Actinomycetes: Role in Biotechnology and Medicine

Actinomycetes: Role in Biotechnology and Medicine

Guest Editors: Neelu Nawani, Bertrand Aigle, Abul Mandal, Manish Bodas, Sofiane Ghorbel, and Divya Prakash

BioMed Research International

Actinomycetes: Role in Biotechnologyand Medicine

BioMed Research International

Actinomycetes: Role in Biotechnologyand Medicine

Guest Editors: Neelu Nawani, Bertrand Aigle, Abul Mandal,Manish Bodas, Sofiane Ghorbel, and Divya Prakash

Copyright 2013 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in BioMed Research International. All articles are open access articles distributed under the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalwork is properly cited.

Contents

Actinomycetes: Role in Biotechnology and Medicine, Neelu Nawani, Bertrand Aigle, Abul Mandal,Manish Bodas, Sofiane Ghorbel, and Divya PrakashVolume 2013, Article ID 687190, 1 page

Actinomycetes: A Repertory of Green Catalysts with a Potential Revenue Resource, Divya Prakash,Neelu Nawani, Mansi Prakash, Manish Bodas, Abul Mandal, Madhukar Khetmalas, and Balasaheb KapadnisVolume 2013, Article ID 264020, 8 pages

Streptomyces misionensis PESB-25 Produces a Thermoacidophilic Endoglucanase Using SugarcaneBagasse and Corn Steep Liquor as the Sole Organic Substrates, Marcella Novaes Franco-Cirigliano,Raquel de Carvalho Rezende, Monica Pires Gravina-Oliveira, Pedro Henrique Freitas Pereira,Rodrigo Pires do Nascimento, Elba Pinto da Silva Bon, Andrew Macrae, and Rosalie Reed Rodrigues CoelhoVolume 2013, Article ID 584207, 9 pages

Anti-Candida Properties of Urauchimycins from Actinobacteria Associated with Trachymyrmex Ants,Thais D. Mendes, Warley S. Borges, Andre Rodrigues, Scott E. Solomon, Paulo C. Vieira,Marta C. T. Duarte, and Fernando C. PagnoccaVolume 2013, Article ID 835081, 8 pages

Identification and Biotechnological Application of Novel Regulatory Genes Involved in StreptomycesPolyketide Overproduction through Reverse Engineering Strategy, Ji-Hye Nah, Hye-Jin Kim, Han-Na Lee,Mi-Jin Lee, Si-Sun Choi, and Eung-Soo KimVolume 2013, Article ID 549737, 10 pages

Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes,Surang Chankhamhaengdecha, Suphatra Hongvijit, Akkaraphol Srichaisupakit, Pattra Charnchai,and Watanalai PanbangredVolume 2013, Article ID 782847, 8 pages

Streptomyces lunalinharesii Strain 235 Shows the Potential to Inhibit Bacteria Involved in BiocorrosionProcesses, Juliana Pacheco da Rosa, Elisa Korenblum, Marcella Novaes Franco-Cirigliano, Fernanda Abreu,Ulysses Lins, Rosangela M. A. Soares, Andrew Macrae, Lucy Seldin, and Rosalie R. R. CoelhoVolume 2013, Article ID 309769, 10 pages

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013, Article ID 687190, 1 pagehttp://dx.doi.org/10.1155/2013/687190

EditorialActinomycetes: Role in Biotechnology and Medicine

Neelu Nawani,1 Bertrand Aigle,2,3 Abul Mandal,4 Manish Bodas,1

Sofiane Ghorbel,5 and Divya Prakash1

1 Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune 411033, India2Universite de Lorraine, UMR 1128, Dynamique des Genomes et Adaptation Microbienne, 54506 Vanduvre-les-Nancy, France3 INRA, Dynamique des Genomes et Adaptation Microbienne, UMR 1128, 54506 Vanduvre-les-Nancy, France4 School of Life Sciences, System Biology Research Center, University of Skovde, Box 408, 541-28 Skovde, Sweden5 Laboratoire de Genie Enzymatique et de Microbiologie, Ecole Nationale DIngenieurs de Sfax, Sfax, Tunisia

Correspondence should be addressed to Neelu Nawani; [email protected]

Received 16 May 2013; Accepted 16 May 2013

Copyright 2013 Neelu Nawani et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Actinomycetes, one of themost diverse groups of filamentousbacteria, are well recognized for their metabolic versatility.The bioactive potential of these bacteria facilitates theirsurvival even in distress and unfavourable ecological con-ditions. This special issue is dedicated to the importance ofmultitude of primary and secondarymetabolites produced byactinomycetes.The six articles published in this issue balancethe biocatalytic and biocidal potential of actinomycetes.

The importance of large repertory of enzymes fromactinomycetes and their potential in replacing chemicalcatalysts is discussed. Successful commercialization of theseenzymes is an important step towards revolutionizing greentechnology. Reduction in the cost of enzyme production isdemonstrated by production of endoglucanases from Strep-tomyces sp. on low-cost substrates. Such low-cost productioninitiatives can be extended to other enzymes andmetabolites.Novel properties like thermal and ionic stabilities and a betterturnover make these systems infallible and regenerative.The activity of enzymes from actinomycetes is not confinedto substrate conversion alone but broadened to biocontrolof quorum-sensing-dependent phytopathogens, as mediatedby acyl-homoserine-lactone-degrading enzymes from endo-phytic actinomycetes.

Unexplored environments often appeal to researchersin the hope of accruing novel bacteria, a continuous questwhich has actually led to discovery of unusually industriousmicrobes. Antimicrobial potential of actinobacteria isolatedfrom the integument of Trachymyrmex fungus-growing ants

is on par with commercial antimicrobials, clearly manifest-ing a new explorable niche actinobacterial symbionts ofplants and animals. The term antimicrobials often leadsour thoughts to medicine-related but its environment-related applications are less contrived. Streptomyces lunal-inharesii produces antimicrobial substances against sulfate-reducing bacteria commonly responsible for corrosion in thepetroleum industry, with an ability to replace the existingbiocides. Making the best out of the already good can beachieved for actinomycetes by strain improvement.Advancedmicroarray-driven reverse engineering strategies for theunderstanding andmodulation of independently functioningregulatory pathways can allow these microfactories to over-produce important antibiotics.

In a nutshell, actinomycetes offer the most promisingsynthesizers of many industrially and commercially mean-ingful metabolites. Novel and unexplored habitats may offerbacterial assemblages not reached hitherto. An integrationof newer habitats, screening, and improvement technologiescan offer promising candidates for biotechnology and health-related applications.

Neelu NawaniBertrand AigleAbul MandalManish Bodas

Sofiane GhorbelDivya Prakash

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013, Article ID 264020, 8 pageshttp://dx.doi.org/10.1155/2013/264020

Review ArticleActinomycetes: A Repertory of Green Catalysts with a PotentialRevenue Resource

Divya Prakash,1 Neelu Nawani,1 Mansi Prakash,1 Manish Bodas,1 Abul Mandal,2

Madhukar Khetmalas,1 and Balasaheb Kapadnis3

1 Dr. D. Y Patil Biotechnology & Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune 411 033, India2 System Biology Research Center, School of Life Sciences, University of Skovde, P.O. Box 408, 541 28 Skovde, Sweden3Department of Microbiology, University of Pune, Pune 411 007, India

Correspondence should be addressed to Neelu Nawani; [email protected]

Received 25 December 2012; Revised 27 March 2013; Accepted 28 March 2013

Academic Editor: Bertrand Aigle

Copyright 2013 Divya Prakash et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Biocatalysis, one of the oldest technologies, is becoming a favorable alternative to chemical processes and a vital part of greentechnology. It is an important revenue generating industry due to a global market projected at $7 billion in 2013 with a growthof 6.7% for enzymes alone. Some microbes are important sources of enzymes and are preferred over sources of plant and animalorigin. As a result, more than 50% of the industrial enzymes are obtained from bacteria. The constant search for novel enzymeswith robust characteristics has led to improvisations in the industrial processes, which is the key for profit growth. Actinomycetesconstitute a significant component of the microbial population in most soils and can produce extracellular enzymes which candecompose various materials. Their enzymes are more attractive than enzymes from other sources because of their high stabilityand unusual substrate specificity. Actinomycetes found in extreme habitats produce novel enzymes with huge commercial potential.This review attempts to highlight the global importance of enzymes and extends to signify actinomycetes as promising harbingersof green technology.

1. Introduction

Biocatalysis offers green and clean solutions to chemicalprocesses and is emerging as a challenging and reverredalternative to chemical technology. The chemical processesare now carried out biologically by biocatalysts (enzymes)which are integral components of any biological system.However, the utility of enzymes is not nave to us, as theyhave been an integral part of our lives from immemorialtimes.Their utility dates back to 1914, when they were used indetergents even before their protein nature was determinedin 1960 [1]. Their use in fermentation processes like wine andbeermanufacture, vinegar production, and breadmaking hasbeen practised for several decades. However, a commercialbreakthrough happened in the later half of 20th century withfirst commercial protease production in 1957 by Novozymes[1]. Since then, due to the advent of newer industries, theenzyme industry has not only seen an enormous growth buthas also matured with a technology-oriented perspective.

Commercially available enzymes are derived from plants,animals, and microorganisms. The enzymes derived fromplants include papain, bromelain, ficin, lipooxygenase,among several others [2], and those derived from animalsources include pepsin and renin. However, a major fractionof commercially available enzymes are derived frommicrobesdue to their ease of growth, nutritional requirements, anddownstream processing. To meet the increasing demandof robust, high turnover, economical and easily availablebiocatalysts, research is always channelized for novelity inenzyme or its source or for improvement of existing enzymesby engineering at gene and protein level [1]. Search fornovel enzymes from unusual ecological niches is often moreattractive option leading to development of high-throughputscreening programs. Enzymes with new physical and phys-iological characteristics like high productivity, specificity,stability at extreme temperature, pH or other physiologicalconditions, low cost of production, and tolerance to inhbitorsare always most sought after properties from an industrial

2 BioMed Research International

Table 1: Commercially relevant enzymes produced by actino-mycetes.

Enzyme Use Industry ofapplication

Protease

Detergents DetergentCheese making FoodClarification- low calorie beer BrewingDehiding LeatherTreatment of blood clot Medicine

CellulaseRemoval of stains DetergentDenim finishing, softening ofcotton Textile

Deinking, modification of fibers Paper and pulp

Lipase

Removal of stains DetergentStability of dough andconditioning Baking

Cheese flavoring DairyDeinking, cleaning Textile

XylanaseConditioning of dough BakingDigestibility Animal feedBleach boosting Paper and pulp

Pectinase Clarification, mashing BeverageScouring Textile

Amylase

Removal of stains DetergentSoftness of bread softness andvolume Baking

Deinking, drainageimprovement Paper and pulp

Production of glucose andfructose syrups

Starch industry

Removal of starch from wovenfabrics Textile

Glucoseoxidase Strengthening of dough Baking

Lipoxygenase Bread whitening BakingPhytase Phytate digestibility Animal feedPeroxidase Removal of excess dye Textile

standpoint. An important criterion for enzymes derived frommicrobes remains that the source microbe should have agenerally regarded as safe (GRAS) status [3].

Many microbes particularly bacteria and fungi are cur-rently employed for the production of various industrialenzymes [4]. Hydrolases cover more than 75% of commer-cially used enzymes and are often in great demand. Theseare however used in a crude form to make the processeconomically viable and also to meet the demand of enzymeat a large scale [5]. Amongst the hydrolases, proteases occupyan important platform, as they are extensively used indetergent industry, followed by starch industry which is thesecond largest user of enzymes and textiles, baking, food, andanimal feed industries. The applications of few commerciallysignificant enzymes are enlisted in Table 1.

This review touches the global and Indian enzymemarketscenario and further highlights the potential of actinomycetesas sources of important industrial enzymes like cellulases,pectinases, proteases, and chitinases which can be employedfor the recovery of numerous value added products withapplications in biomedicine and waste management.

2. Enzyme Market: Global and Indian Scenario

The global market is expected to experience a growth of6% in enzyme requirement with an estimated market of $7billion in 2013 [28]. North America and Western Europe arepredicted to show an increased growth, while the highestgrowth is likely in developing countries of Asian, Africanand Mideast regions, along with Latin America and EasternEurope. China is emerging as an important base and marketfor industrial enzymes due to various R&D activites set up bymany industrial giants which accounts for 10% of the globalscenario [29]. The demand of diagnostic and therapeuticenzymes is expected to increase owing to improvement inmedical care facilities in developing countries and globalhealth care reforms. Few enzyme manufacturing industriesin the world include AB Enzymes GmbH, Advanced EnzymeTechnologies Ltd., Amano Enzyme Inc., Asahi Kasei PharmaCorporation, Cargill Texturizing Solutions, Genencor Inter-national Inc., DSM Food Specialties, Hayashibara Company,Nexgen Biotechnologies Inc., Novozymes A/S, and MapsEnzymes Ltd.

The global industrial enzyme market has evolved con-tinounsly due to numerous mergers and acquisitions. Inthe year 2011, enzyme industry giants like Novozymes andDuPont occupied market shares of 47% and 21%, respectively[30]. Technical enzymes were valued at $1.2 billion in 2011,and this is expected to rise to $2.2 billion in 2016 with thehighest sales predicted in the leather and bioethanol markets[31]. Similarly, food and beverage enzyme sector is expectedto achieve about $2.1 billion by 2016, from a value of $1.3billion in 2011 as shown in Figure 1.This iswell correlatedwiththe numerous patents which have been filed over a periodof years which indicate an increasing trend. From Figure 2,it can be inferred that due to the lack of information onintellectual property rights (IPRs) in the 1970s, there werehardly any patents on any of the industrial enzymes [32]. Butfrom the year 2000 till date, there has been a tremendousincrease in the number of patents filed or obtained forvarious enzymes. The maximum number of patents is forproteases followed by amylases and cellulases perhaps dueto maximum utility of these enzymes. The application andissuing of patents for various enzymes is expected to grow inthe future due to green technologies.

Speciality enzymes due to their unique properties likeextreme thermostability, specific activity, and activity overa wide range of pH are expected to occupy an importantcategory in future due to their robustness. These specialityenzymes also include those useful in medicine and biotech-nology, for example, kinases, polymerases, andnucleases [33].Besides this, their utility in wide range of personal careproducts is revolutionalizing the cosmetic industry too. The

BioMed Research International 3

Other enzymes1.5

Food and beverageenzymes

1.3

(2011)

Technical enzymes1.2

(a)

Other enzymes 2.2

Technical enzymes2.2

Food and beverageenzymes

2.1

(2016)

(b)

Figure 1: Global enzyme industry market in the years 2011 and 2016.

0500

100015002000250030003500400045005000

Amylase

Xylanase

Cellulase

Protease

Amylase

XylanaseCellulase

ProteaseAmylaseXylanaseProtease

AmylaseXylanaseCellulase

Protease

197079 198089 199099 200009 201013Years

Num

ber o

f pat

ents

Figure 2: Growth in number of patents issued for importantindustrial enzymes over past few decades.

speciality sector is expected to reach $4.3 billion by 2015,and industrial enzyme segment is worth $80 million, accord-ing to reports by reputed market researchers and industryanalysts [34]. This growth would be mainly accelerated bythe pharmaceutical and diagnostics industry, the largest end-users of these enzymes which will continue to grow due to theemergence of enzyme replacement therapies and innovationsin thrombolytics.

India imports 70% of the total enzyme consumed by itsmarket which indicates need of indigenous manufacturersand technologies. The most important enzymes in demandare of the pharmaceutical sector consuming more than 50%of the total enzymes. This is followed by detergent enzymes(20%), textile enzymes (20%), and the rest comprises of foodenzymes [35]. Novozymes is one of the leading industriesin the enzyme market in India. The need of the hour is astrong R&D in terms of investment. There is necessity forstrong legislation and IPR regulations which would makeIndia withstand the stiff competition on the global front. Inaddition, the enzyme market is risky and has high captialcosts. India offers excellent human resource power which canlower production costs compared with many other countriesmaking this country an attractive location for investment.Besides, the biodiversity in India is valuable for the screeningof novel enzymes andmetabolites which can be produced andutilized at the industrial level.

3. Actinomycetes as a Source forIndustrial Enzymes

Actinomycetes are one of the ubiquitous dominant groups ofgram positive bacteria. Actinomycetes have been commer-cially exploited for the production of pharmaceuticals, neu-traceuticals, enzymes, antitumor agents, enzyme inhibitors,and so forth [36]. These bioactive compounds are of highcommercial value, and hence actinomycetes are regularlyscreened for the production of novel bioactive compounds.A wide array of enzymes and their products applied inbiotechnological industries and biomedical fields has beenreported from various genera of actinomycetes. Since thereis vital information available due to the advent of genomeand protein sequencing data, actinomycetes have been con-tinuously employed of the production of proteases, cellulases,chitinases, amylases, xylanases, and others. Representativeexamples of industrially important enzymes from actino-mycetes are discussed below, and their enzymatic propertiesare enlisted in Table 2.

3.1. Cellulases. Cellulases convert cellulose to fermentablesugars fit for human consumption and the largest knownproducers are from genus Streptomyces [37]. Cellulases fromStreptomyces sp. are reported to have an alkaline pH opti-mum and high thermostability. Subsequently, the enzymewas used as a supplement in detergents to clean, soften,and restore the color of the fabrics. It was also tested forthe treatment of textiles, processing of paper and pulp,and as an animal feed additive [6]. Besides Streptomyces,several other genera like Thermobifida and Micromonosporaproduce recombinant cellulases that can be commerciallyexploited [7]. A recombinant cellulase with thermal andpH stability is reported from Streptomyces thermoviolaceus;this enzyme retains its activity in the presence of commer-cial detergents highlighting its superiority to the existingcommercial cellulases [8]. Cellulase fromThermomonosporafusca has been used for degradation of cotton and avi-cel [9]. These enzymes not only hold a biotechnologicalpromise but can be economical due to their low costof production. Their production can be carried out oncheap substrates like rice and wheat straw [10] and fruitpeels [38].


Table 2: List of industrially viable enzymes from actinomycetes and their characteristics.

Enzyme Producing Strain pH stability Thermal stability Substrate specificity ReferenceRecombinant Streptomyces sp. 5.012.0 4050C CMC [6]Thermobifida halotolerans 6.08.0 4050C CMC [7]

Cellulase Recombinant Streptomyces sp. 10.0 40C CMC [8]Thermomonospora sp. 7.010.0 50C CMC [9]Streptomyces ruber 5.57.0 3540C CMC [10]Actinomadura sp. 4.0 70C Xylan [11]

Xylanase Recombinant strain 5.07.0 7080C Xylan [12]

Recombinant strain 5.07.0 6070C Birch xylan [13]Streptomyces spp. 8.011.0 4560C Xylan [14]Streptomyces sp. 5.07.0 4550C Starch [15]

Streptomyces erumpens 9.0-10.0 4050C Starch [16]Amylase Nocardiopsis sp. 8.6 7080C Starch [17]

Thermobifida fusca 5.07.0 60C Starch [18]Nocardiopsis sp. 5.010.0 3545C Starch [19]

Pectinase Streptomyces lydicus 4.07.0 45C Polygalacturonic acid [20]Thermoactinomyces sp. 4.0 50C NA [21]

Nocardiopsis sp. 10.0 4050C Casein [22]Protease Streptomyces pactum 7.5 40C Casein [23]

Streptomyces thermoviolaceus 6.5 65C Keratin [24]Streptomyces sp. 4.011.0 3060C Keratin azure [25]

Nocardiopsis prasina 7.0 5060C Colloidal chitin [21]Chitinase Streptomyces thermoviolaceus 6.0 60C Colloidal chitin [26]

Microbispora sp. 3.011.0 3050C Colloidal chitin [27]CMC: Carboxymethyl cellulose; NA: not available.

3.2. Xylanases. Streptomyces spp. are prolific producersof another commercially important enzyme, xylanase.Xylanases from Streptomyces sp. are preferred in treatmentof rice straw pulp to improve the pulp bleachability. Thispreference is due to absence of cellulase contamination inthe xylanase and also due to reduced usage of chemicalsduring bleaching and pulping [39]. Further, xylanases fromactinomycetes are stable on kraft pulps and can be used in thecrude form thereby making the process economical. Highthermostability and specific activity, two desirable propertiesof enzymes to be employed in industrial processes, arereported in xylanases from Actinomadura sp. FC7 andNonomuraea flexuosa [11, 12]. Similarly, fused xylanases fromfungi and actinomycetes have been employed in paper andpulp industries, due to high thermal and pH stability [13].In many higher plants and agricultural wastes, the contentof xylan is almost 2040% of the dry weight. Xylan withhemicelluloses is the second most renewable biopolymer[14]. Since pure xylan is expensive, alternate cheap substrateslike these can serve as potential substrates at industrial level.Streptomyces spp. were able to produce high levels of xylanasewhen untreated rice straw was utilized which resulted insignificant biobleaching [39]. Similarly, Streptomyces sp.was able to hydrolyze various agricultural residues like oilcake and straw waste which resulted in increased biogasproduction [14].

3.3. Amylases. Another important group of enzymes is theamylases which are employed in the starch processing indus-try for the conversion of starch to high fructose syrups [15].One of the focus areas with respect to starch industry isthe production of tailor length maltooligosaccharides whichcan be produced with amylases with a very specific modeof action. Thermophilic and acidophilic amylases which canfind applications in bakery, brewing, and alcohol indus-tries have been studied from Streptomyces erumpens [16].Thermostable amylases are reported from Nocardiopsis sp.which have important applications in bakery and paperindustries [17]. The amylase fromThermobifida sp. producedmaltotriose as the major end product from refined starch andraw sago starch [18]. Such amylases are lucrative catalystsin nutrition and healthcare [18]. Besides this, end-productspecific amylases can be used for the production of mal-tooligosaccharides from low cost starch substrates [40].Manyactinomycetes have also been reported for the productionof cold-active -amylases which can be employed in tex-tile industries, detergents, bioethanol producing industries[19].

3.4. Pectinases. Food industry uses pectinases particularlyin clarification of fruit juices, in degumming of fibres, winemaking, and retting of bast fibres. Pectinases from Strepto-myces sp. are reported [20]; however, reports of pectinases


from other genera of actinomycetes are scanty. The demandof cold-active pectate lyases is increasing due to their abilityto retain the palatability and nutritional characteristics offood products. Occurrence of pectin degrading genes in fewactinomycetes suggests their characterization could perhapsyield pectinases with novel properties.

3.5. Proteases. The quest for novel proteases and theirformulations for industries like detergents, animal feed, andbreweries is observed from several decades. Most of theproteases reported from Streptomyces spp. are alkali-tolerant,and some of them are salt tolerant and belong to genera otherthan the genus Streptomyces [21]. Proteases fromNocardiopsisspp. are employed as detergents additives [22] and for thedepilation of hides and skins in the leather industry.Dehairing of goat skin by proteases from Streptomycessp. makes the process economically and environmentallyfeasible [23]. Keratin-rich wastes like feathers, hair, nails,and horn are waste products of agroindustrial processes.Keratinolytic Streptomyces spp. capable of degrading keratinat temperatures higher than 50C are reported [24]. SomeStreptomyces hydrolyze keratin by pronases as seen for Strep-tomyces griseus [24]. Proteases from other sources are used inconjunction with enzymes from actinomycetes for recoveryof antioxidants from shellfish waste. Protease productionwas also carried by growingMicrobispora sp. on the shellfishwaste [25]. End products of protein hydrolysis rich in aminoacids and peptides serve as a low cost animal feed.

3.6. Chitinases. Chitinases are another class of hydrolaseswhich have gained tremendous importance in the pasttwo decades. They are glycosyl hydrolases that catalyze thedegradation of chitin, which is an insoluble linear -1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin isa major constituent of the shells of crustaceans, exoskeletonsof insects, and cell walls of a variety of fungi [26]. Chiti-nases are useful in protoplast preparation from fungi, asbiocontrol agents against plant pathogenic fungi, nematodes,and so forth and are recently used for the extraction ofchitin oligomers which are important biomedical products.Chitinases occur in several actinomycetes and possess uniqueproperties in terms of thermostability and activity in wide pHrange which makes them suitable for industrial applications[21, 26, 27]. One of their most resourceful applications isthe production of chitin oligosaccharides. Chitin oligosac-charides (COS) have anticoagulant, antimicrobial, antic-holesteremic, anticancer, wound-healing, antitumor, andantioxidant activities which make them bright candidates forbiomedical applications [26]. COS can be recovered fromlow cost substrates like shrimp, crab, and squid pen waste[41, 42]. Chitinase from Microbispora sp. was employed forthe recovery of chitobiose, a potential antioxidant whichcan be used as a food additive and for other biomedicalapplications [25]. This renewable resource can be utilized forthe growth of many chitinolytic organisms as well as for theeffective recovery of COS at the industrial level. The disposalof the waste is also carried out effectively by the biologicalutilization by actinomycetes.

3.7. Other Enzymes from Actinomycetes. An array of otherenzymes with industrial potential reported from actino-mycetes includes lignin peroxidases, laccases, and tyrosi-nases which are effective in the treatment of textile dyes[43] promising their application in waste treatment plants.Esterases and amidases from Nocardia sp. have been usedto increase the hydrophilicity of polyethylene terephthalateand polyamide fibers. This can be an ecofriendly and cost-effective method in textile industries [43]. To get enzymeswith novel properties or functionalities, high throughputscreening (HTS) programs are adopted for choosing rareactinomycetes which are a source of novel compounds. Suc-cess examples include therapeutic enzymes like thrombinaseand L-asparaginase from marine Streptomyces sp. which areused in the treatment of myocardial infarction and leukemia.A sponge associated Streptomyces sp. produced phytoene, acarotenoid with enhanced antioxidant activity making it apromising food additive [44].

Access to advanced technologies has made it possible toobtain untapped microbes with novel properties, and actino-mycetes have been natural reservoirs of excellent enzymes.The existing HTS methods are used for choosing industriallyimportant bacteria and have not been actively extended toactinomycetes. Several HTS methods which can be used forexploring novel enzymes from actinomycetes are discussed.Fluorescence activated cell sorting (FACS) is successfullyemployed for sorting of desired clones from a genomiclibrary, where fluorescent substrate specific for a particularenzyme is used. The positive fluorescence indicates biocat-alytic activity of the clone [45]. Gel MicroDrop technologydetects clones positive for specific enzymes by capturingthe fluorescence emitted due to catalytic breakdown ofbiotinylated substrate by the clone [46].

Metagenomics has offered rapid screening methodswhere the bioactive potential of unculturablemicrobes can beexplored. A clonal library is prepared using the metagenomeobtained from extreme habitats like arid regions, oceanbeds, stratosphere, and others without expecting the actualmicrobes to grow under laboratory conditions which usuallylimits the exploitation of the bioactive potential of thesenonculturable microbes [47]. These technologies also helpto determine the functional aspects of a microenvironment[48]. Althoughmetagenomic approach hasmany advantages,it suffers from a common disadvantage like low or no expres-sion of desired gene(s). Multiple displacement amplificationhas allowed researchers to overcome the problem of low or noexpression. Here whole genome amplification is carried outfrom single cells, which unlocks entire biochemical potentialof an uncultured microbe from a complex habitat. Otherinnovative approaches like substrate-induced gene expres-sion screening (SIGEX), preamplification inverse-PCR (PAI-PCR), and metagenomic DNA shuffling provide insights onthe functional metagenomics of a particular habitat [48].

A smart and diversified technique which acceleratesevolution is the directed evolution approach, where a libraryof genetic variants is created and themutants are screened fordesired enzymatic properties.Thebest variants are shortlistedand reorganized for another round of library creation whichis repeated a number of times to possibly get the best variants,


a process which follows the rule of natural evolution but isperformed at a pace that gives the result of evolution in ashort time [49]. This method is a significant driving forcefor the discovery of next generation biocatalysts [50]. Anadded advantage to this method is the inclusion of ultrahigh-throughput FACS-based screening which aids in the rapidscreening of the variant library [51]. Recent improvements inscreening technologies which can be very useful for enzymesfrom actinomycetes are the Drop-based microfluidics whichwas successfully used to screenmutants with ten times potenthorse peroxidase activity than wild type [52]. HTS in drop-based microfluid platform carries a small foot print chipwith an array of insoluble substrates specific for the enzymeof interest; this gives rapidity, parallel execution, and costeconomics to the screening protocol [53].

Reporter gene technology is another step up in thescreening methods which offer simplicity and sensitivity.Green fluorescent protein (GFP), a reporter of choice, iswidely used due to ease of its detection and acquiescencewith host systems [54], and coupled hexose oxidases are usedfor the detection of reducing sugars released from polymericsubstrates due to enzyme hydrolysis [55]. A workhorse inproteomics, MALDI-LTQ-Orbitrap, is an excellent tool toscreen proteins in complex matrices and suspensions. Thetechnique works on ion trap and MALDI and is interfacedwith liquid and gas chromatography separations [56]. Otherionization techniques include electrospray ionization massspectrometry (ESI-MS) which is used to measure femtomolequantities of proteins [57]. Some in silico techniques to sortpotent enzymes from database are developed. Predictive 3D-QSAR CoMFA and CoMSIA are powerful techniques whichpredict superior enzymes based on structural propertiesand microenvironment provided for in silico reaction withsubstrate. These techniques however require validation inlaboratory to assess the suitability of themodels in prediction[58].The extension of these advanced technologies in screen-ing of bioactive molecules from actinomycetes might providesupreme strains and enzymes with premium properties.

4. Conclusion

The industrial enzyme market is one of the fastest growingrevenue generating sectors in the world. Only 20 enzymes arecurrently utilized on the industrial level indicating the needfor further research and development of low cost enzymesand their production. The application of enzymes in diversebiotechnological industries indicates a positive trend whichneeds to be satisfied with the discovery of novel enzymes andmetabolites. Since very few enzymes have been potentiallyutilized at the industrial level; there is a huge scope for thedevelopment of robust and low cost enzymes. Actinomycetesare a reservoir of important enzymes and metabolites dueto their versatile genetic repertory. However, many of therare genera of actinomycetes have been neither explorednor manipulated for their biotechnological and industrialpotential. Studies on unique ecological environments couldyield molecules that could become future harbingers of greentechnology.

Acknowledgments

The authors are grateful to the Department of Science andTechnology, Government of India, Swedish InternationalDevelopment Cooperation Agency, Sweden, and Dr. D. Y.Patil Vidyapeeth, Pune, for support.

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Research ArticleStreptomyces misionensis PESB-25 Producesa Thermoacidophilic Endoglucanase Using Sugarcane Bagasseand Corn Steep Liquor as the Sole Organic Substrates

Marcella Novaes Franco-Cirigliano,1 Raquel de Carvalho Rezende,1

Mnica Pires Gravina-Oliveira,1 Pedro Henrique Freitas Pereira,1

Rodrigo Pires do Nascimento,2 Elba Pinto da Silva Bon,3 Andrew Macrae,1

and Rosalie Reed Rodrigues Coelho1

1 Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Goes, Centro de Ciencias da Saude (CCS), UniversidadeFederal do Rio de Janeiro (UFRJ), Avenida Carlos Chagas Filho 373, Bloco I, Laboratorio 055, 21941-902 Rio de Janeiro, RJ, Brazil

2 Departamento de Engenharia Bioqumica, Escola de Qumica, Centro de Tecnologia (CT), Universidade Federal do Rio de Janeiro(UFRJ), Avenida Athos da Silveira Ramos 149, Bloco E, sala 203, 21941-909 Rio de Janeiro, RJ, Brazil

3 Departamento de Bioqumica, Instituto de Qumica, Centro de Ciencias Matematicas e Natureza (CCMN), Universidade Federal doRio de Janeiro (UFRJ), Avenida Athos da Silveira Ramos 149, Bloco A, sala 539, 21941-909 Rio de Janeiro, RJ, Brazil

Correspondence should be addressed to Rodrigo Pires do Nascimento; [email protected]

Received 1 October 2012; Revised 8 January 2013; Accepted 4 February 2013

Academic Editor: Divya Prakash

Copyright 2013 Marcella Novaes Franco-Cirigliano et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Streptomyces misionensis strain PESB-25 was screened and selected for its ability to secrete cellulases. Cells were grown in a liquidmediumcontaining sugarcane bagasse (SCB) as carbon source and corn steep liquor (CSL) as nitrogen source, whose concentrationswere optimized using response surface methodology (RSM). A peak of endoglucanase accumulation (1.01 UmL1) was observed ina medium with SCB 1.0% (w/v) and CSL 1.2% (w/v) within three days of cultivation. S. misionensis PESB-25 endoglucanase activitywas thermoacidophilic with optimum pH and temperature range of 3.0 to 3.6 and 62 to 70C, respectively. In these conditions,values of 1.54UmL1 of endoglucanase activity were observed. Moreover, Mn2+ was demonstrated to have a hyperactivating effecton the enzyme. In the presence ofMnSO

4

(8mM), the enzyme activity increased threefold, up to 4.34UmL1. Mn2+ also improvedendoglucanase stability as the catalyst retained almost full activity upon incubation at 50C for 4 h, while in the absence of Mn2+,enzyme activity decreased by 50% in this same period. Three protein bands with endoglucanase activity and apparent molecularmasses of 12, 48.5 and 119.5 kDa were detected by zymogram.

1. Introduction

Enzymatic hydrolysis of cellulose is a challenge worldwide,because currently we lack inexpensive and efficient enzymesto hydrolyse the 1.5 trillion tons of cellulose producedannually [1]. Enzyme blends and optimization are requiredto speed up enzymatic hydrolysis to make the process com-mercially viable. Cellulose is a homopolymer of -1,4 linkedglucose units presenting both amorphous and crystalline

regions. Its hydrolysis is carried out by endo--1,4-glucanase(EC 3.2.1.4), which cleaves internal -1,4-glycosidic bondsat random positions and forms insoluble reducing sugars,and by exo--1,4-glucanase (EC 3.2.1.91) that hydrolysescellulose from its reducing and nonreducing ends releasingsoluble reducing sugars with prevalence of cellobiose. Theenzyme -glucosidase (EC 3.2.1.21) converts cellobiose intoglucose monomers [2]. An increase in the formation of freereducing and nonreducing ends from endo-acting cellulases


could speed up the action of the exoglucanases and the totalcellulose hydrolysis process. A significant amount of researchon new endoglucanases has been done [3, 4].

Cellulolytic organisms are ubiquitous in nature. Theyare mostly bacteria and fungi, aerobic or anaerobic, andmesophilic or thermophilic. Actinomycetes, which areGram-positive filamentous soil bacteria, are well known for theirability to decompose complex molecules, particularly the lig-nocellulose components, which make them important agentsin decomposition processes [5]. They have also been shownto produce thermostable cellulases, with alkalophilic andacidophilic characteristics [6, 7].Thework that has been donein our laboratory with strains from the Streptomyces genusindicates that endoglucanase activity is predominant in thesebacterial cellulases. In previous studies from our laboratory,we reported that the culture supernatant of S. malaysiensisAMT-3, S. drozdowiczii M-7A, and S. viridobrunneus SCPE-09 presented endoglucanase activity with optimal pH in therange of 4.0 to 5.0, optimal temperature around 50C andmolecular masses, according to zymogram analyses, in therange of 37 to 178 kDa [810].

This study investigated cellulase production by an acti-nobacterial strain, S. misionensis PESB-25. Experimentaldesign was performed to optimize endo--1,4-glucanaseproduction using SCB as the main carbon source and CSLas nitrogen source. As seen before, these low-cost materialscan be suitable for cellulases production [810]. The elec-trophoretic profiles of extracted enzymes were determinedby zymogram analyses. Enzymatic activity was investigatedover a range of pH and temperature values in the culturesupernatants (crude enzyme preparation).The effect of metalions, most importantly Mn2+, on the endoglucanase activityand stability was also evaluated.

2. Materials and Methods

2.1. Microorganism Screening, Preservation, and Cultivation.StreptomycesmisionensisPESB-25was collected from a sugar-cane crop soil in the State of Pernambuco, Brazil.The dilutionplate technique was used for the isolation of the bacterialstrain, which was selected as cellulolytic via its cultivation onsolidmedium containing carboxymethylcellulose low viscos-ity (CMClw) as carbon source followed by the identificationof the CMC-degrading zones using the Congo red dye [11].Spore suspensions were prepared according to Hopwoodand colleagues [12] after cultivation at 28C for 15 days inyeast extract-malt extract-agar medium [13]. Spores weremaintained in 20% (v/v) glycerol at 20C.

2.2. Molecular Identification of Bacterial Strain PESB-25.Genomic DNA was extracted using the method describedby Kurtzman and Robnett [14]. PCR amplification of therrs gene was carried out using the GoTaq Green MasterMix kit (Promega Corporation), with primers 27F [15] and1541R [16], in a thermal cycler model Gene Amp PCRSystem 9700 (Applied Biosystems). Amplified fragmentswere purified using the Illustra GFX PCR DNA and GelBand Purification kit (GEHealthcare) and sequenced directly

using ABI Prism dye terminator cycle sequencing reactionkit (Applied Biosystems) in an automatic sequencer (ABImodel 3730; Applied Biosystems). The sequence of rrs geneobtained was compared with sequences online at the Ribo-somal Database Project (RDP) release 10 [17] and GenBank[18] using the NCBI (The National Center for Biotechnol-ogy Information) basic local alignment search tool, BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi) [19].

2.3. Endoglucanase Production Using Experimental Design.Streptomyces misionensis PESB-25 was cultivated in liquidmedium with SCB and CSL as the main carbon and nitrogensources, respectively. SCB consists of 43.8% cellulose, 25.8%hemicellulose, 22.1% lignin, 6.1% extractives, and 1.4% ash[20]. It contains, approximately, 45.3% carbon and 0.5%nitrogen [21]. CSL is a major by-product of the corn wet-milling industry and contains 47% crude protein, 26% lacticacid, 7.8% phytic acid, 2.5% reducing sugars (as dextrose), and17% ash, total nitrogen being 7.5% [22].

Response surface methodology (RSM) was used as atool for the optimization of SCB and CSL concentrations(independent variables) in the range indicated in Table 1.Endoglucanase activity (UmL1) was the dependent variable.A 22 central composite rotational design (CCRD) was used todesign experiments.

Cultivations were carried out in 125mL Erlenmeyer flaskscontaining 25mL of mineral salts solution [23] (in gL1:NaCl, 2.0; KH

2PO4, 3.0; K

2HPO4, 6.0; MgSO

47H2O, 0.5;

CaCl2, 0.05), supplemented with a trace element solution [13]

(in gL1: CuSO45H2O, 6.4; ZnSO

47H2O, 1.5; FeSO

47H2O,

1.1; MnCl24H2O, 7.9), with SCB and CSL at the relevant

concentrations.Themedium start pHwas adjusted to 7.0.Thegrowthmediumwas inoculated with 25 L of a spore suspen-sion (109 sporesmL1) and incubated at 28C, under agitation(200 rpm), for 3 days. The cultures were filtered throughglassmicrofiber filter (Millipore), and the culture supernatant(crude enzyme preparation) was used for endoglucanaseactivity determination.

2.4. Standard Endoglucanase Activity Assay. Endoglucanaseactivity was determined by measuring the release of reducingsugars in a reaction mixture containing 0.5mL of the crudeenzyme preparation and 0.5mL of CMClw (SIGMA) 4.0%(w/v) solution in sodium citrate buffer 50mM (pH 4.8)incubated at 50C for 10min. Reducing sugars were assayedby the dinitrosalicylic acid method [24]. One unit (IU) ofendoglucanase activity corresponded to the formation of1 mol of reducing sugars equivalent per minute under theassay conditions [25].

2.5. Effect of pH, Temperature, and Ions on the EnzymeActivityand Stability. To study the effect of pH and temperatureon the supernatants endoglucanase activity, a CCRD 22 wasused. In the 12 experiments which were carried out, thetemperature ranged from 40 to 70C and the pH values from3.0 to 7.0 as shown inTable 3. Citrate buffer (50mM)was usedfor pH 3.0, 3.6 and 5.0 and phosphate (50mM) for pH 6.4 and7.0 [26]. Statistical analysis of the results was performed using


Table 1: Observed and predicted values of endoglucanase activity for the independent variables SCB and CSL concentrations used in centralcomposite rotational design (CCRD), from the crude enzyme extract of Streptomyces misionensis PESB-25.

Run SCB (%w/v)/Coded level CSL (%w/v)/Coded level Endoglucanase activity(UmL1) ObservedEndoglucanase activity(UmL1) Predicted

1 0.65 (1) 0.77 (1) 0.95 0.11 0.912 1.35 (+1) 0.77 (1) 0.72 0.005 0.673 0.65 (1) 1.63 (+1) 0.86 0.003 0.854 1.35 (+1) 1.63 (+1) 0.98 0.038 0.965 0.5 (1.41) 1.2 (0) 0.93 0.022 0.956 1.5 (1.41) 1.2 (0) 0.83 0.032 0.867 1.0 (0) 0.6 (1.41) 0.66 0.003 0.718 1.0 (0) 1.8 (1.41) 0.87 0.024 0.889 1.0 (0) 1.2 (0) 1.03 0.016 1.0110 1.0 (0) 1.2 (0) 1.03 0.044 1.0111 1.0 (0) 1.2 (0) 1.00 0.003 1.01The statistical analysis of the results was performed using the software Design Expert 7.0 (trial version).Values are based on Mean SD of 3 individual observations.

the software Design Expert 7.0 (trial version), and responsesurface graphics were plotted with STATISTICA 7.0 (trialversion).

The influence of sodium, calcium, potassium, and bariumions in the chloride form and copper, magnesium, cobalt,manganese, and iron in the sulfate formon the endoglucanaseactivity was done by the addition of the relevant salts at2mM final concentration in the enzyme activity assay usingthe previously determined optimal conditions for pH andtemperature. The effect of Mn2+ was studied using at finalconcentrations of 1, 2, 4, 8, and 10mM.

Endoglucanase thermal stability was evaluated at 65Cand 50Cupon incubation at different time intervals. Stabilityexperiments were also performed in the presence of MnSO

4

(8mM or 16mM) in mixtures with 1.5mL of the crudeenzyme plus 1.5mL of MnSO

4solutions. In all cases, residual

enzymatic activity was assayed at optimal conditions for pHand temperature, taking into account the relevant enzymedilutions.

2.6. Zymogram of Endoglucanase Activity. The culture super-natants from optimized growth conditions were analyzedby electrophoresis on denaturing 10% sodium dodecyl sul-phate (SDS)-polyacrylamide gel added of copolymerizedCMClw (SIGMA) 0.2% (w/v) as the zymogram substrate.Electrophoresis was performed at constant voltage (100V)at 4C for 3 h followed by incubation with Triton X-100sodium acetate 1.0% buffer for 30min in ice bath for SDSremoval. The detection of protein bands with endoglucanaseactivity was performed by incubating gels at 50C and pH4.8 (sodium citrate buffer 50mM) for 30min, followed bythe gel immersion in Congo red 0.1% (w/v) for 10min andwashing with NaCl 1M until the visualization of the enzymebands [27]. The molecular masses of the enzyme bands seenin gels were estimated by comparing their position in thegel with a molecular mass ladder using standard molecularmasses ranging from 12 to 225 kDa (Full-Range Rainbow-GE Healthcare), which was run along with the sample andphotographed before Congo red staining.

Table 2: Statistical ANOVA for the model of endoglucanase pro-duction at different levels of concentrations of SCB and CSL.Source ofvariations

Sum ofsquares

Degrees offreedom

Meansquare value

value(prob > )a

Model 0.13 5 0.03 11.74 0.01Residual 0.01 5 0.002Lack of fit 0.01 3 0.003 2.08 0.34Pure error 0.003 2 0.001Total 0.14 10aStatistically significant at 90% of confidence level; 2 = 0.84.

3. Results and Discussion

The sequencing of rrs gene resulted in a 1491 base sequencewhich was 100% similar to Streptomyces misionensis TypeStrain NRRL B-3230, and as such PESB-25 was putativelyidentified as a strain belonging to S.misionensis.The sequenceobtained was submitted to the GenBank database (GenBankID: JN869290). S. misionensis Type Strain NRRL B-3230 wasisolated in Misiones, Argentina, and it produces misionin,an antibiotic active against phytopathogenic fungi, includingHelminthosporium and Alternaria [28]. Strains from thisspecies have been cited in the literature confirming theirpresence in certain soils [29] and their antibiotic productioncapacity [30]; however, there have been no reports that strainsof this species can be cellulolytic.

The use of RSM and CCRD tools for the optimization ofStreptomyces misionensis endoglucanase production resultedin enzyme activity accumulation in the range of 0.67 to1.03UmL1 (Table 1). The fitted response surface for theproduction of endoglucanase is given in Figure 1. Best resultswere obtained at center-point conditions, with SCB 1.0%(w/v) and CSL 1.2% (w/v), although results obtained in someother concentrations were not so different (e.g., 1.35% SCBand 1.03% CSL). The interaction effect evident between SCBand CSL could be related to the C :N proportion necessaryfor microbial growth, and consequently better enzyme pro-duction. The relevant regression equations, resulting from


1.01 1 0.9 0.8

0.7 0.6 0.5 0.4

1.2

1

0.8

0.6

0.4

0.2

1.8

1.5

1.2

0.9

0.6 0.50.76

1

1.241.5

Sugarca

ne bagas

se

(% w/v)

Corn steep liquor

(% w/v)

Endo

gluc

anas

eac

tivity

(U m

L1

)

Figure 1: Response surface on endoglucanase production by Strep-tomyces misionensis PESB-25 using SCB and CSL concentrations asthe independent variables.

the analysis of variance (ANOVA) (Table 2) have shownendoglucanase production as a function of the codified valuesof SCB and CSL. The equation that represented a suitablemodel for endoglucanase production () is given in:

= 1.01 0.03SCB 0.06SCB2 + 0.06 CSL

0.11CSL2 + 0.09SCBCSL 0.024.

(1)

The model value of 11.74 implies that the model issignificant at a high confidence level. The probability valuewas also very low (


70

62.7

55

47.3

40 34

56

7

0.4

0.8

1.2

1.6

pH

1.20.80.4

Endo

gluc

anas

eac

tivity

(U m

L1

)

Temperature ( C)

Figure 2: Response surface for Streptomyces misionensis PESB-25endoglucanase activity by using pH and temperature values as theindependent variables.

and N sources for enzymatic production [(SCB 1.0% (w/v)and CSL 1.2% (w/v)] and one of the pH and conditionssuggested by model, pH 3.0 and 70C, in triplicate. Theresults obtained were 1.54 0.01UmL1 of endoglucanaseactivity that represented an increase of 50% in endoglucanaseactivity in comparison to that observed at pH 4.8 and50C. Based on these results, we can conclude that Strep-tomyces misionensis PESB-25 produces a thermoacidophilicendoglucanase.

Cellulases with maximum activity at the acidic pH rangeare often observed for fungal enzymes [31] as well as for Strep-tomyces. As such, endoglucanase produced by S. malaysiensisAMT-3, S. viridobrunneus SCPE-09, S. drozdowiczii M7A,and Streptomyces sp. J2 presented maximal activity in thepH range from 4.0 to 6.0 [810, 32]. However, optimum pHfor the Streptomyces misionensis PESB-25 endoglucanase wasdetermined as 3.0, which is noteworthy.

In general, the optimum temperature for endoglucanaseactivity for Streptomyces strains is around 50C [810, 33].Our strain showedmaximum activity at 70C, a characteristicthat differs from most other Streptomyces. Jaradat et al.[32] described an optimal endoglucanase activity at 60C,obtained from Streptomyces sp. J2, but as far as we areaware, there are no reports in the literature of an endoglu-canase Streptomyces origin with optimal activity at such ahigh temperature. These unusual results concerning pH andtemperature make our strain a very promising candidatefor biotechnological applications, especially when very acidicand thermophilic conditions will be necessary.

Table 5: Effect of metal ions on endoglucanase activity. Enzymewasproduced by S. misionensis PESB-25 grown on 1.0% (w/v) SCB and1.2% (w/v) CSL.

Iona Relative activity(%)Endoglucanase activity

(UmL1)Control (no addition) 100.0 1.72NaCl 133.2 2.0 2.23CuSO4 140.6 0.3 2.30MgSO4 126.6 0.9 2.18CoSO4 161.2 0.6 2.73MnSO4 201.5 0.1 3.48FeSO4 131.1 0.1 2.34CaCl2 137.6 2.3 2.25KCl 125.3 4.9 2.17BaCl2 109.3 0.9 1.97aThe final concentration in the reaction mixture was 2mM.Values are based on Mean SD of 3 individual observations.

Table 6: Effect of different manganese concentrations on endoglu-canase activity.

Mn2+concentrationa

Relative activity(%)

Endoglucanase activity(UmL1)

Control (no addition) 100.0 1.721mM 182.2 1.5 3.082mM 201.5 0.08 3.484mM 185.4 9.0 3.288mM 243.0 5.7 4.3410mM 233.7 1.6 3.96aFinal Concentration in the reaction mixture.Values are based on Mean SD of 3 individual observations.

Metal ions may be a requirement for enzymatic activityand might even be an integral component of the enzymecomplex [34]. Ions may also be required as cofactors for theirmaximum activity [35]. According to Chauvaux et al. [36],manganese and other metal ions can enhance the substratebinding affinity of the enzyme and stabilize the conformationof the catalytic site. The results for the effect of several metalions on endoglucanase activity of S. misionensis PESB-25 areshown in Table 5. None of the ions studied inhibited theenzyme activity at a concentration of 2mM. The addition ofBa2+ resulted in a small increase in activity (9.3%), whichdiffers from the results reported by Grigorevski-Lima andcolleagues [10], who showed that endoglucanase activity of S.drozdowicziiM7A greatly increased (86%) in the presence ofBa2+. In these experiments, the addition ofMn2+ and Co2+ tothe S. misionensis PESB-25 supernatant resulted in significantincreases in endoglucanase activity (101.5 and 61.2%, resp.).

Considering the significant effect of Mn2+ 2mM onendoglucanase activity, this effect was further evaluated.The results are shown in Table 6 and they show the effectof Mn2+ in the concentration range of 1 to 10mM. Thision had a hyperactivating effect on endoglucanase, with


maximum activity of 4.34UmL1 observedwithMn2+ 8mMwhich corresponded to an increase of 143% in endoglucanaseactivity in relation to when no Mn2+ was added.

Although studies dealing with the activation of cellu-lase activity by manganese in Streptomyces strains have notbeen previously reported, there is a report on the positiveeffect of this ion on Bacillus subtilis cellulase 5A [37]. Alsosome fungal cellulases are activated by Mn2+. Gao et al.[38] studied the influence of several metal ions on activityof a purified endoglucanases from Aspergillus terreus andfound an increase of 43% when using Mn2+ 2mM. Taoet al. [39], studying Aspergillus glaucus, found incrementsof 30% when the final concentration of Mn2+ 4mM wasused for a purified endoglucanases obtained when grow-ing the fungus in SCB medium. Manganese was also ableto increase enzymatic activity of other enzymes, such asendonucleases from Penicillium chrysogenum PCL501, wherean increase of 219.6% in presence ofMn2+ 2mMwas observed[34].

Few articles have been published describing cellulaseproduction by actinomycetes using agroindustrial residuesas substrates, and most of them have given very low valuesfor endoglucanase activity when using wheat straw (WS)[40, 41] or wheat bran (WB) [10] as the main substrate.Our group has obtained values of 0.71 UmL1 when usingbrewer spent grain (BSG) [8], andmore recently 2.00UmL1when using wheat bran [9]. Values as high as 4.34UmL1,obtained in the present research, have not been described yetfor endoglucanase production by actinomycetes using low-cost residues, especially SCB.

The results of the endoglucanase thermal stability areshown in Figure 3. When the enzyme crude extract wasincubated at 65C, the enzyme activity decreased 70% of itsinitial activity within 15min of incubation. However, uponincubation at 50C, activity decreased to 40% within 30min,retaining this activity for 2 h.The enzyme half-life at 50Cwas4 h.

It is known that metal ions play an important role instabilizing proteins, protecting against thermal denaturationby binding at specific sites [36, 42]. Several studies haveshown increased enzyme thermal stability in presence ofcalcium [27, 42, 43], which is known to regulate the stabilityand reactivity of a wide variety of biological proteins [43].Given the strong positive effect of Mn2+ on endoglucanaseactivity, the effect of this ion on the enzyme stability wasfurther investigated. It was observed that in the presence ofMn2+ 8mM, the crude enzyme preparation increased 25% ofits initial activity upon incubation for 30min at 65C, andwhen Mn2+ 16mM was used, the activity increased to over70%. Moreover, at a manganese ion concentration of 16mM,the enzyme half-life at 65C was almost 2 hours.

Results from enzyme stability at 50C were even morepromising. The incubation of crude extract with Mn2+ atfinal concentration of 16mM resulted in an increase inthermal stability of 40% after 4 hours incubation (Figure 3),in comparison to the results for the experiments in theabsence of the ion. Activity retention of over 92% for 5 h,

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9

Rela

tive a

ctiv

ity (%

)

Time (h)

Figure 3: Thermal stability of Streptomyces misionensis PESB-25endoglucanase activity at 65C (- -) and 50C (): crude extract(filled diamond), crude extract + MnSO

4

8mM (filled triangle),and crude extract + MnSO

4

16mM (filled circle). Residual activityis expressed as a percentage of the original activity. Error barsrepresent one standard deviation of each experimental point ( = 3).

and over 70% after 9 h of incubation, shows beyond doubtthe positive effect of Mn2+ 16mM on the enzyme structuralstabilization. According to the overall results, incubation ofthe crude extract with Mn2+ at 50C increased the half-lifeof the enzyme from 4 h (no Mn2+ addition) to more than 8 h(addition of Mn2+ 8mM) or even more than 30 h (additionof Mn2+ 16mM). Values of half-lives of 8 h have beencurrently reported in the literature for Streptomyces strains[9, 10].

These are very promising results for the Streptomycesmisionensis endoglucanase. Its natural thermal stability(which can be significantly enhanced with manganese) indi-cates potential as a biocatalyst for industrial process thatdemands long processing times at elevated temperatures,such as those in the food, sugar, and fuel ethanol industries[33]. Also, additional studies for the determination of itsstability at different pH values and different periods of timewould be interesting for future industrial applications.

The zymogram analysis of the culture supernatant ofStreptomycesmisionensis PESB-25 is shown in Figure 4.Threeprotein bands with endoglucanase activity and estimatedmolecular masses of 12.0, 48.5 and 119.5 kDa are clearlyshown. Cellulose degradingmicroorganisms commonly pro-duce multienzyme systems [44]. As such, and in accordanceto previous reports, Nascimento and colleagues [8] observedthree cellulolytic bands (51, 115, and 178 kDa) in the super-natants of S. malaysiensis AMT-3 when BSG 0.5% (w/v) andCSL 1.2% (w/v) were used. Da Vinha et al. [9], in theirstudy, cultured Streptomyces viridobrunneus SCPE-09 in 2.0%wheat bran (w/v) and 0.19% CSL (w/v). In these conditions,two bands of endoglucanase activity were observed, onewith estimated molecular masses of 37 and the other with119 kDa.

Additional studies about these enzymes are required tobetter evaluate their feasibility for further industrial applica-tions. Purification would enable kinetics studies and also thedetermination of their specific activity.


150

102

52

38

24

(kD

a)

(kD

a)

119.5

48.5

12

Figure 4: Zymogram analysis (duplicate experiment) of the culturesupernatant of S. misionensis PESB-25 showing three bands withendoglucanase activity. Cells were grown on SCB 1.0% (w/v) andCSL 1.2% (w/v). The calculated molecular masses (in kDa) of theendoglucanases are indicated on the right side of the figure. Themolecular masses of the markers Full-Range Rainbow (GE-RPN800E) are indicated on the left side of the figure. For details see text.

4. Conclusions

In this study, S. misionensis PESB-25 was able to grow andproduce endoglucanase in a culture medium containinga salt solution and agroindustrial by-products, specificallysugarcane bagasse and corn steep liquor, as the main car-bon and nitrogen substrates. Characterization of the crudeenzyme showed that the endoglucanases produced wereacidic, thermophilic, and thermotolerant. An optimum pHof 3.0 was reported which is rare. An optimum activitytemperature at 70C was seen and is novel for actinobacterialstrains.The activity of these endoglucanases was also stronglyincreased and more stable in the presence of a number ofmetal ions, especially Mn2+. Activity of 4.34 UmL1 wasobtained under these conditions. This level of activity placesthis study amongst the highest described in the literature forcellulase production by Streptomyces strains using low-costresidues as substrates. The effect of Mn+2 16mM on enzymestability was also important and noteworthy. Manganese atthat concentration increased the enzyme stability half-lifefrom less than 4 h to greater than 30 h at 50C and from lessthan 30 minutes to 2 h at 65C.

The characteristics of thermoacidophiles, thermal sta-bility, and induction by manganese suggest that endoglu-canases from S. misionensis PESB-25 could be consideredas promising alternatives in biotechnological applications.For example they could be used as a complement to fungalenzymatic mixtures improving the lignocellulose hydrolysisfor ethanol production. Combining advantageous enzymecharacteristics with the use of low-cost residues (SCB andCSL), we have the potential for a new low-cost enzymeproduction process.

Abbreviations

ANOVA: Analysis of varianceBLAST: Basic local alignment search toolBSG: Brewer spent grainCCRD: Central composite rotational designCMClw: Carboxymethylcellulose low viscosityCSL: Corn steep liquorNCBI: National center for biotechnology informationRDP: Ribosomal database projectRSM: Response surface methodologySCB: Sugarcane bagasseSDS: Sodium dodecyl sulphateWB: Wheat branWS: Wheat straw.

Acknowledgments

This work was supported by grants from the ConselhoNacional de Desenvolvimento Cientfico e Tecnologico(CNPq), Coordenacao de Aperfeicoamento de Pessoal doEnsino Superior (CAPES), and Financiadora de Estudose Projetos (FINEP). The authors also thank the Postgrad-uate Programme in Plant Biotechnology (Programa depos-graduacao em Biotecnologia Vegetal) and PostgraduateProgramme in Science, Microbiology (Programa de pos-graduacao em Ciencias, Microbiologia) at Universidade Fed-eral do Rio de Janeiro (UFRJ).

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Research ArticleAnti-Candida Properties of Urauchimycins from ActinobacteriaAssociated with Trachymyrmex Ants

Thais D. Mendes,1,2 Warley S. Borges,3,4 Andre Rodrigues,1,5 Scott E. Solomon,6

Paulo C. Vieira,4 Marta C. T. Duarte,7 and Fernando C. Pagnocca1,5

1 Center for the Study of Social Insects, Sao Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil2 EMBRAPA Agroenergy, Parque Estacao Biologica, 70770-901 Braslia, DF, Brazil3 Chemistry Departament, Federal University of Esprito Santo (UFES), 29075-910 Vitoria, ES, Brazil4 Chemistry Department, Federal University of Sao Carlos (UFSCar), 18052-780 Sao Carlos, SP, Brazil5 Department of Biochemistry and Microbiology, Sao Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil6Department of Ecology and Evolutionary Biology, Rice University, Houston, TX, USA7Division of Microbiology, Center for Chemistry, Biology and Agriculture Research (CPQBA/UNICAMP),13081-970 Paulnia, SP, Brazil

Correspondence should be addressed to Fernando C. Pagnocca; [email protected]

Received 12 November 2012; Revised 29 January 2013; Accepted 2 February 2013

Academic Editor: Manish Bodas

Copyright 2013 Thais D. Mendes et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

After decades of intensive searching for antimicrobial compounds derived from actinobacteria, the frequency of isolation of newmolecules has decreased. To cope with this concern, studies have focused on the exploitation of actinobacteria from unexploredenvironments and actinobacteria symbionts of plants and animals. In this study, twenty-four actinobacteria strains isolated fromworkers of Trachymyrmex ants were evaluated for antifungal activity towards a variety of Candida species. Results revealed thatseven strains inhibited the tested Candida species. Streptomyces sp. TD025 presented potent and broad spectrum of inhibition ofCandida and was selected for the isolation of bioactive molecules. From liquid shake culture of this bacterium, we isolated therare antimycin urauchimycins A and B. For the first time, these molecules were evaluated for antifungal activity against medicallyimportant Candida species. Both antimycins showed antifungal activity, especially urauchimycin B. This compound inhibited thegrowth of all Candida species tested, with minimum inhibitory concentration values equivalent to the antifungal nystatin. Ourresults concur with the predictions that the attine ant-microbe symbiosismay be a source of bioactivemetabolites for biotechnologyand medical applications.

1. Introduction

The increased resistance of microorganisms to antibiotics is aproblem of public health [1].The increasing number of fungalspecies that can infect humans, particularly immunocompro-mised individuals, further reinforces this concern. A limitednumber of antifungal agents are commercially available whencompared to antibacterial drugs. This scenario motivates thesearch for new bioactive compounds in various biologicalsystems using several approaches, including metagenomicsand microbial genome-mining.

Actinobacteria are widely known for their ability to pro-duce bioactive secondary metabolites, especially compoundswith antimicrobial activity. These bacteria are responsible forproducing two-thirds of the commercially available antibi-otics [2, 3]. Most actinobacteria species explored commer-cially were isolated from the soil. However, after decades ofbioprospecting actinobacteria from this environment, it isbecoming more difficult to obtain strains producing novelbioactivemetabolites [4].Thus, many companies have turnedthe search for microbial producers of novel antifungal com-pounds to other environments such as hydrothermal vents,


marine environments, tropical rain forests, and micr

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