Punto de Vista de La Industria

Biotechnology Advances 17 (1999) 561594
Research review paper
Microbial alkaline proteases: From a

bioindustrial viewpoint
C. Ganesh Kumara,*, Hiroshi Takagib
a
Dairy Microbiology Division, National Dairy Research Institute, Karnal 132 001, India
Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-11, Japan
Abstract
Alkaline proteases are of considerable interest in view of their activity and stability at alkaline pH.
This review describes the proteases that can resist extreme alkaline environments produced by a wide
range of alkalophilic microorganisms. Different isolation methods are discussed which enable the
screening and selection of promising organisms for industrial production. Further, strain improvement
using mutagenesis and/or recombinant DNA technology can be applied to augment the efficiency of
the producer strain to a commercial status. The various nutritional and environmental parameters affecting the production of alkaline proteases are delineated. The purification and properties of these
proteases is discussed, and the use of alkaline proteases in diverse industrial applications is
highlighted. 1999 Elsevier Science Inc. All rights reserved.
Keywords: Alkaline proteases; Alkalophiles; Microbial; Industrial enzymes
1. Introduction
Enzymes are well known biocatalysts that perform a multitude of chemical reactions and
are commercially exploited in the detergent, food, pharmaceutical, diagnostics, and fine
chemical industries. Of the .3000 different enzymes described to date the majority have
been isolated from mesophilic organisms [1]. These enzymes mainly function in a narrow
range of pH, temperature, and ionic strength. Moreover, the technological application of enzymes under demanding industrial conditions makes the currently known arsenal of enzymes
unrecommendable. Thus, the search for new microbial sources is a continual exercise, but
one must respect biodiversity. The microorganisms from diverse and exotic environments,
Extremophiles, are considered an important source of enzymes, and their specific properties
are expected to result in novel process applications [2,3].
* Corresponding author. Present address: Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VII
M, Calcutta 700054, India.
0734-9750/99/$ see front matter 1999 Elsevier Science Inc. All rights reserved.
PII: S0 7 3 4 - 9 7 5 0 ( 9 9 ) 0 0 0 2 7 -0
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C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594
Alkaline proteases (or Subtilisins, E.C. 3.4.21.14) are a physiologically and commercially
important group of enzymes used primarily as detergent additives. They play a specific catalytic role in the hydrolysis of proteins. In 1994, the total market for industrial enzymes accounted for approximately $400 million, of which enzymes worth $112 million were used
for detergent purposes [4]. In Japan, 1994 alkaline protease sales were estimated at 15 000
million yen (equivalent to $116 million) [5]. There is expected to be an upward trend in the
use of alkaline proteases so that by the turn of the decade the total value for industrial enzymes is likely to reach $700 million or more [4].
2. 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 1/100 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 bioenergetic membrane
properties and transport mechanisms, enabling their survival and multiplication under such
adverse conditions [6,7]. Such microorganisms may also be referred 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 in excess of 10, but have an optimal growth rate nearer to neutrality [8]. 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 neutrality [9].
2.1. Habitat
Alkalophilic microorganisms are widely distributed in nature and can be found in almost
all environments without the restriction of alkalinity. However, a few of the naturally-occurring alkaline environments, namely soda soils, lakes, and deserts, harbor a wide range of
these types [10]. Their ecological and chemical aspects have been studied in detail [11,12].
Others include the dilute alkaline springs, desert soils and soils containing decaying proteins
or forest soil [11,1315]. The pH values of these environments are commonly around 10 and
above. The man-made alkaline environments were found to be the effluents from food, textiles, tanneries, potato processing units, paper manufacture units, calcium carbonate kilns,
detergents and other industrial processes [11,16,17].
Highly saline, alkaline environments are relatively rare in the world compared with high
saline, neutral environments. However, there is a possibility that such environments harbor a
563
unique microbial population [1822]. The best sources for halophilic alkalophiles have been
the extreme soda lakes of the Wadi Natrum in Egypt and Lake Magadi in Kenya [23]. The
study of this unique group of microorganisms has aroused interest because of the extreme
tolerance of haloalkalophiles to the two environmental extremes, salinity and high pH. Moreover, in this category there are also moderate thermophiles, with growth temperatures of approximately 408C.
2.2. Isolation and screening
The isolation of obligate alkalophilic organisms from human and animal feces was first
reported by Vedder 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 depends on, a highly alkaline pH [24]. Today, many of these
alkalophilic Bacillus strains are of considerable industrial importance, particularly for use of
proteases in laundry detergents [25], xylanases for use in paper pulp industry [26], and cyclodextrin glucanotransferase for cyclodextrin manufacture from starch [27,28]. These industrial applications have prompted the isolation of alkalophilic microorganisms from a variety
of natural and man-made alkaline environments [14,23,29]. 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 environments [12]. These organisms were
also isolated from 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 alkalinize neutral medium 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 enzymes [6].
In natural environments, sodium carbonate is generally the major source of alkalinity. Its
addition to the isolation media enhances the growth of alkalophilic microorganisms [30].
Substitutes for sodium carbonate include sodium bicarbonate, sodium sesquicarbonate, potassium carbonate, sodium borate, and sodium orthophosphate or the occasional addition of
sodium hydroxide [17,31]. The addition of Na2CO3 to the medium for the isolation of alkalophilic thermophiles results in brown color and cracking of the medium [32]. At temperatures
of .708C, agar-based media usually lose their gel strength and exhibit water of syneresis,
making them useless for isolation of thermophiles [33]. As a result, the need for gelling
agents with good thermal stability led to the discovery of agents such as Gelrite [34,35]
and an optimized concentration (3%, w/v) of bacteriological grade agar [36].
2.3. 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 prod-
564
ucts is to grow a large number of organisms on agar plate media and to relate each organisms production capability to the radius of the products 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 producers in submerged cultures [37].
A similar observation was made of Aspergillus oryzae and A. sojae by Nasuno and Ohara [38].
The formation of proteases by A. oryzae was associated with a smooth conidial type, whereas
the enzyme producing A. sojae strains had echinulate or tuberculate conidia.
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, amylolytic, lipolytic, or cellulolytic activities using appropriate substrates such as skim milk or casein, starch, tributyrin, butter fat,
or carboxymethylcellulose. The isolates exhibiting desired level of activity are chosen and
maintained on slants for further use. However, as the number of alkalophilic microorganisms
present in soils is generally found to be very low, enrichment of soil samples before screening is often necessary.
The most commonly used general medium for the isolation of alkalophiles was described
by Horikoshi [39]. Several different types of defined media have also been used in the past,
including nutrient agar [17], glucose-yeast extract-asparagine (GYA) agar [40], MYGP agar
[41], peptone-yeast extract-glucose (PPYG) medium [16], and other undefined media, such
as alkaline casein agar medium [15] and wheat meal agar [42]. 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 [23].
2.4. 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. Many of the fungi have also been reported to produce
extracellular alkaline proteases [43]. The different alkaline proteases producing fungal species are summarized in Table 2. The alkaline proteases of Aspergillus sp., in particular, have
been studied in detail. Some alkaline proteases producing strains of imperfect fungi, such as
Dendryphiella sp. and Scolebasidium sp., have found application in detergents [44].
Yeasts reported to produce alkaline proteases include Candida lipolytica [45]; Yarrowia
lipolytica [46], and Aureobasidium pullulans [47]. However, very few studies exist on the alkaline protease producing alkalophilic actinomycetes [48]. The different species of Streptomyces reported to produce alkaline proteases include Streptomyces rectus var. proteolyticus
[49,50]; Streptomyces griseus [51]; Streptomyces sp. [52,53]; Streptomyces moderatus
NRRL 3150 [54]; Streptomyces sp. YSA 2 130 [55]; S. diastaticus SS1 [56,57]; S. corchorusii ST36 [58], and S. pactum DSM 40530 [59]. Other types of alkalophilic actinomycetes
include Nocardiopsis dassonvillei [6062] and Oerskovia xanthineolytica TK-1 [63].
565
Table 1
Alkaline protease-producing Bacillus species
Bacillus spp. and their strains [References]
Bacillus alcalophilus ATCC 21522 (Bacillus sp. No. 221) [39]
B. alcalophilus [181]
B. alcalophilus subsp. halodurans KP1239 [119]
B. amyloliquefaciens [97,290]
B. circulans [291]
B. coagulans PB-77 [292]
B. firmus [82,144]
B. intermedius [293]
B. lentus [294]
B. licheniformis [101,113,123,295297]
B. proteolyticus [160]
B. pumilus [298,299]
B. sphaericus [134,300]
B. subtilis [126,301,302]
B. subtilis var. amylosacchariticus [303]
B. thuringiensis [183]
Bacillus sp. Ya-B [159]
Bacillus sp. NKS-21 [263]
Bacillus sp. B21-2 [42]
Bacillus sp. Y [219]
Bacillus sp. CW-1121 [304]
Bacillus sp. KSM-K16 [179,210]
Bacillus sp. MK5-6 [10]
Some of the Gram-negative bacteria producing alkaline proteases were identified as

Pseudomonas aeruginosa [64]; Pseudomonas maltophila [65]; Pseudomonas sp. strain B45
[66]; Xanthomonas maltophila [67]; Vibrio alginolyticus [68]; and Vibrio metschnikovii
strain RH530 [69].
Alkaline proteases are also produced by some rare microorganisms. Kurthia spiroforme, a
spiral shaped Gram-positive bacterium possessing a distant relationship to genus Bacillus,
was reported to produce alkaline proteases [70]. Further, a bacterial isolate capable of producing alkaline proteases and showing a symbiotic relationship with a marine shipworm,
Psiloteredo healdi, was also reported by Greene et al. [71].
Halophiles that were described to produce alkaline proteases included Halobacterium sp.
[72]; Halobacterium halobium ATCC 43214 [73], and Halomonas sp. ES-10 [74,75]. The alkalopsychrotrophic and alkalopsychrophilic bacteria represent a new potential source for alkaline proteases [76]. These organisms are characterized by their adaptation to both cold temperatures and alkaline conditions. An alkalopsychrotrophic Bacillus sp. capable of producing
alkaline proteases of high activity at low temperatures was isolated by Margesin et al. [77].
Despite the many published reports on alkaline proteases from alkalophilic Bacillus spp., very
few reports exist on thermostable alkaline proteases from alkalophiles. Many of the thermophilic
alkalophiles have growth temperatures of .608C [78,79], with a few exceptions of ,608C
[80,81]. Thermostable alkaline proteases from various thermophilic alkalophiles are listed in Ta-
566
Table 2
Alkaline protease-producing fungal species
Fungal species [References]
Aspergillus candidus [38]
A. flavus [96]
A. fumigatus [305,306]
A. melleus [307]
A. niger [251]
A. oryzae [308310]
A. sojae [311]
A. sulphureus [312]
A. sydowi [313]
Cephalosporium sp. KSM 388 [314]
Chrysosporium keratinophilum [315]
Conidiobolus coronatus [316]
Entomophthora coronata [317]
Fusarium graminearum [115]
Fusarium sp. [121,318]
Paecilomyces marquandii [319]
P. lilacinus [320]
Penicillium griseofulvin [321]
P. liliacinum No. 2093 [322]
Rhizopus oryzae [122]
Scedosporium apiospermum [176]
Tritirachium album Limber [238,323,324]
ble 3. Because alkaline proteases are of great commercial importance, considerable information
has been compiled on the various industrial producer organisms (Table 4).
3. 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 significantly different from the culture conditions promoting cell growth [82]. In the industrial production of alkaline proteases, technical
media were usually employed that contained very high concentrations (100150 g dry
weight/litre) of complex carbohydrates, proteins, and other media components [83]. With a
view to develop an economically feasible technology, research efforts are mainly focused
on: (i) improvement in the yields of alkaline proteases; and (ii) optimization of the fermentation medium and production conditions.
3.1. Improvement of yield
Strain improvement plays a key role in the commercial development of microbial fermentation processes. As a rule, the wild strains usually produce limited quantities of the desired
567
Table 3
Microorganisms producing thermostable alkaline proteases
Organism [References]
Bacillus licheniformis [32,40,104]
Bacillus thermoruber BT2T [135]
Bacillus sp. strain B189 [177]
B. stearothermophilus [79,223]
Thermus aquaticus YT-1 [325]
Thermus sp. strain Rt41A [169]
Thermococcus celer, T. stetteri, T. litoralis [326]
Staphylothermus marinus [327]
Thermobacteroides proteolyticus [328]
Malbranchea pulchella var. sulfurea [110]
Torula thermophila [329]
Thermomonospora fusca [330, 331]
Thermoactinomycetes sp. [332,333]
Thermoactinomyces thalpophilus THM1 [334]
enzyme to be useful for commercial application [84]. However, in most cases, by adopting
simple selection methods, such as spreading of the culture on specific media, it is possible to
pick colonies that show a substantial increase in yield [85]. The yield can be further improved by the use of mutagens or antibiotics and the adoption of special techniques or procedures for detecting useful mutants.
Shah et al. [86] developed a cysteine auxotrophic mutant of B. licheniformis with improved protease production. An advantage imparted by cysteine auxotrophy is that the strain
can be readily reisolated in case of contamination with wild type Bacillus strains. Further, increased yields of alkaline proteases have been achieved by Bacillus mutants that were resistant to antibiotics such as vancomycin and ristocetin [87].
Table 4
Commercial producers of alkaline proteases
Organism
Trade names
Manufacturer
Bacillus licheniformis
Alkalophilic Bacillus sp.
Aspergillus sp.
Protein engineered variant
of Savinase
Protein engineered variant
of alkalophilic Bacillus sp.
Genetic engineered
DonorB. lentus
Expressed in Bacillus sp.
Alcalase
Savinase, Esperase
Maxacal, Maxatase
Opticlean, Optimase
Proleather
Protease P
Novo Nordisk, Denmark

Gist-brocades, The Netherlands
Solvay Enzymes GmbH, Germany
Amano Pharmaceuticals Ltd., Japan
Amano Pharmaceuticals Ltd., Japan
Durazym
Maxapem
Solvay Enzymes GmbH, Germany
Purafect
Genencor International, Inc., USA
568
Asporogenous mutant strains of Bacillus spp. are used industrially. In these strains, extracellular proteases are produced for a longer duration as the end products are not diverted towards sporulation. A fivefold increase in the yield of enzyme was observed by the use of alkaline protease-positive asporogenic mutants [25,88].
The advent of protein engineering and sophisticated molecular technologies has opened
possibilities for screening variants of enzymes and tailor-made proteins from alkalophilic
microorganisms with enhanced production yields which may be of interest for specific commercial applications. New constructions have been made by the transfer of genes between
organisms to produce high yielding variants [8994].
Further, the protein engineering approach [95] can be exploited for the improvement of alkaline proteases and/or subtilisins beyond its current limitations. Currently, two conceptionally different strategies are available for generation of protein-engineered variants: random
and site-directed mutagenesis. With random mutagenesis, a large number of variants are produced, but the success of this approach largely depends on the availability of efficient
screening procedures to identify variants with improved properties. Site-directed mutagenesis depends on the access to structural or biochemical data to reduce the number of variants
to be constructed, as every protein variant is purified and individually tested for improvements. For producing mutated enzymes, the two approaches are optimally used in combination with each other. Promising variants generated and identified by random mutagenesis often can be improved by further site-directed introduction of known advantageous mutations.
3.2. Optimization of fermentation medium
Alkaline proteases are generally produced by submerged fermentation. In addition, solid
state fermentation processes have been exploited to a lesser extent for production of these enzymes [66,96,97]. In commercial practice, the optimization of medium composition is done
to maintain a balance between the various medium components, thus minimizing the amount
of unutilized components at the end of fermentation. Research efforts have been directed
mainly toward: (i) evaluation of the effect of various carbon and nitrogenous nutrients as
cost-effective substrates on the yield of enzymes; (ii) requirement of divalent metal ions in
the fermentation medium; and (iii) optimization of environmental and fermentation parameters such as pH, temperature, aeration, and agitation. In addition, no defined medium has
been established for the best production of alkaline proteases from different microbial
sources. Each organism or strain has its own special conditions for maximum enzyme production.
3.2.1. 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% nitrogen [99] and its production is dependent on the availability of
both carbon and nitrogen sources in the medium [99]. 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 alkaline protease production were reported with the use of inorganic nitrogen sources in the production medium [40,54,56]. Enzyme synthesis was found to be re-
569
pressed by rapidly metabolizable nitrogen sources such as amino acids or ammonium ion
concentrations in the medium [100102]. However, one report indicated no repression in the
protease activity with the use of ammonium salts [103].
An increase in protease production by the addition of ammonium sulphate and potassium nitrate was also observed by Sinha and Satyanarayana [104]. Similarly, sodium nitrate (0.25%)
was found to be stimulatory for alkaline protease production [105]. Substitution of sodium nitrate in the basal medium with ammonium nitrate increased enzyme production even more
[106]. The replacement of soybean flour with ammonium sulphate in a fed-batch process
proved cost-effective, and as well resulted in the elimination of unpleasant odours [37].
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
[42] recorded maximum enzyme yields using a combination of 3% soybean meal and 1.5% bonito extract. Soybean meal was also reported to be a suitable nitrogen source for protease production [40,54,107,108]. In addition, by using an acid hydrolysate of soybean in place of conventional soymeal, a threefold increase in total enzyme activity was observed [109].
Corn steep liquor (CSL) was found to be a cheap and suitable source of nitrogen by some
workers [40,42,96]. Apart from serving as a nitrogen source, CSL also provided several micronutrients, vitamins, and growth-promoting factors. However, its use is limited by its seasonal and interbatch variability. Suitable nitrogen sources as substitutes for CSL are still being evaluated. Tryptone (2%) and casein (12%) also serve as excellent nitrogen sources
[106,110].
Addition of certain amino compounds were shown to be effective in the production of extracellular enzymes by alkalophilic Bacillus sp. [111]. However, glycine appeared to have
inhibitory effects on both amylase and protease production. Casamino acids were also found
to inhibit protease production [110]. In some studies, use of oil cakes as a nitrogen source did
not favor enzyme production [40,104].
3.2.2. Carbon source
Studies have also indicated a reduction in protease production due to catabolite repression
by glucose [99,101,112,113]. On the other hand, Zamost et al. [88] correlated the low yields
of protease production with the lowering of pH brought about by the rapid growth of the organism. In commercial practice, high carbohydrate concentrations repressed enzyme production. Therefore, carbohydrate was added either continuously or in aliquots throughout the
fermentation to supplement the exhausted component and keep the volume limited and
thereby reduce the power requirements [83].
Increased yields of alkaline proteases were reported by several workers who used different sugars such as lactose [96], maltose [114], sucrose [106] and fructose [40]. However, a
repression in enzyme synthesis was observed with these ingredients at high concentrations.
Whey, a waste byproduct of the dairy industry containing mainly lactose and salts, has been
demonstrated as a potential substrate for alkaline protease production [47,115]. Similarly,
maximum alkaline protease secretion was observed in Thermomonospora fusca YX, which
used pure cellulose (Solka-floc) as the principal carbon source [116].
Various organic acids, such as acetic acid [117], methyl acetate [118] and citric acid or sodium citrate [119,120] have been demonstrated to increase production of proteases at alka-
570
line pHs. The use of these organic acids was interesting in view of their economy as well as
their ability to control pH variations. n-paraffins were also found to be used by Fusarium sp.
for the production of increased amounts of alkaline proteases [121].
3.2.3. Metal ion requirement
Divalent metal ions such as calcium, cobalt, copper, boron, iron, magnesium, manganese,
and molybdenum are required in the fermentation medium for optimum production of alkaline proteases. However, the requirement for specific metal ions depends on the source of enzyme. The use of AgNO3 at a concentration of 0.05 mg/100 ml or ZnSO4 at a concentration
of 125 mg/100 ml resulted in an increase in protease activity in Rhizopus oryzae [105,122].
Potassium phosphate has been used as a source of phosphate in most studies
[37,78,82,123]. 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 [82]. When the phosphate concentration was more than 4 g/l, precipitation of the medium on autoclaving was observed [124]. This problem, however, could be overcome by the
supplementation of the disodium salt of EDTA in the medium [125]. In at least one case, the
salts did not have any effect on the protease yields [106].
3.2.4. pH and temperature
The important characteristic of most alkalophilic microorganisms is 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 period [83]. The advantage in the use of carbonate in the medium for an
alkaline protease has been well demonstrated [14].
The culture pH also strongly affects many enzymatic processes and transport of various
components across the cell membrane [82]. When ammonium ions were used, the medium
turned acidic, while it turned alkaline when organic nitrogen, such as amino acids or peptides
were consumed [124]. The decline in the pH may also be due to production of acidic products [82]. 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 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 [127]. However, studies by Frankena et al. [101] showed that a link existed between enzyme synthesis and energy metabolism in bacilli, which was controlled by temperature and oxygen uptake. The optimum temperature values reported for maximum protease
production are given in Table 5.
3.2.5. 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) variations in the agitation speed of the bioreactor; or (iii) use of oxy-
571
Table 5
Optimum temperature values for maximum protease production
Optimum temperature (8C)
Organism [Reference]
28
30
Penicillum griseofulvin [321]

Bacillus sp. B21-2 [42]
Streptomyces diastaticus [56]
Aspergillus flavus [96]
Bacillus sp. Y [219]
Bacillus sp. MK5-6 [120]
Bacillus licheniformis [37]
Bacillus sp. strain GX6638 [80]
Bacillus sp. no. AH-101 [81]
B. alcalophilus subsp. halodurans KP1239 [119]
B. firmus [82]
Bacillus licheniformis [123]
Bacillus sp. strain B189 [177]
Bacillus thermoruber BT2T [135]
Bacillus lichenformis [40]
Thermoactinomycetes sp. HS682 [332]
B. stearothermophilus AP-4 [223]
B. stearothermophilus F1 [79]
32
35
36
37
39.5
40
45
52
55
60
gen-rich or oxygen-deficient gas phase (appropriate air-oxygen or air-nitrogen mixtures) as

the oxygen source [82,128]. The variation in the agitation speed influences the extent of mixing in the shake flasks or the bioreactor and will also affect the nutrient availability.
Optimum yields of alkaline protease are produced at 200 rpm for B. subtilis ATCC 14416
[126] and B. licheniformis [40]. In one study, Bacillus sp. B21-2 produced increased enzyme
titres when agitated at 600 rpm and aerated at 0.5 volume per volume per min [42]. Similarly, Bacillus firmus exhibited maximum enzyme yields at an aeration rate of 7.0 l min21
and an agitation rate of 360 rpm. However, lowering the aeration rate to 0.1 l min21 caused a
drastic reduction in the protease yields [82]. This indicates that a reduction in oxygen supply
is an important limiting factor for growth as well as protease synthesis.
3.3. Correlation between growth and protease production
The production of an enzyme exhibits a characteristic relationship with regard to the
growth phase of that organism. In general, the synthesis of protease in Bacillus species is
constitutive or partially inducible and is controlled by numerous complex mechanisms operative during the transition state between exponential growth and the stationary phase
[129,130]. The production of extracellular proteases during the stationary phase of growth is
characteristic of many bacterial species [129]. The sequence as well as the rate of enzyme
production is, however, variable with the specific organism. At early stationary phase, two or
more proteases are secreted and the ratio of the amount of the individual proteases produced
also varied with the Bacillus strains [129,131]. In several cases, the function of the enzyme is
not very clear, but its synthesis is correlated with the onset of a high rate of protein turnover
and often sporulation [126,132].
Further, the growth environment can also influence enzyme synthesis, since protease pro-
572
duction in Bacillus sp. is extracellular in nature. In one study, the effect of light on growth
and alkaline protease production in Bacillus fermentation process showed that fluorescent
light (9000 lux) induced greater cell mass with lower protease yields compared with dark
growth conditions [133].
There is little or no enzyme production during the exponential growth phase [113]. However, in the case of B. subtilis ATCC 14416 [126], and B. sphaericus BSE 18 [134], enzyme
production was growth associated and in the mid-exponential phase, and often a rapid autodeactivation process was observed after the culture reached the maximum enzyme activity
[126]. In other cases, the synthesis and secretion of the protease was initiated during the exponential growth phase, with a substantial increase near the end of the growth phase and with
maximum amounts of protease produced in the stationary growth phase [80,82,107,
119,135,136]. In addition, a requirement for a high concentration of sodium ions was observed for alkaline protease production [114].
During alkaline protease production, it was also observed that the pH of the fermented
medium dropped from alkaline to acidic; from pH 10.1 to 8.5 in the case of an alkalophilic
Bacillus strain YaB [107] and from pH 9.6 to 8.5, in the case of an alkalophilic Bacillus sp.
MK5-6 [137]. A similar observation of decline in pH was also reported by Takagi et al. [109].
3.4. 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 [138]. Furthermore, immobilized
enzymes render continuous production processes possible via packed bed reactors and may
lead to more stable biocatalysts [139]. The two main methods for immobilization are wholecell immobilization and cell-free immobilization.
3.4.1. 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. [140] and Sutar et al. [141]. Batch [142] and repeated batch [143]
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 [144].
3.4.2. 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 the purpose include bentonite [145], porous glass [146149], nylon [150] and ver-
573
miculite [151,152]. Although porous glass has been widely used, the relatively high cost of
this support has been the limiting factor for industrial application. 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 [153,154]. In one study, Srokova and Cik [155] 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 studies have addressed an increase in the thermostability profile and the
pH-activity profile of the enzyme toward the alkaline side [146,148,152]. 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 [156,157].
4. Purification of alkaline proteases

Crude preparations of alkaline proteases 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 [95,107].
4.1. 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 rapid
lowering of the temperature of the fermented medium (to below 58C) becomes indispensable to
prevent microbial contamination as well as to maintain enzyme activity and stability.
The removal of the cells, solids, and colloids from the fermentation broth is the primary
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 [83,158].
Changes in pH may also be suitable for better separation of solids [159]. 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 [160].
Flocculating agents are generally employed to effect the formation of larger flocs or agglomerates, which in turn accelerate the solidliquid separation. Cell flocculation [161] 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 flocculating agent at 150 ppm and pH 7.09.0, and gave 74% yield of alkaline
protease activity [162]. In some cases, it becomes necessary to add a bioprocessing filter aid,
such as diatomaceous earth, before filtration [160,163].
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4.2. Isolation and purification

When isolating enzymes on industrial scale for commercial purposes, the prime consideration has been the cost of production in relation to the value of the end product.
4.2.1. Concentration
Because the amount of enzyme present in the cell-free filtrate is usually low, the removal
of water is a primary objective. Recently, membrane separation processes have been widely
used for downstream processing [164]. Ultrafiltration (UF) is one such membrane process
that has been largely used for the recovery of enzymes [165167] and formed a preferred alternative to evaporation.
This pressure-driven separation process is inexpensive, results in little loss of enzyme activity, and offers both purification and concentration [168], as well as diafiltration, for salt
removal or for changing the salt composition [135,160,169]. 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.
Han and associates [170] used a temperature-sensitive hydrogel ultrafiltration for concentrating an alkaline protease. This hydrogel comprised poly (N-isopropyl-acrylamide), which changed
its volume reversibly by the changes in temperature. The separation efficiency of the enzyme was
dependent on the temperature and was 84% at temperatures of 158C and 208C. However, at temperatures above 258C, a decrease in the separation efficiency was observed.
4.2.2. Precipitation
Precipitation is the most commonly used method for the isolation and recovery of proteins
from crude biological mixtures [171]. 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 lowers 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 found wide utility
only in acidic and neutral pH values and developed ammonia under alkaline conditions [83].
Hence, the use of sodium sulphate or an organic solvent formed 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 [172].
Many reports revealed the use of acetone at different volume concentrations: 5 volumes
[39], 3 volumes [61,173], and 2.5 volumes [174], as a primary precipitation agent for the recovery of alkaline proteases. Precipitation was also reported by various workers with acetone
at different concentrations: 80% (v/v) [15,69], 66% (v/v) [175]; or 44, 66, and 83% (v/v)
[58], 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 [176].
4.2.3. Ion-exchange chromatography (IEC)
Alkaline proteases are generally positively charged and are not bound to anion exchangers
[137,159,177]. However, cation exchangers can be a rational choice and the bound molecules are eluted from the column by an increasing salt or pH gradient [332].
575
4.2.4. 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 [178] as well as purify the alkaline protease from a Bacillus sp. [179]. Other affinity matrices used were Sephadex-4-phenylbutylamine [110], casein agarose [59,135], or
N-benzoyloxycarbonyl phenylalanine immobilized on agarose adsorbents [176]. However,
the major limitations of affinity chromatography are the high cost of enzyme supports and
the labile nature of some affinity ligands, which make them unrecommendable for use at
process scale.
4.2.5. 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 H3PO4, MgSO4 [180183].
In addition, other methods, such as the use of reversed micelles for liquidliquid extraction [184], affinity precipitation [185], and foam fractionation [186] have also been employed for the recovery of alkaline proteases.
4.3. 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. Because loss of activity is encountered during storage in the factory, shipment to client(s) and/
or storage in clients 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 [187
190]. Also, to prevent contamination of the final commercial crude preparation during storage,
addition of sodium chloride at 1820% concentration has been suggested [83,191]. In certain
cases, for the purpose of convenience in handling and storage, the liquid enzyme preparations are
often brought to powder form. However, the handling of dry enzymes poses 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 [192,193]. Site-directed mutagenesis is often based on specific protein design strategies, including change of electrostatic
potential [194,195], introduction of disulfide bridges [196,197], replacement of oxidation
labile residues [198], modification of side chain interactions [199], improvement of internal packaging [200], strengthening of metal ion binding [201], reduction in unfolding entropy [202,203], residue substitution or deletion based on homology [204,205] and modification of substrate specificity [206,207].
576
5. Properties of alkaline proteases

The enzymatic and physicochemical properties of alkaline proteases from several microorganisms have been studied extensively.
5.1. Optimum pH and temperature
The optimum pH range of alkaline proteases is generally between pH 9 and 11, with a few
exceptions of higher pH optima of 11.5 [55,208,209], pH 1112 [39,137], pH 12.3 [52,210]
and pH 1213 [177]. They also have high isoelectric points and are generally stable between
pH 6 and 12 [14]. The optimum temperatures of alkaline proteases ranges from 50 to 708C.
In addition, the enzyme from an alkalophilic Bacillus sp. B189 showed an exceptionally high
optimum temperature of 858C. Alkaline proteases from Bacillus sp., Streptomyces sp. and
Thermus sp. are quite stable at high temperatures, and the addition of Ca21 further enhanced
enzyme thermostability.
5.2. Molecular masses
The molecular masses of alkaline proteases ranges from 15 to 30 kDa [211] with few reports of higher molecular masses of 31.6 kDa [212], 33 kDa [176,213]; 36 kDa [61] and 45
kDa [69]. However, an enzyme from Kurthia spiroforme had an extremely low molecular
weight of 8 kDa [70]. In some Bacillus sp., multiple electrophoretic forms of alkaline proteases were observed [137,179,214]. The multiple forms of these enzymes were the result of
nonenzymatic, irreversible deamination of glutamine or asparagine residues in the protein
molecules, or of autoproteolysis [179].
5.3. Metal ion requirement and inhibitors
Alkaline proteases requires a divalent cation like Ca21, Mg21 and Mn21 or a combination
of these cations, for maximum activity. These cations were also found to enhance the thermal
stability of a Bacillus alkaline protease [215]. It is believed that these cations protect the enzyme against thermal denaturation and play a vital role in maintaining the active conformation of the enzyme at high temperatures [47,70,216]. In addition, specific Ca21 binding sites
that influence the protein activity and stability apart from the catalytic site were described for
Proteinase K [217].
Inhibition studies give insight into the nature of the enzyme, its cofactor requirements, and
the nature of the active site [218]. In some of the studies, catalytic activity was inhibited by
Hg21 ions [79,219]. In this regard, the poisoning of enzymes by heavy metal ions has been
well documented in the literature [220].
Alkaline proteases are completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and
diisopropyl fluorophosphate (DFP). In this regard, PMSF sulfonates the essential serine residue
in the active site and results in the complete loss of activity [221]. This inhibition profile classifies these proteases as serine hydrolases [222]. In addition, some of the alkaline proteases were
found to be metal ion dependent in view of their sensitivity to metal chelating agents, such as
EDTA [70,223,224]. Thiol inhibitors have little effect on alkaline proteases of Bacillus spp., although they do affect the alkaline enzymes produced by Streptomyces sp. [55,58].
577
5.4. Substrate specificity

Although alkaline proteases are active against many synthetic substrates as well as native
proteins, reaction rates vary widely. The alkaline proteases 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 [222]. With the B-chain of insulin as substrate, the bonds most frequently cleaved by a number of alkaline proteases were
Glu4- His5, Ser9-His10, Leu15-Tyr16, Tyr16-Leu17, Phe25-Tyr26, Tyr26-Thr27 and Lys29-Ala30
[163,169,175,176,225227]. In addition, Tsai et al. [226] 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. [228] observed that the alkaline elastase from Bacillus sp. Ya-B also hydrolysed
elastin and elastase-specific substrates like succinyl-Ala3-p-nitroanilide and succinyl-AlaPro-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 enzymesubstrate 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 [229].
In keratin, the disulfide bonds form an important structural feature and prevent the proteolytic degradation of the most compact areas of the keratinous substrates. Until now, an
ability to reduce disulfide bonds has not been described for any keratinolytic enzyme
[230,231]. However, by the use of disulfide-reducing agents like thioglycolic acid or dithiothreitol (DTT), the enzymatic clevage of keratin can be accompanied by a simultaneous reduction of disulfide bonds. A thermostable alkaline protease from an alkalophilic Bacillus
sp. no. AH-101 exhibiting keratinolytic activity showed degradation of human hair keratin
with 1% thioglycolic acid at pH 12 and 708C, and the hair was solubilized within 1 h [232].
Similarly, enhanced keratin degradation after addition of DTT has also been reported for alkaline proteases of Streptomyces sp. [59,233].
6. Industrial application
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 applications currently using alkaline proteases are:
6.1. Detergent additives
Microbial alkaline proteases dominate commercial applications with a significant share of
the market captured by subtilisins and/or alkaline proteases from Bacillus spp. for laundry
detergent applications [234]. Alkaline proteases added to laundry detergents enable the re-
578
lease of proteinaceous material from stains [235]. The increased usage of these proteases as
detergent additives is mainly due to the cleaning capabilities of these enzymes in environmentally acceptable, nonphosphate detergents. In addition to improved washing efficiency,
the use of enzymes allows lower wash temperatures and shorter periods of agitation, often
after a preliminary period of soaking [236].
Ideally, proteases and other enzymes used in detergent formulations should have high activity and stability over a broad range of pH and temperature. The enzymes used should be
effective at low levels (0.40.8%) and should also be compatible with various detergent
components along with oxidizing and sequestering agents. They must also have a long shelf
life [234]. Very few published reports are available on the compatibility of the alkaline proteases with detergents [15,106,237,324]. Some cleaning applications are less demanding
than others. For instance, presoak formulations and contact lens cleaning solutions do not require the same enzyme thermal stability as an all-temperature laundry detergent.
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 forms one of the most important aspects of 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 [239,240]. The enzyme detergent preparations presently
marketed for cleaning of membrane systems are Alkazym (Novodan A/S, Copenhagen, Denmark), Terg-a-zyme (Alconox, Inc, New York, USA), Ultrasil 53 (Henkel KGaA, Dusseldorf, Germany) and P3-paradigm (Henkel-Ecolab GmbH, Dsseldorf, Germany). These enzyme-based cleaners rely on the proteases to cleave and solubilize the protein foulant. The
use of thermophilic proteases from Thermus sp. strain Rt41A and alkaline proteases from
Bacillus sp. strain MK5-6 has also been successful [137,241]. The use of a cocktail of proteases and lipases to degrade and solubilize protein and fat foulants have also proven beneficial. In addition, contact lens cleaning solutions using an alkaline protease from a marine
shipworm bacterium cleaned the contact lens at low temperatures [71,242]. In India, one
such enzyme-based optical cleaner in the form of tablets containing Subtilopeptidase A is
presently marketed by M/s Bausch and Lomb (India) Ltd.
6.2. Tannery industry
Alkaline proteases possessing elastolytic and keratinolytic activity offer an effective
biotreatment of leather, especially the dehairing and bating of skins and hides [243]. The alkaline conditions enable the swelling of hair roots and subsequent attack of proteases on the hair
follicle protein allow for easy removal of the hair. Despite the strong alkaline conditions, this
process is pleasant and safer than traditional methods using sodium sulfide treatment, which
contributes to 100% of sulfide and over 80% of the suspended solids in tannery effluents [96].
The bating following the dehairing process involves the degradation of elastin and keratin, removal of hair residues, and the deswelling of collagen, which produces a good, soft leather
mainly used for making leather clothes and goods. In addition, studies carried out by different
workers have demonstrated the successful use of alkaline proteases in leather tanning from Aspergillus flavus [96], Streptomyces sp. [244], B. amyloliquefaciens [97], and B. subtilis [245,246].
579
6.3. 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 % (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
X-ray film enables not only the silver, but also the polyester film base, to be recycled.
The alkaline proteases from Bacillus sp. B21-2 [247], Bacillus sp. B189 [78] and B. coagulans PB-77 [248] 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
[249] on the basis of kinetic studies and mechanism of enzymatic hydrolysis of the gelatin
layers on X-ray film and the resulting release of silver particles [250].
6.4. Medical uses
Collagenases with alkaline protease activity are increasingly used for therapeutic applications in the preparation of slow-release dosage forms. A new semi-alkaline 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 [251]. Similarly, Elastoterase, a preparation with high
elastolytic activity from Bacillus subtilis 316M, was immobilized on a bandage for therapeutic application in the treatment of burns and purulent wounds, carbuncles, furuncles, and
deep abscesses [252]. Furthermore, Bacillus spp. have been recognized as being safe to humans [253] and an alkaline protease having fibrinolytic activity has been used as a thrombolytic agent [173].
6.5. Food industry
Alkaline proteases can hydrolyze proteins from plants, fish, or animals to produce hydrolysates of well-defined 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 [254] and a debittered enzymatic whey protein hydrolysate
[255] were produced. In addition, the hydrolysates obtained had between two and six amino
acid residues with molecular weights not exceeding 100 kDa [256,257].
Very recently, another alkaline protease from B. amyloliquefaciens resulted in the production of a methionine-rich protein hydrolysate from chick pea protein [258]. The protein hydrolysates commonly generated from casein, whey protein and soyprotein find major application in hypoallergenic infant food formulations [259]. They can also be used for the
fortification of fruit juices or soft drinks and in manufacturing protein-rich therapeutic diets
[147,254,260,261].
In addition, protein hydrolysates having angiotensin I-converting enzyme inhibitory activity were produced from sardine muscle by treatment with a B. licheniformis alkaline protease. These protein hydrolysates could be used effectively as a physiologically functional
food that plays an important role in blood pressure regulation [262].
Further, proteases play a prominent role in meat tenderization, especially of beef. An alkaline elastase [263] and thermophilic alkaline protease [264] have proved to be successful and
580
promising meat tenderizing enzymes, as they possess the ability to hydrolyze connective tissue proteins as well as muscle fibre proteins. The tenderization process can be achieved by
sprinkling the powdered enzyme preparation or by immersion in an enzyme solution and/or
by injecting the concentrated protease preparation into the blood stream or meat. A method
has been developed in which the enzyme is introduced directly into the circulatory system of the
animal shortly before slaughter [265] or after stunning the animal to cause brain death [266].
Soluble meat hydrolysates can also be derived from lean meat wastes and from bone residues after mechanical deboning by solubilization with proteolytic enzymes. However, the
hydrolysates are usually bitter when the degree of hydrolysis is above 10%, which is needed
for sufficient solubilization. Alcalase has been found to be the most appropriate enzyme in
terms of cost, solubilization, and other relevant factors. In an optimized process with Alcalase at a pH of 8.5 and temperature of 55608C, a solubilization of 94% was achieved
[267,268]. The resulting meat slurry is further pasteurized to inactivate the enzyme, and
finds wide application in canned meat products, soups, and seasonings. The cleaned bones
may also be used as an excellent raw material for the production of gelatin.
A patented method used a specific combination of neutral and alkaline proteases for hydrolyzing raw meat. The resulting meat hydrolysate exhibited excellent organoleptic properties and can be used as a meat-flavored additive to soup concentrates. Hydrolysis of over
20% did not show any bitterness when such combinations of enzymes were used. The reason
for this may be that the preferential specificity was favorable when metalloproteinase and
serine protease were used simultaneously [269].
6.6. Waste treatment
Alkaline proteases provide potential application for the management of wastes from various food processing industries and household activities. These proteases can solubilize proteins in wastes through a multistep process to recover liquid concentrates or dry solids of nutritional value for fish or livestock [270,271].
Dalev [272] reported an enzymatic process using a B. subtilis alkaline protease in the processing of waste feathers from poultry slaughterhouses. Feathers constitute approximately
5% of the body weight of poultry and can be considered a high protein source for food and
feed, provided their rigid keratin structure is completely destroyed. Pretreatment with NaOH,
mechanical disintegration, and enzymatic hydrolysis resulted in total solubilization of the
feathers. The end product was a heavy, grayish powder with a very high protein content
which could be used as a feed additive.
Similarly, many other keratinolytic alkaline proteases were used in feed technology [273
275] for the production of amino acids or peptides [149,276], for degrading waste keratinous
material in household refuse [277], and as a depilatory agent to remove hair in bath tub
drains, which caused bad odors in houses and in public places [232].
6.7. Chemical industry
It is now firmly established that enzymes in organic solvents can expand the application of
biocatalysts in synthetic chemistry [278280]. However, a major drawback of this approach
is the strongly reduced activity of enzymes under anhydrous conditions. Thus, it is of practi-
581
cal importance to discover ways to activate enzymes in organic solvents. Some studies have
demonstrated the possibility of using alkaline proteases to catalyze peptide synthesis in organic solvents [281283]. In addition, many efforts to synthesize peptides enzymatically
have employed proteases immobilized on insoluble supports [146,147,284].
A sucrose-polyester synthesis was carried out in anhydrous pyridine using Proleather, a
commercial alkaline protease preparation from Bacillus sp. [285]. The polyester, which is
extremely water-soluble and also soluble in polar organic solvents, finds its application as a
biodegradable plastic. The Proleather also catalyzes the transesterification of D-glucose with
various acyl donors in pyridine [286].
Further, the enzyme Alcalase acted as catalyst for resolution of N-protected amino acid esters [287] and alkaline proteases from Conidiobolus coronatus were found to replace subtilisin
Carlsberg in resolving the racemic mixtures of DL-phenylalanine and DL-phenylglycine [288].
6.8. Other uses
Alkaline proteases from Conidiobolus sp. were also able to act as a substitute for trypsin
used in the preparation of animal cell cultures [289]. Further, Kwon et al. [69] reported the
use of alkaline proteases from Vibrio metschnikovii RH530 as an alternative for Proteinase K
in DNA isolation.
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Biotechnology Advances 17 (1999) 561594

Research review paper

Microbial alkaline proteases: From a

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

Some of the Gram-negative bacteria producing alkaline proteases were identified as

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

Novo Nordisk, Denmark

Novo Nordisk, Denmark

Solvay Enzymes GmbH, Germany

Genencor International, Inc., USA

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

Penicillum griseofulvin [321]

gen-rich or oxygen-deficient gas phase (appropriate air-oxygen or air-nitrogen mixtures) as

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

4. Purification of alkaline proteases

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

4.2. Isolation and purification

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

4.2.4. Affinity chromatography

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

5. Properties of alkaline proteases

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

5.4. Substrate specificity

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

6.3. Silver recovery

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

[166] Bohdziewicz J. Ultrafiltration of technical proteolytic enzymes. Process Biochem 1994;29:10918.

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561594

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