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Inc.causediseasesanddamageinagriculturalcrops.Chitinasesalsoplayanimportantphysio-logicalandecologicalroleinecosystemsasrecyclersofchitin,bygeneratingCandNsources.Someproducersofchitinases,includingTrichodermaspp.,arealsosourcesofmycolyticenzymepreparations(51,59,60).ChitinolyticenzymesaredefinedasenzymesthatcleaveabondbetweentheC1andC4oftwoconsecutiveGlcNAcunits.Onthebasisofaminoacidsequencesimilarities,allchitinaseshavebeengroupedintofamilies18,19 ,and2 0,underthemainclassofglycosylhydrolases.Mostofthemicrobialchitinasesbelongtofamily18(61,62).Evenwithinthesamefamily,chitinasesshowwidelydifferingpropertieswithrespecttosub-stratespecificity,reactionspecificity,andpHoptimum.Thechitinolyticenzymesaredi-videdintothreeprincipaltypesdependingontheiractiononchitinsubstrates.AccordingtothenomenclaturesuggestedbySahaiandManocha(59),endochitinases(EC3.2.1.14)aredefinedasenzymescatalyzingtherandomhydrolysisof1, 4- linkagesofGlcNAcatinternalsitesovertheentirelengthofthechitinmicrofibril.Theproductsofthereactionaresoluble,low-molecular-massmultimersofGlcNAcsuchaschitotetraose,chitotriose,anddiacetylchitobiose.Exochitinases,alsotermedchitobiosidasesorchitin-1, 4- -chito-biosidases(63),catalyzetheprogressivereleaseofdiacetylchitobioseunitsinastepwisefashionasthesoleproductfromthechitinchains,suchthatnomonosaccharidesoroligo-saccharidesareformed.Thethirdtypeofchitinolyticenzymeischitobiasealsotermedashexosaminidase(EC3.2.1.52)orN-acetyl- -1 ,4-d-glucosaminidase(EC3.2.1.30)belongstofamily20andalsoactsinexosplittingmodeondiacetylchitobioseandhigheranalogsofchitin,includingchitotrioseandchitotetraose,toproduceGlcNAcmonomers.RapidandspecificmethodshavebeendevelopedfordetectionandquantitativeassaysofN-acetyl-β-gluco-saminidase,chitobiosidase,andendochitinaseinsolutionsusingp-nitrophenyl-N-acetyl-β-d-glucosaminide,p-nitrophenyl-β-d-N,N′-diacetylchitotriose,andp-nitrophenyl-β-d-N,N′,N″-triacetylchitotrioseorcolloidalchitinassubstrates,respectively(64).Proceduresalsoaredescribedforthedirectassayofthesethreeenzymesaftertheirseparationbysodiumdodecylsulfate(SDS)-polyacrylamidegelelectrophoresis(PAGE)inwhichtheenzymesarevisualizedasfluorescentbandsbyusinganagaroseoverlaycontaining 4- methyl-umbelliferylderivativesofN-acetyl-β-d-glucosaminide,β-d-N,N′-diacetyl-chitobioside,orβ-d-N,N,N″-triacetylchitotriose,respectively(65).AsetofchitinolyticenzymessecretedbyvariousstrainsofT.harzianum(e.g.,TM,T-Y,39.1,CECT2413,P1ϭT.atroviride),whengrownonchitinasthesoleCsource,consistsofN-acetylglucosaminidases,endochitinases,andexochitinases(chitobiosidases).Intotal,10separatedchitinolyticenzymeswerelistedbyLorito(50);onlyonestepinthemicroparasiticprocessofT.harzianum,whichisthedissolutionofthecellwallofthetargetfungusbyenzymeactivity,mayinvolvemorethan20separategenesandgeneproductssynergisticonetoanother(Table1).TwoN-acetylglucosaminidaseswithappar-ent ... Inc.causediseasesanddamageinagriculturalcrops.Chitinasesalsoplayanimportantphysio-logicalandecologicalroleinecosystemsasrecyclersofchitin,bygeneratingCandNsources.Someproducersofchitinases,includingTrichodermaspp.,arealsosourcesofmycolyticenzymepreparations(51,59,60).ChitinolyticenzymesaredefinedasenzymesthatcleaveabondbetweentheC1andC4oftwoconsecutiveGlcNAcunits.Onthebasisofaminoacidsequencesimilarities,allchitinaseshavebeengroupedintofamilies18,19 ,and2 0,underthemainclassofglycosylhydrolases.Mostofthemicrobialchitinasesbelongtofamily18(61,62).Evenwithinthesamefamily,chitinasesshowwidelydifferingpropertieswithrespecttosub-stratespecificity,reactionspecificity,andpHoptimum.Thechitinolyticenzymesaredi-videdintothreeprincipaltypesdependingontheiractiononchitinsubstrates.AccordingtothenomenclaturesuggestedbySahaiandManocha(59),endochitinases(EC3.2.1.14)aredefinedasenzymescatalyzingtherandomhydrolysisof1, 4- linkagesofGlcNAcatinternalsitesovertheentirelengthofthechitinmicrofibril.Theproductsofthereactionaresoluble,low-molecular-massmultimersofGlcNAcsuchaschitotetraose,chitotriose,anddiacetylchitobiose.Exochitinases,alsotermedchitobiosidasesorchitin-1, 4- -chito-biosidases(63),catalyzetheprogressivereleaseofdiacetylchitobioseunitsinastepwisefashionasthesoleproductfromthechitinchains,suchthatnomonosaccharidesoroligo-saccharidesareformed.Thethirdtypeofchitinolyticenzymeischitobiasealsotermedashexosaminidase(EC3.2.1.52)orN-acetyl- -1 ,4-d-glucosaminidase(EC3.2.1.30)belongstofamily20andalsoactsinexosplittingmodeondiacetylchitobioseandhigheranalogsofchitin,includingchitotrioseandchitotetraose,toproduceGlcNAcmonomers.RapidandspecificmethodshavebeendevelopedfordetectionandquantitativeassaysofN-acetyl-β-gluco-saminidase,chitobiosidase,andendochitinaseinsolutionsusingp-nitrophenyl-N-acetyl-β-d-glucosaminide,p-nitrophenyl-β-d-N,N′-diacetylchitotriose,andp-nitrophenyl-β-d-N,N′,N″-triacetylchitotrioseorcolloidalchitinassubstrates,respectively(64).Proceduresalsoaredescribedforthedirectassayofthesethreeenzymesaftertheirseparationbysodiumdodecylsulfate(SDS)-polyacrylamidegelelectrophoresis(PAGE)inwhichtheenzymesarevisualizedasfluorescentbandsbyusinganagaroseoverlaycontaining 4- methyl-umbelliferylderivativesofN-acetyl-β-d-glucosaminide,β-d-N,N′-diacetyl-chitobioside,orβ-d-N,N,N″-triacetylchitotriose,respectively(65).AsetofchitinolyticenzymessecretedbyvariousstrainsofT.harzianum(e.g.,TM,T-Y,39.1,CECT2413,P1ϭT.atroviride),whengrownonchitinasthesoleCsource,consistsofN-acetylglucosaminidases,endochitinases,andexochitinases(chitobiosidases).Intotal,10separatedchitinolyticenzymeswerelistedbyLorito(50);onlyonestepinthemicroparasiticprocessofT.harzianum,whichisthedissolutionofthecellwallofthetargetfungusbyenzymeactivity,mayinvolvemorethan20separategenesandgeneproductssynergisticonetoanother(Table1).TwoN-acetylglucosaminidaseswithappar-ent ... involved in triggering the expression of the 42-kD endochitinase and the 73-kD N-acetyl-β-d-glucosaminidase. This last enzyme revealed high similarityto N-acetyl-glucosaminidases from other eukaryotes,...