Collagenolytic enzymes produced by fungi: a systematic review

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brazilian journal of microbiology 48(2017)13–24

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Review

Collagenolytic

enzymes

produced

by

fungi:

a

systematic

review

Maria

Carolina

de

Albuquerque

Wanderley

a

_,

_José

_Manoel

_Wanderley

_Duarte

_Neto

a

_,

José

Luiz

de

Lima

Filho

a

_,

_Carolina

_de

_Albuquerque

_Lima

b

_,

_José

_António

_Couto

_Teixeira

c

_,

Ana

Lúcia

Figueiredo

Porto

d,∗

a_Universidade_Federal_de_Pernambuco_(UFPE),_Laboratório_de_{Imunopatologia}_Keizo_Asami_(LIKA),_Recife,_PE,_Brasil b_Universidade_de_Pernambuco_(UPE),_Faculdade_de_Ciências,_Educac¸ão_e_Tecnologia_de_Garanhuns,_Garanhuns,_PE,_Brasil c_Universidade_do_Minho_(UMinho),_Centro_de_Engenharia_Biológica,_Braga,_Portugal

d_Universidade_Federal_Rural_de_Pernambuco_(UFRPE),_Departamento_de_Morfologia_e_Fisiologia_Animal,_Recife,_PE,_Brasil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29December2015 Accepted15August2016

Availableonline30September2016 AssociateEditor:AdalbertoPessoaJr

Keywords: Collagenase Fungus Characterization Puriﬁcation Production

a

b

s

t

r

a

c

t

Specific proteasescapableofdegradingnativetriplehelicalordenaturedcollagenhave beenrequiredformanyyearsandhavealargespectrumofapplications.Therearefew completereportsthatfullyuncoverproduction,characterizationandpurificationoffungi collagenases.Inthisreview,authorssearchedthroughfourscientificonlinedatabases usingthefollowingkeywords(collagenolyticORcollagenase)AND(fungiORfungusOR fun-gal)AND(productionORsynthesisORsynthesize)AND(characterization).Scientificcriteria wereadoptedinthisreviewtoclassifyfoundarticlesbyscore(from0to10).After exclu-sioncriteria,21articleswereselected.Noneobtainedthemaximumof10pointsdefinedby themethodology,whichindicatesadeficiencyinstudiesdealingsimultaneouslywith pro-duction,characterizationandpurificationofcollagenasebyfungi.Amongmicroorganisms studiedthenon-pathogenicfungiPenicilliumaurantiogriseumandRhizoctoniasolanistoodout involumetricandspecificcollagenaseactivity.Theonlyarticlefoundthatmade sequenc-ingofatruecollagenaseshowed100%homologywithseveralmetalloproteinasesfungi.A cleargapinliteratureaboutcollagenaseproductionbyfungiwasverified,whichprevents furtherdevelopmentintheareaandincreasestheneedforfurtherstudies,particularlyfull characterizationoffungalcollagenaseswithhighspecificitytocollagen.

licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author_at:_Department_of_Morphology_and_Animal_Physiology,_Federal_Rural_University_of_Pernambuco,_Av._Dom_Manoel

deMedeiros,s/n,52171-900Recife,PE,Brazil. E-mail:analuporto@yahoo.com.br(A.L.Porto).

http://dx.doi.org/10.1016/j.bjm.2016.08.001

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brazilian journal of microbiology48(2017)13–24

Introduction

Collagenisaﬁbrousproteinfoundinskin,tendons,bones, teeth,bloodvessels,intestinesandcartilage,correspondingto 30%ofthetotalprotein,whosemainfunctionisstructural.1,2

There are morethan 26 geneticallydistinct types of colla-gens,characterizedbyconsiderablecomplexityanddiversity intheirstructure,theirsplicevariants,presenceofadditional, non-helical domains, their assembly and their function.3,4

Eachcollagenmoleculeisasmall,hardstickformedby inter-lacinginatriplehelixofthreepolypeptidechainscalledalpha chains(Fig.1).

Speciﬁcproteasescapableofdegradingnativetriple heli-calordenaturedcollagenhavebeenrequiredformanyyears.5

Collagenaseshavebeenisolatedandcharacterizedfrom dif-ferent sources,as digestivetracts offish and invertebrates including:tadpoletailfin,6,7_Atlantic_cod,8_land_snail_(Achatina fulica),9_tropical_shrimp_(Penaeus_vannamei),10,11_catfish (Parasil-urusasotus),10,12_mackerel_(Scomber_japonicas)13_;_besides_plants

(Zingiberofﬁcinale)14_;_bacteria_as:_Bacillus_cereus_and _Klebsiella pneumoniae,15 _Bacillus_pumilus,16 _Bacillus_{licheniformis}17–19 _and

fungi,showninthisreview.

Proteases,ingeneral,frommicrobialsourcesarepreferred tothe enzymes fromplant andanimal sourcesforits bio-chemicaldiversityand geneticmanipulationpossibility.20,21

Microbialcollagenasehavebeenrecoveredfrompathogenic micro-organisms,especiallyClostridiumhistolyticum,whichis the most widely used commercial source.22 _Other _studies

reported collagenase producing fungi ofgenera Aspergillus, Cladosporium,PenicilliumandAlternaria.23

Among microorganisms that produce collagenolytic enzymes,ﬁlamentousfungihave greatadvantagessuchas highproductivityandlowproductioncost,rapiddevelopment, and the resulting enzyme may be modiﬁed and recovered more easily.24 _Enzyme _production _occurs _{extracellularly,}

which makesit particularly easier torecover afterwards.25

Asfungalproteases are capableofhydrolyzing manyother proteins besides collagen, the demand for collagenases fromfungiwithsuitablecharacteristics,namelyhigh speci-ﬁcity,is avery signiﬁcantresearch directiontobetaken.26 Collagenases are capable of hydrolyzing both native and denaturedcollagen,andarebecomingincreasinglyimportant commercially.27

Collagenaseshavebeenusedinmedical,pharmaceuticals, food,cosmeticsandtextilessegmentsandhaveapplications infurandhidetanningtohelpensuretheuniformdyingof leathers.28,29_In_medical_{applications,}_it_can_be_used_in_burns

andulcerstreatment,30,31_to_eliminate_scars,32_for_{Dupuytren’s}

diseasetreatmentinadditiontovarioustypesofﬁbrosissuch asliver cirrhosis, topreparing samplesfor diagnosis,33 _for

productionof peptides with antioxidant and antimicrobial

Alpha chains

Fig.1–Collagenmolecule:intertwiningthreealphachains triplehelix.

activities,34_and_play_an_extremely_important_role_in_the

trans-plantsurgerysuccessofsomespeciﬁcorgans.32

Therulesforvertebratecollagenaseclassificationarevery clear, but the same does notapply to microbial enzymes. It isdifficult todistinguish betweentrue collagenases and gelatinases or other proteases,which leadsto controversy and imprecision inthe classificationand nomenclature of theseenzymes.Microbialcollagenasesarecapableof degrad-ingtriple-helicalcollagenanddenaturedfragmentsinvarious sites and are less specific. Althoughseveral proteases can hydrolyzedenaturedcollagen,theycannotbemistakenwith true collagenases, ableto hydrolyzethe native collagen as foundinconnectivetissues.35,36

Thesearchfornewmicrobialcollagenaseshasincreased over the years and its productioncurrentlyrepresentsone of the biggestenzyme industries.37,38 _The _development_of

newproductionmethods,includingthesearchforproducing micro-organisms,alternativesourcesofsubstrates,and bet-terextractionconditionsandpuriﬁcationofcollagenase,has beenofgreatimportance,sinceithasawideapplication spec-trumwithhighbiotechnologicalpotential.Besides,themain publishedreviewpapersconcerningmicrobialcollagenolytic enzymesarelimitedtobacterialsource.22,35,39_In_this_context,

theauthorsfelttheneedtobetterunderstandthestateofthe artregardingproduction,characterizationandpuriﬁcationof collagenolyticenzymesbyfungi.

Material

and

methods

The ﬁrst step on this process, was to make electronic searches in the Scopus (http://www.scopus.com/), Sci-enceDirect (http://www.sciencedirect.com/), ISI Web of Science (http://apps.isiknowledge.com) and PubMed

(http://www.ncbi.nlm.nih.gov/pubmed)databases,using the

following keywords: (collagenolytic OR collagenase) AND (fungi ORfungusORfungal)AND (productionORsynthesis ORsynthesize)AND(characterization).

Thisprocedureallowedselectingpublishedpapersonthe productionandcharacterizationofcollagenolyticenzyme pro-duced byfungi. Papersthat did notreport onthe enzyme production process were excluded. There were no limita-tionsregardingtheyearanddateofpublication,duetolack ofpublicationsabout thisissue. Norestrictionswere made formethodology used, typesofanalysis andquantiﬁcation ofresults.In addition,therewere norestrictionon typeof micro-organism,collagenolyticactivitymethodology,culture conditionsandcharacterizationassays.

Twoindependentsearchesweremadeandtheconformity of the selected papersvalidated,considering the inclusion criteria described.Incaseofdivergenceamongthepapers, allofthecriteriawerereviewedanddiscussed.Wheninthe article title onlyprotease productionwasmentioned, lack-ingcollagenrelatedterms,researchersproceededtosummary evaluation,lookingformethodologiesforactivity determina-tioninvolvingcollagenorgelatinassubstrate.

Papersselectioncriteriaweredeﬁnedtoevaluateboth bet-terconditionsforcollagenolyticenzymeproductionbyfungus withbiotechnologicalpotentialapplicabilityand methodolog-icalqualityinthecharacterizationoftheenzyme.Scientiﬁc

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15

criteria adoptedin this reviewwere according to the ones proposedbyGreenhalgh.40_The_parameters_were_classiﬁed_on

thescale:adequate(score:2),partiallyadequate(score:1)and inadequate(score:0)oradequate(score:1)andinadequate(0).

Productionprocess:Papersthatstudiedthebestgrowing con-ditionsforproducingcollagenolyticenzymereceivedascore of2,papersthat didnotconduct studiestoimprove grow-ingconditions,usingcollagenorgelatinassubstrate,received scoreof1,andthosewhichusednonspecificmeansfor col-lagenproductionreceivedascoreof0.Characterizationofthe enzyme:papersthatreportedbiochemicalcharacterizationof enzymeandincludedothertestsaswellasoptimumpHand temperatureandenzymeinhibitiontests,receivedascoreof 2.ThosewhichevaluatedonlyoptimumpHandtemperature andtheeffectofinhibitorsreceivedascoreof1.Papersthat didnothaveatleastthesethreefactorsinenzyme charac-terizationwere consideredinadequateandreceivedascore of 0. Quantification method of collagenolytic activity: methods thatusedchromogenicsubstrates(OrangeCollagenorAzocoll) forquantificationofcollagenolyticactivity,receivedascore of2.Paperswithother quantitativemethodologiesfor col-lagenolyticactivity,receivedascoreof1,andthosethatheld onlyqualitativeanalysisactivity,receivedascoreof0. Purifica-tion:purificationbychromatographymethodsreceivedascore of2,thosewhichusedotherpurificationmethods,receiveda scoreof1,andthosethatdidnotdoanykindofpurification, received0.Micro-organism:articlesthatusednon-pathogenic fungiforcollagenolyticenzymeproductionreceivedascoreof 1,whilethoseusingpathogenicfungiwereconsidered inade-quateandreceivedascoreof0.Substratespecificity:enzymes withspecificactivityovercollagen,receivedascoreof1;those whopresentedawidehydrolysisspectrumorhavenotbeen tested,receivedascoreof0.

Maximumoverallscorewas10points.Otherparameters suchasproductiontime,yearofpublication,satisfactory col-lagenolyticactivity,amongothers,didnotscored but were takenintoconsideration,astheywererelevanttosubsequent discussion.TheparametersscoredaresummarizedinTable1. Atablewasassembledwithasummaryofselected arti-clesrelevantdataaccordingtocriteriaadoptedonthereview, including somefeatures as optimumpH and temperature, inhibitors, enzyme nature (true collagenase or not) and enzymesequence.

Results

and

discussion

Byapplyingtheestablishedsearchprocedure,atotalof1346 articleswerefoundinScienceDirectdatabase,678articlesin Scopusdatabase,45articlesinPubMed,and5articlesinWeb ofScience,totaling2074articles.Basedondeﬁnedinclusion andexclusioncriteria,21articleswereselectedforthisreview, distributedasshowninFig.2.

Regarding the scores obtained for each selected article, none obtained the maximum of 10 points deﬁned by the methodology.According tothedistributioninTable 2, only onearticlehit ascoreof9(4.77% ofselectedarticles),two articles obtained the score of 8 (9.52%), and three articles reachedthescoreof7(14.29%).71.43%ofthearticlesachieved scoresbelow7,whichindicatesadeﬁciencyinstudiesdealing

simultaneouslywithproduction,characterizationand purifi-cationofcollagenasebyfungi.Wheretheenzymeobtained shouldpresent specificitytosubstrate andhaveitsactivity quantified bythe methodadoptedasthe mostappropriate (Azocoll).

Asdescribedinthemethodology,notimeintervalhasbeen deﬁned.However,only11articleshavebeenpublishedinthe last10 years.Ofthese11 articles,only4werepublishedin thelast5years,clearlyindicatinganeedforfurtherresearch relatedtotheproductionofcollagenasebyfungi.

Microorganism

Based onthis systematicreview, 21 articleswere selected, of which 17 were carried out with 10 different genera of ﬁlamentous fungi (Penicillium, Aspergillus, Arthrobotrys, Monacrosporium,Trichophyton,Microsporum,Lecanicillium, Ento-mophthora, Micromycetes and Lagenidium).Two generafound wereclassiﬁedasdimorphic(CoccidioidesandParacoccidioides),

andonlyonehadayeastmorphology(Zygosaccharomyces). From the industrial point of view, pathogenicity can negatively inﬂuence microorganism choice for bioprocess development.Interestingly,approximately40%offungicited inselected articlesaredescribed asclassicpathogens.The non-pathogenicspeciesthatwereassociatedwithgood col-lagenolyticenzymeproductionwereRhizoctoniasolaniwitha productionof212.3U/mL55_and_Penicillium_{aurantiogriseum}_with

231U/mL24_and₁₆₄_U/mL58_.

A great diversity of collagenolytic enzymes producing fungicouldbeobserved(morethan20differenttaxa).Most belonging tophylum Ascomycota, other to phyla Basidiomy-cota (R. solani), Entomophthoromycota (Conidiobolus coronatus)

andOomycetes(Lagenidiumgiganteum).Filamentousfungiare clearlymorestudiedincomparisontoyeastsforcollagenolytic enzymeproduction.Manyarticlescontainpathogenicfungi inordertobetterunderstanditspathogenesismechanisms and notinorder tostudy enzymaticproductionitself.The genus Aspergillus was the most frequent, followed by Peni-cilliumandEntomophthoragenres.Consideringpathogenesis, enzymeactivityandspecificity,thefungibetterqualifiedfor enzymeproductionwerethefilamentousfungusP. aurantiogri-seumandZygosaccharomycesrouxiiyeast.

Culturemedium

Culture mediumselectionisofgreat importancefor colla-genaseproduction,sincethisfactorwilldirectlyaffectﬁnal processcost.Assaidearlier,oneoftheadvantagesofworking with microorganisms isthe possibility to vary the compo-sition of the culture medium, using lower cost materials, suchasbyproducts oftheﬁshingindustry,forexample,as substrate. Nine ofthe selected papers presenteda culture medium containing collagen or gelatin in its composition, other studies used other sources of carbon and nitrogen, mainlyyeastextract.Somestudiesinvolvingbacteriaindicate thataddinggelatinorcaseininthemediumincreasesthe col-lagenaseyield.However,theworkofOkandHashinaga48_with Z.rouxiiyeast,observedthataddinggelatininYPGmedium was not essential for the production of collagenase. Lima etal.24_reported_the_use_of_a_inexpensive_culture_medium_for

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Table1–Scoreofselectedparametersforcriticalevaluationofthesystematicreview.

Criteriafordeterminingthescores Pointing

2 1 0

(A)Production Speciﬁcforcollagenase,with controlledvariables

Speciﬁcforcollagenase,with uncontrolledvariables

Nospeciﬁcforcollagenase

(B)Characterization Complete Partial Absent

(C)Microorganism Non-pathogenic Pathogenic

(D)Collagenolyticactivitymethod AzocollorOrangeCollagen Others Absent

(E)Puriﬁcation Complete Partial Absent

(F)Substrate Collagenase(speciﬁc) Non-Speciﬁc

Search Science direct Total: 1346 Selected: 7 Excluded: 1339 Scopus Total: 678 Selected: 11 Excluded: 667 Pubmed Total: 45 Selected: 1 Prev. selected: 1 Excluded: 43 Web of science Total: 5 Selected: 2 Prev. selected: 2 Excluded: 1

Fig.2–Totalarticlesselectedinfourdifferentdatabasesusingthedescribedmethodology.

Table2–Scoresdistributionofselectedarticles.

Authors (A) (B) (C) (D) (E) (F) Total

Hurionetal.(1977)41 ₁ ₀ ₀ ₂ ₂ ₀ ₄

Hurionetal.(1979)42 ₁ ₀ ₀ ₂ ₂ ₀ ₄

OlutiolaandNwaogwugwu(1982)43 ₀ ₂ ₁ ₀ ₀ ₀ ₄

DeanandDomnas(1983)44 ₀ ₂ ₁ ₁ ₁ ₀ ₆

Zhuetal.(1990)45 ₀ ₀ ₀ ₀ ₀ ₀ ₀ Tomeeetal.(1994)46 ₁ ₀ ₀ ₀ ₀ ₀ ₃ Ibrahim-Granetetal.(1996)47 ₀ ₁ ₀ ₂ ₂ ₀ ₄ OkandHashinaga(1996)48 ₂ ₁ ₁ ₁ ₁ ₁ ₇ Benitoetal.(2002)49 ₀ ₂ ₁ ₂ ₂ ₀ ₇ Minglianetal.(2004)50 ₁ ₂ ₁ ₂ ₂ ₀ ₈ Yangetal.(2005)51 ₁ ₂ ₁ ₁ ₁ ₀ ₆ Wangetal.(2006)52 ₁ ₂ ₁ ₂ ₂ ₀ ₇ Mahmoudetal.(2007)53 ₂ ₁ ₀ ₂ ₂ ₀ ₆ Vianietal.(2007)54 ₁ ₀ ₁ ₀ ₀ ₀ ₃ Hamdy(2008)55 ₂ ₂ ₁ ₂ ₂ ₀ ₈ Lopesetal.(2008)56 ₀ ₁ ₀ ₀ ₀ ₀ ₁ Voltanetal.(2008)57 ₁ ₀ ₀ ₁ ₁ ₀ ₄

Limaetal.(2011a)24 ₂ ₂ ₁ ₁ ₁ ₁ ₉

Limaetal.(2011b)58 ₂ ₀ ₁ ₀ ₀ ₀ ₅

deSiqueiraetal.(2014)59 ₀ ₂ ₁ ₁ ₁ ₀ ₆

Sharkovaetal.(2015)26 ₀ ₀ ₁ ₀ ₀ ₀ ₃

(A)Production:Specificforcollagenaseproductionwithcontrolledvariables(score2),specificforcollagenaseproductionwithuncontrolled variables(score1),non-specificforcollagenase(score0).

(B)Characterization:Completecharacterization(score2),partialcharacterization(score1),absent(score0). (C)Microorganism:Non-pathogenicmicroorganism(score1),pathogenicmicroorganism(score0).

(D)Collagenolyticactivity:Chromogenicsubstrateforcollagenolyticactivitymethod(score2),othersquantitativemethods(score1),qualitative (score0).

(E)Purification:Purificationbychromatography(score2),partialpurification(score1),absent(score0). (F)SubstrateSpecificity:Collagenasewithspecificityforcollagen(score1),non-specific(score0)

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P.aurantiogriseumcollagenaseproduction,usingsoyﬂouras mainsubstrate,andthesamemediumwasusedbyauthors Limaetal.,58_reaching_one_of_the_best_{collagenolytic}_activity

valuesfoundduringthisreview(Table3).

AccordingtoHamdy,55_the_use_of_different_batch_or_collagen

typesmayinterfereinenzymesproduction(enzymeactivity) andcollagenasesfromdifferentmicroorganismshave afﬁn-ityforspeciﬁctypesofcollagen.60_The_production_of_different

fungiindifferentmediamustbethesubjectofextended stud-ies.

Cultureconditions

Processdevelopmentisafactortobeconsideredsince opti-mizationofcultureconditionscanpromoteanincreaseinthe yieldsofproteaseandreductioninproductioncosts,amajor issuefromanindustrialpointofview.58,61

Culture medium initial pH inﬂuences many enzymatic processes,suchasenzymeproduction,celltransportacross membranesandextracellularproteasesexpression.62,63 _The

pH of the culture medium used in the selected articles rangedfrom5.5to8.0,whiletemperaturerangedfrom18to 37◦C.Regardingagitation,onlyHurionetal.42 _showed

non-mixedenzymeproduction,withmicroorganismE. coronata.

Inmostoftheworks,rangedanagitationwasintherange 100–200rpm.

Fermentation time to collagenase production varied widely,from24hto14days,atimeof6–7daysbeingreported by8papers.Severalstudiesshowedactivitydecayafterthe 7thdayoffermentation.Zhu etal.45 _demonstrated_that, _in

mediumcontaininginsolublecollagen,after2weeks,fungus growsonlytohalfthemassobtainedinmilkmediumfor1 week.Articlesthatstudiedtimeinﬂuenceonenzyme produc-tionreportedhigherproductionduringstationaryphase.

The work of Lima et al.24 _presented _a _factorial _design

todefinethebest growingconditionsfortheproductionof collagenase.AuthorsstatedthatinitialpH,temperatureand concentration ofsubstrate are significant factors for colla-genaseproductionbyP.aurantiogriseumusingsoybean flour medium.

Temperatureinﬂuenceonproteaseproductionby microor-ganismsisanimportantfactor.64 _Temperature_can_regulate

somecomponentsasenzymaticsynthesis,enzymesecretion andlengthoftheenzyme’ssynthesisphase,besidesthe prop-ertiesofcellwall63,65_._In_general,_studies_used_temperatures

between 18 and 37◦C during production. The papers that studieddifferenttemperaturesshowed30◦Castheoptimum temperatureforcollagenolyticproteaseproduction. Accord-ingtodeSiqueiraetal.,59_incubation_temperature_interferes

withfungusgrowthandmetabolism,andconsequently, pepti-daseproduction,thebesttemperaturebeing30◦C,according toHamdy.55 _Lima_et _al.24 _reported_that_the _best_conditions

forvolumetriccollagenolyticactivityandbiomassproduction were24◦CandpH7.0.

Among works that discriminated the shaking speed, 150–200rpmweremostused,exceptforYangetal.,51_that_used

100rpm.Hamdy55_showed_in_his_results_that_although_there

islittledifference,theagitationof175rpmwasthebestfor enzymeproduction.

Collagenolyticactivity

Collagenolyticactivitycanbedescribedascollagenhydrolysis bycollagenasewithpeptidesoraminoacidsrelease.Different methods are described in literatureto measure this activ-ity: colorimetric, ﬂuorescence, turbidity and viscometry or radioactivity,amongothers.Allthesemethodsarequite time-consuming,thetimeneededrangingfrom3to18h.Onthe otherhand,theirmajoradvantageisthatmostofthemuse nativecollagens.22,66

The radioactive or fluorescent methods require more time to produce substrate and more specific measuring equipment, as well as immunological methods. Moreover, synthetic oligopeptide is not an entirely specific substrate forcollagenase.66_Another_used_technique_was_developed_by

Mandletal.,60_using_collagen_in_natura_as_substrate_and

ninhy-drinascoloringreagent.Theninhydrinmethodmeasuresfree aminoacidsrelease,whichdifﬁcultcontinuousactivity mon-itoringormayunderestimateenzymesactivityifitreleases peptidesandnotfreeaminoacids.Besides,inthismethodthe ninhydrincanreactwithfreeaminoacidsexistinginsolution, whichlimitsthetechniquesensitivity.67

Amongcolorimetricmethods,thereistheAzocollbased.68

TheAzocollisanazodye-impregnatedcollagen,whichisa speciﬁc substrate forcollagenase,sinceit allowsobserving hydrolysisbyreleaseofdye-impregnatedsolublepeptidesthat aremeasuredbyspectrophotometry,increasingthemethod sensitivity.

All 21 articles selected in this review have different methodologiestoquantifycollagenaseactivity.Eightofthe articlesusedAzocollasasubstrateformeasurementof col-lagenolytic activity. Other papers used other quantitative methods,suchas:ninhydrin(4items),Folin(1item),synthetic peptide(4items)andOrangeCollagen(1item).

Regarding the speciﬁc activity, less than half the articles quantify this parameter. Interestingly Hamdy55

reported a speciﬁc activity value well above the others (18,064.7×103_U/mg)._Another _article_that _presented_a _good

speciﬁc activity was Lima et al.,24 _with ₃₁₉_U/mg. _In

gen-eral, the speciﬁc activity varied signiﬁcantly (from 0.37 to 18,064.7×103_U/mg)._The_highest_activities_were_observed_in

studies involving production optimization. However, effec-tiveness ofproductiontendstobeevaluatedbyvolumetric collagenolyticactivityduetotheindustrialrelevanceofthis parameterLimaetal.58

Enzymecharacterization

Isoeletricpoint

From selectedarticles, onlytwovaluesforisoelectricpoint ofcollagenolyticenzymewerereported.Thevaluesfoundby Minglianetal.50_and_Wang_et_al.52_were_respectively_4.9_to_an

enzymeproducedbyA.oligosporaand6.8toanotherproduced byM.microscaphoides.However,inthesestudiesnosigniﬁcant collagenolyticactivitywasreportedwhencomparedtoother activitiesfound,ascanbeseeninTable3.

pHandtemperatureoptimal

TheoptimumpHforenzymeactivityvariedconsiderably(pH 5–10). For the best results regardingcollagenolytic activity,

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 13–24

Table3–Summaryofselectedarticlesrelevantdataaccordingtothecriteriaadoptedonthereview.

Purif. Enzymenature Enzymesequence Substrate Speciﬁc

activity

Inhibitors pHand

temper.

Isoelectric point Chromatography Gelatinolytic X BAE(trypsin-like),Elastin,

SyntheticPeptides 0.088 nkat/mg X X X Ultraﬁltration,Sephadex G-25column

Gelatinolytic X Casein,Elastin,SyntheticPeptides X EDTA,DFP,TLCK,TPCK X X

Ammoniumsulfate Collagenolytic X Casein,Elastin,Collagen,Gelatin, p-nitrophenolcaprylate

3.6U/mg Ca2+_,_Na+_,_EDTA,_2,4-DNP _pH_7,₃₅◦ _X

X Collagenolytic X BAPA,TAME X PMSF,TPCK,

IAA2-mercaptoethanol, cysteineHCl,Zn,Ca,Mg, EDTA,Ca,Mg pH8.4,60◦ _X (NH4)2SO4Ammonium Sulfate,SephadexG25, BiogelA

Collagenolytic X Azocasein,TypeICollagen,Elastin X EDTA,fenantrolin,PA, PMSF,elastina,NEM

X X

Cationexchange chromatography

Collagenolytic X Casein,Elastin,OrangeCollagen 0.39U/mg X X X

GF,Orange3,Yellow1,HA, TSK

Collagenolytic VFLGREPKPDAFY SyntheticPeptides,Collagen X Fenantrolina,EDTA, X X

X Collagenolytic X SyntheticCollagen,Peptides 70.4U/mg X pH8.2 X

Ammoniumsulfateand cationexchange chromatography

X AEQTDSTWGL Casein,BSA,Skimmedmilk,

Gelatin,Collagen,Denatured Collagen,Nematodecuticle

0.37U/mg PMSF,EDTA,PepstatinA, Leupeptin,Aprotinins

pH10,55◦ X

Ammoniumsulfate,Q SepharoseFF,Sephacryl S-100

Gellatonolytic X Casein,Gelatin,Nematodecuticle, Azocoll

1.12U/mg PMSFeSSI pH6–8,45◦ 4.9

Ultraﬁltration,HITrapSPFF, HiPrepphenylFF

Gellatonolytic AITQQQGAPW Casein,BSA,Gelatin,Collagen, Nematodecuticle

48U/mg Leupeptin,Aprotinin,EDTA, PepstatinA,PMSF

pH10,70◦ _X

Source15Q,Phenyl Superose

Gellatonolytic AEQLDSTWGL Casein,BSA,Skimmedmilk, Gelatin,HydrolyzedCollagen

X PMSF pH9,60◦ 6.8

AmmoniumSulfate, SephadexG-25e DEAE-cellulose

Collagenolytic X X 92.17U/mg Cetrimide X X

X Gelatinolytic X Keratin,Elastase,SyntheticPeptide X X X X

Ammoniumsulfate, DEAE-cellulose,Sephadex G150

Collagenolytic X Collagen,Casein,elastin 18,064.7x103

U/mg

EDTA,Iodoacetate,Sodium arsenate,arsenito,Cysteine

pH5,40◦ X

X X X Casein,Gelatin,Keratin,Albumin,

Hemoglobin

X PMSF X X

X Collagenolytic X Casein,Elastase.Azocoll X PMSF,EDTA,

Phenanthroline

X X

X Collagenolytic X Azocoll,TypeIcollagen,Gelatin,

Azocasein

319U/mg PMSF,iodoaceticacid,EDTA ePepstatinA

pH9,37◦ X

X Collagenolytic X X X X X X

X Collagenolytic X Casein,Keratin X PMSF,EDTA,IAA pH6.5,55◦ _X

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 13–24

19

Table3–(Continued). Purif. Molecular weight (kDa)

Col.activ. Col.activ.method. Culture conditions

Culture medium

Microorg. Authors

Chromatography 23–40 X Syntheticpeptide pH5.6,30◦C,

withoutagitation,15 days

Casaminoacids, Dextrose,CaCl2,YEand Berthelotsolution

E.coronata Hurionetal.(1977)41

Ultraﬁltration, SephadexG-25 column X X Syntheticpeptide pH5.6,30◦C, without,15days Casaminoacids, Dextrose,CaCl2,YEand Berthelotsolution

E.coronata Hurionetal.(1979)42

Ammoniumsulfate X X Achillestendon

bovine

7days,30◦_C _Glucose,_salts,

l-cysteine,tryptone, biotin,thiamine

A.aculeatus Olutiolaand Nwaogwugwu (1982)43

X X 8U/mL Azocoll Gyrotoryshaker

(20–24◦C),sobluzes ﬂuorescents

Peptone,yeastextract andglucose(PYG)broth

L.giganteum DeanandDomnas (1983)44

(NH4)2SO4Ammonium

Sulfate,Sephadex G25,BiogelA

X X SDS-PAGE 2weeks,T.A. M9(without

NH4Cl)+collagen

A.ﬂavus Zhuetal.(1990)45

Cationexchange chromatography 32 X Orangecollagene SyntheticPeptide 5days,37◦C, 150rpm Yeastcarbon base+collagentypeI

Aspergillus Tomeeetal.(1994)46 GF,Orange3,Yellow1,

HA,TSK

82 X RatnativetypeI

collagen

25◦C,pH6,7days Sabouraud T.schoenleinii Ibrahim-Granetetal.

(1996)47

X X 70.4U/mL Ninhydrin pH7,25◦C,50h,

withagitation

YPG Z.rouxii OkandHashinaga

(1996)48 Ammoniumsulfateand

cationexchange chromatography 35 1%and2% Azocoll 26◦C,200rpm,7 days LMZ P. chrysogenum Benitoetal.(2002)49 Ammoniumsulfate,Q SepharoseFF, SephacrylS-100 38 0.0134U/mL/m Azocoll 25–18◦_C,₆ days,150-200rpmpH 6.5

LMZ–withgelatin A.oligospora Minglianetal.

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20

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 13–24 Table3–(Continued). Purif. Molecular weight (kDa)

Col.activ. Col.activ.method. Culture conditions Culture medium Microorg. Authors Ultraﬁltration,HITrapSP FF,HiPrepphenylFF 32 14% Nondescribed 26◦C,100rpm,6 days

Glucose,gelatinand salts

L.psalliotae Yangetal.(2005)51 Source15Q,Phenyl Superose 39kDa Collagen15.9%, Denatured Collagen48.1%, Folin 6days,26◦C, 200rpm

Proteaseinducing–with gelatin M. microscaphoides Wangetal.(2006)52 AmmoniumSulfate, SephadexG-25e DEAE-cellulose

72–92kDa 82.95U/mL Ninhydrin 6days,37◦_C _Gelatin,_glucose,_yeast

extract,andnative bovinecollagen

A.ﬂavus Mahmoudetal. (2007)53

X X 1Unitof

collagenase

Syntheticpeptide 14days MediumwithtypeI

collagen

M.canis Vianietal.(2007)54 Ammoniumsulfate,

DEAE-cellulose, SephadexG150

66kDa 212.33U/mL Ninhydrin 108h,175rpm,pH

5.5,30◦C

Sabouraud-glucose-collagen

R.solani Hamdy(2008)55

X 25kDa X Zymogram pH5.5,9days,T.A. Czapek C.immitis Lopesetal.(2008)56

X 20–200kDa 1.2U/mL Azocoll 150rpm,35◦C,for7,

14,21e28days

Yeastcarbon

base+collagen+vitamin solution;

neopeptoneBHI+elastin

P.brasiliensis Voltanetal.(2008)57

X X 164U/mL Azocoll 0.75%gelatin,

200rpm,pH8.0and 28◦C

Soybeanﬂour,glucose andmineralsolution

P. aurantiogri-seum

Limaetal.(2011a)24

X X 231U/mL Azocoll pH7.0,24◦C,24h Soybeanﬂour,Glucose

andmineralsolution

P. aurantiogri-seum

Limaetal.(2011b)58

X X 0.165OD/mL Azocoll 2.0×105_espores,

72h,30◦C,75% humidity

Solidmediumofwheat bran

A.terreus deSiqueiraetal. (2014)59

X X 113.2and

332×10−3U/mL

Azocoll 200rpm,28◦C,4

days

Several Micromycetes Sharkovaetal.

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21

Limaetal.58 _and_Mahmoud_et_al.,53 _the_optimum_pH_of_the

enzymewasnotevaluated.OkandHashinaga48_evaluated_the

optimalpH(8.2)oftheenzymeproducedbyZ.rouxiiyeast. Limaetal.24_found_that_pH_of_9.0_was_the_best_for_{collagenolytic}

enzymeproducedbyP.aurantiogriseum.Onlytheenzyme pro-ducedbyR.solanipresentedanacidoptimumpH,5.0.55 _As

pH,optimumenzymeactivitytemperaturealsovariedgreatly (from35to70◦C).Onlyoneoftheworkshaveproducedain naturacollagenspeciﬁccollagenaseandevaluatedoptimum temperature,37◦C.24

Inhibitors

Enzymeinhibitorsaremoleculesthatinteractwithenzyme or compounds that chelate metal ions required by the enzymetomaintainitsconformation.22Somecompoundscan inactivateirreversiblyto collagenase,suchasdithiothreitol (DTT)and mercaptoethanol.69,70 _Other_inhibitors _tested_are

phenylmethylsulphonylﬂuoride(PMSF)forserineproteases, ethylenediaminetetraaceticacid(EDTA)formetalloproteases, andiodoaceticacid(IAA)forcysteineproteases.58

Of the 21 selected articles, most conducted inhibitors tests (14 articles). Six concluded that the enzyme belongs toserine proteases group, four concludedbelongs to met-alloproteinases, two articles to both of the groups and in theremainderarticlesnoconclusionwereobtained.The col-lagenolytic enzymeproduced by R.solani was inhibited by Hg2+_, _iodoacetate, _arsenate, _arsenite, _cystein _and _EDTA.55

Limaetal.24_reported_the_inhibition_of_the_collagenase_enzyme

producedbyP. aurantiogriseum byPMSF, indicating thatthe enzymeisaserineprotease.

Substratespeciﬁcity

For certain industrial applications, such as medical and cosmetic areas, the enzyme specificity is one ofthe most importantparameterstoconsider.Fromthe21selected arti-cles, 15 conducted substrate specificity tests using other proteinsources.Noneperformedspecificitytestsusing differ-enttypesofcollagen.Hamdy55_tested_the_enzyme_produced_by R.solanioncollagen,caseinandgelatin,andthebestresults wereobtainedwithcollagen.Limaetal.24_reported_enzyme

speciﬁcitytestsproducedbyP.aurantiogriseumonAzocoll,type Icollagen,gelatinandazocasein,wherethebestresultswere foundfortheﬁrstsubstrate,Azocoll.

Molecularweight

Theidentiﬁedsizeofcollagenolyticenzymesfoundinthe dif-ferentpapersrangedfrom25to82kDa.However,themajority ofthevalues(5of11papers)arebetween32and39kDa.None ofthetwostudiesthathavespeciﬁcactivityforcollagen suc-ceeded inobtaining the preciseenzymemolecular weight. Amongthearticlesthatpresentedlargestenzymaticactivity, onlyHamdy55_determined_the_enzyme_size_by

electrophore-sis,reportingavalueof66kDa,with212.33U/mLofenzyme activity.

Molecularanalysis

Only two articles found performed sequencing of gene responsibleforenzymeproduction.Bothstudieswereabout collagenolyticproteases from nematode-trappingfungi.50,52

However,theseenzymeshavelowactivityfornativecollagen, whichpreventsitscharacterizationasatruecollagenase.

Theenzymesequenceoftruecollagenaseproducedby Tri-cophytonschoenleinii(VFLGREPKPDAFY)hadhomologywithrat proteasethimetoligopeptidaseandYscDoligopeptidasefrom yeastSaccharomycescerevisiaeandwasclassiﬁedassubfamily ofzinc-metalloproteinases.Itwasfoundhomologytovarious fungi,suggestingthattheenzymemaybeinvolvedincellular mechanismforconserved.47

It was conducted a Standard Protein BLAST, available on the NCBI (National Center for Biotechnology Informa-tion) website, waspossible toﬁnd, with100% homology,a widevarietyofsequencesoffungalproteasesfrom the fol-lowing genres:Trichophyton(accessionnumbersOAL69080.1, EGD96548.1,XP003234056.1),Coccidioides(accessionnumbers XP012213938.1, KMU73771.1, XP003065029.1), Microsporum

(accession number XP 003170328.1)and Arthroderma (acces-sion number: XP002849330.1) and Paemoniella (accession number KKY24142.1). In addition, a putative conserved domain of Peptidase Gluzincin family (thermolysin-like proteinase,TLPs)couldbefound,thatincludesSeveral zinc-dependentmetallopeptidases(accessionnumbercl14813),as Fungalysinthathydrolyzesextracellularmatrixproteins,such as elastin, keratin and collagen.71 _Family _of _Gluzincin _is

included amongfamiliesdependent zincmetalloproteinase withskills tohydrolyzecollagenandpresent waste critical roleinassistingtheconnectionandopening(unwinding)of collagen.35

Enzymenature

Bacterialproteasescanbedividedintotwogroupsaccording to the ability to hydrolyze native or denatured colla-gen, being considered as gelatinolytic and collagenolytic, respectively.35,60 _With_regard _to_the _fungal_collagenase,_this

classiﬁcationisnotwellunderstood.However,adoptingthe sameparametersusedbyDuarteetal.,35_systematic_review

foundarticles13(61.10%)whodescribedproducedenzymesas truecollagenases,sixarticles(28.57%)withenzymesclassified onlyasgelatinolyticandonlytwoarticles(9.52%)couldnotbe identifythenatureoftheenzyme(Table3).Aproperenzyme characterizationmustincludeconfirmationofthisactivity,so itcanbeidentifiedtherealpotentialofthestudiedenzymes.

Puriﬁcation

Onceacrudecollagenaseextractisrecovered,itmustbe puri-fiedusingoneofseveralchromatographicmethodsthatcanbe classifiedas:gelfiltration,ionexchange,hydrophobic interac-tionoraffinity.22_Furthermore,_there_are_traditional_enzymatic

extractionmethods,suchasammoniumsulfateprecipitation, ultraﬁltration,Tris–HClbufferextraction,withsodium bicar-bonatebuffer,amongothers.22,72

Fromthe21articlesselected,12hadsomekindof puriﬁ-cation, 11 ofthem using chromatographic techniques and onlyoneexclusivebyammoniumsulfate.43_Mahmoud_et_al.53

puriﬁedtheenzymeproducedbyA.ﬂavususingthe DEAE-Cellulosecolumnand obtainedayieldof39.43%. Hamdy55

could yield60.49%withthe puriﬁcation usinggel ﬁltration chromatography, but the enzyme activity had reduced the amountto128.4U/mL.

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22

Theotherspapersreportinggoodenzymaticactivitiesdid notundergoanypuriﬁcationactivities.24,48,58_Other_selected

articlesshowednosigniﬁcantamountofenzymenorquantify thecollagenaseproduced.

Conclusions

Fromthe21selectpapers,11were publishedinthelast10 years and only four in the last 5 years. According to the scoringmethodologycriteria,onlyfivestudiesshowedscore ≥7. This papersummarized the main findings on produc-tionoffungalcollagenase.Onlytwostudiesreportedenzymes withhighspecificitytocollagenoverotherproteinsubstrates. AmongmicroorganismsstudiedtheP.aurantiogriseumandR. solanistoodoutinvolumetricandspecificcollagenaseactivity, and are non-pathogenic filamentous fungi and extracellu-larenzymeproducers. Inthe culturemediumcomposition the use of collagen-basedcompounds seems notessential forcollagenolyticenzymesproduction.Forenzymes charac-terization,articlesfounddifferedalotregardingparameters analyzed. The articles with better scores did not undergo anappropriatepurification process.Six ofselected articles presentedenzymesthatcouldnotbeconsideredtrue colla-genases.Althoughtwo ofthearticleshave foundthegene responsibleforenzymeproduction,bothenzymesshowedlow activityagainstnativecollagen. Theonlyarticlefoundthat madesequencingofatruecollagenaseshowed100% homol-ogywithseveralmetalloproteinasesfungi.Itwaspossibleto observeagapinliteratureaboutcollagenaseproductionby fungianditscharacterization,whichpreventsfurther devel-opmentintheareaandincreasestheneedforfurtherstudies, particularly forfull characterizationof fungal collagenases withhighspecificity.Itwasalsoobservedthatstudiedfungal collagenasespresentspromisingandcompetitive biotechnol-ogycharacteristicswhencomparedwithbacterialenzymes, mostusedcommercially.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

ThisworkwassupportedbyFundaçãodeAmparoàCiência eTecnologia doEstado dePernambuco (FACEPE), Conselho NacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES).

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