Commercial Potential Of Fungal Protease: Past, Present And .
218Journal of Pharmaceutical, Chemical and Biological SciencesISSN: 2348 -7658Impact Factor (SJIF): 2.092December 2014 -February 2015 ; 2(4):218 -234Available online at http://www.jpcbs.infoOnline published on December 27, 2014Review ArticleCommercial Potential of Fungal Protease: Past, Present and FutureProspectsJ. Srilakshmi, J. Madhavi, Lavanya S, Ammani K*Departments of Biotechnology, Acharya Nagarjuna University, Guntur-522510, A.P., India* Corresponding AuthorReceived: 30 November 2014Revised: 14 December 2014Accepted: 14 December 2014ABSTRACTProteases are one of the most important classes of proteolytic enzymes widely distributed in the animalkingdom, plant and as well as microbes. These enzymes possess the enormous commercial potential and havebeen used in several industrial processes, including food industry, leather processing, silk processing, detergentindustry and therapeutic applications. The major source of commercial enzymes including protease in microbialworld not only yields high quality of enzyme but also enzyme with diverse substrate specificity. Bacterial andfungal enzymes including proteases are widely accepted for different industrial process in last few decades.However, fungal protease are more promising for commercial application as these microorganism are moreresistant to harsh climatic conditions and produces proteins/enzymes in their habitats. Fungal species arecompetent in expression of enzymes in psychrophilic, mesophilic and thermophilic conditions. In last fewdecades, psychrophilic and thermophilic enzymes were identified for their commercial potential worldwide.Several fungal strains have been isolated and characterized for ideal source of protease production. In thisauthor has given emphasis on commercial potential of protease from fungal source.Keyword: Protease; thermophilic and psychrophilic protease; fungal protease; food processing; silk degumming;leather processingINTRODUCTIONProteins are the most versatile molecules inbiological world meant for several function includingstructural organization and catalytic capabilities [1].Enzymes are the biocatalyst with higher precessionand accuracy while performing biochemicalreactions. The entire biological world has beenevolved with diverse enzymes and microbialpopulation shown tremendous potential inJ Pharm Chem Biol Sci, December 2014 -February 2015; 2(4): 218-234
J. Srilakshmi et alproduction of these amazing molecules. Bothbacterial and fungal species are competent inproduction of these versatile molecules which haveserved mankind since several decades [2-3]. Inrecent years there has been a phenomenal increasein the use of proteases as industrial catalysts. Thereare several advantages while using enzyme asbiocatalyst over conventional chemicals. Mostsignificant achievement made using thesebiocatalysts in last one decade is chemical freeindustrial process which led to environmentaldamage and pollution. These amazing moleculesoffer a high degree of substrate specificity whichleads to efficient biochemical process withnegligible error in product formation [4]. Further,enzymes obtained from diverse biological sourcesperform several catalytic reaction ideal for modernrevolutionary industrial demand. Thermal andchemical stability further boosted significance ofthese molecules as these enzymes can catalysereaction in different temperature and in thepresence of various chemicals [5].Another aspect which efficiently drives modernindustrial operation is reuse of catalyst to cut downproduction cost. Here enzyme emerged as keyplayer which can be used for several times incontinuous reaction after immobilization onappropriate material [6]. These enzymes had maderemarkable land mark in several industries includingfood processing, detergent industry, leather andtextile industry [7-8]. To meet commercial demandnumerous biological sources were explored andtonnes of enzyme had been produced in last fewdecades. Among these potential sources used forenzyme production microbial (bacterial and fungal)species lies on the top with enormous capability ofenzyme production [9]. Further, advancement inbiological science especially bioengineering,molecular biology and protein engineering enabledto produce enzyme in large scale and specific toreaction [10]. Significance in using enzyme frommicrobial sources over conventional catalyst is ease219in downstream process where purification ofproduct became easier. Food processing, leathermaking and silk degumming are driven by microbialprotease and fungal protease constitutes more than30% of enzyme in all these process. It is widelyaccepted that protease are contributing theirpotential in modern therapeutics includingdevelopment of anti-inflammatory drugs, clotdissolving agents, antimicrobial and cancertreatment [11]. Finding a novel and suitable sourcefor such amazing molecule has been dauntingchallenges for decades which led to exploration ofmicrobial diversity and use of modern recombinantDNA technology to fulfil commercial demand.ProteaseProteases are abundantly and widely distributed inbiological world including plant, animal andmicrobes[12].A protease alsocalledaspeptidase or proteinase is group of enzyme thatperforms proteolysis known as hydrolysis ofthe peptide bonds that link amino acids together inthe polypeptide chainformingtheprotein[13]. Proteases constitute more than 70% ofindustrial enzyme alone and microbial sources(bacterial and fungal) are leading supplier of theseenzyme. These enzymes possess catalytic activity inbroad range of temperature and pH [14-15]. Theentire protease group has been evolved duringorganic evolution where catalytic site for eachenzyme became key factor in driving biochemicalreaction. Such catalytic promiscuity has emerged askey tool for modern commercial industry whereseveral biochemical reactions can be catalysed bysingle group of enzyme [16].However, discovery of psychrophilic andthermophilic enzyme including proteases enhancedtheir catalytic spectrum. In current prospect, severalproteases have been isolated and produced in arecombinant way to catalyse reactions intemperature ranges 50C-1000C and in pH range 2-10[17-18]. The physiological role of protease has beenJ Pharm Chem Biol Sci, December 2014 -February 2015; 2(4): 218-234
J. Srilakshmi et alestablished and these molecules participate invarious physiological reactions including digestion,defence mechanism. All the protease exhibitscommon mechanism of action as acting oncarbonyl- carbon bond of peptide group. However,different protease utilize different strategies togenerate nucleophile to attack of carbonyl-carbon(O C-C) bond. The most acceptable classification ofenzyme is based on the molecular mechanism ofenzyme. The core amino acids in process of catalysisas active site of enzyme define specificity andcatalytic efficiency of any biocatalyst [19].Classification of ProteaseThe protease group is vast and constitute more than70% of commercial enzymes with diverse substrateand catalytic capabilities. The proteases have beenclassified based on several criteria such as targetedamino acid for hydrolysis, chemical environmentand type of substrate [20]. However, mostconvincing mode of classification is based on aminoacid involve in hydrolysis and on that basisproteases have been classified into six majorgroups:1. Serine Proteases (EC 3.24.21)2. Threonine proteases (EC 3.4.25)3. Cysteine proteases (EC 3.4.22)4. Aspartate proteases (EC 3.4.23)5. Glutamic acid proteases (EC 3.4.19)6. Metalloproteases (MMPs) (EC 3.4.24)Serine proteases (EC 3.24.21)The serine proteases contribute major industrial andtherapeutic protease where serine serves as thenucleophilic amino acid. In this class of ally available serine protease [21]. Withtheir tremendous scope in industry and medicineseveral recombinant serine protease have beenproduced and are in commercial use. Till date, 16proteases superfamilies’ have been reportedand MEROPS database which is protease database220was developed as repository of all serine proteasesfrom various sources [22]. Each proteasesuperfamily uses the catalytic triad or dyad in adifferent protein fold and so represents convergentevolution of the catalytic mechanism. Among allthese protease super-families, four distinct andmost significant from commercial point of view aretrypsin-like, chymotrypsin-like, elastase-like andsubtilisin-like proteases [23].Threonine proteases (EC 3.4.25)However, threonine protease is another industriallysignificant protease where threonine (Thr) residuelies on catalytic site. These proteases have muchsignificance in physiology and proteasome andacyltransferases are classical example [24]. Till date,fivefamiliesalongwithtwoseparate superfamilies identifiedfromvarioussources. The two major threonine proteasesuperfamiliesclassifiedastheNtnfold proteosomes (superfamily PB) and the DOMfold ornithine acyltransferases (superfamily PE). Thisclassification is based on organic evolution ofthreonineproteasedenotesanindependent, convergent evolutions of the sameactive site [25].Cysteine proteases (EC 3.4.22)Further, cysteine proteases also known as thiolprotease possess great importance in industrialapplications. These proteases perform catalysisassociated with nucleophilic thiol in a catalytic triador dyad. These proteases are primarily present in allthe fruits including papaya, pineapple, fig and kiwifruit. Cysteine proteases had shown their potentialin poultry industry and are key competent of meattenderizers. Additionally, cysteine proteases havebeen employed as therapeutics enzyme incontrolling viral infection [26]. The 14 differentsuperfamilies have been reported under cysteineprotease and as per MEROPS protease database theJ Pharm Chem Biol Sci, December 2014 -February 2015; 2(4): 218-234
J. Srilakshmi et alevolution of all these families denotes convergentevolution of catalytic site [27].Aspartate proteases (EC 3.4.23)Aspartic acid proteases are quite different fromother proteases as their contribution in maintainingphysiological functions. The classical examples arepepsins, cathepsins, and renins key enzyme whichmaintain physiology [28]. This group of enzymeshows their activity in lower pH and showingresemblance with acid proteases. Till date fourdifferent families under aspartase proteases havebeen identified from various sources and aredenoted as Family A01 (Pepsin family), Family A02,Family A22 and Family Ax1. The organic evolution ofaspartate protease families as per MEROPSdatabase is due to ancestral gene duplication [29].Glutamic acid proteases (EC 3.4.19)Most important glutamic acid proteases which arewidely present in fungal species are key tenzyme forfood processing and modern therapeutics such asantitumor and anticancer [30]. Recently, theglutamic protease family re-classified as a sixthcatalytic type of peptidase (family G1) in theMEROPs database. [31]. The presence of glutamicacid protease in fungi is unclear as there is completelack of such enzyme in fungal physiology. But withlarge number of fungal isolates and the divergenceof glutamic acid protease suggested concept of geneconservation and the evolution of protein families.Metallo-proteases (MMPs) (EC 3.4.24)A novel protease group called metalloproteasewhich enormously used in drug developmentinvolve metal ion for catalysis. Due to wide range ofsubstrate affinity and diverse sources of proteasesthe applications of proteases are unique and widelyapplicable in different industries [32-33]. Further,thermostable protease discovery solve manyproblems encountered while using conventionalproteases such as storage and industrial operation221running in higher temperatures. Further,immobilization techniques of modern enzymeengineering technology enhanced commercialsignificance of these enzymes [34]. Apart from thesemajor groups proteases also classified based onreaction environment. The catalytic capability ofenzyme including protease affected by pH in largerextent and hence proteases have been classifiedbased on maximum catalytic activity shown inparticular pH. The three major sub classificationshave been made as acid proteases, alkalineproteases and neutral proteases. This classificationof protease lies on principle of reaction condition ofenzymes, acid proteases perform enzymaticcatalysis in lower pH range 2-6, while alkalineprotease in higher pH 8-10. However, neutralprotease shows their maximum activity near a pH of7.0. This classification does not fall in any scientificcriteria precisely used for enzyme classificationhowever it offer ease in selection of enzyme fordesiredindustrial process.Further, suchclassification is also useful in designing reactionconditions in order to avoid production loss [35].Historical background and present demandSince the ancient time period, the enzymetechnology was associated in fermented products aspart of food and medicine. Over the last fewcenturies, bakery products, alcohol and vinegarhave served mankind as part of conventional foodand liquor across the globe [36]. Fungi played acrucial role to produce these products available forhuman consumption, even without knowing exactmechanism. However, several mushrooms specieswere known as food stuff with high nutritionalvalues [37-38]. Apart from these applications,numerous fugal species were used as part of folkmedicine to combat several life threateningdisorders. As for concern of industrial applications,the use of fungal extract of hunting animals (toxin),cleaning natural fabric such as silk and recycling ofwaste material was known since ancient timeJ Pharm Chem Biol Sci, December 2014 -February 2015; 2(4): 218-234
J. Srilakshmi et al222period. In the last century with advancingtechnology in molecular biology these potentialenzymes for the commercial application fromvarious fungal sources were defined [39-40]. Severalnovel species of fungi were isolated andcharacterized for large scale production ofproteases. Among these species Aspergilous, Yeast,Candida, Penicillium and Cephalosporium are majorone and were employed for large scale productionof proteases and other enzymes [41-42]. Over thelast few decades, several fungal strains wereisolated and characterized for the source of theproteases which can catalyze biochemical reactionsin different pH, temperatures [43-44]. Further,recombinant DNA technology and enzymeengineering technology has played crucial role inlarge scale production and precise reuse of theproteases produced [45]. Proteases have a largevariety of applications in various industries andmany investigations are focused on the discoveryand characterization of novel naturally occurringproteases from sources that have been overlooked.The proteases produced from different fungalspecies are in the use of several industrialoperations, including, food processing, making ofleather, fabric industry, meat tenderazation and astherapeutics [46].Psychrophilic FungiThe microbial world has been key source of variousenzymes for industrial and therapeutic applicationsince many decades. Bacterial species are on the topconcern for large scale production of enzymeworldwide [47].However, fungi also havecontributed tremendous scope as potential sourceof proteases and other enzymes. Fungi are widelydistributed organism are capable in producingseveral class of proteases including cold tolerantprotease, protease acting on normal temperatureand thermostable protease acting in highertemperature [48]. Several deep sea fungi arecapable in producing psychrophilic proteaseincluding Aspergillus terreus, Beauveria brigniartiiand Acremonium butyri. Another class of fungicalled as thermophiles such as aromycesthermophiles,Myriococcumthermophilum and Dactylomyces thermophiles arecapable in producing commercial enzyme includingprotease that act in higher temperature (table 3)[49]. Several strain of yeast including Candidalipolytica, Yarrowia lipolytica and Aureobasidiumpullulans have been reported in large scaleproduction of protease [50].Sources of Commercially Significant ProteasesTo meet commercial demands, large scaleproduction of these enzymes became necessary andhence several sources were explored includingfungi. The fungi possess diverse habitat, growubiquitously and produce various proteins/enzymesfor their survival. Among these enzyme proteasesconstitutes a major class along with other enzymessuch as lipase, cellulase, xylinase and pectinase.Based on habitat fungi are classified aspsychrophilic, mesophilic and thermophilic emergedas potential sources of commercial enzymesincluding proteases.Mesophililic fungiThe mesophilic fungal strains are also competent inproduction of large scale of commercial proteaseenzymes. The major class of mesophilic fungi isAspergillus which contribute more than 25% ofprotease produced from fungal source whichincludes Aspergillus candidus, A. flavus, A.fumigatus, A. melleus, A. niger, A. oryzae, A. sojae,A. sulphurous and A. sydowi. Such diverse fungalbiodiversity results in various kinds of proteasecatalyse numerous biochemical reactions [51-52].Industries such as food processing basically utilizedcold tolerant enzymes including protease andmarine environment is enormous sources ofJ Pharm Chem Biol Sci, December 2014 -February 2015; 2(4): 218-234
J. Srilakshmi et alprotease producing enzymes. Similarly, hot springsand marshy area are the key source for thermophilicfungi ideally produces protease and other enzymefor commercial application (table 3) [53]. It is widelyaccepted that mesophilic fungi are distributedubiquitously and need to explore for ideal candidatesource [54].Thermophilic fungiAmong the eukaryotic organisms, only a few speciesof fungi have the ability to thrive at highertemperature. Such fungi comprise thermophilic andthermotolerant forms, which are arbitrarilydistinguished on the basis of their minimal andmaximum temperature growth. In such habitat,these strains produces heat shock proteins (HSPs)allow organism to sustain in hostile environment.Thermophilic proteases are produced bythermophilic microbes including bacteria and fungipossess great scope for commercial application [5556]. These proteases withstand with theirproteolytic activity at higher temperature ideal forseveral industrial process. Deep Ocean and hotspring are key geographical area for hunting theseproteases which meet commercial demands [57-58].Several fungal strains naturally build their habitat insuch harsh condition and produce thermostableproteases. A list of fungal strains isolated from suchharsh conditions and protease produced forcommercial application is shown in table 2. Till datemore than 150 different fungal strains have beenisolated and characterised that are capable inproducing such enzymes [59].These proteases not only offer catalytic activity inhigher temperature but also possess longer shelf lifeand provide ease in storage [60]. There istremendous potential in fungal biodiversity toisolate such proteases with higher catalyticpotential. Enzymes of thermophilic fungi have beenstudied primarily to explore their suitability inbioprocesses and, to a lesser extent, to probesimilarities and differences in physicochemical223properties between enzymes from mesophilic andthermophilic fungi [61]. Thermophilic fungi alsosynthesize heat shock proteins (HSPs) and acquirethermo tolerance. They observed that conidia ofThermomyces lanuginosus, germinated at 50 C andheat shocked at 55 C for 60 min prior to exposure to58 C (Table 3) [62]. Later, Thermomyces lanuginosusstrains were characterized as excellent source ofxylanase production another commercial significantenzyme.Present studyWith the increasing potential of fungal derivedenzymes, a study was carried out to isolated novelfungal strains and enzyme production by solid statefermentation. To isolate novel fungal strain varioussamples were explored including cow dung, snuff,birds n
However, fungal protease are more promising for commercial application as these microorganism are more heir habitats. Fungal species are competent in expression of enzymes in psychrophilic, mesophilic and thermophilic conditions. In last few decades, psychrophilic and thermophilic enzymes were identified for their commercial potential worldwide. Several fungal strains have been isolated and .
Microbes account a two-thirds share of commercial protease around the globe. Twelve Aspergillus sp were isolated from stressed soil samples from different areas in Bangalore. Isolated fungal strains were screened for Protease production by plate assay (casein plate). Aspergillus sp KP 10were selected as best protease producer (1.08 cm) and were confirmation carried out by Thin layer .
The fungal culture was spread onto a slide by using second needle in order to tease out the fungal structures. The needles sterilised in bunsen flame (Holt et al., 1994; Leck, 1999; Mohanasrinvasan et al., 2012). Screening for protease production Skim milk agar medium is used for screening of protease production by fungi
Optimization of Alkaline protease production by fungal isolate using solid state fermentation (Department of Microbiology, V.E.S College of Arts, Science and Commerce, Chembur, Mumbai-400071, India) Abstract :The purpose of the present study focuses on production of extracellular alkaline proteases by a local fungal isolate through solid state fermentation. The influence of process parameters .
Mangrove microflora as potential source of hydrolytic enzymes for commercial applications Bhavya Kachiprath, Solly Solomon, G. Jayanath, . than 50% of the fungal isolates showed amylase, protease and lipase activity. Among yeasts, 80% showed lipase production, 40% showed cellulase and protease production and 20% strains exhibited glutaminase and ligninase production (Fig. 1). Molecular .
Protease is the most valuable commercial enzyme and it covers the 60% of total enzyme market. Plants, animals and microbes, viruses, and archea are the main sources for protease production (Rao et al., 1998). Alkaline proteases hold a great potential for application in the detergent and leather industries (Kumar and Takagi, 1999; Oberoi et al., 2001) because of the increasing tendency to .
An alkalophilic fungal strain, Aspergillus versicolor PF/F/107 isolated from poultry farm produced alkaline protease at 40 C and 9.0 pH. Protease showed compatibility with commercial detergents and retained 50-76% of its original activity at 40 C in the presence of detergents in the following order: Ghadi, Ujalla, Surf
Fungal community composition correlated with gross nitrification rate, with 43% of the variation in gross nitri-fication rate attributable to soil fungal abundance. Conclusions Changes in soil fungal community caused by bamboo invasion into broadleaf forests were closely linked to changed soil organic C chemical composition
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