Journal of Scientific & Industrial ResearchVol. 61. September 2002. pp 6<)0-70-1

A Review on Microbial Alkaline Proteases

P Ellaiah''', 13 Srinivasulu and K Adinarayana'

Ph.umuccuticul Hiotcchnolog y Division, Department of Phannuceuticut Scicncc,.Audhra University, Visakhap.nnam 530 om

Alkaline prorcasex are of considcruhlc interest in view of their activity and stability at alkaline pll. This review describes theprotcascs that can resist extreme alkaline environments produced by a wide range of ulk alophilic microorg anisms. Differentisol.uion methods which enable the screening and selection of promising organisms 1'01' industrial production are discussed. Thevarious nutritional and cnvironmcntul parameters affecting the production of alkaline protcuscs are dclinctucd. The production ofprorcuscs by free and immobilized whole cells is discussed. The purification. properties. and applications of these pretenses arealso discussed.

IntroductionAlkaline proteases are a physiologically and

commercially important group of enzymes used prima-rily as detergent additives. They playa specific catalyticrole in the hydrolysis of proteins. In 1994 the total mar-ket for industrial enzymes accounted for approximately$4(}O mill ion, of wh ich. cnzy mes worth $112 million wereused for detergent purposesl. In Japan, 1094 alkalineprotease sales were estimated at IS,OOO million yen(equivalent to Silo million)'. This enzyme accounts for4() per cent of the total worldwide enzyme sales. Thereis expected to he an upward trend in the use of alkalinepro teases in the future.

Proicascs catalyze the cleavage of pcpt iele bondsin proteins. They arc enzymes of class the hydroluscsand subclass the peptide hydrolascs or pcptidascs. Pro-teases may be either cxopeptidscs, whose actionx aredirected by the amino or carboxyl terminus of proteins.or endopeptidascs. which cleave internal peptide bonds.Endopcoridases arc also termed as protcinascs. t\ morerational system of prorcascs classification is based Oil acomparison of active sites, mechanism of action and3-~ structure:'.

Protcases can also be classified Oil the basis or:(a) pH(b) Substrate specificity

"' Corresponding author. E-Illail: adikunamncni ·Cr.!- rcdiurnail.com

(c) Similarity in action to well characterized enzymeslike trypsin, chymotrypsin & elastase, and

(d) Active site amino acid residue & catalytic mecha-nism.

More conventionally, proteases are classifiedinto four important groups like serine, cysteine, asparticand metallo proreases.

Serine ProteasesSerine proreases arc the most widely distributed

group of proteolytic enzymes of both microbial and ani-mal origin". The enzymes have a reactive serine residuein the active site and arc generally inhibited bydiisopropyl fluorophosph.ue (DlP) and phenyl methylsulphonyl fluoride (PMSF). Most or the protcascs arealso inhibited by some thiol reagents, such as !J-ch lororncrcuric benzoate (pCM 13). prohabl y due to thepresence or cysteine residue near the active sill'. whichprobably docs not participate in the catalytic mechanismof the enzyme. These arc genet'ally active at neutral andalkaline pl-l, with an optimum pH between 7-1/. Theyhave broad substrate specificities, including consider-able cstcrcolytic activity towards many ester substrates,and are generally of low molecular weight (18.5-35 kDa),

Cvsteine ProteasesCysteine prorcascs arc sensitive to sulphydryl

reagents, such aspCM13, Na-tosyl-L-Iysine chlorornethylketone (TLCK), iodoacetic acid, iodoacetamidc, heavy

ELLAIAH et al.: MICROBIAL ALKALINE PROTEASES 691

metals, and are activated by reducing agents such aspotassium cyanide or cysteine, dithiothreitol, and ethyl-ene diaminetetraacetic acid (EDTA). The occurrence ofcysteine proteases has been reported in only a few fungi".Intracellular enzymes with properties similar to cysteineproteinases have been reported in Trichosporon species,Oidiodendron kalrai, and Nannirzia fulva. Extracellularcysteine proteases have been observed in Microsporiumspecies, Aspergillus oryzae, and Sporotrichumpulverulentum', Most of these enzymes are active at pH5-8. Some are stimulated by reducing agents'.

Aspartic Pro teases

Aspartic proteases are characterized by maxi-mum activity at low pH (3-4) and insensitivity to inhibi-tors of the other three groups of enzymes". They arewidely distributed in fungi, but are rarely found in bac-teria or protozoa. Most aspartic proteases are sensitiveto epoxy and diazo-ketone compounds in the presenceof copper cations. They are also inhibited by pepstatinor streptomyces pepsin inhibitor.

Most aspartic proteases have molecular weightsin the range 30-45 kDa, and their isoelectric points areusually in the range pH 3.4-4.6. These enzymes are spe-cific against aromatic or bulky amino acid residues onboth sides of the cleavage point. Catalytic activities in-volve two aspartic acid residues. The catalytic mecha-nism of the aspartic proteases requires the initial bind-ing of a water molecule at the active site before nucleo-philic attack on the substrate peptide bond. Most of thefungal aspartic pro teases are unstable above neutral pHand are not found in cultures growing at neutral or alka-line pH.

M etalloproteases

All these enzymes have pH optima between pH5-9 and are sensitive to metal-chelating reagents, suchas EDTA, but are unaffected by serine protease inhibi-tors or sulphydryl agents". Many of the EDTA- inhibitedenzymes can be reactivated by ions, such as zinc, cal-cium, and cobalt. These are widespread, but only a fewhave been reported in fungi. Most of the bacterial andfungal metalloproteases are zinc-containing enzymes,with one atom of zinc per molecule of enzyme. The zincatom is essential for enzyme activity. Calcium is requiredto stabilize the protein structure.

Alkalophilic MicroorganismsAll microorganisms follow a normal distribu-

tion pattern based on the pH dependence for their opti-mal growth, and the majority of these microorganismsare known to proliferate well at near neutral pH values.As the pH moves away from this neutral range the num-ber of microorganisms decreases. The number ofalkalophilic bacteria found in the soil is about 1/10 to 11100 of that of neutrophilic bacteria. However, some neu-trophilic organisms are capable of growth even at ex-treme pH conditions. This is primarily due to the specialphysiological and metabolic systems, which they haveadopted by altering the bioenergitic membrane proper-ties and transport mechanisms, enabling their survivaland multiplication under such adverse conditions. Suchmicroorganisms may also be refereed to as pH depen-dent extremophiles.

Alkalophilic microorganisms constitute a diversegroup that thrives in highly alkaline environments. Theyhave been further categorized into two broad groups,namely alkalophiles and alkalotolerants. The termalkalophiles is used for those organisms that were ca-pable of growth above pH 10, with an optimal growtharound pH 9, and are unable to grow at pH 7 or less. Onthe other hand, alkalotolerant organisms are capable ofgrowing at pH values 10, but have an optimal growthrate at pH 7 (ref. I). The extreme alkalophiles have beenfurther subdivided into two groups, namely facultativeand obligate alkalophiles. Facultative alkalophiles haveoptimal growth at pH 10 or above but can growwell at neutrality, while obligate alkalophiles fail togrow at pH 7.

Isolation and Screening

Vedder" has reported the isolation of obligatealkalophilic organisms from human and animal feces in1934. He briefly described these organisms and proposedthe name Bacillus alcalophilus for his strains and alsostated that he had been able to prove that life exists thatnot only tolerates, but also depends on, a highly alkalinepH. Today, many of these alkalophilic Bacillus strainsare of considerable industrial importance, particularlyfor use of proteases in laundry detergent. Normal gar-den soil was reported to be a preferred source for isola-tion, presumably because of the various biological ac-tivities that generate transient alkaline conditions in suchenvironment? These organisms were also isolated from

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692 J SCI IND RES VOL 61 SEPTEMBER 2002

nonalkaline habitats, such as neutral and acidic soils,and thus appear to be fairly widespread.

One of the most important and noteworthy fea-tures of many alkalophiles is their ability to modulatetheir environment. They can convert neutral medium toalkaline or acidify high alkaline medium to optimizeexternal pH for growth. However, their internal pH isbetween pH 7 and 9, always lower than the externalmedium. Thus, alkalophilicity is maintained by theseorganisms through bioenergetic membrane properties andtransport mechanisms, and does not necessarily rely onalkali resistant intracellular enzyme.

In natural environments, sodium carbonate isgenerally the major source of alkalinity. Its addition tothe isolation media enhance the growth of alkalophilicmicroorganisms. The addition of sodium carbonate tothe medium for the isolation of alkalophilic thermopilesresults in brown colour and cracking of the medium. Attemperatures of> 70°C, agar based media usually losetheir gel strength and exhibit water of syneresis, makingthem useless for isolation of thermopiles. As a result,the need for gelling agents with good thermal stabilityled to the discovery of agents, such as Gelrite" 8 and anoptimized concentration (3 per cent w/v) of bacterio-logical grade agar".

Enrichment and SelectionThe primary stage in the development of an in-

dustrial fermentation process is to isolate strain(s) ca-pable of producing the target product in commercialyields. This approach results in intensive screening pro-grams to test a large number of strains to identify highproducers having novel properties. The conventionalpractice with many extracellular microbial products isto grow a large number of organisms on agar plate me-dia and to relate each organism's production capabilityto the radius of the product's zone of diffusion aroundthe colony. In the course of designing a medium, forscreening proteases, it is essential that the medium shouldcontain likely inducers of the product and be devoid ofconstituents that may repress enzyme synthesis. It hasbeen reported that B.licheniformis produces very nar-row zones of hydrolysis on casein agar despite beingvery good protease producer in submerged culture. Nor-mally, alkalophilic organisms are isolated by surface plat-ing on a high alkaline medium and subsequent screen-ing for the desired characteristics. The organisms arefurther grown on specific media for estimating pro-teolytic activities using appropriate substrates such skim

milk or casein. The isolates, exhibiting desired level ofactivity are chosen and maintained on slants for furtheruse. The most commonly used general medium for theisolation of alkalophiles has been described byHorikoshi 10. Several types of defined media have alsobeen used for their isolation which include nutrient agar,glucose-yeast extract-asparagine agar (GYA), MYGPagar, peptone-yeast extract-glucose (PPYG) medium, andother undefined media, such as wheat meal agar. Themedium composition was varied by several workers toisolate microorganisms of choice, such as those with highproteolytic activity or those that were thermostable. Forany type of medium, a high pH value is essential to iso-late the obligate alkalophiles.

Alkalophilic Microorganisms Exhibiting ProteaseActivity

Of all the alkalophilic microorganisms that havebeen screened for use in various industrial applications,members of the genus Bacillus were found to be pre-dominant and a prolific source of alkaline proteases. Thedifferent alkaline protease-producing Bacillus speciesand strains are summarized in Table 1. Several fungi havealso been reported to produce extracellular alkaline pro-teases. The different alkaline proteases producing fun-gal species are summarized in Table 2.

Alkaline proteases are also produced by somerare actinomycetes. Kurthia spiroforme, a spiral shapedGram-positive bacterium possessing a distant relation-ship to genus Baillus, was reported to produce alkalineproteases. Further, a bacterial isolate capable of produc-ing alkaline pro teases and showing a symbiotic relation-ship with a marine shipworm, Psiloteredo healdi, wasalso reported by Greene et al. II .

Table 1- Alkaline protease producing Bacillus speciesRef.

B·firmus

B. alcalophilus

B. amyloliquefaciens

B. proteolyticus

Bacillus alcalophilus ATCC 21522

(Bacillus sp. No. 221)

B. subtilis

B. thuringiensis

Bacillus sp. Y

Bacillus sp. KSM-K 16

86878840

10

8955

90

50

ELLAIAH et al.: MICROBIAL ALKALINE PROTEASES

Table 2 - Alkaline protease producing fungal species

Ref.

Ac flavus

A. fumigatus

A. melle us

9192,93

9495

96

97

989910048

A. niger

Chrysosporium keratinophilum

Fusarium graminearum

Penicillium griseofulvim

Fusarium sp.

P. lilac inus

Scedosporium apiosermum

693

Table 3 - Microorganisms producing thermostable alkalineproteases

Ref.

Bacillus licheniformis 27

13

5261

10124

10247

A. stearothermophilus

Bacillus thermoruber BT2T

Bacillus sp. strain B 189Thermomonospora fusca

Thermoactinomycetes sp.

Staphylothermus marinus

Malbranchea pulchella var. sulfurea

Bacillus licheniformis Alcalase

Table 4 - Commercial producers of alkaline proteases

Novo Nordisk, Denmark

Protein engineeredvariant of Savinase® Durazym

Protein engineeredvariant of alkalophilicBacillus sp. Maxapem

Savinase, esperase

Gist-brocades, The Netherlands

Alkulophilic Bacillus sp.

Alkalophilic Bacillus sp.

Novo Nordisk, Denmark

Solvay Enzymes GmbH, German

Novo Nordisk, Denmark

Maxacal, maxatase

Solvay Enzymes GmbH, GermanyAlkalophilic Bacillus sp.

Alkalophilic Bacillus sp. Proleather

Opticiean, optimase

Amano Pharmaceuticals Ltd, Japan

Aspergillus sp. Protease P Amano Pharmaceuticals Ltd. Japan

Halophiles that were described to produce alka-line proteases included Halobacterium sp.,Halobacterium ATCC 43214, and Halomonas sp. ES-10. Alkalopsychrotropic and alkalopsychrophilic bacte-ria represent a new potential source for alkaline proteases.These organisms are characterized by their adaptationto both cold temperatures and alkaline conditions. Analkalopsychrotropic Bacillus sp. capable of producingalkaline proteases of high activity at low temperatureswas isolated by Margesin et al. 12.

Despite many published reports on alkaline pro-teases from alkalophilic Bacillus sp., very few reportsexist on thermostable alkaline proteases fromalkalophiles. Many of the thermophilic alkalophiles grow

at 60°C (ref.13), with a few exceptions". Thermostablealkaline proteases from various thermophilic alkalophilesare listed in Table 3. Because alkaline proteases are ofgreat commercial importance, considerable informationhas been compiled on various industrially important pro-ducing organisms (Table 4).

Production of Alkaline ProteasesMost alkalophilic microorganisms produce al-

kaline proteases, though interest is limited only to thosethat yield substantial amounts. It is essential that theseorganisms be provided with optimal growth conditionsto increase enzyme production. The culture conditionsthat promote protease production were found to be sig-

J SCI IND RES VO l. 6 1 SEPTEM BER 2002

nificantly different from the cu lture condi ti ons promot­

ing ce ll growth. In the industrial production of alkaline

proteases, techni cal media were u.- ually employed that

contained very high concentrations ( I 00 - I 50 g dry wt

I L) of complex carbohyd rates, proteins, and other me­

dia components. With a view to develop an economi­

ca ll y feasi ble technology, efforts arc mainl y focused on: (i) Improvement in the y ields of alkaline proteases and

(ii ) Optimizati on of the fermentati on medium and pro­

duct ion conditi ons.

flllp rol'e ll /enl ol Yield

Strain improvement plays a key role in the com­

mercial deve lopment or microb ial fermentati on pro­

cesses . As a rul e the w ild strains usuall y produce lim­

i ted quantiti es of the des ired enzy me to be use ful for commercial appli cation15

• Howc,·er, in most cases , by

adopting simple selecti on methods, such as spreading o f

the culture on specifi c media, it is possible to pick co lo­nies that show a substantial increase in y ield . Conven­

tional ph ys ical and chemical mutagens arc used for

screening of high y ielding strain s. Shah e1 a /. 1r' have developed a cyste in e

auxotropi c mutant of /J . Licl1 enUrnmis w ith improved

protease producti on. A n ad vantage imparted by cys teine auxotrophy is that the strain can be readil y rcisolatcd in

the case of contamination wi th w ild type 11ocil/us mu­tant s that were res istant to antibiotics such as vancomy­

cin and ri stocctin. Asporogenous mutant strains of Ba­cillus sp. are used industrially. A five-fold increase in the y ield of enzyme was observed by the usc of alkaline

protease pos iti ve asporogenic mutants. The adven t of protein eng inee ri ng and sophi sti ­

cated molecular technologies ha ve opened poss ibiliti es

fo r screen ing high y ielding variants of enzy mes and tai ­lor made proteins from alkalophilic microorgani sms w ith enhanced producti on in y ields, which may be of interest for spec ifi c commercial applicati ons. 1 ew constructi ons

ha ve been made by the trans fer of genes between organ­isms to produce high yie lding variant s17 Further the pro­te in eng inee ri ng approach can be exploited for the im­

provement of alkaline proteascs and I or subtil is ins be­yo nd it s current limit ati o ns. C urrentl y. t wo

conccpti onall y di ffe rent strategies arc ava ilable for gen­eration of protein enginee red vari an ts: random and site­

directed mutagenesis. With random mutagenes is, a large number of va ri ants arc produced, but the success o f thi s approach largely depends on the avai labilit y o f efficient

properti es. Site-di rec ted mutagenesis depends on the access to structural or bioche'11 ical data to reduce the

number of variants to be cons truc:ecl . as every protein var iant is purified and indi vidually tes ted for i mprove­

ments. Promising variants generated and identified by

random mutagenes is, o rten can be improved by further

si te-di reeled introduction o f known ad vantagcous muta­ti ons.

Opti111i::,o!ion ol F ennenlalion M edi 11 111

A I kal i ne proteascs are general! y produced by submerged fermentati on. In addition , so lid state fermen­tation processes have also been exploited to a lesser ex ­

ten t for production o f these enzymes 1x. Efforts have been direc ted mainl y towards: ( i ) Eva luallon of the effec ts of

va ri ous carbon and nitrogenous nutrients as cost-cllec­

ti vc substrates on the y ield of enzymes: ( ii ) Requirement of divalent metal ions in the fermen tati on medium; and

(iii ) Optimi za tion of environ mental and fermentat ion

parameters such as pH , temperature, aeration , and agita­tion. In addition. no defined med ium has been established

for the best producti on o f alkaline pro teases from eli ffer­

cnt microbial sources. Each organi . m or strain has its own spec ial condit ions for maxim u 1 enzyme produc­

ti on.

Co rhon Source Studies have indicated a reduction in protease

production clue to cataboli te re pre~: s i o n by glucose 1'J·

20

On the other hand, Zamost e / a/. 21 have correl ated the low y ields or protease procluct ,on w ith the lowering of pH brought abou t by the rapid growth of the organi sm .

In commercial prac ti ce, high carbohyclratG concentrations

repressed enzyme production. T herefore, carbohydrate was added, ci ther coni i nuously or in al iquots through­

out the fermentation to supplement th l' exhausted com­ponent and keep the volume limited and thereby red uce the power requ irements.

! ncrcased y ields of al bl ine pro teases were re­ported by several workers who used di ffcrent sugars such

as lactose22, maltosc2

\ sucrose2·', and fructose25

. How­ever, a repression in enzyme synthesis was observed with

these ingredients at hi gh concen trati ons. Whey, a was te

byproduct of the dairy industry, containing mainl y lac­tose and sa lts, has been demonstrated as a potential sub­

strate for alkali ne pro tease product ion. Vari ous organ ic

acids, such as aceti c :JC id , methyl acetate, and citric ac id

or sod ium citrate have been demon tra tecl to increase

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ELLAIAH et [II.: MICROBIAL ALKALINE PROTEASES 695

these organic acids was interesting in view of theireconomy as well as their ability to control pH variations.

Nitrogen SourceIn most microorganisms, both inorganic and or-

ganic forms of nitrogen are metabolized to produce aminoacids, nucleic acids, proteins, and cell wall components.The alkaline protease comprises 15.6 per cent nitrogenand its production is dependent on the availability ofboth carbon and nitrogen sources in the medium 19. Al-though complex nitrogen sources are usually used foralkaline protease production the requirement for a spe-cific nitrogen supplement differs from organism to or-ganism. Low levels of alkal ine protease production werereported with the use of inorganic nitrogen sources inthe production medium. Enzyme synthesis was found tobe repressed by rapidly metabolizable nitrogen sources,such as amino acids or ammonium ion concentrations inthe medium": However, one report indicated no repres-sion in the protease activity with the use of ammoniumsalts":

Sinha and Satyanarayana " have observed anincrease in protease production by the addition of am-monium sulphate and potassium nitrate. Similarly, so-dium nitrate (0.25 per cent) was found to be stimulatoryfor alkaline protease production. On the contrary, sev-eral reports have demonstrated the use of organic nitro-gen sources leading to higher enzyme production thanthe inorganic nitrogen sources. Fujiwara and Yamamoto"have recorded maximum enzyme yields using a combi-nation of 3 per cent soybean meal and 1.5 per cent bo-nito extract. Soybean meal was also reported to be a suit-able nitrogen source for protease production?".

Corn steep liquor (CSL) was found to be acheapand suitable source of nitrogen by some workers".Tryptone (2 per cent) and casein (1-2 per cent) also serveas excellent nitrogen sources". Addition of certain ami nocompounds was shown to be effecti ve in the productionof extracellular enzymes by alkalophilic Bacillus sp.However, glycine appeared to have inhibitory effects onboth amylase and protease production. Casamino acidswere also found to inhibit protease production?". Oilcakes (as nitrogen source) were found to stimulate theproduction of enzymes. In some studies, use of oil cakesdid not favour enzyme production".Metal Ion Requirement

Divalent metal ions, such as calcium, cobalt,copper, boron, iron, magnesium, manganese, and mo-lybdenum are required in the fermentation medium for

optimum production of alkaline proteases. However therequirement for specific metal ions depends on the sourceof enzyme. The use of AgN03 at a cone. of 0.05 mg/I 00mL or ZnSO 4 at a concentration of 0.125 mg/1 00 mLresulted in an increase in protease activity in Rhizopusoryzae. Potassium phosphate has been used as a sourceof phosphate in most studies. This was shown to be re-sponsible for buffering the medium. Phosphate at theconcentration of 2 g/L was found optimal for proteaseproduction. However, amounts in excess of this concen-tration showed an inhibition in cell growth and repres-sion in protease production. When the phosphate con-centration was 4 g/L, precipitation of the medium onautoclaving was observed". This problem, however,could be overcome by the supplementation of the diso-diurn salt of EDTA in the medium. In at least one casethe salts did not have any effect on the protease yields.

pH and TemperatureThe important characteristic of most alkalophilic

microorganisms show their strong dependence on theextracellular pH for cell growth and enzyme production.For increased protease yields from these alkalophiles,the pH of the medium must be maintained above 7.5throughout the fermentation process. The culture pH alsostrongly affects many enzymatic processes and transportof various components across the cell membrane. Whenammonium ions were used the medium turned acidic,while it turned alkaline when organic nitrogen, such asaminoacids or peptides were consumed". The declinein the pH may also be due to production of acidic prod-ucts. In view of a close relationship between proteasesynthesis and the utilization of nitrogenous compounds,pH variations during fermentation may indicate kineticinformation about the protease production, such as thestart and end of the protease production period.

Temperature is yet another critical parameter thathas to be controlled and varied from organism to organ-ism. The mechanism of temperature control of enzymeproduction is not well understood. However, studies byFrankena et al.co have shown that a link existed betweenenzyme synthesis and energy metabolism in Bacilli,which was controlled by temperature and oxygen up-take.Aeration and Agitation

During fermentation the aeration rate indirectlyindicates the dissolved oxygen level in the fermentationbroth. Different dissolved oxygen profiles can be ob-tained by: (i) Variations in the aeration rate; (ii) Varia-

696 J SCI IND RES VOL 61 SEPTEMBER 2002

tions in the agitation speed of the bioreactor; or (iii) Useof oxygen rich or oxygen deficient gas phase (appropri-ate air oxygen or air-nitrogen mixtures) as the oxygensource". The variation in the agitation speed influencesthe extent of mixing in the shake flasks or the bioreactorand also affects the nutrient availability.

Optimum yields of alkaline protease are pro-duced at 200 rpm for B. subtilis ATCC 14416 and B.licheniformis. In one study, Bacillus sp.B21-2 producedincreased enzyme titres when agitated at 600 rpm andaerated at 0.5 vvm. Similarly, Bacillus firmus exhibitedmaximum enzyme yields at an aeration rate of 7.0 LImin and an agitation rate of 360 rpm. However, lower-ing the aeration rate to 0.1 L'min caused a drastic reduc-tion in the protease yields. This indicates that a reduc-tion in oxygen supply is an important limiting factor forgrowth as well as protease synthesis.

Immobilization of Alkaline ProteasesThe interest in the use of immobilized enzymes

in industry is based on the potential advantages theyconfer over their soluble counterparts, including in-creased stability to temperature, pH, and organic solvents;recovery and reuse of the enzyme; and, in the case ofproteases, removal or reduction of autolysis or denatur-ation. Furthermore, immobilized enzymes render con-tinuous production processes possible via packed bedreactors and may lead to more stable biocatalysts. Thetwo main methods for immobilization are whole cellimmobilization and cell-free immobilization.

Whole Cell ImmobilizationBecause alkaline protease is an extracellular

enzyme, whole cell immobilization is the method ofchoice. By using immobilized cells the protease can beproduced in a shorter reaction time. Further the rate ofprotease production can be improved over that of sub-merged batch fermentation. The long-term stability ofthe immobilized cells during the course of fermentationand the easy separation of enzyme also make them prom-ising candidates for commercial exploitation. Physicalentrapment of whole cells in polymeric gel matrices wasused as an immobilized method by Kokubu et al." andSutar et al?'. Batch" and repeated batch" fermentationprocesses were also demonstrated using urethane foamas an immobilization carrier. Further, Bacillus firmuscells were immobilized on cellulose triacetate fibres andfilms, followed by cross-linking with a bifunctional re-agent, glutaraldehyde, which improved alkaline proteasebiosynthesis.

Cell-free Immobilization

Attachment of alkaline proteases to an insolublecarrier (by either physical adsorption or covalent cou-pling) is the most prevalent method of immobilization.Various carriers employed for this purpose include ben-tonite, porous glass, nylon and vermiculite. Althoughporous glass has been widely used the relatively highcost of this support has been the limiting factor for in-dustrial applications. The method of immobilization ofthe alkaline proteases on these supports using glutaral-dehyde involves covalent attachment of the amino groupsof the enzyme to the available aldehyde groups presentin the glutaraldehyde activated support. In one study,Srokova and Cik37 successfully immobilized an alkalineprotease onto a gel of o-hydroxyethylcellulose throughphotochemical polymer carrier crosslinking induced bythe photolysis of aromatic azides. Some immobilizationstudies38.39 have addressed an increase in thethemostability profile and the pH activity profile of theenzyme towards the alkaline side. The increase in ther-mal stability is mainly due to the multipoint covalentattachment and the stabilization of the weak ionic forcesand hydrogen bonds between the protease and the sup-port, which protects the enzyme from inactivation andautolysis. Further the change in the pH values may beattributed to the partition effects that cause different con-centrations of hydrogen ions in the microenvironmentof the immobilized enzyme when coupled to a carrierpossessing electrostatic interactions.

Isolation and Purification of Alkaline Proteases

When isolating enzymes on industrial scale forcommercial purposes the prime consideration is the costof production in relation to the value of the end product.

Crude preparations of alkaline pro teases are gen-erally employed for commercial use. Nevertheless thepurification of alkaline proteases is important from theperspective of developing a better understanding of thefunctioning of the enzyme.

RecoveryAfter successful fermentation, when the fer-

mented medium leaves the controlled environment ofthe fermenter, it is exposed to a drastic change in envi-ronmental conditions. The removal of the cells, solids,and colloids from the fermentation broth is the primary

ELLAIAH et al.: MICROBIAL ALKALINE PROTEASES 697

step in enzyme downstream processing, for whichvacuum rotary drum filters and continuous disc centri-fuges are commonly used. To prevent the losses in en-zyme activity caused by imperfect clarification or to pre-vent the clogging of filters, it is necessary to performsome chemical pretreatment of the fermentation brothbefore commencing separation. Changes in pH may alsobe suitable for better separation of solids. Furthermorethe fermentation broth solids are often colloidal in na-ture and are difficult to remove directly. In this case,addition of coagulating or flocculating agents becomesvital:". Flocculating agents are generally employed toeffect the formation of larger floes or agglomerates,which, in turn, accelerate the solid-liquid separation. Celltlocculation can be improved by neutralization of thecharges on the microbial cell surfaces, which includeschanges in pH and the addition of a range of compoundsthat alter the ionic environment. The flocculating agents,commonly used are inorganic salts, mineral hydrocol-loids, and organic polyelectrolytes. For example the useof a polyelectrolyte Sedipur TF 5 proved to be an effec-tive tlocculating agent at 150 ppm and pH 7.0-9.0, andgave 74 per cent yield of alkaline protease activity. Insome cases, it becomes necessary to add a bioprocessingfilter aid, such as diatomaceous earth. before filtration".

ConcentrationAs the amount of enzyme present in the cell free

filtrate is usually low therefore the removal of water is aprimary objective. Recently, membrane separation pro-cesses have been widely used for downstream process-ing. Ultrafiltration (UF) is one such membrane processthat has been largely used for the recovery of enzymes"and formed a preferred alternative to evaporation. Thispressure driven separation process is expensive, resultsin Iittle loss of enzyme activity, and offers both puri fica-tion and concentration, as well as diafiltration, for saltremoval or for changing the salt composition". How-ever, a disadvantage underlying this process is the foul-ing or membrane clogging due to the precipitates formedby the final product. This clogging can usually be alle-viated or overcome by treatment with detergents, pro-teases, or acids and alkalies.

Hand et al," have used a temperature-sensitivehydrogel ultrafiltration for concentrating an alkaline pro-tease. This hydrogel comprised poly (N-isopropylacrylamide), which changed its volume revers-ibly by the changes in temp~rature. The separation effi-ciency of the enzyme was dependent on the temperature

and was 84 per cent at J 5° C and 20° C, respectively.However, above 25° C, a decrease in the separation effi-ciency was observed.

PrecipitationPrecipitation is the most commonly used method

for the isolation and recovery of proteins from crude bio-logical mixtures. It also performs both purification andconcentration steps. It is generally effected by the addi-tion of reagents such as salt or an organic solvent, whichlower the solubility of the desired proteins in an aque-ous solution. Although precipitation by ammonium sul-phate has been used for many years, it is not the precipi-tating agent of choice for detergent enzymes. Ammo-nium sulphate has found wide utility only in acidic andneutral pH values and it formed ammonia under alka-line conditions. Hence the use of sodium sulphate or anorganic solvent gave the preferred choice. Despite bet-ter precipitating qualities of sodium sulphate over am-monium sulphate the poor solubility of the salt at lowtemperatures, restricted its use for this purpose.

Many reports have revealed the use of aceroneat different vol umc concentrations: 5 volumes Ill, 3 vol-umes ", and 2.5 volumes", as a primary precipitationagent for the recovery of alkaline proteases. Precipita-tion was also reported by various workers with acetoneat different concentrations' 80 per cent (v/v) 4),66 percent (v/v)4<>;or 44,66, and 83 per cent (v/v)47, followedby centrifugation and/or drying. Precipitation of enzymescan also be achieved by the use of water soluble, neutralpolymers, such as polyethylene glycol ".

ion-exchange Chromarography (lEe)Alkaline proteases are generally positively

b d . h 49 Hcharged and are not oun to anion exc angers. ow-ever, cation exchangers can be a rational choice and thehound molecules are el uted from the column by an in-crease in salt or pH gradient.

Affinity ChromatographyReports on the purification of alkaline proteases

by different affinity chromatographic methods showedthat an affinity adsorbent hydroxyapatite was used toseparate the neutral protease as well as purify the alka-line protease from a Bacillus sp.:". Other affinity matri-ces used were Sephadex-a-phenylbutylamine", caseinagarose", or N-benzoyloxycarbonyl phenylalanine, im-mobilized on agarose adsorbents". However the majorlimitations of affinity chromatography are the high cost

698 J SCI IND RES VOL 61 SEPTEMBER 2002

of enzyme supports and the labile nature of some affin-ity ligands, which do not recommend them for use as aprocess scale.

Aqueous Two-phase SystemsThis technique has been applied for purifica-

tion of alkaline proteases using mixtures of polyethyl-ene glycol (PEG) and dextran or PEG and salts such asH3P04 and MgSO/4,55. In addition, other methods, suchas the use of reversed micelles for liquid-liquid extrac-tion, affinity precipitation, and foam fractionation havealso been employed for the recovery of alkaline proteases.

StabilizationThe enzyme preparations, used commercially,

are impure and are standardized to specified levels ofactivity by the addition of diluents and carriers. Furtherthe conditions for maximum stability of crude prepara-tions may be quite different than for purified enzymes.As loss of activity is encountered during storage in thefactory therefore shipment to client(s) and / or storagein client's facilities, storage stability is of prime concernto enzyme manufacturers. Protease solutions are subjectto proteolytic and autolytic degradation that results inrapid inactivation of enzymatic activity. To maintain theenzyme activity and provide stability, addition of stabi-lizers, like calcium salts, sodium formate, borate, pro-pylene glycol, glycerine or betaine polyhydric alcohols,protein preparations, and related compounds has provedsuccessful". Also, to prevent contamination of the finalcommercial crude preparation during storage, additionof sodium chloride at 18-20 per cent concentration hasbeen suggested". The handling of dry enzymes possesspotential health hazards and, therefore, it is customaryto maintain the enzyme preparations in stabilized liquidform. The stabilization of alkaline proteases and/or sub-tilisins has also been made possible through use of pro-tein engineering and numerous examples have been citedin literature. The alkaline and thermal stabilities of sub-tilisin BPN9 were improved by random mutagenesis fol-lowed by application of proper screening assays. Site-directed mutagenesis is often based on specific proteindesign strategies, including change of electrostatic po-tential, introduction of disulphide bridges, replacementof oxidation labile residues, modification of side chaininteractions, improvement of internal packaging,strengthening of metal ion binding, reduction in unfold-ing entropy, residue substitution or deletion based onhomology and modification of substrate specificity'F".

-

Properties of Alkaline ProteasesThe enzymatic and physiochemical properties

of alkaline proteases from several microorganisms havebeen extensively studied.

Optimum pH and TemperatureThe optimum pH range of alkaline proteases is

generall y between pH 9 and 11, with a few exceptionsof higher pH optima of 11.5 (ref. 60), pH 11-12 (ref. 10,49), and pH 12-13 (ref. 61). They also have high iso-electric points and are generally stable between pH 6and 12 (ref. 62). The optimum temperatures of alkalineproteases range between 50 and 70°C. In addition theenzyme from an alkalophilic Bacillus sp. B 18 showedan 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 Ca2+ further enhanced enzymethemostability.

Molecular Masses

The molecular masses of alkaline proteasesrange between 15 and 30 kDa63 with a few reports ofhigher molecular masses of 31.6 kDa64 , 33 kDa53 , 36kDa65, and 45 kDa45. However, an enzyme from Kurthiaspiroforme had an extremely low molecular weight of 8kDa66. In some Bacillus sp., multiple electrophoreticforms of alkaline proteases were observedv". The mul-tiple forms of these enzymes were the result of nonen-zymatic, irreversible deamination of glutamine or aspar-agine residues in the protein molecules, or ofautoproreolysis'",

Metal Ion Requirement and Inhibitors

Alkaline proteases require a divalent cation likeCa+2, Mg+2,and Mn+2 or a combination of these cations,for maximum activity. These cations were also found toenhance the thermal stability of a Bacillus alkaline pro-tease". It is believed that these cations protect the en-zyme against thermal denaturation and playa vital rolein maintaining the active conformation of the enzyme athigh temperatures. In addition, specific Ca2+binding sitesthat influence the protein activity and stability, apart fromthe catalytic site were described for proteinase K. Inhi-bition studies give insight into the nature of the enzyme,its cofactor requirements, and the nature of the activesite. In some of the studies, catalytic activity was inhi-

ELLAIAH et al.: MICROBIAL ALKALINE PROTEASES 699

bited by Hg+2ions. In this regard the poisoning of en-zymes by heavy metal ions has been well documented inthe literature".

Alkaline proteases are completely inhibited byphenylmethylsulphonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP). In this regard, PMSF sulphonatesthe essential serine residue in the active site and resultsin the complete loss of activity. This inhibition profileclassifies these proteases as serine hydrolases. In addi-tion, some of the alkaline pro teases were found to bemetal ion dependent in view of their sensitivity to metalchelating agents, such as EDTA66.69.Thiol inhibitors havelittle effect on alkaline pro teases of Bacillus sp., althoughthey do affect the alkaline enzymes produced by Strep-tomyces Sp.47,60,

Substrate Specificity

Although alkaline proteases are active againstmany synthetic substrates, as well as native proteins,reaction rates vary widely. The alkaline pro teases and/or subtilisins are found to be more active against caseinthan against haemoglobin or bovine serum albumin. Al-kaline proteases are specific against aromatic or hydro-phobic amino acid residues, such as tyrosine, phenyla-lanine, or leucine at the carboxyl side of the splittingpoint, having a specificity similar to, but less stringentthan a chymotrypsin. With the B-chain of insulin as sub-strate the bonds most frequently cleaved by many alka-line proteases were Glu 4 - His 5, Ser 9 - His 10, Leu 15- Tyr 16, Tyr 16 -Leu 17, Phe 25 - Tyr 26, Tyr 26 - Thr27, and Lys 29 -Ala 30 (ref. 42,44). In addition, Tsai etal." have elucidated that an alkaline elastase from Ba-cillus sp. Ya-B cleaved both the oxidized insulin A- andB-chains in a block cutting manner.

Tsai et al." observed that the alkaline elastasefrom Bacillus sp. Ya-B also hydrolysed elastin andelastase specific substrates, like succinyl-Ala3 -p-nitroanilide and succinyl-Ala-Pro- Ala-p-nitroanilide, ata faster rate. This enzyme showed a preference for ali-phatic amino acid residues, such as alanine that arepresent in elastin. It is considered that the elastolysiswas initiated by the formation of an enzyme substratecomplex through electrostatic interaction between posi-tively charged residues of the elastase and negativelycharged residues of the elastin in a pH range below 10.6.In keratin the disulphide bonds form an important struc-tural feature and prevent the proteolytic degradation ofthe most compact areas of the keratinous substrates. A

thermostable alkaline protease from an alkalophilic Ba-cillus sp. no. AH-101 exhibiting keratinolytic activityshowed degradation of human hair keratin with I percent thioglycolic acid at pH 12 and 70°C, and the hairwas solubilized within 1 h. Similarly, enhanced keratindegradation after addition of DTT has also been reportedfor alkaline proteases of Streptomyces sp. 71,

Applications of Alkaline ProteasesAlkaline proteases are robust enzymes with con-

siderable industrial potential in detergents, leather pro-cessing, silver recovery, medical purposes, food process-ing, feeds, and chemical industries, as well as waste treat-ment. These enzymes contribute to the development ofhigh value added applications or products by using en-zyme aided (partial) digestion. The different areas ofapplications currently using alkaline proteases are givensubsequently.

Detergent IndustryThe detergent industry has now emerged as the

single major consumer of several hydrolytic enzymesacting in the alkaline pH range. Detergents containingdifferent enzymes; proteases, amylases, and lipases areavailable in the international markets under several brandnames. The use of different enzymes as detergent addi-tives arises from the fact that proteases can hydrolyseproteinaceous strains, amylases are effective againststarch and other carbohydrate stains while lipases areeffective against oily or fat stains. For an enzyme to beused as a detergent additive. It should have two quali-ties :(i) An alkaline pH and (ii) It should also be compat-ible with detergents. The major use of detergent com-patible proteases is in laundry detergent formulations.Detergents available in the international market, suchas Dynamo, Era plus (Procter & Gamble), Tide (ColgatePalmolive) contain proteolytic enzymes derived mostlyfrom the genus Bacillus.

The interest in using alkaline enzymes in auto-matic dishwashing detergents has also increased recently.The in-place cleaning of ultrafiltration (UF) and reverseosmosis (RO) membranes form one of the most impor-tant aspects IJf modern dairy and food industries. TheUF and RO membranes are put to a variety of uses, in-cluding concentration, fractionation, clarification and/or sterilization of liquid foods, such as milk, whey, eggwhite, fruit juices, wines, and other beverages. The en-zyme detergent preparations presently marked for clean-ing of membrane systems are Alkazym (Novodan A/S,

700 J SCI IND RES VOL 61 SEPTEMBER 2002

Copenhagen, Denmark), Terg-A-Zyme (Alconox, Inc,ew York, USA) and Uitrasil 53 (Henkel kGaA ,

Dusseldorf, Germany). In addition, contact lense clean-ing solutions containing alkaline protease derived froma marine shipworm bacterium was used for the cleaningof contact lens at low temperatures". In India, one suchenzyme based optical cleaner (available in the form oftablets containing Subtilopeptidase A) is presently mar-keted by Mis Bausch and Lomb (India) Ltd.

Leather IndustryAnother industrial process, which has received

attention, is the enzyme-assisted dehairing of animalhides and skin in the leather industry. Traditionally, thisprocess is carried out by treating animal hides with asaturated solution of lime and sodium sulphide, besidesbeing expensive and particularly unpleasant to carry out,a strongly polluting effluent is produced. The alterna-tive to this process is enzyme-assisted dehairing. En-zyme-assisted dehairing is preferentially possible ifpro-teolytic enzymes can be found that are stable and activeunder the alkaline conditions (pH 12) of tanning.

Early attempts, using a wide variety of enzymeswere largely unsuccessful, but proteases from certainbacteria which are alkalophilic in nature have been shownto be effective in assisting the hair removal process. Sev-eral alkaline proteases from alkalophilic actinomyceteshave also been investigated for this purpose. Some ofthese have been shown to be particularly active againstkeratinous proteins, such as hair, feather, and wool atalkaline pH and may have commercial applications.

Silver RecoveryAlkaline proteases find potential application in

the bioprocessing of used X-ray films for silver recov-ery. Used X-ray film contains approximately 1.5 to 2.0per cent (by weight) silver in its gelatin layers. The con-ventional practice of silver recovery by burning film,causes a major environmental pollution problem. Thusthe enzymatic hydrolysis of the gelatin layers on the X-ray film enables not only the silver, but also the polyes-ter film base, to be recycled.

The alkaline proteases from Bacillus sp. B21-2(ref. 73) and B. coagulans PB-77 (ref. 74) decomposedthe gelatinous coating on the used X-ray films from whichthe silver was recovered. Further, a continuous processfor silver recovery was also reported" on the basis of

•

kinetic studies and mechanism of enzymatic hydrolysisof gelatin layers on X-ray film and the resulting releaseof sil ver panicles.

Medicinal UsesCollagenases with alkaline protease activity are

increasingly used for therapeutic applications in thepreparation of slow-release dosage forms. A new semi-alkaline protease with high collagenolytic activity wasproduced by Aspergillus niger LCF9. The enzyme hy-drolyzed various collagen types without amino acid re-lease and liberated low molecular weight peptides ofpotential therapeutic use. Further more, Bacillus spp.have been recognized as being safe to humans" and analkaline protease having fibrinolytic activity has beenused as a thrombolytic agent",

Food IndustryAlkaline proteases can hydrolyse proteins from

plants, fish or animals to produce hydrolysates of well-defined peptide profile. The commercial alkaline pro-tease, Alcalase has a broad specificity with some prefer-ence for terminal hydrophobic amino acids. Using thisenzyme, a less bitter hydrolysate and a debittered enzy-matic whey protein hydrolysate were produced.

Recently, another alkaline protease from B.aniyloliquefaciens resulted in the production of a me-thionine-rich protein hydrolysate from chickpea protein",The protein hydrolysates commonly generated fromcasein, whey protein and soyprotein find major applica-tion in hypoallergenic infant food formulations. Theycan also be used for the fortification of fruit juices orsoft drinks and in manufacturing protein-rich therapeu-tic diets".

In addition, protein hydrolysatcs having angio-tensin I-converting enzyme inhibitory activity were pro-duced from sardine muscle by treatment with a B.lichinijormis alkaline protease. These protein hydroly-sates could be used effectively as a physiologically func-tional food that play an important role in blood pressureregulation.

Further, pro teases playa prominent role in meattendarization, especially of beef. An alkaline elastase"and thermophilic aikaline protease" have proved to besuccessful and promising meat tenderizing enzymes, asthey possess the ability to hydrolyze connective tissueproteins as well as muscle fibre proteins. A method hasbeen developed in which the enzyme is introduced di-rectly in the circulatory system of the animal, shortly

ELLAIA H eta/. : MI CRO BI AL ALKALI NE PROT EASES 70 1

before slaughter o r after stunning the animal to cause brain death .

A potenti al method used a specific combination of neutral and alkaline proteases for hydro lys ing raw meat. The resul ting meat hydro lysate exhibi ted excel­lent organoleptic properties and can be used as a meat­fl avoured additi ve to soup concentrates. Hydro lys is of over 20 per cent did not show any bitterness when such combin ations of enzy mes were used. The reason for thi s may be that the preferenti al spec ificity was favourable when metalloproteinase and serine proteinase were used simultaneously.

Waste Treatment Alkaline proteases prov ide potenti al appli cation

fo r the management of wastes from various food pro­cessing industries and household acti viti es. These pro­teases can solubili ze proteins in wastes through a multi­step process to recover liquid concentrates or dry solids of nutritional value fo r fi sh or li vestock.

Dalev8 1 has reported an enzy mati c process, us­ing a B. subtilis alkaline protease in the processing of waste feathers from poultry slaughter houses. The end product was a heavy, gray ish powder with a very high prote in content, which could be used as a feed additi ve.

Similarl y, many other keratino lytic alkaline pro­teases have been used in feed technology82 fo r the pro­duction of amino ac ids or peptides83

, fo r degrading was te keratinous material in household refu se, and as a depil a­tory agent to remove hair in bath tub drains, which caused bad odors in houses and in public pl aces .

Clzemicallndustry It is now firmly establi shed that enzymes in or­

ganic solvents can expand the application of biocatalysts in synthetic chemi stry. However, a major drawback of thi s approach is the strongly reduced acti vity of enzymes under anhydrous conditions. Thus, it is of practical im­portance to discover ways to acti vate enzymes in organic solvents . Some studies have demonstrated the poss ibil­ity of using alkaline proteases to catalyze peptide syn­thes is in organic solvents84

. In additi on, many efforts to synthes ize peptides enzymatically have employed pro­teases immobilized on insolubl e supports .

A sucrose-po lyester synthes is was carried out in anhydrous pyridine us ing Proleather, a commercial alkaline protease preparati on from Bacillus sp .. The Proleather also catalyzes the transes terification of D-

Further the enzy me Alcalase whi ch act as cata­lyst for resolution of N-protected amino ac id es ters and alkaline proteases fro m Conidiobolus coronatus replaced subtili sin Carl sberg in resolving the race mic mixtures of DL-phenylalanine and DL-phenylg lyc ine.

Conclusions Al kaline proteases are important in view of their

acti vity and stability at a lkaline pH . This review descri bes the proteases that can res ist extreme alkaline environ­ments produced by a wide range of alkalophili c micro­organi sms. Di fferent screening programs are described for the selection of promi sing isolates for the industri al production of alkaline proteases and their applications. With a vi ew to develop an economi cally feas ible tech­no logy, effo rts are mainly focused on improvement in the y ie lds of alka line proteases by strain improvement, optimization of the fermentati on medi a, and producti on conditi ons. The purificati on and properties of alkaline proteases from a wide vari ety of microorgani sms are di scussed.General view of the wide and complex matter on alkaline proteases is presented . It is hoped that such a review will prove to be a comprehensive introducto ry

guide on alkaline proteases.

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