proteases alcalinas

ELSEVIER P I I : S O 9 6 0 - 8 5 2 4 ( 9 7 ) O O I 8 2 - X

Bioresource Technology 64 (1998) 175-183 © 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain 0960-8524/98 $19.00

ALKAI.INE PROTEASES: A REVIEW

Adil Anwar* & Mohammed Saleemuddin

Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India

(Received 22 September 1997; revised version received 1 November 1997; accepted 10 November 1997)

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

Key words: Alkaline proteases, industrial catalysts, stabilization, natural and engineered stabilized enzymes.

INTRODUCTION

Proteases constitute one of the most important groups of industrial enzymes, accounting for at least a quarter of the total global enzyme production sales (Layman, 1986). Proteases are by far the most important group of enzymes produced commercially and are used in the detergent, protein, brewing, meat, photographic, leather and dairy industries (Kalisz, 1988). Fibrous proteins, such as horn, feather, nails and hair, are abundantly available in nature as wastes, but these can be converted to

*Author to whom correspondence should be addressed at: Department of Microbiology B172, University of Colorado Medical School, 4200 E 9th Avenue, Denver, Co 80262, USA.

useful biomass, protein concentrate or amino acids using proteases derived from cerain micro- organisms.

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

175

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

176 A. Anwar, M. Saleemuddin

Among the inorganic nitrogen sources, 0-2% sodium nitrate was found to be the best. Metal ions (Cu 2÷, Zn 2÷, Ca 2÷, Ag 3÷, Pb 2÷, Hg 2÷) and surfactants (Tween 80, SDS, Triton X-100) acted as inducers for protease production, while fungicides and phenolics were found to be inhibitory.

Maximum protease production was obtained from the fungus Conidiobolus coronatus when the basal medium contained 1% sucrose, 0"3% sodium nitrate, 0.1% dipotassium phosphate, 0.05% magnesium sulphate, 0.1% potassium chloride and 0.001% ferrous sulphate at pH 7.0 (Phadatare et al., 1993). Cell growth and the production of the desired metabolites can be continued over a prolonged period in continuous cultures. Feasibility of steady- state operation makes continuous cultures very attractive for industrial production of cell mass and non-biomass products, using freely suspended or immobilized cells.

Studies on protease production via continuous bioreactor operations have included suspension cultures of Bacillus subtilis (Heineken & O'Connor,

1972), B. f i rmus (Helmo et al., 1985), immobilized cultures of B. lichenformis (Okita & Kirwan, 1988) and Serrata marcescens (Vuillemard et aL, 1988). Proteases produced by Bacillus sp. are by far the most important group of enzymes being exploited commercially. Some observations on protease production in continuous suspension-cultures of B. firmus NRS 783 have been documented (Moon & Parulekar, 1993). These investigators have reported a strategy providing useful guidelines in the designing of bioreactors for the mass production of alkaline protease by B. f irmus. Continuous-culture experiments were conducted by varying a single parameter at a time. It was found that an increase in dilution rate led to decreased utilization of principal carbon, nitrogen and phosphorus sources (glucose, ammonium chloride, potassium dibasic phosphate) resulting in an increase in the specific production rate of protease. Moon & Parulekar (1993) felt that the approach would be of significant benefit to industrial production on a large scale and that the use of bioreactors could also lead to a substantial

Table 1. Some industrially important alkaline proteases

Species Source pH Optimum/ Industrial application(s) Reference stability

Streptococcus sp. Bacterial 8.0 Bacillus stearothermophilus Bacterial 9.5

Tritirachium album Fungal 9.0-12.0 (proteinase T)

Tritirachium album Fungal 7"0-10"0 (proteinase R)

Conidiobolus coronatus Fungal 9"7 (alkaline proteinase B)

Bacillus sp. Y. (BYA) Bacterial 10.0-12.5 Bacillus lichenformis Bacterial 8.2

(Alcalase) Bacillus sp. (AH-101) Bacterial 12.0-13-0 Rhizopus oryzae (RO, liT, Fungal 3.0-11.0

KGP) Conidiobolus coronatus (NCI Fungal 8.5

86.8.20) Bacillus firmus Bacterial 8.0 Bacillus sp. (P-001A) Bacterial 9.5

Bacillus sp. (B 18) Bacterial 12.0 Bacillus sp. Bacterial 12.0 Bacillus sp. Bacterial 8.5 Thermus Rt 41A Bacterial 11"0 Bacillus sp. (Savinase/ Bacterial 9.0-11-0

Durazym) Bacillus licheniformis Bacterial 8"2

(Alcalase) Bacillus subtilis Bacterial 8-5

Bacillus sp. Bacterial 8.5 Bacillus subtilis Bacterial - Amycolata/Amycolatopsis - 8-11.0

Spilosoma obliqua Insect larvae 11-0 (Lepidoptera)

Dairy/cheese production Detergents and heavy duty

laundry powders Laundry detergents

formulations Laundry detergent

formulations Resolution of racemic

mixtures of D,L-phenyl alanine and glycine

Detergent formulations Catalyst for N-protected

amino acids Dehairing/leather industry ?

Commercial detergents

Detergent industry Production of biomass from

natural waste ? ? Dehairing/leather industry ? Detergent formulations

Synthesis of biologically active peptides

Bating agent in leather industry

? Contact lens cleansing agent Cheese and detergents

Commercial detergents and stain remover formulations

Van Boven et al. (1988) Sato et al. (1990)

Samal et al. (1990)

Samal et al. (1990)

Sutar et al. (1991)

Shimogaki et al. (1991) Chen et al. (1991)

Takami et al. (1992) Banerjee & Bhattacharya

(1992) Phadatare et al. (1993)

Moon & Parulekar (1993) Atalo & Gashe (1993)

Fujiwara et al. (1993) Masui et al. (1994) Loperena et al. (1994) Wilson et al. (1994) Bossi et al. (1994)

Chen etaL (1995)

Hameede taL (1996)

Sinha et al. (1996) Sanyo et al. (1996) Denmark patent no. 04, 082

(1997) Anwar & Saleemuddin

(1997)

Alkaline proteases: A review 177

improvement in protease production in a continuous-culture operation.

USES OF ALKALINE PROTEASES

Alkaline proteases in the dairy industry Lactic acid bacteria constitute a very important group in the dairy industry for the production of fermented milk products such as cheese (Thomas & Mills, 1981). For their supply of amino acids in milk, these bacteria are solely dependent on their proteolytic system for the degradation of casein, which also meets their demand for a supply of peptides. These processes contribute significantly to the development of flavour during cheese ripening (Schmidt et al., 1976). In the cheese industry, Lactobacillus sp. and Streptococcus cremoris are the two most important bacteria (De Man et al., 1960; Van Boven et al., 1988). Lactic acid bacteria have two main functions as starter bacteria in the manufacturing of cheese: (i) the acidification of milk; and (ii) the development of flavour during ripening. In both the processes proteolytic activity plays a central role (Hugenholtz et al., 1987). Owing to the importance of proteases in cheese production, in recent years much attention has been focused on these enzymes in the study of starter bacteria (Law, 1979; Doi et al., 1981; Thomas & Mills, 1981; Hugenholtz et aL, 1984; Geis et al., 1985).

The number of proteases among the strains has been found to vary between one (Law, 1979; Geis et al., 1985) and four (De Man et aL, 1960; Hugenholtz et aL, 1984) and the localization of proteases in the cell has been found to be either extracellular (Exter- kate, 1976), cell-wall associated (Schmidt et al., 1976; Law, 1978; Exterkate, 1981; Hugenholtz et aL, 1984; Geis et al., 1985), membrane bound (Otto et al., 1982), or cytoplasmic (Cliffe & Law, 1979; Cliffe & Law, 1985; Law, 1979). However, most of the investigators agree that Ca 2+ ions are important for the activity of these proteases (Exterkate, 1981; Gels et aL, 1985), although in one case Mn 2+ and Co 2+ ions were found to be necessary as co-factors (Doi et aL, 1981).

The protease from Streptococcus cremoris used in cheese production was found to be optimally active at pH 8"0, and at pH values lower than pH 5"0 no hydrolysing activity could be detected whereas at pH 10 only 50% of the activity was found (Van Boven et al., 1988), which implies the importance of the alkaline nature of ,proteases in the dairy industry. The role of lactic acid bacteria in the fermentation of different foods, the composition of the fermentation, flora, metabolic activities and their use as starters in cheese production have been reviewed recently (Luecke et al., 1996). It is noteworthy to mention that proteolytic enzymes from Amycolata sp. and Amycolatopsis sp., having alkaline pH optima

(8-11), are also currently in use for the production of cheese (Denmark patent No. 1997, 04,082).

Degradation of proteinaceous waste into useful biomass by alkaline proteases Fibrous proteins such as horn, feather, nail and hair are abundantly available in nature as waste. This waste can be converted into useful biomass, protein concentrate or amino acids using proteases from certain micro-organisms. Venugopal et al. (1989) immobilized cells from B. megaterium in calcium alginate and the extra-cellular proteases secreted by these cells were useful to solubilize fish meat. Dalev & Simeonova (1992) employed alkaline protease from B. subtilis for the complete utilization of the main waste of the leather industry for the production of useful products Such as animal glue, which could be used as a high quality glue in carpentry, and protein concentrate for fodder and animal tallow. An alkaline protease from a thermophilic Bacillus sp. (P 001A) has been isolated (Atalo & Gashe, 1993). The protease from this bacterium was found to be stable over a pH range of 4"5-11.5 with a pH optimum of 9.5. It was maximally active at 55°C and could hydrolyse 90, 60, 50% of skin, feather and horn. This protease appears to have all the properties of a good industrial catalyst for the production of amino acids from protein concentrate. More recently Dalev (1994) reported the use of alkaline protease from B. subtilis in processing waste feathers from poultry slaughter houses. Feathers constitute about 5% of the body weight of poultry and can be considered as a high protein source for food and feed provided their rigid keratin is destroyed completely. The solubilization of feathers was achieved by pretreatment with NaOH, mechan- ical disintegration and enzymic hydrolysis, resulting in a useful end product with a very high protein content which could be used mainly as a feed constituent.

Alkaline proteases in the leather industry Another industrial process which has received attention is the enzyme assisted de-hairing of animal hides and skin in the leather industry. Traditionally, this process is carried out by treating animal hides with a saturated solution of lime and sodium sulphide. Besides being expensive and particularly unpleasant to carry out, a strongly polluting effluent is produced. The alternative to this process is enzyme-assisted de-hairing. Enzyme-assisted de-hairing is preferentially possible if proteolytic enzymes can be found that are stable and active under the alkaline conditions (pH 12) of tanning.

Early attempts using a wide variety of enzymes were largely unsuccessful, but proteases from certain bacteria which are alkalophilic in nature have been shown to be effective in assisting the hair-removal process (Horikoshi & Akiba, 1982; Sharpe & Munster, 1986). Several alkaline proteases from


alkalophilic actinomycetes have also been investigated for this purpose. Some of these have been shown to be particularly active against keratinous proteins such as hair, feather, wool, etc. at alkaline pH and may have commercial applications (Sharpe & Munster, 1986).

The substitution of chemical depilatory agents in the leather industry by proteolytic enzymes produced by Bacillus sp., could have important economical and environmental impacts. A Bacillus sp. exhibiting promising depilatory activity has been isolated and optimal conditions for the maximum protease production have been worked out in laboratory bioreactors (Loperena et al., 1994). The alkalophilic Bacillus sp. No. AH-101 produces an extremely thermostable alkaline serine protease that has a high pH optimum (12-13) and possesses keratinolytic activity (Takami et al., 1992). Using a different approach, these workers have cloned the gene encoding the protease in Escherchia coli and expressed it in B. subtilis. The cloned protease was identical to AH-101 protease in its optimum thermostability in highly alkaline environments and suitable for use in tanneries. In a recent study, Masui et al. (1994) investigated the rational shift of the optimum pH and the enhancement of thermostability of Bacillus sp. B18'. The protease gene was cloned and an even more thermostable mutant enzyme was created by introducing a point mutation when the position in j~-turn Thr-203 was replaced by proline. The enzyme was optimally active in alkaline conditions (pH 12-13) hence the protease has the potential of replacing chemical depilatory agents in the leather industry. In a more recent study, Hameed et al. (1996) employed an alkaline protease from a B. subtilis isolate as bating agent for the production of high quality leather. Increase in the tensile, bursting and tear strengths, along with elongation at breaking of leathers is indicated with the increasing amounts of alkaline protease used in the bating process.

Alkaline proteases in detergent formulations The detergent industry has now emerged as the single major consumer of several hydrolytic enzymes acting in the alkaline pH range. Detergents containing different enzymes; proteases, amylases and lipases are available in the international markets under several brand names. The use of different enzymes as detergent additives arises from the fact that proteases can hydrolyse proteinaceous stains, amylases are effective against starch and other carbohydrate stains while lipases are effective against oily or fat stains. F o r an enzyme to be used as a detergent additive it should have two qualities: it should have an alkaline pH and it should also be compatible with detergents. The major use of detergent-compatible proteases is in laundry detergent formulations (Anstrup & Andersen, 1974; Van T//burg, 1984). Detergents available in the international market such as Dynamo ® , Era plus ®

(Procter & Gamble), Tide ® (Colgate Palmolive) contain proteolytic enzymes the majority of which are produced by members of the genus Bacillus. Although subtilisins have been the enzymes of choice in detergent formulations (US patent Nos 1240058, 370482, 374971, 4266031 and UK patent No. 13155937), these are not ideal detergent enzymes due to their low stability in presence of detergents (Samal et al., 1990).

To overcome the problem of low stability of subtilisins in detergent formulations Von der Osten et al. (1993) successfully employed site-directed mutagenesis in the construction of subtilisin variants with improved storage and oxidation stabilities. Sato et al. (1990) investigated the effects of various poly- (styrene sulfonate methacrylate) copolymers, poly- acrylate and tripolyphosphate, anionic builders, as well as poly-(vinyl alcohol vinyl acetate) non-ionic co-polymers, on the proteolytic activity of B. stearothermophilus (Toyozyme NP), which is a detergent additive. It was found that the addition of anionic builders lowered the activity, while the non-ionic co-polymeric builders enhanced the activity. In an interesting study, Samal et al. (1990) separated two novel proteases from the fungus Tritirachium album and the proteases, termed as 'R' and 'T', were checked for their thermal and detergent stabilities. The pH optimum of the protease R was between 7.0 and 10.0, while the protease T was maximally active between pH 9.0 and 12.0. The optimal temperature for both proteases R and T was 55 to 60°C. Stability of these two proteases in the presence of commercial detergents was investigated by incubating the enzyme at 50°C in the presence of laundry detergents of different compositions. The stability was determined in three laundry detergents containing proteases (Eraplus ~, Tide ®, Dynamo ®) and in detergent (Wisk ®) that does not contain any protease. The stability of protease T was excellent in all the detergent formulations in which the endogenous enzyme had been destroyed by heat prior to the addition of protease T. Protease R was also found to be extremely stable, retaining 90% of its original activity after 1 h incubation at 50°C in Eraplus ® or Dynamo ~. The stability of protease R was, however, found to be less in detergent Tide ® , where it lost 50% of its activity after 1 h. The activity of protease T tested in the presence of laundry detergent Wisk ®, which does not contain any endogenous enzyme, was found to be 90% after 1 h at 50°C, while in similar conditions protease R lost up to 50% of its activity after 10 min. The use of protease T in all the cases and that of R in most of the cases would lead to use of less enzyme in detergent formulations. Because of the thermal stability and detergent compatibility, these two novel proteases, especially protease T, could have diverse industrial applications in addition to use in detergent formulations (Samal et al., 1990).


In the course of a search for an alkaline-stable protease for industrial use, an alkaline protease was isolated from the Bacillus sp. Y (Shimogaki et al., 1991). The protease from the Bacillus sp. Y was found to have an optimum pH of 10.0-12.5. In addition, it was also very stable towards surface- active agents, such as sodium dodecyl sulfate (SDS) and sodium linear alkyl benzene sulfonate, implying its potential for use in detergent formulations.

Although bacterial proteases have long been used in detergents, the main drawback in their use is that they require cost-intensive filtration methodologies to obtain a microbe-free enzyme preparation. However, proteases from fungal origin offer an advantage that mycelium can be easily removed by filtration techniques. Phadatare et al. (1993) investigated the properties of an alkaline protease from Conidiobolus coronatus (NCL 86.8.20) related to enzyme production and compatibility with commercial detergents. The optimized fermentation conditions for maximum protease production in order to make its industrial application feasible economically, particularly in detergent formulations have also been reported (Phadatare et al., 1993).

An ideal detergent enzyme should be stable at high pH and temperatures up to 40°C, withstand oxidizing and chelating agents, and be effective at low enzyme levels in detergent solutions. Moreover, it should also have broad substrate specificity. The alkaline protease from C. coronams showed a high level of stability up to pH 8.5, no loss in activity was detected up to 40°C and it was also compatible with most of the detergents tested. The protease was also able to hydrolyse various protein substrates tested. The data obtained in the study (Phadatare et al., 1993) implies that the protease of C. coronatus has all the potential to be used as a detergent enzyme.

An alternative source of detergent compatible enzymes could be insect guts. The advantages being that cost-intensive filtrations are not involved as in the case of bacterial and fungal sources. Taking these things into consideration, in a more recent study from our laboratory (Anwar & Saleemuddin, 1997) we reported on the alkaline pH-acting digestive enzymes from the polyphagus insect pest Spilosoma obliqua. Their stability and potential as detergent additives have also been discussed. The protease from the insect source was found to be active at pH 11.0 and had a temperature optimum of 50°C. An ammonium sulfate (60% saturation) fraction from the larval guts of S. obliqua was found to be highly effective in facilitating the removal of old blood stains from cotton cloth both in the presence and absence of detergents.

Anwar (1996) also showed that the purified protease from the S. obliqua larval guts was compatible with most of the commercial detergents tested, albeit to differing extents, while its broad substrate specificity, as demonstrated by the cleavage of

different proteins, implied its effectiveness against a wide variety of stains. The data obtained from both studies imply that the larval protease has all the potential to be used as a detergent additive and as a stain remover. Although the economics of using an enzyme from the insect source may not appear feasible there is always a possibility of cloning the enzyme (Von der Osten et al., 1993). A low-foaming liquid detergent containing proteolytic enzymes with longer shelf-life has been reported (Okamoto et al., 1997). This liquid detergent is currently in use as a dish-washing detergent and is available commercially in the international market.

Studies on the possible use of enzymes in detergents should also be directed towards their stability when formulated into detergent liquids or powders in concentrated form as sold in the market. However, caution must also be exercised in the source of enzymes to be used as detergent additives as some of these might cause allergic reactions in humans.

Alkaline proteases in the synthesis and resolution of o,L-amino acids Amino acids are of increasing importance as dietary supplements for both humans and domestic animals. Only the L-amino acids can be assimilated by living organisms and since the chemical synthesis of amino acids produces a racemic mixture it is all the more necessary to separate the isomers before commercial use. Resolution is one of the best ways to produce optically pure unnatural amino acids. An extracellular low molecular weight protease (6800) has been purified to homogeneity from the culture filtrate of C. coronatus (NCIM 1328) by Sutar et al. (1991). The protease was found to have a pH optimum of 9"7 and exhibited esterolytic acitivity on N-benzoyl-L-tyrosine ethyl ester (BTEE) and it was successfully employed to resolve the racemic mixture of D,L-phenyl alanine and D,L- phenyl glycine and can thus potentially replace subtilisin carlsberg in resolving the racemic mixture of amino acids (Sutar et al., 1991).

Kinetic resolution of N-protected amino acid esters in organic solvents catalysed by a stable industrial alkaline protease 'Alcalase' has been reported (Chen et al., 1991). Alcalase is a proteolytic enzyme isolated from a selected strain of B. licheniformis, its major component being subtilisin carlsberg. It was found that Alcalase was stable in organic solvents and capable of use as a catalyst in the resolution of N-protected amino acids having unusual side chains. Two resolution procedures, hydrolysis in aqueous solution with dioxan as co-solvent and hydrolysis in t-butanol containing 5% phosphate buffer have been discussed in detail elsewhere (Chen et al., 1991).

In a recent study, Tsuchiya et al. (1993) found that a serine alkaline protease from Cephalosporum sp. KM 388 was specific against esters of aromatic


and hydrophobic amino acids. The cleavage specificity of KM 388 protease D was broader than those of other alkaline proteases and the site of Arg-Gly (22-23) was cleaved, which is a specific site for trypsin-like proteases. In a more recent study Chen et al. (1995) reported a useful procedure for the synthesis of biologically active peptides using (subtilisin carlsberg) Alcalase.

LIMITATIONS

The overall potential of alkaline proteases in industrial process is yet to be exploited fully. The inherent disadvantages in the use of proteases, in particular, are related to thermal, operational and storage problems as they are easily prone to inactivation by self-digestion (autolysis), whereas a good industrial catalyst should be stable under the toughest operating conditions and for long durations. To overcome such limitations great attention has been devoted to the problem of enzyme stabilization.

STABILIZATION OF ALKALINE PROTEASES

Stabilization of enzymes by immobilization can be categorized according to whether the protein becomes immobilized by chemical binding or by physical retention. The most widely used immobilization techniques are based on the binding of enzyme molecules to carriers by covalent bonds, or by adsorptive interactions, entrapment into gels/ beads/fibres, cross-linking or co-cross-linking with bifunctional reagents, encapsulation in micro- capsules or membranes.

Based on these, numerous methods have been employed to immobilize proteolytic enzymes and stabilize them against pH, thermal, chemical denaturants and storage inactivation (Martinek et al., 1977; Church et al., 1992; Kumakura et al., 1984; Dua et al., 1984, 1985; Kovalenko & Sokolovsky, 1987; Blanko & Guisan, 1988). Information related to the stabilization of proteases such as carboxy- peptidase (Dua et al., 1985), chymotrypsin (Matveeva et al., 1985), pronase (Church et al., 1992), trypsin (Reddy et al., 1986) and other proteases (Johnson et al., 1990; Gusek & Kinsella, 1990) is available in the literature.

Certain factors such as cost, reuse and stability profoundly influence the applications of immobilized enzymes as industrial catalysts by giving an edge over their soluble counterparts, as immobilization confers additional stability to an enzyme and it can also be reused. It is possible to recover the immobilized enzymes by centrifugation or filtration. This procedure is especially recommended when the enzyme used is expensive as its reuse would reduce operational cost. A process involving the use of immobilized papain to eliminate the chill haze caused by the proteins present in beer is in vogue. An industrially important alkaline protease from Trichoderma koningii immobilized by cross-linking with glutaraldehyde could be stored in distilled water at 4°C for 60 days or recycled eight times in enzymic reaction with practically no loss in activity (Manonmani & Joseph, 1993). However, the data available on the stabilization of alkaline proteases is scanty (Table 2). One possible reason which can be attributed to this discrimination is that alkaline proteases are industrially important and are prefer- ably used in soluble forms.

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

Enzyme Stabilizing support/ Stabil i ty/improvement Reference modification against

Alkaline protease (EC. 3.4. (a) Acylation with succinic Autolysis Maneepun & Klibanov group) and maleic anhydrides (1982)

(b) Aluminium oxide/silica Kovalenko & Sokolovsky gel (1987)

Hydroxyl-ethyl-acrylate Kumakura et al. (1984) Thermolysin (EC. 3.4.24.4. group)

Subtilisin BPN' Subtilisin (EC 3.4. group) Subtilisin BPN' Subtilisin carlsberg Alkaline protease (Thermus

Rt 41A) Alkaline protease

( Trichoderma koningii) Subtilisin

Bacillus sp. B19' Alkaline protease Alkaline protease Alkaline protease

tetradeca-ethylene glycol Site-directed mutagenesis

Porous chitosan beads Porous chitosan beads Controlled pore glass beads

Glutaraldehyde

Site-directed mutagenesis

Site-directed mutagenesis Cellulose co-polymer Polyurethane Glutaraldehyde

Temperature

Temperature

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

pH/Temperature

Storage and oxidation stabilities

Thermal stability Improved shelf-life ? Temperature

Pantoliano et al. (1987) Zaks & Klibanov (1988) Kise & Hayakawa (1991) Kise & Hayakawa (1991) Wilson et al. (1993)

Manonmani & Joseph (1993)

Von der Osten et al. (1993)

Masui et al. (1994) Virnik et al. (1996) Grajek et al. (1996) Anwar & Saleemuddin

(1997)


Detrimental aspects of stabilization Although enzyme stabilization is now a pressing problem in biotechnological applications, stabilization of enzymes in soluble form has always been an important desirable goal. Increased attention has been focussed on the stabilization of enzymes in soluble forms, and many efforts have been made in search of new and different methods to obtain soluble, but stabilized, enzymes (Schmidt, 1979; Gray, 1988), as it is impossible to use insoluble enzymes in several biotechnological applications including detergent, food, cosmetic and textile industries.

FUTURE STRATEGIES

The future strategies in the identification of industrially important, stable enzymes, particularly proteases could be to search for naturally occurring enzymes with intrinsic stability or to produce stable enzymes by means of protein engineering. These strategies are based upon two different but corre- lated approaches.

Naturally occurring, stable enzymes The simplest approach to obtain a stable enzyme is to look for the desired enzyme in a readily available organism. During the last few years many enzymes have been isolated from alkalophilic/thermophilic organisms which are capable of surviving in extremes of pH and temperature and are also stable against a wide variety of denaturants such as urea and detergents (Cowan & Daniel, 1982; Wojtczak, 1987; Takii et al., 1987). Hence great interest has been generated in the search for new thermophilic and alkalophilic strains for industrial applications.

Production of stable enzymes by enzyme engineering There seems litle doubt that new organisms capable of secreting industrially important enzymes will continue to be discovered and those which are already known have to be exploited practically. Subtle alterations in the amino acid sequences, using techniques such as site-directed mutagenesis, could be beneficial in producing enzymes for industrial applications. Indeed site-directed mutagenesis experiments have produced subtilisins (Wells & Powers, 1986; Pantoliano et al., 1987; Von der Osten et al., 1993) and other enzymes (Liao et al., 1986; Matsumura et al., 1986; Bae et al., 1987; Heslot et al., 1987; Scrutton et aL, 1988; Masui et al., 1994) with enhanced stability in soluble form.

CONCLUSIONS

In the present review we have tried to present a general view of the wide and complex matter of the industrial applications of alkaline proteases. It is hoped that such a review will be an introduction to

the problem of alkaline proteases in industry for newcomers to the field and will also encourage those who have already thought about the problem to consider the new experimental approaches discussed. Our apologies if just a part of the existing ocean of literature has been mentioned and for other unintentional omissions.

ACKNOWLEDGEMENTS

Financial assistance provided by the University Grants Commission, Government of India, in the form of a fellowship to one of us (A.A.) is thankfully acknowledged.

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Bae, M., Hwang, J. & Park, S. (1987). Molecular cloning and expression of alkaline amylase gene of alkalophilic Bacillus sp. AI-8 and enzyme properties in E. coli. Korean J. Appl. Microbiol. Bioeng., 15, 441-445.

Banerjee, R. & Bhattacharyya, B. (1992). Extracellular alkaline protease by newly isolated Rhizopus oryzae. Biotechnol. Lett., 14, 301-304.

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