have been
direc ted mainl y towa rd s: ( i) Eva luallo n o f th e effec ts o f
va ri ous carbon and nitrogenou s nutri ent s as cost-cllecti vc substrat es on th e y ield o f enzy mes : ( ii ) Requirement
of divalent metal ions in th e fermen tati on medium; and
(iii ) Optimi za tion of environ mental and ferm entat ion
pa ram eters such as pH , temperature, aeration , and agitati on. In addition . no defined med ium has been establi shed
for th e best producti on o f alkaline pro teases from eli ffercnt mi crob ial sources. Each organi . m or strain has its
ow n spec ial condit ions for ma x im u 1 enzy me producti on.

Co rhon So urce
Studi es have indicated a reduction in prot ease
production clue to cataboli te re pre~: s i o n by glucose 1'J 20
On th e oth er hand , Z am os t e / a/. 21 have co rrel at ed th e
low y ield s or protease procluct ,on w ith th e lowerin g of
pH brou ght abou t by th e rapid grow th of th e orga ni sm .
In co mmercial prac ti ce, hi gh ca rbohyclratG concentrati ons
repressed enzy me prod uction. T herefore, carbohydrate
was added , ci ther co ni i nu ously or in al iquots throughout th e fermentation to suppl ement th l' exhau sted component and keep the volume limited and th ereby red uce
th e power requ irements.
! ncrcased y ields of al bl ine pro teases were reported by several wo rk ers w ho used di ffcre nt sugars such
as lactose 22 , maltosc 2 \ sucrose 2', and fru ctose 25 . However, a repression in enzyme sy nthesis was observed with
th ese in gredi ents at hi gh co ncen trati ons. Wh ey, a was te
byproduct of th e dairy indu stry, co nt ainin g mainl y lactose and sa lts, has been demonstrated as a potential substrate for alkali ne pro tease product i on. Va ri ous organ ic
acids, such as aceti c :JC id , meth yl aceta te, and citric ac id
or sod ium citrate have been demon tra tecl to increase

._---

----.. --.----~----.-------~--.. ------

ELLAIAH et [II.: MICROBIAL ALKALINE PROTEASES

these organic acids was interesting

in view of their
economy as well as their ability to control pH variations.
Nitrogen

Source
In most microorganisms,
both inorganic and organic forms of nitrogen are metabolized to produce amino
acids, nucleic acids, proteins, and cell wall components.
The alkaline protease comprises 15.6 per cent nitrogen
and its production is dependent on the availability of
both carbon and nitrogen sources in the medium 19. Although complex nitrogen sources are usually used for
alkaline protease production the requirement for a specific nitrogen supplement differs from organism to organism. Low levels of alkal ine protease production were
reported with the use of inorganic nitrogen sources in
the production medium. Enzyme synthesis was found to
be repressed by rapidly metabolizable
nitrogen sources,
such as amino acids or ammonium ion concentrations
in
the medium": However, one report indicated no repression in the protease activity with the use of ammonium

salts":
Sinha and Satyanarayana " have observed an
increase in protease production by the addition of ammonium sulphate and potassium nitrate. Similarly, sodium nitrate (0.25 per cent) was found to be stimulatory
for alkaline protease production. On the contrary, several reports have demonstrated the use of organic nitrogen sources leading to higher enzyme production than
the inorganic nitrogen sources. Fujiwara and Yamamoto"
have recorded maximum enzyme yields using a combination of 3 per cent soybean meal and 1.5 per cent bonito extract. Soybean meal was also reported to be a suitable nitrogen source for protease production?".
Corn steep liquor (CSL) was found to be acheap
and suitable source of nitrogen by some workers".
Tryptone (2 per cent) and casein (1-2 per cent) also serve
as excellent nitrogen sources". Addition of certain ami no
compounds was shown to be effecti ve in the production
of extracellular
enzymes by alkalophilic
Bacillus sp.
However, glycine appeared to have inhibitory effects on
both amylase and protease production. Casamino acids
were also found to inhibit protease production?". Oil
cakes (as nitrogen source) were found to stimulate the
production of enzymes. In some studies, use of oil cakes
did not favour enzyme production".
Metal Ion Requirement
Divalent metal ions, such as calcium, cobalt,
copper, boron, iron, magnesium, manganese, and molybdenum are required in the fermentation medium for

695

optimum production of alkaline proteases. However the

requirement for specific metal ions depends on the source
of enzyme. The use of AgN03 at a cone. of 0.05 mg/I 00
mL or ZnSO 4 at a concentration
of 0.125 mg/1 00 mL
resulted in an increase in protease activity in Rhizopus
oryzae. Potassium phosphate has been used as a source
of phosphate in most studies. This was shown to be responsible for buffering the medium. Phosphate at the
concentration
of 2 g/L was found optimal for protease
production. However, amounts in excess of this concentration showed an inhibition in cell growth and repression in protease production. When the phosphate concentration was 4 g/L, precipitation
of the medium on
autoclaving
was observed".
This problem, however,
could be overcome by the supplementation
of the disodiurn salt of EDTA in the medium.
In at least one case
the salts did not have any effect on the protease yields.
pH and Temperature
The important characteristic of most alkalophilic
microorganisms
show their strong dependence on the
extracellular pH for cell growth and enzyme production.
For increased protease yields from these alkalophiles,
the pH of the medium must be maintained above 7.5
throughout the fermentation process. The culture pH also
strongly affects many enzymatic processes and transport
of various components across the cell membrane. When
ammonium ions were used the medium turned acidic,
while it turned alkaline when organic nitrogen, such as
aminoacids or peptides were consumed".
The decline
in the pH may also be due to production of acidic products. In view of a close relationship between protease
synthesis and the utilization of nitrogenous compounds,
pH variations during fermentation may indicate kinetic
information about the protease production, such as the
start and end of the protease production period.
Temperature is yet another critical parameter that
has to be controlled and varied from organism to organism. The mechanism of temperature control of enzyme
production is not well understood. However, studies by
Frankena et al.co have shown that a link existed between
enzyme synthesis and energy metabolism
in Bacilli,
which was controlled by temperature and oxygen uptake.
Aeration and Agitation
During fermentation the aeration rate indirectly
indicates the dissolved oxygen level in the fermentation
broth. Different dissolved oxygen profiles can be obtained by: (i) Variations in the aeration rate; (ii) Varia-

696

J SCI IND RES VOL 61 SEPTEMBER 2002

tions in the agitation speed of the bioreactor; or (iii) Use

of oxygen rich or oxygen deficient gas phase (appropriate air oxygen or air-nitrogen mixtures) as the oxygen
source". The variation in the agitation speed influences
the extent of mixing in the shake flasks or the bioreactor
and also affects the nutrient availability.
Optimum yields of alkaline protease are produced at 200 rpm for B. subtilis ATCC 14416 and B.
licheniformis. In one study, Bacillus sp.B21-2 produced
increased enzyme titres when agitated at 600 rpm and
aerated at 0.5 vvm. Similarly, Bacillus firmus exhibited
maximum enzyme yields at an aeration rate of 7.0 LI
min and an agitation rate of 360 rpm. However, lowering the aeration rate to 0.1 L'min caused a drastic reduction in the protease yields. This indicates that a reduction in oxygen supply is an important limiting factor for
growth as well as protease synthesis.
Immobilization of Alkaline Proteases
The interest in the use of immobilized enzymes
in industry is based on the potential advantages they
confer over their soluble counterparts, including increased stability to temperature, pH, and organic solvents;
recovery and reuse of the enzyme; and, in the case of
proteases, removal or reduction of autolysis or denaturation. Furthermore, immobilized enzymes render continuous production processes possible via packed bed
reactors and may lead to more stable biocatalysts. The
two main methods for immobilization are whole cell
immobilization and cell-free immobilization.
Whole Cell Immobilization
Because alkaline protease is an extracellular
enzyme, whole cell immobilization is the method of
choice. By using immobilized cells the protease can be
produced in a shorter reaction time. Further the rate of
protease production can be improved over that of submerged batch fermentation. The long-term stability of
the immobilized cells during the course of fermentation
and the easy separation of enzyme also make them promising candidates for commercial exploitation. Physical
entrapment of whole cells in polymeric gel matrices was
used as an immobilized method by Kokubu et al." and
Sutar et al?'. Batch" and repeated batch" fermentation
processes were also demonstrated using urethane foam
as an immobilization carrier. Further, Bacillus firmus
cells were immobilized on cellulose triacetate fibres and
films, followed by cross-linking with a bifunctional reagent, glutaraldehyde, which improved alkaline protease
biosynthesis.

Cell-free Immobilization
Attachment of alkaline proteases to an insoluble
carrier (by either physical adsorption or covalent coupling) is the most prevalent method of immobilization.
Various carriers employed for this purpose include bentonite, porous glass, nylon and vermiculite. Although
porous glass has been widely used the relatively high
cost of this support has been the limiting factor for industrial applications. The method of immobilization of
the alkaline proteases on these supports using glutaraldehyde involves covalent attachment of the amino groups
of the enzyme to the available aldehyde groups present
in the glutaraldehyde activated support. In one study,
Srokova and Cik37 successfully immobilized an alkaline
protease onto a gel of o-hydroxyethylcellulose through
photochemical polymer carrier crosslinking induced by
the photolysis of aromatic azides. Some immobilization
studies38.39 have addressed
an increase
in the
themostability profile and the pH activity profile of the
enzyme towards the alkaline side. The increase in thermal stability is mainly due to the multipoint covalent
attachment and the stabilization of the weak ionic forces
and hydrogen bonds between the protease and the support, which protects the enzyme from inactivation and
autolysis. Further the change in the pH values may be
attributed to the partition effects that cause different concentrations of hydrogen ions in the microenvironment
of the immobilized enzyme when coupled to a carrier
possessing electrostatic interactions.
Isolation and Purification

of Alkaline Proteases

When isolating enzymes on industrial scale for

commercial purposes the prime consideration is the cost
of production in relation to the value of the end product.
Crude preparations of alkaline pro teases are generally employed for commercial use. Nevertheless the
purification of alkaline proteases is important from the
perspective of developing a better understanding of the
functioning of the enzyme.
Recovery
After successful fermentation, when the fermented medium leaves the controlled environment of
the fermenter, it is exposed to a drastic change in environmental conditions. The removal of the cells, solids,
and colloids from the fermentation broth is the primary

ELLAIAH

et al.: MICROBIAL

step in enzyme downstream

processing,
for which
vacuum rotary drum filters and continuous disc centrifuges are commonly used. To prevent the losses in enzyme activity caused by imperfect clarification or to prevent the clogging of filters, it is necessary to perform
some chemical pretreatment
of the fermentation
broth
before commencing separation. Changes in pH may also
be suitable for better separation of solids. Furthermore
the fermentation
broth solids are often colloidal in nature and are difficult to remove directly. In this case,
addition of coagulating or flocculating agents becomes
vital:". Flocculating agents are generally employed to
effect the formation of larger floes or agglomerates,
which, in turn, accelerate the solid-liquid separation. Cell
tlocculation
can be improved by neutralization
of the
charges on the microbial cell surfaces, which includes
changes in pH and the addition of a range of compounds
that alter the ionic environment. The flocculating agents,
commonly used are inorganic salts, mineral hydrocolloids, and organic polyelectrolytes.
For example the use
of a polyelectrolyte
Sedipur TF 5 proved to be an effective tlocculating agent at 150 ppm and pH 7.0-9.0, and
gave 74 per cent yield of alkaline protease activity. In
some cases, it becomes necessary to add a bioprocessing
filter aid, such as diatomaceous earth. before filtration".

Concentration
As the amount of enzyme present in the cell free
filtrate is usually low therefore the removal of water is a
primary objective. Recently, membrane separation processes have been widely used for downstream processing. Ultrafiltration
(UF) is one such membrane process
that has been largely used for the recovery of enzymes"
and formed a preferred alternative to evaporation. This
pressure driven separation process is expensive, results
in Iittle loss of enzyme activity, and offers both puri fication and concentration,
as well as diafiltration, for salt
removal or for changing the salt composition".
However, a disadvantage underlying this process is the fouling or membrane clogging due to the precipitates formed
by the final product. This clogging can usually be alleviated or overcome by treatment with detergents, proteases, or acids and alkalies.
Hand et al," have used a temperature-sensitive
hydrogel ultrafiltration for concentrating an alkaline protease.
This
hydrogel
comprised
poly
(Nisopropylacrylamide),
which changed its volume reversibly by the changes in temp~rature. The separation efficiency of the enzyme was dependent on the temperature

ALKALINE

697

PROTEASES

and was 84 per cent

at J 5 C and 20 C, respectively.

However, above 25 C, a decrease

in the separation

effi-

ciency was observed.

Precipitation
Precipitation is the most commonly used method
for the isolation and recovery of proteins from crude biological mixtures. It also performs both purification and
concentration steps. It is generally effected by the addition of reagents such as salt or an organic solvent, which
lower the solubility of the desired proteins in an aqueous solution. Although precipitation by ammonium sulphate has been used for many years, it is not the precipitating agent of choice for detergent enzymes. Ammonium sulphate has found wide utility only in acidic and
neutral pH values and it formed ammonia under alkaline conditions. Hence the use of sodium sulphate or an
organic solvent gave the preferred choice. Despite better precipitating qualities of sodium sulphate over ammonium sulphate the poor solubility of the salt at low
temperatures,
restricted its use for this purpose.
Many reports have revealed the use of acerone
at different vol umc concentrations:
5 volumes Ill, 3 volumes ", and 2.5 volumes",
as a primary precipitation
agent for the recovery of alkaline proteases. Precipitation was also reported by various workers with acetone
at different concentrations'
80 per cent (v/v) 4),66 per
cent (v/v)4<>;or 44,66, and 83 per cent (v/v)47, followed
by centrifugation and/or drying. Precipitation of enzymes
can also be achieved by the use of water soluble, neutral
polymers, such as polyethylene glycol ".

ion-exchange Chromarography (lEe)

Alkaline proteases
are generally
positively
. exc hangers. 49 H owcharged and are not boun d to anion
ever, cation exchangers can be a rational choice and the
hound molecules are el uted from the column by an increase in salt or pH gradient.

Affinity Chromatography
Reports on the purification of alkaline proteases
by different affinity chromatographic
methods showed
that an affinity adsorbent hydroxyapatite
was used to
separate the neutral protease as well as purify the alkaline protease from a Bacillus sp.:". Other affinity matrices used were Sephadex-a-phenylbutylamine",
casein
agarose", or N-benzoyloxycarbonyl
phenylalanine,
immobilized on agarose adsorbents".
However the major
limitations of affinity chromatography
are the high cost

698

J SCI IND RES VOL 61 SEPTEMBER 2002

of enzyme supports and the labile nature of some affinity ligands, which do not recommend them for use as a
process scale.
Aqueous Two-phase Systems
This technique has been applied for purification of alkaline proteases using mixtures of polyethylene glycol (PEG) and dextran or PEG and salts such as
H3P04 and MgSO/4,55. In addition, other methods, such
as the use of reversed micelles for liquid-liquid extraction, affinity precipitation, and foam fractionation have
also been employed for the recovery of alkaline proteases.
Stabilization
The enzyme preparations, used commercially,
are impure and are standardized to specified levels of
activity by the addition of diluents and carriers. Further
the conditions for maximum stability of crude preparations may be quite different than for purified enzymes.
As loss of activity is encountered during storage in the
factory therefore shipment to client(s) and / or storage
in client's facilities, storage stability is of prime concern
to enzyme manufacturers. Protease solutions are subject
to proteolytic and autolytic degradation that results in
rapid inactivation of enzymatic activity. To maintain the
enzyme activity and provide stability, addition of stabilizers, like calcium salts, sodium formate, borate, propylene glycol, glycerine or betaine polyhydric alcohols,
protein preparations, and related compounds has proved
successful". Also, to prevent contamination of the final
commercial crude preparation during storage, addition
of sodium chloride at 18-20 per cent concentration has
been suggested". The handling of dry enzymes possess
potential health hazards and, therefore, it is customary
to maintain the enzyme preparations in stabilized liquid
form. The stabilization of alkaline proteases and/or subtilisins has also been made possible through use of protein engineering and numerous examples have been cited
in literature. The alkaline and thermal stabilities of subtilisin BPN9 were improved by random mutagenesis followed by application of proper screening assays. Sitedirected mutagenesis is often based on specific protein
design strategies, including change of electrostatic potential, introduction of disulphide bridges, replacement
of oxidation labile residues, modification of side chain
interactions,
improvement
of internal packaging,
strengthening of metal ion binding, reduction in unfolding entropy, residue substitution or deletion based on
homology and modification of substrate specificity'F".

Properties of Alkaline Proteases

The enzymatic and physiochemical properties
of alkaline proteases from several microorganisms have
been extensively studied.
Optimum pH and Temperature
The optimum pH range of alkaline proteases is
generall y between pH 9 and 11, with a few exceptions
of higher pH optima of 11.5 (ref. 60), pH 11-12 (ref. 10,
49), and pH 12-13 (ref. 61). They also have high isoelectric points and are generally stable between pH 6
and 12 (ref. 62). The optimum temperatures of alkaline
proteases range between 50 and 70C. In addition the
enzyme from an alkalophilic Bacillus sp. B 18 showed
an exceptionally high optimum temperature of 85C.
Alkaline proteases from Bacillus sp., Streptomyces sp.,
and Thermus sp. are quite stable at high temperatures,
and the addition of Ca2+ further enhanced enzyme
themostability.
Molecular Masses
The molecular masses of alkaline proteases
range between 15 and 30 kDa63 with a few reports of
higher molecular masses of 31.6 kDa64 , 33 kDa53 , 36
kDa65, and 45 kDa45. However, an enzyme from Kurthia
spiroforme had an extremely low molecular weight of 8
kDa66. In some Bacillus sp., multiple electrophoretic
forms of alkaline proteases were observedv". The multiple forms of these enzymes were the result of nonenzymatic, irreversible deamination of glutamine or asparagine residues in the protein molecules,
or of
autoproreolysis'",
Metal Ion Requirement and Inhibitors
Alkaline proteases require a divalent cation like
Ca+2, Mg+2,and Mn+2 or a combination of these cations,
for maximum activity. These cations were also found to
enhance the thermal stability of a Bacillus alkaline protease". It is believed that these cations protect the enzyme against thermal denaturation and playa vital role
in maintaining the active conformation of the enzyme at
high temperatures. In addition, specific Ca2+binding sites
that influence the protein activity and stability, apart from
the catalytic site were described for proteinase K. Inhibition studies give insight into the nature of the enzyme,
its cofactor requirements, and the nature of the active
site. In some of the studies, catalytic activity was inhi-

ELLAIAH et al.: MICROBIAL ALKALINE PROTEASES

bited by Hg+2ions. In this regard the poisoning of enzymes by heavy metal ions has been well documented in
the literature".
Alkaline proteases are completely inhibited by
phenylmethylsulphonyl fluoride (PMSF) and diisopropyl
fluorophosphate (DFP). In this regard, PMSF sulphonates
the essential serine residue in the active site and results
in the complete loss of activity. This inhibition profile
classifies these proteases as serine hydrolases. In addition, some of the alkaline pro teases were found to be
metal ion dependent in view of their sensitivity to metal
chelating agents, such as EDTA66.69.Thiol inhibitors have
little effect on alkaline pro teases of Bacillus sp., although
they do affect the alkaline enzymes produced by Streptomyces Sp.47,60,
Substrate Specificity
Although alkaline proteases are active against
many synthetic substrates, as well as native proteins,
reaction rates vary widely. The alkaline pro teases and/
or subtilisins are found to be more active against casein
than against haemoglobin or bovine serum albumin. Alkaline proteases are specific against aromatic or hydrophobic amino acid residues, such as tyrosine, phenylalanine, or leucine at the carboxyl side of the splitting
point, having a specificity similar to, but less stringent
than a chymotrypsin. With the B-chain of insulin as substrate the bonds most frequently cleaved by many alkaline proteases were Glu 4 - His 5, Ser 9 - His 10, Leu 15
- Tyr 16, Tyr 16 -Leu 17, Phe 25 - Tyr 26, Tyr 26 - Thr
27, and Lys 29 -Ala 30 (ref. 42,44). In addition, Tsai et
al." have elucidated that an alkaline elastase from Bacillus sp. Ya-B cleaved both the oxidized insulin A- and
B-chains in a block cutting manner.
Tsai et al." observed that the alkaline elastase
from Bacillus sp. Ya-B also hydrolysed elastin and
elastase specific substrates, like succinyl-Ala3 -pnitroanilide and succinyl-Ala-Pro- Ala-p-nitroanilide, at
a faster rate. This enzyme showed a preference for aliphatic amino acid residues, such as alanine that are
present in elastin. It is considered that the elastolysis
was initiated by the formation of an enzyme substrate
complex through electrostatic interaction between positively charged residues of the elastase and negatively
charged residues of the elastin in a pH range below 10.6.
In keratin the disulphide bonds form an important structural feature and prevent the proteolytic degradation of
the most compact areas of the keratinous substrates. A

699

thermostable alkaline protease from an alkalophilic Bacillus sp. no. AH-101 exhibiting keratinolytic activity
showed degradation of human hair keratin with I per
cent thioglycolic acid at pH 12 and 70C, and the hair
was solubilized within 1 h. Similarly, enhanced keratin
degradation after addition of DTT has also been reported
for alkaline proteases of Streptomyces sp. 71,
Applications of Alkaline Proteases
Alkaline proteases are robust enzymes with considerable industrial potential in detergents, leather processing, silver recovery, medical purposes, food processing, feeds, and chemical industries, as well as waste treatment. These enzymes contribute to the development of
high value added applications or products by using enzyme aided (partial) digestion. The different areas of
applications currently using alkaline proteases are given
subsequently.
Detergent Industry
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 strains, amylases are effective against
starch and other carbohydrate stains while lipases are
effective against oily or fat stains. For an enzyme to be
used as a detergent additive. It should have two qualities :(i) An alkaline pH and (ii) It should also be compatible with detergents. The major use of detergent compatible proteases is in laundry detergent formulations.
Detergents available in the international market, such
as Dynamo, Era plus (Procter & Gamble), Tide (Colgate
Palmolive) contain proteolytic enzymes derived mostly
from the genus Bacillus.
The interest in using alkaline enzymes in automatic dishwashing detergents has also increased recently.
The in-place cleaning of ultrafiltration (UF) and reverse
osmosis (RO) membranes form one of the most important aspects IJf modern dairy and food industries. The
UF and RO membranes are put to a variety of uses, including concentration, fractionation, clarification and/
or sterilization of liquid foods, such as milk, whey, egg
white, fruit juices, wines, and other beverages. The enzyme detergent preparations presently marked for cleaning of membrane systems are Alkazym (Novodan A/S,

700

J SCI IND RES VOL 61 SEPTEMBER 2002

Copenhagen,
Denmark),
Terg-A-Zyme (Alconox, Inc,
ew York, USA) and Uitrasil 53 (Henkel kGaA ,
Dusseldorf, Germany). In addition, contact lense cleaning solutions containing alkaline protease derived from
a marine shipworm bacterium was used for the cleaning
of contact lens at low temperatures".
In India, one such
enzyme based optical cleaner (available in the form of
tablets containing Subtilopeptidase A) is presently marketed by Mis Bausch and Lomb (India) Ltd.

Leather Industry
Another industrial process, which has received
attention, is the enzyme-assisted
dehairing 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
dehairing. Enzyme-assisted dehairing is preferentially possible ifproteolytic 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. Several alkaline proteases from 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, and wool at
alkaline pH and may have commercial applications.

Silver Recovery
Alkaline proteases find potential application in
the bioprocessing
of used X-ray films for silver recovery. Used X-ray film contains approximately
1.5 to 2.0
per cent (by weight) silver in its gelatin layers. The conventional practice of silver recovery by burning film,
causes a major environmental
pollution problem. Thus
the enzymatic hydrolysis of the gelatin layers on the Xray film enables not only the silver, but also the polyester film base, to be recycled.
The alkaline proteases from Bacillus sp. B21-2
(ref. 73) and B. coagulans PB-77 (ref. 74) decomposed
the gelatinous coating on the used X-ray films from which
the silver was recovered. Further, a continuous process
for silver recovery was also reported" on the basis of

kinetic studies and mechanism of enzymatic hydrolysis

of gelatin layers on X-ray film and the resulting release
of sil ver panicles.

Medicinal Uses
Collagenases with alkaline protease activity are
increasingly
used for therapeutic
applications
in the
preparation of slow-release dosage forms. A new semialkaline protease with high collagenolytic
activity was
produced by Aspergillus niger LCF9. The enzyme hydrolyzed various collagen types without amino acid release and liberated low molecular weight peptides of
potential therapeutic use. Further more, Bacillus spp.
have been recognized as being safe to humans" and an
alkaline protease having fibrinolytic activity has been
used as a thrombolytic agent",

Food Industry
Alkaline proteases can hydrolyse proteins from
plants, fish or animals to produce hydrolysates of welldefined peptide profile. The commercial alkaline protease, Alcalase has a broad specificity with some preference for terminal hydrophobic amino acids. Using this
enzyme, a less bitter hydrolysate and a debittered enzymatic whey protein hydrolysate were produced.
Recently, another alkaline protease from B.
aniyloliquefaciens resulted in the production of a methionine-rich protein hydrolysate from chickpea protein",
The protein hydrolysates
commonly
generated
from
casein, whey protein and soyprotein find major application in hypoallergenic
infant food formulations.
They
can also be used for the fortification of fruit juices or
soft drinks and in manufacturing
protein-rich therapeutic diets".
In addition, protein hydrolysatcs having angiotensin I-converting enzyme inhibitory activity were produced from sardine muscle by treatment
with a B.
lichinijormis alkaline protease. These protein hydrolysates could be used effectively as a physiologically functional food that play an important role in blood pressure
regulation.
Further, pro teases playa prominent role in meat
tendarization, especially of beef. An alkaline elastase"
and thermophilic aikaline protease" have proved to be
successful and promising meat tenderizing enzymes, as
they possess the ability to hydrolyze connective tissue
proteins as well as muscle fibre proteins. A method has
been developed in which the enzyme is introduced directly in the circulatory system of the animal, shortly

ELL A IA H eta/. : MI C RO BI AL ALK A LI NE PROT EASES

before slaughter o r after stunning the animal to cause

brain death .
A potenti al meth od used a spec ific co mbin ati on
of ne utral and alkalin e proteases for hydro lys ing raw
meat. The res ul tin g meat hydro lysate ex hibi ted excellent organo leptic prope rti es and ca n be used as a meatfl avoured additi ve to soup co ncentrates. Hydro lys is of
over 20 per cent did not show a ny bitterness w hen such
combin ati ons of enzy mes were used . The reason for thi s
may be th at the prefe re nti al spec ifi city was favourabl e
when metall oprotein ase and serine proteinase were used
simultaneo us ly.

Waste Treatment
Alkaline proteases prov ide potenti al appli cati o n
fo r the management of wastes from variou s food processing industri es and househo ld acti viti es. These proteases ca n so lubili ze proteins in wastes through a multistep process to recover liquid co ncentrates or dry so lids
of nutriti onal value fo r fi sh or li vestoc k.
Dalev 8 1 has reported an e nzy mati c process, using a B. subtilis alkaline protease in the process ing of
waste feathers from poultry slaughte r houses. The end
product was a heavy, gray ish powde r w ith a very hi gh
prote in co nte nt, whi ch could be used as a feed additi ve.
Similarl y, many other keratino lyti c alkaline proteases have been used in feed techno logy 82 fo r the production of amino ac ids or pe ptides 83 , fo r degrading was te
keratinous mate ri al in household refu se, and as a depil atory agent to re move hair in bath tub drains, whi ch caused
bad odors in houses and in public pl aces .

Furthe r th e e nzy me Alcalase whi ch act as catalyst for reso luti o n of N-protected amino ac id es ters and
alkalin e proteases fro m Conidiobolus coronatus re pl aced
subtili sin Carl sberg in reso lvin g the race mi c mixtures
of DL-phe nylalanine a nd DL-phe nylg lyc ine.

Conclusions
Al kaline proteases are important in view of their
acti vity and stability at alkaline pH . This rev iew descri bes
the proteases th at can res ist e xtre me alkaline environme nts produced by a w ide range of alkalophili c mi croorgani sms. Di ffe rent screening programs are desc ribed
for the selecti o n of promi sin g iso lates for the industri al
produ cti o n of alk aline proteases and their applicati o ns.
With a vi ew to develop an econo mi call y feas ible techno logy, effo rts are mainly foc used o n improve me nt in
the y ie lds of alka line proteases by strain improve ment,
optimi zati on of the ferm e ntati on medi a, a nd produ cti on
conditi ons. The purificati on and properti es of alkaline
proteases from a wide vari ety of mi croorgani sms are
di sc ussed .Ge nera l view of the wi de and complex matter
on alkalin e proteases is presented . It is hoped th at such a
rev iew will prove to be a comprehensive introducto ry
guide on alkalin e proteases.

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2
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