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Biotechnology Advances 17 (1999) 561–594 Research review paper Microbial alkaline proteases: From a bioindustrial

Biotechnology Advances 17 (1999) 561–594

Biotechnology Advances 17 (1999) 561–594 Research review paper Microbial alkaline proteases: From a bioindustrial

Research review paper

Microbial alkaline proteases: From a bioindustrial viewpoint

C. Ganesh Kumar a, *, Hiroshi Takagi b

a Dairy Microbiology Division, National Dairy Research Institute, Karnal 132 001, India b 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 af- fecting 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 en- zymes 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: S0734-9750(99)00027-0

562 C.G. Kumar, H. Takagi / Biotechnology Advances 17 (1999) 561–594

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 cata- lytic role in the hydrolysis of proteins. In 1994, the total market for industrial enzymes ac- counted 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 en- zymes 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 mi- croorganisms 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 ex- tremophiles. Alkalophilic microorganisms constitute a diverse group that thrives in highly alkaline en- vironments. 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 ex- cess 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 neutral- ity, 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-occur- ring 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 [11,13–15]. 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, tex- tiles, 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

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unique microbial population [18–22]. 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. More- over, in this category there are also moderate thermophiles, with growth temperatures of ap- proximately 40 C.

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 ex- ists 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 cyclo- dextrin glucanotransferase for cyclodextrin manufacture from starch [27,28]. These indus- trial 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 or- ganisms 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, po- tassium carbonate, sodium borate, and sodium orthophosphate or the occasional addition of sodium hydroxide [17,31]. The addition of Na 2 CO 3 to the medium for the isolation of alkalo- philic thermophiles results in brown color and cracking of the medium [32]. At temperatures of 70 C, 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-

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ucts is to grow a large number of organisms on agar plate media and to relate each organ- ism’s production capability to the radius of the product’s zone of diffusion around the colony. In the course of designing a medium for screening proteases, it is essential that the medium

should contain likely inducers of the product and be devoid of constituents that may repress en- zyme synthesis. It has been reported that B. licheniformis produces very narrow zones of hy- drolysis 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 me- dium and subsequent screening for the desired characteristics. The organisms are further

grown on specific media for estimating proteolytic, amylolytic, lipolytic, or cellulolytic ac- tivities 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 screen- ing 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 indus- trial applications, members of the genus Bacillus were found to be predominant and a pro- lific 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 spe- cies 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 al- kaline protease producing alkalophilic actinomycetes [48]. The different species of Strepto- myces 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 130 [55]; S. diastaticus SS1 [56,57]; S. corcho- rusii ST36 [58], and S. pactum DSM 40530 [59]. Other types of alkalophilic actinomycetes include Nocardiopsis dassonvillei [60–62] and Oerskovia xanthineolytica TK-1 [63].

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Table 1 Alkaline protease-producing Bacillus species

565

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 [101,113,123,295–297]

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 pro- ducing 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 al- kalopsychrotrophic and alkalopsychrophilic bacteria represent a new potential source for alka- line proteases [76]. These organisms are characterized by their adaptation to both cold temper- atures 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 60 C [78,79], with a few exceptions of 60 C [80,81]. Thermostable alkaline proteases from various thermophilic alkalophiles are listed in Ta-

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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 [308–310]

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 condi- tions promoting cell growth [82]. In the industrial production of alkaline proteases, technical media were usually employed that contained very high concentrations (100–150 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 fermenta- tion medium and production conditions.

3.1. Improvement of yield

Strain improvement plays a key role in the commercial development of microbial fermen- tation processes. As a rule, the wild strains usually produce limited quantities of the desired

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Table 3 Microorganisms producing thermostable alkaline proteases

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Organism [References]

Bacillus licheniformis [32,40,104] Bacillus thermoruber BT2T [135] Bacillus sp. strain B18 [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 im- proved by the use of mutagens or antibiotics and the adoption of special techniques or proce- dures for detecting useful mutants. Shah et al. [86] developed a cysteine auxotrophic mutant of B. licheniformis with im- proved 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, in- creased yields of alkaline proteases have been achieved by Bacillus mutants that were resis- tant to antibiotics such as vancomycin and ristocetin [87].

Table 4 Commercial producers of alkaline proteases

Organism

Trade names

Manufacturer

Bacillus licheniformis Alkalophilic Bacillus sp. Alkalophilic Bacillus sp. Alkalophilic Bacillus sp. Alkalophilic Bacillus sp. Aspergillus sp. Protein engineered variant of Savinase ® Protein engineered variant of alkalophilic Bacillus sp. Genetic engineered Donor—B. lentus Expressed in Bacillus sp.

Alcalase

Novo Nordisk, Denmark Novo Nordisk, Denmark Gist-brocades, The Netherlands Solvay Enzymes GmbH, Germany Amano Pharmaceuticals Ltd., Japan Amano Pharmaceuticals Ltd., Japan

Savinase, Esperase

Maxacal, Maxatase

Opticlean, Optimase

Proleather

Protease P

Durazym

Novo Nordisk, Denmark

Maxapem

Solvay Enzymes GmbH, Germany

Purafect

Genencor International, Inc., USA

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Asporogenous mutant strains of Bacillus spp. are used industrially. In these strains, extra- cellular proteases are produced for a longer duration as the end products are not diverted to- wards sporulation. A fivefold increase in the yield of enzyme was observed by the use of al- kaline 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 com- mercial applications. New constructions have been made by the transfer of genes between organisms to produce high yielding variants [89–94]. Further, the protein engineering approach [95] can be exploited for the improvement of al- kaline proteases and/or subtilisins beyond its current limitations. Currently, two conception- ally different strategies are available for generation of protein-engineered variants: random and site-directed mutagenesis. With random mutagenesis, a large number of variants are pro- duced, but the success of this approach largely depends on the availability of efficient screening procedures to identify variants with improved properties. Site-directed mutagene- sis 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 improve- ments. For producing mutated enzymes, the two approaches are optimally used in combina- tion with each other. Promising variants generated and identified by random mutagenesis of- ten 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 en- zymes [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 parame- ters 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 pro- duction.

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 pro- tease 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 sup- plement differs from organism to organism. Low levels of alkaline protease production were reported with the use of inorganic nitro- gen sources in the production medium [40,54,56]. Enzyme synthesis was found to be re-

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pressed by rapidly metabolizable nitrogen sources such as amino acids or ammonium ion concentrations in the medium [100–102]. 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 ni- trate 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 ni- trate 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 lead- ing 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% bo- nito extract. Soybean meal was also reported to be a suitable nitrogen source for protease pro- duction [40,54,107,108]. In addition, by using an acid hydrolysate of soybean in place of con- ventional 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 mi- cronutrients, vitamins, and growth-promoting factors. However, its use is limited by its sea- sonal and interbatch variability. Suitable nitrogen sources as substitutes for CSL are still be- ing evaluated. Tryptone (2%) and casein (1–2%) also serve as excellent nitrogen sources

[106,110].

Addition of certain amino compounds were shown to be effective in the production of ex- tracellular 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 or- ganism. In commercial practice, high carbohydrate concentrations repressed enzyme produc- tion. 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 differ- ent 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 so- dium citrate [119,120] have been demonstrated to increase production of proteases at alka-

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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 alka- line proteases. However, the requirement for specific metal ions depends on the source of en- zyme. The use of AgNO 3 at a concentration of 0.05 mg/100 ml or ZnSO 4 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 ex- cess of this concentration showed an inhibition in cell growth and repression in protease pro- duction [82]. When the phosphate concentration was more than 4 g/l, precipitation of the me- dium 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 depen- dence 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 through- out 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 prod- ucts [82]. In view of a close relationship between protease synthesis and the utilization of ni- trogenous 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 organ- ism 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 be- tween enzyme synthesis and energy metabolism in bacilli, which was controlled by tempera- ture 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-

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Table 5 Optimum temperature values for maximum protease production

Optimum temperature ( C)

Organism [Reference]

28

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]

30

32

35

36

Bacillus sp. strain GX6638 [80] Bacillus sp. no. AH-101 [81]

37

B.

alcalophilus subsp. halodurans KP1239 [119]

B.

firmus [82]

39.5

Bacillus licheniformis [123] Bacillus sp. strain B18 [177] Bacillus thermoruber BT2T [135] Bacillus lichenformis [40]

40

45

52

Thermoactinomycetes sp. HS682 [332]

55

B.

stearothermophilus AP-4 [223]

60

B.

stearothermophilus F1 [79]

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 mix- ing 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]. Simi- larly, Bacillus firmus exhibited maximum enzyme yields at an aeration rate of 7.0 l min 1 and an agitation rate of 360 rpm. However, lowering the aeration rate to 0.1 l min 1 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 oper- ative 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-

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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]. How- ever, 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 au- todeactivation 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 ex- ponential 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 ob- served 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 ad- vantages they confer over their soluble counterparts, including increased stability to temper- ature, pH, and organic solvents; recovery and reuse of the enzyme; and, in the case of pro- teases, 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 whole- cell 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 fermenta- tion. 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 im- proved 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 em- ployed for the purpose include bentonite [145], porous glass [146–149], nylon [150] and ver-

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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 immobili- zation of the alkaline proteases on these supports using glutaraldehyde involves covalent at- tachment 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] success- fully immobilized an alkaline protease onto a gel of O-hydroxyethylcellulose through photo- chemical 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 par- tition 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. Nev- ertheless, the purification of alkaline proteases is important from the perspective of develop- ing a better understanding of the functioning of the enzyme [95,107].

4.1. Recovery

After successful fermentation, when the fermented medium leaves the controlled environ- ment 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 5 C) 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 continu- ous 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 ag- glomerates, which in turn accelerate the solid–liquid 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 or- ganic polyelectrolytes. For example, the use of a polyelectrolyte Sedipur TF 5 proved to be an effective flocculating agent at 150 ppm and pH 7.0–9.0, 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 consider- ation 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 [165–167] and formed a preferred al- ternative to evaporation. This pressure-driven separation process is inexpensive, results in little loss of enzyme ac- tivity, 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 un- derlying 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 de- tergents, proteases, or acids and alkalies. Han and associates [170] used a temperature-sensitive hydrogel ultrafiltration for concentrat- ing 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 15 C and 20 C. However, at tem- peratures above 25 C, 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 solubil- ity 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 re- covery 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 mole- cules are eluted from the column by an increasing salt or pH gradient [332].

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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 af- finity 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 H 3 PO 4 , MgSO 4 [180–183].

In addition, other methods, such as the use of reversed micelles for liquid–liquid extrac- tion [184], affinity precipitation [185], and foam fractionation [186] have also been em- ployed 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 maxi- mum stability of crude preparations may be quite different than for purified enzymes. Be- cause loss of activity is encountered during storage in the factory, shipment to client(s) and/ or storage in client’s facilities, storage stability is of prime concern to enzyme manufacturers. Protease solutions are subject to proteolytic and autolytic degradation that results in rapid inac- tivation 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 18–20% 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 haz- ards 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 BPN were improved by random mutagen- esis followed by application of proper screening assays [192,193]. Site-directed mutagene- sis 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 inter- nal packaging [200], strengthening of metal ion binding [201], reduction in unfolding en- tropy [202,203], residue substitution or deletion based on homology [204,205] and modifi- cation of substrate specificity [206,207].

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5. Properties of alkaline proteases

The enzymatic and physicochemical properties of alkaline proteases from several micro- organisms 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 11–12 [39,137], pH 12.3 [52,210] and pH 12–13 [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 70 C. In addition, the enzyme from an alkalophilic Bacillus sp. B18 showed an exceptionally high optimum temperature of 85 C. Alkaline proteases from Bacillus sp., Streptomyces sp. and Thermus sp. are quite stable at high temperatures, and the addition of Ca 2 further enhanced enzyme thermostability.

5.2. Molecular masses

The molecular masses of alkaline proteases ranges from 15 to 30 kDa [211] with few re- ports 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 pro- teases 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 Ca 2 , Mg 2 and Mn 2 or a combination of these cations, for maximum activity. These cations were also found to enhance the thermal stability of a Bacillus alkaline protease [215]. It is believed that these cations protect the en- zyme against thermal denaturation and play a vital role in maintaining the active conforma- tion of the enzyme at high temperatures [47,70,216]. In addition, specific Ca 2 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 Hg 2 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 classi- fies 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., al- though they do affect the alkaline enzymes produced by Streptomyces sp. [55,58].

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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, -chymotrypsin [222]. With the B-chain of in- sulin as substrate, the bonds most frequently cleaved by a number of alkaline proteases were Glu 4 - His 5 , Ser 9 -His 10 , Leu 15 -Tyr 16 , Tyr 16 -Leu 17 , Phe 25 -Tyr 26 , Tyr 26 -Thr 27 and Lys 29 -Ala 30 [163,169,175,176,225–227]. 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-Ala 3 -p-nitroanilide and succinyl-Ala- Pro-Ala-p-nitroanilide at a faster rate. This enzyme showed a preference for aliphatic amino acid residues, such as alanine, that are present in elastin. It is considered that the elastolysis was initiated by the formation of an enzyme–substrate complex through electrostatic interac- tion 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 pro- teolytic 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 dithio- threitol (DTT), the enzymatic clevage of keratin can be accompanied by a simultaneous re- duction 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 70 C, and the hair was solubilized within 1 h [232]. Similarly, enhanced keratin degradation after addition of DTT has also been reported for al- kaline 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 differ- ent 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-

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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 environ- mentally 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 ac- tivity and stability over a broad range of pH and temperature. The enzymes used should be effective at low levels (0.4–0.8%) 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 pro- teases 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 re- quire the same enzyme thermal stability as an all-temperature laundry detergent. The interest in using alkaline enzymes in automatic dishwashing detergents has also in- creased 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, fraction- ation, 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, Den- mark), Terg-a-zyme (Alconox, Inc, New York, USA), Ultrasil 53 (Henkel KGaA, Dussel- dorf, Germany) and P3-paradigm (Henkel-Ecolab GmbH, Düsseldorf, Germany). These en- zyme-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 pro- teases and lipases to degrade and solubilize protein and fat foulants have also proven benefi- cial. 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 alka- line 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, re- moval 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 As- pergillus flavus [96], Streptomyces sp. [244], B. amyloliquefaciens [97], and B. subtilis [245,246].

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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. B18 [78] and B. coag- ulans 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 applica- tions 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 hydro- lyzed 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 therapeu- tic 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 hu- mans [253] and an alkaline protease having fibrinolytic activity has been used as a throm- bolytic agent [173].

6.5. Food industry

Alkaline proteases can hydrolyze proteins from plants, fish, or animals to produce hy- drolysates of well-defined peptide profile. The commercial alkaline protease, Alcalase, has a broad specificity with some preference for terminal hydrophobic amino acids. Using this en- zyme, 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 produc- tion of a methionine-rich protein hydrolysate from chick pea protein [258]. The protein hy- drolysates commonly generated from casein, whey protein and soyprotein find major appli- cation 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 activ- ity were produced from sardine muscle by treatment with a B. licheniformis alkaline pro- tease. 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 alka- line elastase [263] and thermophilic alkaline protease [264] have proved to be successful and

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promising meat tenderizing enzymes, as they possess the ability to hydrolyze connective tis- sue 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 resi- dues 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 Alca- lase at a pH of 8.5 and temperature of 55–60 C, 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 hy- drolyzing raw meat. The resulting meat hydrolysate exhibited excellent organoleptic proper- ties 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 vari- ous food processing industries and household activities. These proteases can solubilize pro- teins in wastes through a multistep process to recover liquid concentrates or dry solids of nu- tritional value for fish or livestock [270,271]. Dalev [272] reported an enzymatic process using a B. subtilis alkaline protease in the pro- cessing 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 [278–280]. However, a major drawback of this approach is the strongly reduced activity of enzymes under anhydrous conditions. Thus, it is of practi-

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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 or- ganic solvents [281–283]. 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 es- ters [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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