Katı substrat fermentasyonu (KSF) ile trichoderma harzianum ve trichoderma viride türü funguslarda ksilanaz üretimi, saflaştırılması ve biyokimyasal özelliklerin belirlenmesi

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(1)T.C. %$/,.(6ø5h1ø9(56ø7(6ø )(1%ø/ø0/(5ø ENSTøTÜSÜ %ø<2/2-ø$1$%ø/ø0'$LI. .$7,68%675$7)(50(17$6<218 .6)

(2) ø/( 75ø&+2'(50$+$5=ø$180 VE 75ø&+2'(50$9ø5ø'( 7h5h)81*86/$5'$.6ø/$1$=h5(7ø0ø 6$)/$ù7,5,/0$6,9(%ø<2.ø0<$6$/g=(//ø./(5ø1 %(/ø5/(10(6ø. D2.725$7(=ø. +h6(<ø1$/3(5ø57(0. %$/,.(6ø5, MAYIS - 2018.

(3) T.C. %$/,.(6ø5h1ø9(56ø7(6ø )(1%ø/ø0/(5ø(167ø7ÜSÜ %ø<2/2-ø$1$%ø/ø0'$LI. KATI SUBSTRAT FERMENTASYONU (KSF) ø/( 75ø&+2'(50$+$5=ø$180 VE 75ø&+2'(50$9ø5ø'( 7h5h)81*86/$5'$.6ø/$1$=h5(7ø0ø 6$)/$ù7,5,/0$6,9(%ø<2.ø0<$6$/g=(//ø./(5ø1 %(/ø5/(10(6ø. DOKTORA TEZø. +h6(<ø1$/3(5ø57(0. Jüri Üyeleri : 3URI'U$\úH'LOHN$=$= 7H]'DQÕúPDQÕ

(4) Prof. Dr. Hilmi NAMLI Prof. Dr. *YHQg='(0ø5 Prof. Dr. Ataç UZEL Prof. Dr. Olga SAK. BALI.(6ø5, MAYIS - 2018.

(5)

(6) %X WH] oDOÕúPDVÕ %DOÕNHVLU hQLYHUVLWHVL 5HNW|UO÷ %LOLPVHO $UDúWÕUPD Projeleri Birimi WDUDIÕQGDQ2014-094 QROXSURMHLOHGHVWHNOHQPLúWLU.

(7) ÖZET KATI SUBSTRAT FERMENTASY218 .6)

(8) ø/(75ø&+2'(50$ +$5=ø$180 VE 75ø&+2'(50$9ø5ø'( TÜRÜ FUNGUSLARDA .6ø/$1$=h5(7ø0ø6$)/$ù7,5,/0$6,9(%ø<2.ø0<$6$/ g=(//ø./(5ø1%(/ø5/(10(6ø '2.725$7(=ø +h6(<ø1$/3(5ø57(0 %$/,.(6ø5h1ø9(56ø7(6ø)(1%ø/ø0/(5ø(167ø7h6h Bø<2/2-ø$1$%ø/ø0'$/I (TEZ '$1,ù0$1,352)'5$<ù('ø/(. AZAZ) %$/,.(6ø5, MAYIS - 2018 %X oDOÕúPDGD .DWÕ 6XEVWUDW )HUPHQWDV\RQ .6)

(9) NOWU RUWDPÕQGD JHOLúWLULOHQ Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL ¶GHQ ȕ-ksilosidaz enzLPL VDIODúWÕUÕOPÕú YH EL\RNLP\DVDO NDUDNWHUL]DV\RQX \DSÕOPÕúWÕU %LUoRNEL\RWHNQRORMLNX\JXODPDODUGDWLFDUL|QHPHVDKLSȕ-ksilosidaz enzimi, .6) RUWDPÕQGD VXEVWUDW RODUDN EX÷GD\ NHSH÷LQLQ NXOODQÕOPDVÕ\OD JHOLúWLULOHQ Trichoderma harzianum .6) RUWDPÕQÕQ QHPOHQGLUPH VÕYÕVÕ S+ Sitrik asit monohidrat (C6H8O7H2O), RSWLPXP VÕFDNOÕN oC ve inkübasyon süresi 7 gün) ve Trichoderma viride .6)RUWDPÕQÕQ QHPOHQGLUPHVÕYÕVÕS+Di-sodyum hidrojen fosfat dihidrat (Na2HPO4.2H2O) tamponu ile nemlendirilerek, optimum VÕFDNOÕN 25oC ve inkübasyon süresi 7 gün) VXúODUÕQGDQ elde HGLOPLúWLU ȕ-ksilosidaz enzimi, amonyum sülfat çöktürmesi ve Sepharose-4B-L-Tyrosine-1-Napthylamine NXOODQÕODUDN+LGURIRELN(WNLOHúLP.URPDWRJUDILVLLOHLNLEDVDPDNWD VDIODúWÕUÕOPÕúWÕU SaflDúWÕUÕODQ ȕ-ksilosidaz enzim aktivitesi, p-nitrofenil-ȕ-D-ksilopiranosit (p13;

(10) VXEVWUDWÕ NXOODQÕODUDN EHOLUOHQPLúWLU Trichoderma harzianum ȕ-ksilosidaz enzimi (Thȕ) %50,48 verimle 215,76 kat; Trichoderma viride ȕ-ksilosidaz (Tvȕ) ise %33,43 verimle 92,39 NDW VDIODúWÕUÕOPÕúWÕU 6'6-PAGE ve Native-PAGE ile Thȕ ve Tvȕ HQ]LPOHULQLQPROHNOD÷ÕUOÕNODUÕVÕUDVÕ\ODN'DYHN'DRODUDN EHOLUOHQPLú olup tek DOWQLWHGHQROXúWXNODUÕWHVSLWHGLOPLúWLUThȕ ve Tvȕ enzimlerinin optimum pH GH÷HUOHUL 6,0; optimum VÕFDNOÕN GH÷HUOHUL LVH VÕUDVÕ\OD & YH & RODUDN VDSWDQPÕúWÕU Thȕ enzimin Km ve Vmax GH÷HUOHUL VÕUDVÕ\OD P0 YH (8 Tvȕ HQ]LPLQLQ LVH P0 YH (8 RODUDN EHOLUOHQPLúWLU $\UÕFD VDIODúWÕUÕODQ ȕksilosidaz enzimlerinin aktiviteleri bir ȕ-ksilosidaz inhibitörü olan D-(+)-ksiloza NDUúÕ EHOLUOHQPLúWLU D-(+)-ksiloz inhibitörünün Thȕ enzimini NDUÕúÕN Tvȕ enzimini LVH\DUÕúPDOÕRODUDNLQKLEHHWWL÷L EHOLUOHQPLúWLU'-(+)-ksilozun Thȕ enzim aktivitesi üzerine IC50 ve Ki GH÷HUOHUL VÕUDVÕ\OD 1,1x10-1 mM ve 1,10x10-3/5,57x10-3; Tvȕ enziminin IC50 ve Ki GH÷HUOHUL LVH 7,1x10-2 mM ve 2,64x10-4±1x10-5 olarak KHVDSODQPÕúWÕU. $1$+7$5 .(/ø0(/(5 Trichoderma harzianum, Trichoderma viride ȕ-ksilosidaz, NDWÕ VXEVWUDW IHUPHQWDV\RQX RSWLPL]DV\RQ VDIODúWÕUPD EL\RNLP\DVDO elektroforetik ve kinetik özellikler i.

(11) ABSTRACT XYLANASE PRODUCTION, PURIFICATION AND IDENTIFICATION OF BIOCHEMICAL CHARACTERISTICS OF 75ø&+2'(50$+$5=ø$18M AND 75ø&+2'(50$9ø5ø'(SPECIES FUNGI THROUGH SOLID STATE FERMENTATION PH.D THESIS +h6(<ø1$/3(5ø57(0 BALIKESIR UNIVERSITY INSTITUTE OF SCIENCE BIOLOGY (SUPERVISOR: 352)'5$<ù('ø/(. AZAZ ) %$/,.(6ø5, MAY 2018 ,Q WKLV VWXG\ SXULILFDWLRQ DQG ELRFKHPLFDO FKDUDFWHUL]DWLRQ RI ȕ-xylosidase enzyme purified from Trichoderma harzianum NRRL 13019 and Trichoderma viride NRRL 6418 growth in solid state fermentation (SSF) was performed. ȕ-xylosidase enzyme that has many biotechnological applications, was primarily obtained from Trichoderma harzianum (SSF conditions moistening citric acid monohydrate (C6H8O7H2O) pH 4.0, temperature 35°C and incubated 7 days) and Trichoderma viride (SSF conditions moistening disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) pH 8.5, temperature 25°C and 7 days) grown in SSF using wheat bran DV D VXEVWUDWH ȕ-xylosidase enzyme was purified using two-step procedures, namely ammonium sulfate precipitation and Sepharose-4B-L-Tyrosine1-Napthylamine Hydrophobic Interaction Chromatography. ,QRXUVWXG\SXULILHGȕ-xylosidase enzyme activity was determined by using para-Nitrophenyl-beta-D-xylopyranoside (pNPX) substrate. The purification rate was IRXQG IROG ZLWK \LHOG RI IRU WKH REWDLQHG ȕ-xylosidase from Trichoderma harzianum 7Kȕ

(12) DQG\LHOGIRUWKHREWDLQHGȕ-xylosidase from Trichoderma viride 7Yȕ

(13) 0ROHFXODU ZHLJKWV RI 7Kȕ DQG 7Yȕ HQ]\PHV ZHUH determined 20kDa and 25kDa, respectively, using SDS and Native PAGE analysis, and are formed in sub-XQLW2SWLPXPS+YDOXHVRI7KȕDQG7YȕHQ]\PHVZHUH respectively, optimum temprature values were determined as 75°C and 65°C. The Km and Vmax YDOXHV RI 7Kȕ HQ]\PH ZHUH GHWHUPLQHG DV P0 DQG (8UHVSHFWLYHO\ZKLOHIRUWKH7YȕHQ]\PHP0DQG(8,QDGGLWLRQ WKHDFWLYLWLHVRISXULILHGȕ-xylosidase enzymes were determined against D-(+)-xylose ZKLFK LV D ȕ-xylosidase inhibitor. It is determined that D-(+)-xylose inhibitor LQKLELWHGWKH7KȕHQ]\PHPL[HGDQGWKH7YȕHQ]\PHFRPSHWLWLYHO\7KH ,& DQG Ki values for D-(+)-[\ORVH 7Kȕ HQ]\PH DFWLYLW\ ZHUH FDOFXODWHG DFFording to an order as 1.1x10-1 mM and 1.10x10-3/5.57x10-3; respectively IC50 and Ki values of 7YȕHQ]\PHDV[-2 mM and 2.64x10-4±1x10-5.. KEYWORDS: Trichoderma harzianum, Trichoderma viride, ȕ-xylosidase, solid substrate fermentation, optimization, purification, biochemical, electrophoretic and kinetic properties ii.

(14) ødø1'(.ø/(5 Sayfa ÖZET....................................................................................................................... i ABSTRACT ........................................................................................................... ii ødø1'(.ø/(5 .................................................................................................... iii ù(.ø//ø67(6ø................................................................................................... vi 7$%/2/ø67(6ø ............................................................................................... viii 6(0%2//ø67(6ø ................................................................................................x ÖNSÖZ.................................................................................................................. xi 1. *ø5øù..................................................................................................................1 1.1 Enzimlerin özellikleri....................................................................................3 1.1.1 Enzimler ve sÕQÕIODQGÕUPD......................................................................4 1.2 Ksilan ve kVLODQD]ODUÕQgenel özellikleri .......................................................6 1.2.1 Ksilan .....................................................................................................6 1.2.2 Ksilanazlar .............................................................................................7 1.2.3 Ksilanaz kD\QDNODUÕ................................................................................9 1.2.4 .VLODQD]ODUÕQoHúLWOLOL÷LYHVÕQÕIODQGÕUPDVÕ.............................................9 1.2.5 .VLODQD]ODUÕQJenel özellikleri .............................................................10 1.2.6 .VLODQD]ODUÕQEiyoteknoloji ve endüstriyel uygulama aODQODUÕ.............11 1.2.6.1 .VLODQD]ODUÕQbiyoteknolojideki uygulama aODQODUÕ ...................11 1.2.6.2 .VLODQD]ODUÕQendüstriyel uygulama aODQODUÕ .............................11 1.3 Ksilanaz üretimi ..........................................................................................14 1.3.1 .DWÕVubstrat fermentasyon (KSF) yöntemi .........................................15 1.3.1.1 .DWÕVubstrat fermentasyonunda avantajlar ve dezavantajlar ..............................................................................17 1.4 Trichoderma cinsinin genel özellikleri .......................................................18 2. MATERYAL VE METOT .............................................................................20 2.1 Materyal ...................................................................................................20 2.1.1 Ksilanaz aktivitesine sahip mikrofunJXVODUÕQEelirlenmesi .................20 2.1.2 Deneylerde kXOODQÕODQTrichoderma türleri ve genel özellikleri..........20 2.1.2.1 Trichoderma harzianum Rifai 1969 ..........................................20 2.1.2.2 Trichoderma viride Pers. Ex Gray 1821....................................21 2.1.3 .XOODQÕODQWamponlar ve çözeltiler.......................................................22 2.1.3.1 Mikrofungus kültürlerini gHOLúWLUPHNLçin hD]ÕUODQDQ besiyerleri ..................................................................................22 2.1.3.2 Hidroliz zonu belirlenirken kXOODQÕODQçözeltiler .......................23 2.1.3.3 0LNURIXQJXVODUÕQVpor sVSDQVL\RQODUÕelde edilirken kXOODQÕODn çözelti........................................................................23 2.1.3.4 KSF ortaPÕQGDQemlendirme sÕYÕVÕolarak kXOODQÕODFDN tampon çözeltilerin hD]ÕUODQPDVÕ ...............................................24 2.1.3.5 Ksilanaz aktivitesinin belirlenmesinde kXOODQÕODQVubstrat çözeltisi ve tampon çözeltilerin hD]ÕUODQPDVÕ............................24 2.1.3.6 Hidrofobik eWNLOHúLPNromatografisi kolonunda kXOODQÕODQ jelin sentezlenmesi.....................................................................25 2.1.3.6.1 Sefaroz (Sepharose) 4B jelini aNWLIOHúWLUPH ...................25 2.1.3.6.2 L-Tirozin bD÷ODPD..........................................................25 2.1.3.6.3 1-Naftilamin bLOHúL÷LQLQED÷ODQPDVÕ ..............................26 iii.

(15) 2.1.3.7 Lowry yöntemiyle proteinlerin kantitatif tayininde kXOODQÕODQoözeltiler ...................................................................26 2.1.3.8 Hidrofobik eWNLOHúLPNromatografisi (HEK) yönteminde kXOODQÕODQoözeltiler ...................................................................27 2.1.3.9 SDS-PAGE tHNQL÷LQGHNXOODQÕODQçözeltiler .............................27 2.1.3.10 NATIVE-PAGE tHNQL÷LQGHkulODQÕODQoözeltiler ......................29 2.2 Metot ...................................................................................................30 2.2.1 Ksilanaz aktivitesine sahip olan mLNURIXQJXVODUÕQEelirlenmesi .........30 2.2.2 0LNURIXQJXVODUÕQretimi ve spor eldesi..............................................30 2.2.3 .DWÕsubstrat fermentasyon (KSF) oUWDPÕQÕQhD]ÕUODQPDVÕ .................31 2.2.4 .DWÕsubstrat fermentasyonu (KSF) kültür oUWDPÕQDekim ve kÕVPLVDIODúWÕUÕOPÕúNsilanaz enziminin eldesi......................................31 2.2.5 Üretim kRúXOODUÕQÕQNDWÕVubstrat fermentasyon (KSF) kültür kRúXOODUÕQGDJHOLúWLULOHQPLNURIXQJXVȕ-ksilosidaz enzim sentezi üzerine etkisinin belirlenmesi ..................................................32 2.2.5.1 Optimum pH ve nemlendirme sÕYÕODUÕQÕQ belirlenmesi .............32 2.2.5.2 Optimum inkübasyon sÕFDNOÕ÷ÕQÕQ belirlenmesi ........................32 2.2.5.3 Optimum inkübasyon süresinin belirlenmesi ............................33 2.2.6 ȕ-ksilosidaz aktivitesinin belirlenmesi.................................................33 2.2.7 Protein tayini........................................................................................35 2.2.7.1 Kalitatif protein tayini................................................................35 2.2.7.2 Lowry metodu ile proteinlerin kantitatif tayini .........................35 2.2.7.3 BSA standart gUDIL÷LQLQ hD]ÕUODQPDVÕ........................................36 2.2.8 Enzim sDIODúWÕUPD.................................................................................37 2.2.8.1 Amonyum sülfat çöktürme aUDOÕ÷ÕQÕQbelirlenmesi ...................37 2.2.8.2 Hidrofobik eWNLOHúLP kURPDWRJDILVLLOHȕ-ksilosidaz enziminin sDIODúWÕUÕOPDVÕ............................................................38 2.2.9 NATIVE/SDS-PAGE yöntemi kXOODQÕODUDNȕ-ksilosidaz enziminin sDIOÕ÷ÕYHalt birimlerinin belirlenmesi................................38 2.2.9.1 SDS-PAGE yöntemi kXOODQÕODUDNȕ-ksilosidaz enziminin sDIOÕ÷ÕQÕQYHalt birimlerinin kontrolü........................................39 2.2.9.2 NATIVE-PAGE yöntemi kXOODQÕODUDNȕ-ksilosidaz enziminin sDIOÕ÷ÕQÕQYHalt birimlerinin kontrolü.......................40 2.2.10 6DIODúWÕUÕODQȕ-ksilosidaz enziminin biyokimyasal özelliklerinin belirlenmesi ...................................................................41 2.2.10.1 6DIODúWÕUÕODQȕ-ksilosidaz enziminine fDUNOÕS+¶ODUÕQetkisi .......41 2.2.10.2 6DIODúWÕUÕODQȕ-ksilosidaz enziminin maksimum aktivite g|VWHUGL÷LsÕFDNOÕNdH÷HULQLQ belirlenmesi .................................42 2.2.10.3 6DIODúWÕUÕODQȕ-ksilosidaz enziminin termal kDUDUOÕOÕ÷ÕQÕn belirlenmesi................................................................................42 2.2.10.4 ȕ-ksilosidaz enziminin p-nLWURIHQLOȕ-D-ksilopiranosit (pNPX) sXEVWUDWÕQDkDUúÕ.M ve Vmax dH÷HUOHULQLQ belirlenmesi................................................................................43 2.2.10.5 øQKLELW|Uolarak kXOODQÕODQ'-(+)-ksilozun IC50 dH÷HULQLQ belirlenmesi................................................................................43 2.2.10.6 øQKLELW|Uolarak kXOODQÕODQ'-(+)-ksilozun Ki dH÷HUinin bXOXQPDVÕ...................................................................................43. iv.

(16) 3. BULGULAR ....................................................................................................45 3.1 ȕ-ksilosidaz aktivitesine VDKLSPLNURIXQJXVODUÕQ belirlenmesi...................45 3.2 Üretim NRúXOODUÕQÕQNDWÕVXEVWUDWIHUPHQWDV\RQ (KSF) oUWDPÕQGD Üretilen fXQJXVȕ-ksilosidaz enzimi sentezi üzerine etkisi.........................45 3.2.1 Optimum pH ve QHPOHQGLUPHVÕYÕODUÕQÕQEHOLUOHQPHVL .......................46 3.2.2 Optimum LQNEDV\RQVÕFDNOÕ÷ÕQÕQ belirlenmesi ..................................48 3.2.3 Optimum inkübasyon süresinin belirlenmesi.......................................49 3.3 ȕ-ksilosidaz enziminin VDIODúWÕUPDVÕ ...........................................................50 3.4 Amonyum sülfat tuzu kulODQÕODUDNo|NWUPHDUDOÕ÷ÕQÕQ belirlenmesi.................................................................................................51 3.5 Hidrofobik HWNLOHúLPNURPDWRJUDILVLLOH ȕ-ksilosidaz enziminin sDIODúWÕUÕOPDVÕ ..............................................................................................54 3.6 Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den HOGHHGLOHUHNVDIODúWÕUÕODQȕ-ksilosidaz enziminin NATIVE/SDS PAGE WHNQL÷LNXOODQÕODUDNVDIOÕ÷ÕQÕQYHDOW birimlerinin kontrolü...................................................................................60 3.7 6DIODúWÕUÕODQȕ-ksilosidaz enziminin biyokimyasal özelliklerinin belirlenmesi.................................................................................................62 3.7.1 Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den HOGHHGLOHUHNVDIODúWÕUÕODQ ȕ-ksilosidaz enzim aktivitesine IDUNOÕ S+¶ODUÕQetkisi ..............................................62 3.7.2 Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den HOGHHGLOHUHNVDIODúWÕUÕODQȕ-ksilosidaz enziminin maksimum aktivite g|VWHUGL÷LVÕFDNOÕN GH÷HUOHULQLQbelirlenmesi .....................................................................63 3.7.3 Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den elde edilHUHNVDIODúWÕUÕODQ ȕ-ksilosidaz HQ]LPLQLQWHUPDONDUDUOÕOÕ÷ÕQÕQEHOLUOHQPHVL........................................64 3.7.4 Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den HOGHHGLOHUHNVDIODúWÕUÕODQȕ-ksilosidaz enziminin pNPX sXEVWUDWÕQDkDUúÕ.M ve Vmax GH÷HUOHULQLQ belirlenmesi......................................................................66 3.7.5 Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den sDIODúWÕUÕODQȕ-ksilosidaz enziminin inhibitörü olan D-(+)- ksilozun IC50 dH÷HUOHULQLQ belirlenmesi ...........70 3.7.6 Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den sDIODúWÕUÕODQȕ-ksilosidaz enziminin inhibitörü olan D-(+)-ksilozun inhibisyon tipinin ve Ki dH÷HUOHULQLQbelirlenmesi ......................................................................74 4. 7$57,ù0$9(6218d................................................................................83 5. KAYNAKLAR...............................................................................................101. v.

(17) ù(.ø/ /ø67(6ø Sayfa ùHNLO ùHNLO. .VLODQÕQtam hidrolizinde gerekli enzimler ve etki bölgeleri............ (a) Trichoderma harzianum NRRL 13019 petri (10x10); (b) Trichoderma harzianum NRRL 13019 preparat (10x40)............ ùHNLO (a) Trichoderma viride NRRL 6418 petri (10x10); (b) Trichoderma viride NRRL 6418 preparat (10x40)...................... ùHNLO (Q]LPDNWLYLWHWD\LQLQGHNXOODQÕODQ/KDFLPOLpNPX VWDQGDUWJUDIL÷L................................................................................... ùHNLO Lowry yöntemi ile protein miktarÕQÕQEHOLUOHQPHVLQGHNXOODQÕODQ standart grafik.................................................................................... ùHNLO (a) Trichoderma harzianum NRRL 13019; (b) Trichoderma viride NRRL 6418. ................................................ ùHNLO .DWÕVXEVWUDWIHUPHQWDV\RQNOWURUWDPÕQGDIDUNOÕS+¶ODUGDNL QHPOHQGLUPHVÕYÕODUÕQÕQTrichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den elde edilen ȕ-ksilosidaz enzim aktivitesi üzerine etkisi. .......................................................... ùHNLO .DWÕVXEVWUDWIHUPHQWDV\RQNOWURUWDPÕQGDIDUNOÕLQNEDV\RQ VÕFDNOÕNODUÕQÕQTrichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6418’den elde edilen ȕ-ksilosidaz enzim aktivitesi üzerine etkisi. .......................................................... ùHNLO .DWÕVXEVWUDWIHUPHQWDV\RQNOWURUWDPÕQGDIDUNOÕLQNEDV\RQ sürelerinin Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 64¶GHQHOGHHGLOHQȕ-ksilosidaz enzim aktivitesi üzerine etkisi. .......................................................... ùHNLO Trichoderma harzianum NRRL 13019 amonyum sülfat o|NWUPHDUDOÕ÷Õ-enzim aktivite-SURWHLQPLNWDUÕJUDIL÷L.................... ùHNLO Trichoderma viride NRRL 6418 amonyum sülfat çöktürme DUDOÕ÷Õ-enzim aktivite-SURWHLQPLNWDUÕ JUDIL÷L.................................... ùHNLO Hidrofobik HWNLOHúLPNURPDWRJUDILVL NXOODQÕODUDNVDIODúWÕUÕODQ Trichoderma harzianum NRRL 13019’dan elde edilen ȕ-NVLORVLGD]HQ]LPLQLQHOV\RQJUDIL÷L ............................................ ùHNLO Hidrofobik HWNLOHúLPNURPDWRJUDILVLNXOODQÕODUDNVDIODúWÕUÕODQ Trichoderma viride NRRL 64¶GHQHOGHHGLOHQȕ-ksilosidaz HQ]LPLQLQHOV\RQJUDIL÷L................................................................. ùHNLO Trichoderma harzianum NRRL 13019’GDQVDIODúWÕUÕODQ ȕ-ksilosidaz enziminin poliakrilamid jel elektroforez görüntüleri (a): SDS-poliakrilamid jel elektoroforezi, (b): Native-poliakrilamid jel elektroforezi. ....................................... ùHNLO: Trichoderma viride NRRL 6418¶GHQVDIODúWÕUÕODQȕ-ksilosidaz enziminin poliakrilamid jel elektroforez görüntüleri (a): SDS-poliakrilamid jel elektoroforezi, (b): Native-poliakrilamid jel elektroforezi. ........................................ vi. 8 21 22 34 36 45. 47. 49. 50 52 53 55 58. 61. 61.

(18) ùHNLO Trichoderma harzianum NRRL 13019 ve Trichoderma viride 155/¶GHQVDIODúWÕUÕODQȕ-ksilosidaz enzim aktivitesine IDUNOÕpH larÕQetkisi. ......................................................................... ùHNLO Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 6¶GHQVDIODúWÕUÕODQȕ-ksilosidaz enzim aktivitesine SÕFDNOÕ÷ÕQ etkisi. ................................................................................ ùHNLO: Trichoderma harzianum NRRL 13019’dan elde edilen SDIODúWÕUÕOPÕú ȕ-NVLORVLGD]HQ]LPLQLQWHUPDONDUDUOÕOÕ÷Õ ................... ùHNLO: Trichoderma viride NRRL 6418¶GHQHOGHHGLOHQVDIODúWÕUÕOPÕú ȕ-NVLORVLGD]HQ]LPLQLQWHUPDONDUDUOÕOÕ÷Õ.......................................... ùekil 3.15: Trichoderma harzianum NRRL 13019¶GDQVDIODúWÕUÕODQ ȕ-ksilosidaz enziminin KM ve VPD[GH÷HUOHULQLJ|VWHUHQ Lineweaver-Burk JUDIL÷L .................................................................. : Trichoderma viride NRRL 6418’den VDIODúWÕUÕODQȕ-ksilosidaz ùHNLO enziminin KM ve VPD[GH÷HUlerini gösteren Lineweaver-Burk JUDIL÷L ............................................................................................... ùHNLO: Trichoderma harzianum NRRL 13019’daQVDIODúWÕUÕODQ ȕ-ksilosidaz enzim inhibitörü olan D-(+)-ksiloza ait IC50 JUDIL÷L.... ùHNLO: Trichoderma viride NRRL 6418¶GHQVDIODúWÕUÕODQȕ-ksilosidaz enzim inhibitörü olan D-(+)-ksiloza ait IC50 JUDIL÷L. ...................... ùHNLO: Lineweaver-%XUNJUDIL÷L]HULQGHTrichoderma harzianum NRRL 13019 ȕ-ksilosidaz enzimine D-(+)-NVLOR]XQIDUNOÕ NRQVDQWUDV\RQODUÕQÕQLQKLELV\RQHWNLVL............................................. ùHNLO: Lineweaver-%XUNJUDIL÷L]HULQGHTrichoderma viride NRRL 6418 ȕ-ksilosidaz enzimine D-(+)-ksilozuQIDUNOÕ NRQVDQWUDV\RQODUÕQÕQLQKLELV\RQHWNLVL.............................................. vii. 63 64 65 65. 69. 70 73 73. 78. 82.

(19) 7$%/2/ø67(6ø Sayfa Tablo 2.1: SDS-3$*( GHNLMHOOHULQKD]ÕUODQPDVÕ .............................................. Tablo 2.2: Native-3$*(¶GHNLMHOOHULQKD]ÕUODQPDVÕ .......................................... Tablo 3.1: .DWÕVXEVWUDWIHUPHQWDV\RQNOWURUWDPÕQGDIDUNOÕS+¶ODUGDNL QHPOHQGLUPHVÕYÕODUÕQÕQTrichoderma harzianum NRRL 13019 ve Trichoderma viride 155/¶GHQHOGHHGLOHQȕ-ksilosidaz enzim aktivitesi üzerine etkisi ........................................................... Tablo 3.2: .DWÕVXEVWUDWIHUPHQWDV\RQNOWURUWDPÕQGDIDUNOÕVÕFDNOÕNODUÕQ Trichoderma harzianum NRRL 13019 ve Trichoderma viride 155/¶GHQHOGHHGLOHQȕ-ksilosidaz enzim aktivitesi Üzerine etkisi..................................................................................... Tablo 3.3: .DWÕVXEVWUDWIHUPHQWDV\RQNOWURUWDPÕQGDIDUNOÕLQNEDV\RQ sürelerinin Trichoderma harzianum NRRL 13019 ve Trichoderma viride NRRL 64¶GHQHOGHHGLOHQȕ-ksilosidaz enzim aktivitesi üzerine etkisi ........................................................... Tablo 3.4: Trichoderma harzianum NRRL 13019’dan HOGHHGLOHQNÕVPL VDIODúWÕUÕOPÕúȕ-ksilosidaz enziminin amonyum sülfat çöktürme \|QWHPLVRQXQGDNLȕ-ksilosidaz enzimaktivitesi ve protein mLNWDUÕ ............................................................................................... Tablo 3.5: Trichoderma viride NRRL 6418’den eOGHHGLOHQNÕVPL VDIODúWÕUÕOPÕúȕ-ksilosidaz enziminin amonyum sülfat çöktürme yöntemi sonundaki ȕ-ksilosidaz enzimaktivitesi ve SURWHLQPLNWDUÕ ................................................................................... Tablo 3.6: Trichoderma harzianum NRRL 13019’ dan elde edilen ȕ-NVLORVLGD]HQ]LPLQLQVDIODúWÕUPDWDEORVX ....................................... Tablo 3.7: Trichoderma viride NRRL 64¶GHQHOGHHGLOHQȕ-ksilosidaz HQ]LPLQLQVDIODúWÕUPDWDEORVX............................................................ Tablo 3.8: Trichoderma harzianum NR5/ȕ-ksilosidaz enziminin kinetik parametreleri ......................................................................... Tablo 3.9: Trichoderma viride 155/ȕ-ksilosidaz enziminin kinetik parametreleri...................................................................................... Tablo 3.10: Trichoderma harzianum 155/¶GDQVDIODúWÕUÕODQ ȕ-ksilosidaz enziminin KM, Vmax ve Vmax/KM GH÷HUOHUL .................... Tablo 3.11: Trichoderma viride 155/¶GHQVDIODúWÕUÕODQȕ-ksilosidaz enziminin KM, Vmax ve Vmax/KM GH÷HUOHUL ........................................ Tablo 3.12: Trichoderma harzianum 155/¶GDQVDIODúWÕUÕODQ ȕ-ksilosidaz enzimi üzerinde inhibisyon etkisi gösteren D-(+)-ksilozun, IC50 GH÷HULQLQKHVDSODQPDVÕQGDNXOODQÕODQ veriler ................................................................................................ Tablo 3.13: Trichoderma viride NRRL 6418¶GHQVDIODúWÕUÕODQȕ-ksilosidaz enzimi üzerinde inhibisyon etkisi gösteren D-(+)-ksilozun, IC50 GH÷HULQLQKHVDSODQPDVÕQGDNXOODQÕODQYHULOHU .................................... viii. 28 29. 47. 48. 50. 51. 53 56 59 67 68 69 70. 71. 72.

(20) Tablo 3.14: Trichoderma harzianum NRRL 13019’dan VDIODúWÕUÕODQ ȕ-ksilosidaz enzimi üzerinde inhibisyon etkisi gösteren D-(+)-ksilozun Ki GH÷HULQLQKHVDSODQPDVÕQGDNXOODQÕODQYHULOHU ..... 75 Tablo 3.15: Trichoderma viride NRRL 6418’den VDIODúWÕUÕODQȕ-ksilosidaz enzimi üzerinde inhibisyon etkisi gösteren D-(+)-ksilozun Ki GH÷HULQLQKHVDSODQPDVÕQGDNXOODQÕODQYHULOHU ................................... 79. ix.

(21) 6(0%2//ø67(6ø Sembol. 6HPERO$oÕNODPDVÕYH\D$GÕ. g. Gram. mM. Milimolar. M. Molar. ȝ/. Mikrolitre (10-6 litre). ȝP. Mikrometre (10-6 metre). ȝJ. Mikrogram (10-6 gram). rpm. Rotary Per Minute. kDA. Kilodalton. KSF. .DWÕ6XEVWUDW Fermentasyonu. SKF. 6ÕYÕ.OWU Fermentasyonu. ȕ. Beta. °C. Santigrat Derece. pNPX. p-1LWURIHQLOȕ-D-ksilopiranosit. EU. Enzim Ünitesi. E.C. Enzyme Commission. SDS. Sodyum Dodesil Sülfat. PAGE. Poliakrilamid Jel Elektroforezi. TEMED. N,N,N’,N’, -tetrametil etilen diamin. APS. Amonyum Persülfat. BSA. %RYLQ6HUXP$OEXPLQ 6Õ÷ÕU6HUXP Albumini). [S]. Substrat Konsantrasyonu. [I]. øQKLELW|U Konsantrasyonu. KM. Michaelis-Menten Sabiti. Vmax. Maksimum +Õ]. IC50. øQKLEH(GLFi Konsantrasyonun Yüzdesi. pNPA. p-Nitrofenil Į-L-Arabinofuranosit. oNPX. o-NLWURIHQLOȕ-D-ksilopiranosit. x.

(22) ÖNSÖZ %DQD DUDúWÕUPD RODQD÷Õ VD÷OD\DQ WH] oDOÕúPDPÕQ KHU DúDPDVÕQÕ SODQOD\DUDN bana yol gösteren, engin bilgi ve tecrübelerini benden esirgemeyen, yönlendirme ve GHQH\LPOHULQL EHQLPOH SD\ODúDQ GHVWH÷LQL KHS KLVVHWWL÷LP oDOÕúPDPÕ ELOLPVHO WHPHOOHU ÕúÕ÷ÕQGD úHNLOOHQGLUHQ GDQÕúPDQ KRFDP 3URI'U $\úH 'LOHN $=$=¶D WHúHNNUlerimi bir borç bilirim. dDOÕúPDPÕQ KHU DúDPDVÕQGD \DUGÕPODUÕQÕ HVLUJHPH\HQ ELOJL ELUikimi ve GHVWH÷L\OH \DQÕPGD RODQ <UG'Ro'U 6HOPD d(/(1 <h&(7h5.¶H WHúHNNU ederim. 7H]LQ GHQH\VHO DúDPDODUÕQÕ JHUoHNOHúWLUGL÷LP ODERUDWXYDU RUWDPÕQGD HNLS ROPDQÕQ |QHPLQL ELUOLNWH KDUHNHW HWPHQLQ JFQ VUHNOL RODUDN YXUJXOD\DQ KRFDODUÕPDoDOÕúPDDUNDGDúODUÕP$VOÕ7$1*h1hYH<DSUDN$/3,1$5¶DWHúHNNU ederim. <R÷XQ oDOÕúPD VUHFLPGH EHQL KHU ]DPDQ GHVWHNOH\HQ DQOD\Õú J|VWHUHQ \DQÕPGD RODQ (úLP EL\RORJ <DVHPLQ ø57(0 YH R÷OXP *|NKDQ ø57(0¶H EHQL \HWLúWLUHQ YH EXJQOHUH JHOPHPL VD÷OD\DQ DQQH YH EDEDPD HQ LoWHQ GX\JXODUÕPOD WHúHNNUHGHULP. xi.

(23) 1. *ø5øù Enzimler, hayvansal ve bitkisel hücrelerde biyokimyasal tepkimeleri NDWDOL]OH\HQ SURWHLQ \DSÕVÕQGD RUJDQLN PROHNOOHUGLU +FUHGH |QHPOL PHWDEROLN J|UHYOHUH VDKLS RODQ HQ]LPOHU IDUNOÕ DPDoODUOD NXOODQÕOPDN ]HUH ekonomik ve JQONKD\DWDJLUPLúWLU>@ (Q]LP WHULPL LON NH] : .KQH WDUDIÕQGDQ IHUPHQW \HULQH NXOODQÕOPÕúWÕU .HOLPHQLQ RULMLQL <XQDQFD ROXS ³PD\D´ DQODPÕQÕ WDúÕPDNWDGÕU øQVDQODU HQ]LPDWLN UHDNVL\RQODUGDQ oRN HVNL WDULKOHUGHQ LWLEDUHQ \DUDUODQPÕúODUGÕU gUQH÷LQ úDUDS \R÷XUW HNPHN ER]D YH NÕPÕ] \DSPÕúODUGÕU 7P EX JÕGD PDGGHOHULQL UHWLUNHQ HQ]LPOHUGHQ HQ]LPOHULQ NDWDOLWLN HWNLOHULQGHQ QDVÕO ROGX÷XQX ELOPHGLNOHUL KDOGH \DUDUODQPÕúODUGÕU>@ øON GHID ¶WH 6SDOODQ]L DWPDFD PLGH |] VX\XQXQ HWL HULWWL÷LQL J|VWHUHUHN KFUH GÕúÕQGD GD HQ]LPOHULQ ELU DNWLYLWH\H VDKLS ROGX÷XQX NDQÕWODPÕúWÕU Kirchoff ¶GHEX÷GD\QLúDVWDVÕQÕQúHNHUHG|QúW÷QWHVSLWHWPLúWLU>5]. øON NH] \ÕOÕQGD 3HUVR] YH 3D\HQ DONRO NXOODQDUDN PDOW HNVWUHVLQGHQ QLúDVWD\Õ VLQGLUHQ HQ]LPL D\ÕUW HGHUHN EXQD ³'L\DVWD]´ LVPLQL YHUPLúOHUGLU >6]. \ÕOÕQGD 6FKZDQ PLGH VX\XQGDQ pHSVLQ HQ]LPLQL HOGH HWPLúWLU .ULVWDO KDOGHNL LON HQ]LP RODQ UHD] DQFDN ¶GD 6XPPHU WDUDIÕQGDQ L]ROH HGLOPLúWLU 7ULSVLQ pepsin ve kimotripsin enzimlerinin kristal halleri ise 1930- \ÕOODUÕ DUDVÕQGD 1RUWKURSWDUDIÕQGDQHOGHHGLOPLúWLU>@ (Q]LPOHUKD\YDQVDOELWNLVHOYHPLNURRUJDQL]PDND\QDNOÕRODELOPHNOHELUOLNWH endüstriyel X\JXODPDODUGD NXOODQÕODQODU genellikle mikroorganizmalardan elde HGLOPHNWHGLU %X GXUXPXQ EDúOÕFD VHEHSOHUL PLNURRUJDQL]PDODUGDQ HOGH HGLOHQ enzimlerin hayvansal veya bitkisel kaynaklardan elde edilen enzimlere oranla yüksek NDWDOLWLN DNWLYLWHOHULQLQ ROPDVÕ XFX] YH GDKD VWDELO ROPDODUÕ LVWHQPH\HQ \DQ UQ ROXúWXUPDPDODUÕ\NVHNVDIOÕNWDYHoRNPLNWDUGDUHWLOHELOPHOHULGLU>7]. (QGVWUL\HO DODQGD NXOODQÕP LoLQ PLNURRUJDQL]PDODU oRN X\JXQ FDQOÕODUGÕU 0LNURRUJDQL]PDODUÕQ oHúLWOLOL÷LQin NRUXQPDVÕ YH WHVSLW HGLOPHVL EL\RHQGVWULQLQ JHOLúLPLQH|QHPOLNDWNÕODUGDEXOXQPDNWDGÕU*HQHWLNRODUDNGH÷LúWLULOPLúoRN\|QO 1.

(24) \DSÕGDNL \NVHN E\PH KÕ]ÕQD VDKLS PLNURRUJDQL]PDODU \HQL HQGVWULOHUL\HO DODQODUÕQ ROXúWXUXOPDVÕQGD \NVHN NXOODQÕP SRWDQVL\HOL WDúÕPDNWDGÕUODU >8,9]. 0LNURRUJDQL]PDODUÕQJHQHWLNYHPHWDEROLNSRWDQVL\HOOHULQHLOLúNLQRODUDNHOGH edilen ELOJLOHU WÕEEL YH HQGVWUL\HO DoÕGDQ |QHPOL HQ]LPOHULQ UHWLOPHVL JÕGD YH EL\RLQVHNWLVLWOHULQUHWLPLYHVDNODQPDODUÕoHYUHNLUOLOL÷LQLQHQJHOOHQPHVLNLP\DVDO \ROODUOD UHWLPL PPNQ ROPD\DQ VWHURLG KRUPRQODUÕ YH ED]Õ DQtibiyotikleri içine alan farmasötiklerin üretilmesi gibi pek çok sahada biyoteknolojik proseslerin NXOODQÕPÕQÕYHJHOLúWLULOPHVLQLDUWÕUPÕúWÕU>9]. 7LFDUL DPDoOD NXOODQÕODQ YH UHWLPL \DSÕODQ HQ]LPOHULQ E\N NÕVPÕQÕ mikroorganizmalar üretmekte ve endüstrL\HO DODQGD HQ]LPOHULQ NXOODQÕOPDVÕ GQ\D oDSÕQGD KÕ]OÕ ELU úHNLOGH DUWPDNWDGÕU 0LNURRUJDQL]PDODUGDQ HOGH HGLOHQ HQ]LPOHULQ EL\RWHNQRORMLGHQ \DUDUODQÕODUDN UHWLOPHVL RQODUÕQ oHúLWOL PDWULNVOHUH ED÷ODQDUDN NDUDUOÕ KDOH JHOPHOHUL IDUNOÕ endüstrilerde kullDQÕOPDODUÕQGDNL DUWÕúÕQ ana sebeplerindendir [9]. Enzimler, hücrede bütün metabolik tepkimeleri idare eden ve son derece özel pURWHLQ\DSÕGDNLNDWDOL]|UOHUGLU2UJDQLNNLP\DDODQÕQGDNXOODQÕODQ\|QWHPOHULOHoRN VD\ÕGD NLP\DVDO UHDNVL\RQXQ GR÷UX HQ]LP YDUOÕ÷ÕQGD |]Jül. ve. basitçe. JHUoHNOHúHELOPHVL FDQOÕ KFUHGHQ HQ]LPLQ L]RODV\RQX YH FDQOÕ GÕúÕQGD IDUNOÕ KHGHIOHUH\|QHOLNNXOODQÕPÕQDLOLúNLQoDOÕúPDODUÕQ|QHPLQLDUWÕUPÕúWÕU>]. Enzimatik proseslerin oHYUH NLUOLOL÷LQH GDKD D] QHGHQ ROPDVÕ NLP\DVDO proseslHUGHQ GDKD X\JXQ úDUWODUGD YH GDKD D] PDOL\HWOH JHUoHNOHúPHVLQGHQ GROD\Õ GHUL WHNVWLO YH GHWHUMDQ HQGVWULVL LOH DWÕNODUÕQ JLGHULOPHVL VUHFLQGHNL NXOODQÕPODUÕ E\N|OoGHDUWPÕúWÕU>]. 6RQ \ÕOODUGD HQGVWUL\HO DODQGD NXOODQÕODQ HQ]LPOHULQ \DNODúÕN ¶Õ PLNURRUJDQL]PDODUGDQ IHUPHQWDV\RQ \|QWHPL NXOODQÕODUDN UHWLOPHNWHGLU >10]. 'Q\DJHQHOLQHEDNÕOGÕ÷ÕQGDHQGVWUL\HOHQ]LPOHULQWLFDULSD]DUSD\ÕWDKPLQLRODUDN PLO\DUGRODUGÕU%XHQ]LPOHULQNXOODQÕOGÕ÷ÕDODQODUJHQHODPDoOÕWHNQLNDODQODU JÕGa ve %15 hayvan yemi endüstrisidir [11,12]. EQ]LPOHU ELUoRN HQGVWUL\HO X\JXODPDGD \D\JÕQ RODUDN NXOODQÕOPDNWDGÕU 'DKD NDUDUOÕ \NVHN DNWLYLWHOL YH VSHVLILN HQ]LPOHU LoLQ WDOHS KÕ]OD DUWPDNWDGÕU 'Q\DJHQHOLQGHHQGVWUL\HODODQGDNXOODQÕODQHQ]LPOHULQ Õ$YUXSD GDLVH Japonya ve ABD'de üretilmektedir [13]. 2.

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(37) . ȕ-ksilosidaz. (EC. 3.2.1.37),. a-L-. arabinofuranozidaz (EC 3.2.1.55), a-Glukuronidaz (EC 3.2.1.139), Asetil Ksilan Esteraz (EC 3.1.1.72) ve Ferulik asit (Ferulik ve p-Kumarik asit) esteraz (EC

(38) \HUDOPDNWDGÕU .VLODQD]ODUGR÷DGD\D\JÕQRODUDNEXOXQDQELUHQ]LPJUXEXGXU.VLODQGDNLȕ1,4-glikR]LGLN ED÷ÕQÕ ]LQFLUGH Lo E|OPGHQ NÕUDQ JOLNRVLGD]ODUGÕU R-glikozid hLGUROD]ODU (&

(39) +FUH\H JHUHNOL NDUERQX VD÷ODU YH bitki hücrelerinin SDWRMHQOHUWDUDIÕQGDQHQIHNWHHGLOPHVLGXUXPXQGD|QHPOLURODOÕU>15]. Endo-1,4-ȕ-ksilanaz. ((&.

(40) . NVLODQ. LVNHOHWLQL. JHOLúL. J]HO. KLGUROL]OH\HUHNNÕVDROLJRVDNNDULWOHULQROXúXPXQXVD÷ODPDNWDGÕU ȕ-ksilosidaz (EC 3.2.1.37); (ksilobiD]ȕ-ksilosidaz; ekzo-1,4-ȕ-NVLORVLGD]ȕD-ksilopiranosidaz; ekzo-1,4-ksilosidaz; ekzo-1,4-ȕ-D-ksilosidaz; 1,4-ȕ-D-ksilan NVLORKLGUROD]

(41) NÕVDROLJRVDNNDULWOHULQYHNVLOREL\R]XQLQGLUJHQROPD\DQXoODUÕQDHWNL HGHUHNNVLOR]ROXúWXUPDNWDGÕU a-L-Arabinofuranozidaz (EC 3.2.1.55), a-glukuronidaz (EC 3.2.1.139), asetil ksilan esterazlar (EC 3.1.1.72), Ferulik asit esteraz (EC 3.1.1.73) ve p-Kumarik asit esteraz (EC 3.1.1.-

(42) WDUDIÕQGDQ WHPHO ]LQFLUH ED÷OÕ \DQ JUXSODUÕQ KLGUROL]LQGH etkilidirler. ȕ-ksilosidaz LQGLUJHQROPD\DQXoNÕVÕPGDQ'-NVLOR]ODUÕX]DNODúWÕUDUDN-ȕD-NVLORROLJRVDNNDULWOHULQ KLGUROL]LQL NDWDOL]OHPHNWHGLU .VLODQ KLGUROL]L HVQDVÕQGD NVLOR]XNRSDUDQHQGRNVLODQD]ODUÕQȕ-NVLORVLGD]WDUDIÕQGDQNROD\ELUúHNLOGHKLGUROL]H edilen ksilobioz aktivitesi EXOXQPDPDNWDGÕUD-Arabinofuranozidazlar (EC 3.2.1.55), DUDELQRJDODNWDQODUÕQ DUDELQDQODUÕQ YH DUDELQRNVLODQODUÕQ LQGLUJHQPH\HQ D-LDUDELQRIXUDQRVLOXoNÕVPÕQÕKLGUROL]HHWPHNWHGLUOHU Į-D-glukuronidazlar (EC 3.2.1.1) ksiloz ve D-glukuronik asit ya da 4-0PHWLOHWHU DUDVÕQGD \HU DODQ Į-1,2-JOLNR]LGLN ED÷ODUÕQ KLGUROL]H HGLOPHVLQGH URO. 7.

(43) R\QDUODU .VLODQÕQ HQ]LPOHUOH KLGUROL]H HGLOPHVLQGH Į- ED÷ODUÕ KLGUROL] KÕ]ÕQÕ GúUHQNÕVÕPODUGÕU 'R÷DOJOXNXURQLGD]ODUÕQKLGUROL]DV\RQLúOHPLVÕUDVÕQGDHWNLOLRODELOPHOHULLoLQ HVWHUD]ODU JHUHNOLGLU %XQODU NVLODQGDNL IHQROLN DVLW YH DVHWLN DVLWOHUL NRSDUÕUODU Feruloyl, Asetil, ve p-NXPRU\OOHULQ NVLODQ LVNHOHWLQGHQ X]DNODúWÕUÕOPDVÕ OLJQLQ NRSDUÕOPDVÕQGD |QHPOL ELU EDVDPD÷Õ ROXúWXUDUDN OLJQLQ YH KHPLVHOOR] DUDVÕQGD \HU DODQHVWHUED÷ODUÕQÕQNRSDUÕOPDVÕ\ODOLJQLQLQGHJUHGDV\RQXQDNDWNÕGDEXOXQPDNWDGÕU [25-27].. ùHNLO1.1: .VLODQÕQ7DP+LGUROL]LQGH*HUHNOL(Q]LPOHUve Etki Bölgeleri [21].. .VLODQ KLGUROL]DV\RQXQGD J|UHY DODQ HQ]LPOHUGH VLQHUMLN HWNLOHúLPGHQ EDKVHGLOHELOLU+LGUROL]DV\RQHVQDVÕQGDDNWLIROGX÷XJ|]OHQPHNWHRODQHQ]LPOHUȕ-D-NVLODQ LVNHOHWLQGH HWNLOL ROXUODU YH \DQ ]LQFLUOHUL NRSDUWÕUODU (QGRNVLODQaz ve DVHWLONVLODQHVWHUD]DUDVÕQGDNLVLQHUMLDVHWLONVLODQODUÕQGHJUHGDV\RQXQGDHWNLOLGLU $VHWLNDVLWLQDVHWLONVLODQHVWHUD]HQ]LPLWDUDIÕQGDQX]DNODúWÕUÕOPDVÕNVLODQÕQ LVNHOHWLQL HQGRNVLODQD]ÕQ HWNL HGHELOPHVLQH KD]ÕU KDOH JHWLUPHNWHGLU %XQXQ sonucXQGD KLGUROL] ROGXNoD KÕ]OÕ ELU úHNLOGH JHUoHNOHúPHNWHGLU ȕ-ksilosidazlar, HQGRNVLODQD] LQKLELV\RQXQX VD÷OD\DFDN VRQ UQOHUL SDUoDODU YH NVLODQÕQ KLGUROL]DV\RQXQXQ VHYL\HVLQL DUWÕUÕU %HQ]HU RODUDN Į-DUDELQRIXUDQR]LGD]ODUÕQ endoksilanazlara ilavesi, araELQRNVLODQODUGDúHNHUOHúWLULOPHGXUXPXQXDUWÕUÕU. 8.

(44) 1.2.3 .VLODQD].D\QDNODUÕ (QGRNVLODQD]ODUÕQ JHQHOOLNOH fungus YH EDNWHULOHU WDUDIÕQGDQ UHWLOGLNOHUL VDSWDQPÕúWÕU $\UÕFD ELWNLOHUGHQ HOGH HGLOHQ HQGRNVLODQD]ODU GD EXOXQPDNWDGÕU %X enzimlerin meyvelerde olgunlDúPDG|QHPLQLQELWLPLQGHUHWLOGL÷LEHOLUOHQPLúWLU .VLODQD]ODU oR÷XQOXNOD NVLODQ \D GD NVLODQÕQ EROFD EXOXQGX÷X RUWDPODUGD JHOLúHELOHQ. PLNURRUJDQL]PDODUGD. EXOXQDQ. HQ]LPOHUGLU. .VLODQD]ODU. ED]Õ. ksilooligosakkaritler, L-sorboz, lignoselüloz ve ksiloz biriPOHUL JLEL ELOHúLNOHU WDUDIÕQGDQ LQGNOHQdikleri; bakteriler, mayalar, protozoalar, funguslar, algler, DUWURSRGODUYHNDUÕQGDQEDFDNOÕODUÕQGDLoLQGHEXOXQGX÷XRUJDQL]PDJUXEXWDUDIÕQGDQ UHWLOHELOGLNOHULUDSRUHGLOPLúWLU>21,28].. 1.2.4 .VLODQD]ODUÕQdHúLWOLOL÷LYH6ÕQÕIODQGÕUPDVÕ .VLODQD]ODU ELU RUJDQL]PDGDQ GL÷HULQH IDUNOÕOÕN J|VWHUHELOGL÷L JLEL D\QÕ WUH ait mikroorganizmalarda da ELUGHQ oRN NVLODQD] HQ]LPLQLQ ROGX÷X GD VDSWDQPÕúWÕU Örnek olarak; Aspergillus niger’de oHúLW HNVWUDVHOOHU NVLODQD] YH Trichoderma viride’de WLS NVLODQD] L]RHQ]LPL WHVSLW HGLOPLúWLU .VLODQD]ODUÕQ oHúLWOLOL÷L \DSÕODUÕQGDEXOXQDQVHOOR]ED÷ODPDWHUPDOVWDELOLWHNVLODQED÷ODPDYHIRQNVL\RQX KHQ]EHOLUOHQHPHPLúIDUNOÕE|OJHOHULQYDUOÕ÷Õ\ODDoÕNODQPDNWDGÕU [15]. Ksilanazlar hDNNÕQGD\DSÕODQLONVÕQÕIODQGÕUPDoDOÕúPDODUÕQGDIL]LNRNLP\DVDO |]HOOLNOHUL HVDV DOÕQDUDN LNL IDUNOÕ JUXS |QHULOPLúWLU %X JUXSODU GúN PROHNO D÷ÕUOÕ÷ÕQD VDKLS N'D

(45) -240 amino asit birimi olan ksilanazlar ve yüksek PROHNOD÷ÕUOÕ÷ÕQDVDKLS > 30kDa ), 270-DPLQRDVLWELULPLQLRODQNVLODQD]ODUGÕU [22]. <DSÕODQ VÕQÕIODQGÕUPD oDOÕúPDVÕQGD IXQJDO NVLODQD]ODUÕQ ¶X EX LNL JUXS LoHULVLQGH VÕQÕIODQGÕUÕODPDPÕú eQ]LP DLOHOHUL ROXúWXUXOXUNHQ HQ]LPGH EXOXQDQ NDWDOLWLNDODQODUÕQSULPHU\DSÕODUÕNDUúÕODúWÕUÕOPÕúWÕU2OXúWXUXODQVLVWHPGHNVLODQD]ODU IDPLO\D IDPLO\D )

(46) YH IDPLO\D *

(47) GH VÕQÕUODQPÕúWÕU )DNDW OLWHUDWUOHU LQFHOHQGL÷LQGH IDPLO\D YH ¶WH GH HQGR-1,4-ȕ-ksilanaz aktivitesine sahip oHúLWOL NDWDOLWLN DODQODUÕQ EXOXQGX÷X J|Uülmektedir. 5, 7, 8, 10, 11 ve 43 IDPLO\DODUÕQGDEXOXQDQNVLODQD]\DSÕODUÕQÕQIL]LNRNLP\DVDO|]HOOLNOHULQLQHWNLHWPH 9.

(48) WDU]ODUÕQÕQ YH VXEVWUDW VSHVLILNOLNOHULQLQ ELUELULQGHQ IDUNOÕ ROGX÷X EHOLUOHQPLúWLU [15,28].. 1.2.5 .VLODQD]ODUÕQ*HQHOg]HOOLNOHUL Ksilanazlarda, hidrolitik aktiviteden sorumlu katalitik bölümlerle, substrat ED÷ODPD YH WHUPDO VWDELOLWHGHQ VRUXPOX NDWDOLWLN HWNLVL EXOXQPD\DQ E|OPOHU PHYFXWWXU *HQHOOLNOH IXQJDO ND\QDNOÕ RODQODUÕQ S+ - EDNWHUL ND\QDNOÕ RODQODUÕQ ise pH 5- DUDOÕ÷ÕQGD HWNLOL ROGXNODUÕ ND\QDNODUÕ IDNOÕ RODQ HQGRNVLODQD]ODUÕQ L]RHOHNWULNQRNWDODUÕQÕQS,-DUDOÕ÷ÕQGDGH÷LúLNOLNJ|VWHUGL÷LEHOLUOHQPLúWLU)XQJDl NVLODQD]ODU JHQHOOLNH EDNWHUL ND\QDNOÕ ksilanazlardan daha GúN ÕVÕO GLUHQFLQH sahiptir. .VLODQD]ODUÕQ RSWLPXP VÕFDNOÕN DUDOÕ÷Õ -& JLEL ROGXNoD JHQLú DUDOÕNWD GH÷LúLU 2SWLPXP VÕFDNOÕN GH÷HUL -& DUDOÕ÷ÕQGD ED]Õ NVLODQD]ODU GD EXOXQPDNWDGÕU. .VLODQD]ODUÕQ. GúN. DPRQ\XP. VOIDW. NRQVDQWUDV\RQXQGD. o|]QUONOHUL VÕFDNOÕNOD ELUOLNWH DUWÕú J|VWHUPHNWHGLU )DUNOÕ ND\naklardan elde HGLOHQNVLODQD]ELOHúLPOHULQGHD÷ÕUOÕNOÕRODUDN JOXWDPLNYHDVSDUWLNDVLWVHULQJOLVLQ YHWUHRQLQEXOXQPDNWDGÕUȕ-ksilosidaz ve ksilanaz aktiviteleri, ksilooligosakkaritlerin ]LQFLU X]XQOX÷XQD ED÷OÕGÕU .VLODQD] DNWLYLWHVL ]LQFLU X]XQOXNODUÕQÕQ D]DOPDVÕ\OD JHQHOOLNOHD]DOPDNWDLNHQȕ-NVLORVLGD]DNWLYLWHVLQLQDUWÕúJ|VWHUGL÷L\NVHNGDOODQPD GHUHFHVLQH VDKLS NVLOR]XQ SROLVDNNDULWOHULQ GH NVLODQD]ÕQ DNWLYLWHVLQL JHQHOOLNOH HQJHOOHGL÷LYHNVLODQD]ODUÕQoHúLWOLOL÷LQLQLQFHOHQGL÷LDUDúWÕUPDODUGDS+GH÷HUOHULYH PROHNOOHULQD÷ÕUOÕNODUÕDUDVÕQGDEHOLUOLELULOLúNLQLQROPDVÕGLNNDWHGH÷HUELUGXUXP ROPXú PROHNO D÷ÕUOÕNODUÕ NoN NVLODQD]ODUÕQ ED]LN PROHNO D÷ÕUOÕNOÕODUÕ E\N RODQODUÕQLVHDVLGLNROGX÷XEHOLUOHQPLúWLU>21,22,28].. 10.

(49) 1.2.6 KsiODQD]ODUÕQ%L\RWHNQRORMLYH(QGVWUL\HO8\JXODPD$ODQODUÕ. 1.2.6.1 .VLODQD]ODUÕQ%L\RWHNQRORMLdeki 8\JXODPD$ODQODUÕ 0LNURRUJDQL]PD ND\QDNOÕ NVLODQROLWLN HQ]LPOHU oRN VD\ÕGD HQGVWUL\HO LúOHPGH EL\RWHNQRORMLN SRWDQVL\HOH VDKLS ROPDODUÕQGDQ GROD\Õ LOJL RGD÷Õ KDOLQH JHOPLúWLU Bakteri ve mayalardan daha güçlü üreticiler olarak kabul edildiklerinden ö]HOOLNOHILODPHQWOLIXQJXVODUHOOL\ÕOGDQGDKDX]XQELUVUHGHQEHULHQGVWUL\HOHQ]LP UHWLPLQGH NXOODQÕOPDNWDGÕUlar [21,29@ 6RQ \ÕOODUGD NVLODQ YH NVLODQD]ODUÕQ biyoteNQRORMLGH NXOODQÕPÕ GLNNDWH GH÷HU ELU RUDQGD DUWÕú J|VWHUPLúWLU >13,21,30]. (QGVWUL\HO úLUNHWOHU KHPLVHOOo] LoHUHQ PDGGHOHUH \DSÕODQ LúOHPOHUGH DVLW KLGUROL]L \HULQH HWNLOL HQ]LPDWLN VUHoOHULQ JHOLúWLULOPHVLQH |QHP YHUPHNWHGLUOHU 7LFDUL ksilanazlar FinODQGL\D-DSRQ\DøUODQGD&XPKXUL\HWL$Omanya, Danimarka, Kanada ve ABD’de endüstriyel olarak üretilmektedir. Bu enzimlerin elde edilmesinde NXOODQÕODQ EDúOÕFD PLNURRUJDQL]PDODU Aspergillus niger, Trichoderma sp. ve Humicola insolens¶GLU $\QÕ ]DPDQGD WLFari ksilanazlar bakterilerden de elde HGLOPHNWHGLU .VLODQD]ODU VHOOD] YH SHNWLQD]ODUOD ELUOLNWH GQ\D HQ]LP SD]DUÕQÕQ ¶VLQL ROXúWXUPDNWDGÕUODU >31@ g]HOOLNOH ND÷ÕW KDPXUX YH ND÷ÕW ROPDN ]HUH KD\YDQ \HPL YH JÕGD JLEL IDUNOÕ HQGVWUL\HO DODQODUGD WHPel enzim olma yolunda NVLODQD]ODUÕQ|QHPLDUWPÕúWÕU [21,30].. 1.2.6.2 .VLODQD]ODUÕQ(QGVWUL\HO8\JXODPD$ODQODUÕ .D÷ÕWKDPXUXYHND÷ÕWHQGVWULVL dHYUHVHOG]HQOHPHOHUND÷ÕWKDPXUXYH ND÷ÕW HQGVWULVLQGHki D÷DUWPD LúOHPL VÕUDVÕQGD NORUun NXOODQÕPÕQÕ VÕQÕUODQGÕUPÕúWÕU [32,33@ *QP]GH GR÷D\Õ HQGVWUL\HO DWÕNODUÕQ ]DUDUOÕ HWNLOHULQGHQ NRUXPDN DPDFÕ\OD ND÷ÕW KDPXUX YH ND÷ÕW HQGVWULVLQGH PLNUREL\DO HQ]LPOHULQ NXOODQÕOPDVÕ E\N |QHP ND]DQPÕúWÕU >4@ %XQGDQ GROD\Õ oHYUH NLUOLOL÷LQLQ D]DOWÕOPDVÕ \ROODUÕQGDQ ELULVL GH ND÷ÕW KDPXUXQXQ NVLODQD] HQ]LPL NXOODQÕODUDN |Q LúOHPGHQ JHoLULOPHVLGLU %X LúOHP D÷DUWPDGD NXOODQÕODQ NLP\DVDOODUÕQ |]HOOLNOH GH NORU ELOHúLNOHULQLQ E\N RUDQGD LQGLUJHQPHVLQH YH NLUOLOL÷LQD]DOWÕOPDVÕQÕVD÷ODPDNWDGÕU [35@ øON olarak \ÕOÕQGD ND÷ÕW KDPXUXQX D÷DUWPD LúOHPLQGH HQGRNVLODQD]ODUÕQ 11.

(50) NXOODQÕOPDVÕQÕQ NLP\DVDOODUÕ LQGLUJHGL÷L ELOGLULOPLúWLU >36@ dRN VD\ÕGDNL DUDúWÕUPD EXQXGR÷UXODPÕúYHEXWHNQRORML\LWLFDULNRQXPDJHWLUPLúWLU>7]. (Q]LPOH \DSÕODQ X\JXODPDODUVRQXFXQGD Nk÷ÕW KDPXUXILEUilasyonu ve suyu WXWPDNDSDVLWHVLDUWDULúOHPJ|UPHPLúNk÷ÕWKDPXUXQXQoÕUSPDG|YPHVD\ÕVÕD]DOÕU DWÕN Nk÷ÕW KDPXUXQXQ ED÷ODUÕ RQDUÕOÕU o|]QHQ ND÷ÕW KDPXUXQGDQ NVLODQÕQ X]DNODúWÕUÕOPDVÕ VD÷ODQÕU $\UÕFD NVLODQD]ODUÕQ YDUOÕ÷ÕQGD RGXQ KDPXUXQGD biobeyazlDWPDVHQWHWLNLSHNUHWLPLKDPXUGDQVHOOR]HOGHVLJHUoHNOHúWLWLOPHNWHGLU [21,35-38]. .k÷ÕWKDPXUXQXQD÷DUWÕOPDVÕQGDNXOODQÕODQEL\RORMLN\|QWHPOHUGHNXOODQÕODQ NVLODQD] NVLODQÕQ ¶VLQL VHoLFL RODUDN X]DNODúWÕUDUDN NORUlu. D÷DUWPD. NLP\DVDOODUÕQÕQNXOODQÕPÕQÕRUDQÕQGDD]DOWPDNWDGÕU>9]. .VLODQD]ODU Nk÷ÕW KDPXUX YH Nk÷ÕW HQGVWULVLQGH \R÷XQ RODUDN NXOODQÕOPDNOD EHUDEHU OLJQRVHOOR]LN PDWHU\DOOHULQ G|QúPQGH WDUÕPVDO DWÕN UQOHULQ IHUPDQWDWLI UQOHUH SDUoDODQPDVÕQGD PH\YH VX\XQXQ EHUUDNODúWÕUÕOPDVÕQGD ELUDGDNL NÕYDPÕQ ROJXQODúPDVÕQGD KD\YDQ EHVLQOHULQLQ VLQGLULPLQLQ DUWÕUÕOPDVÕQGD GD E\N |QHP WDúÕPDNWDGÕU >40,41]. Ksilanazlar ekonomik olarak önemli birçok \DUDUOÕUQQLVWHQLOHQVHYL\HGHUHWLPLQGH|QHPOLELUSRWDQVL\HOHVDKLSWLU>21]. *ÕGa endüstrisi: .VLODQD]ODUÕQ JÕGD HQGVWULVLQGH GH ROGXNoD JHQLú ELU NXOODQÕP DODQÕ EXOXQPDNWDGÕU )ÕUÕQFÕOÕNWD ¶OHUGHQ LWLEDUHQ NXOODQÕOPD\D EDúODQDQ NVLODQD]ODUÕQ HNPHN YH KDPXU NDOLWHVLQLQ DUWÕUÕOPDVÕQGD KDPXUXQ \DSÕúNDQOÕ÷ÕQÕQ D]DOPDVÕQGD UDI |PUQQ X]DPDVÕQGD ROGXNoD HWNLOL ROGX÷X ELOLQPHNWHGLUg]HOOLNOHKDPXUXQ LúOHQHELOLUOL÷LQL YH UQ KDFPLQLJHOLúWLUHUHNQLKDL UQ NDOLWHVLQL HWNLOHPHNWHGLU %X÷GD\ XQXQGDNL NVLODQD] LoLQ VXEVWUDW RODQ DUDELQRNVLODQ KDNNÕQGD oRN VD\ÕGD oDOÕúPD\D \DSÕOPÕúWÕU >39,42,43@ .VLODQD]ÕQ HNPHN NDOLWHVLQL DUWÕUPDGDNL URO HNPHN KDFPLQLQ DUWÕúÕ YH GDKD \XPXúDN HNPHN eldesi LOH NHQGLQL DoÕNoD J|VWHUPHNWHGLU %X LúOHP NVLODQD]OD ELUOLNWH DPLOD]ÕQGD NXOODQÕPÕVD\HVLQGHGDKDGDDUWÕUÕOPDNWDGÕU>44@0H\YHVX\XYHúDUDSHQGstrisi de HQ]LPSD]DUÕQÕQROGXNoD|QHPOLELUE|OPQROXúWXUPDNWDGÕU>31]. .VLODQD]ODU JÕGD HQGVWULVLQGH VHE]H YH PH\YHOHULQ VÕYÕODúWÕUÕOPDVÕQGD YH PH\YHVXODUÕQÕQEHUUDNODúWÕUÕOPDVÕQGDVHOOD]YHSHNWLQD]ODEHUDEHUNXOODQÕOPDNWDGÕU Į-L-ArabinofuranosiGD] YH ȕ-D-JOXNRSLUDQR]LGD]ODU úDUDS PH\YH VXODUÕ YH DURPDWLN|]WOHULQLúOHQPHVLQGHNXOODQÕOPDNWDGÕU>20,21,45@%LUDUHWLPLQGHDUSDQÕQ 12.

(51) \DSÕVÕQGDEXOXQDQX]XQ]LQFLUOLDUDELQRNVLODQODUÕQSDUoDODQPDVÕQDRODQDNVD÷OD\DUDN DUSDQÕQKLGUROL]HROPDVÕQÕNROD\ODúWÕUÕUODU>20]. Hayvan yemi endüstrisi: .VLODQD]ODUÕQ GL÷HU ELU NXOODQÕP DODQÕ \HP HQGVWULVLGLUg]HOOLNOHSLOLoOHULQoDYGDULoHUHQ\HPOHULQHNVLODQD]ÕQLODYHHGLOPHVL\OH LQWHVWLQDO YLVNRVLWH LQGLUJHQLU YH EX VD\HGH \HPOHULQ HWNLQOL÷LQL DUWÕUarak piliçlerin D÷ÕUOÕN ND]DQFÕQa etkili olur [20,41]. Ksilanazlar hayvan beslenmesi süresince glukonaz, pektinaz, selülaz, proteaz, amilaz, fitaz, galaktosidaz ve lipaz ile birlikte NXOODQÕOPDNWDGÕU>6]. .VLODQWDUÕPYHJÕGDHQGVWULVLQHDLWDWÕNODUGDoRNID]ODPLNWDUGDEXOXQPDVÕ VHEHEL\OH NVLODQD] DWÕN VXGDNL NVLODQÕQ NVLOR]D G|QúWUOPHVL LoLQ GH NXOODQÕOPDNWDGÕU>47@%LWNLKFUHOHULQGHNVLODQD]X\JXODPDODUÕ\ODILWRVWHUROOHULQ\D÷ açilasyonu ve glikozilasyonu indüklenmektedir [48]. Tekstil endüstrisi: Ksilanazlar kendir ya da keten gibi bitkisel liflerin LúOHQPHVL SURVHVLQGH WHNVWLO HQGVWULVLQGH NXOODQÕOPDNWDGÕU %XQXQ LoLQ NVLODQD]ÕQ VHOOROLWLNHQ]LPOHUGHQDUÕQGÕUÕOPÕúROPDVÕJHUHNPHNWHGLU>31]. Ksilooligosakkaritlerin UHWLPLQGH. VDKLS. .VLORROLJRVDNNDULWOHU. üretimi:. .VLODQD]ODUÕQ NVLORROLJRVDNNDritlerin. ROGXNODUÕ. SRWDQVL\HO. sayesinde. NVLOR]. QLWHOHULQGHQ. ROXúDQ. önemleri úHNHU. DUWPDNWDGÕU. ROLJRPHUOHULQGHQ. ROXúPDNWDGÕUODU YH HQGVWUL\HO RODUDN OLJQRVHOOR]LN ELyokütleden üretilmektedirler [49].. KsilooligosakkDULWOHU NROHVWHURO YH NRORQ NDQVHUL ULVNLQL D]DOWPDVÕ. JDVWURLQWHVWLQDO VLVWHPL NRUXPDVÕ YH WLS-II diyabet üzerinde olumlu etkiye sahip ROPDVÕQHGHQL\OHVD÷OÕN]HULQGHROXPOXHWNLOHUHVDKLS\DUDUOÕUQOHUGHQGLU>50,51]. .VLODQD]ODU NVLODQÕ KLGUROL] HWPH \HWHQHNOHULQGHQ GROD\Õ OLJQRVHOOR]LN DWÕNODUGDQ ksilooligosakkaritler üretiminde önemli role sahiptirler [49]. 'L÷HU WDUDIWDQ NVLODQD]ODU NVLOR] NVLOR-ROLJRPHUOHU YH \DSD\ ELU WDWODQGÕUÕFÕ RODQNVLOLWROUHWLPLQGHGHNXOODQÕOPDNWDGÕU.VLORROLJRVDNNDUitler prebiyotik özellik J|VWHUPHNWH ROXS JÕGD \HP LODo JLEL HQGVWULOHUGH NXOODQÕP DODQÕ EXOPDNWDGÕUODU 6RQ\ÕOODUGDIRQNVL\RQHONVLORROLJRPHUOHULQUHWLPLQHLOJLE\NWU%DúNDELUEDNÕú DoÕVÕ\OD GD NVLODQD]ODU OLJQRVHlOR]LN DWÕNODUÕQ GH÷HUOHQGLULOLS NDWPD GH÷HUL \NVHN UQOHUHG|QúWUOPHVLDoÕVÕQGDQGDE\N|QHPWDúÕPDNWDGÕUODUgUQH÷LQEX÷GD\ NHSH÷LSLULQoNHSH÷LPÕVÕUNRoDQÕúHNHUSDQFDUÕNVSHVLJLELHQGVWUL\HODWÕNODUYH \DQUQOHUNVLODQD]UHWLPLQGHNXOODQÕOPDNWDGÕU>20]. 13.

(52) Pektinolitik enzim sistemlerinin ksilanolitik enzim sistemleri ile birlikte X\JXODQGÕ÷Õ EDúOÕFD DODQODU DUDVÕQGD NHQHYLU YH MW ELWNLOHULQLQ UHoLQHGHQ DUÕQGÕUÕOPDVÕ EXOXQPDNWDGÕU 3HNWLQD]-NVLODQD] HQ]LP VLVWHP ELUOLNWHOL÷L RGXQXQ LúOHQPHVLQGHLONDúDPDRODQNDEXNWDQDUÕQGÕUPDGDGDNXOODQÕOPDNWDGÕU.VLODQD]ODUÕQ ED]ÕODUÕELWNLKFUHOHULQGHQSURWRSODVWUHWLPLQGHKFUHGXYDUÕQÕQ\XPXúDWÕOPDVÕQGD GDNXOODQÕOPDNWDGÕU>15,21]. Biyo-etanol ve Biyo-\DNÕWUHWLPL Ksilanazlar, ligninaz, mannaz, glukanaz, ksilosidaz,. glikoVLGD] JLEL GL÷HU IDUNOÕ HQ]LPOHUOH EHUDEHU OLJQRVHOOR]LN. PDWHU\DOOHUGHQ HWDQRO UHWLPLQGH NXOODQÕOPDNWDGÕU (WDQROQ UHWLOPHVLQGH NXOODQÕODFDNVHUEHVWúHNHUOHULQHOGHHGLOPHVLLoLQNDUERQKLGUDWSROLPHU]LQFLUOHULQLQ GHSROLPHUL]DV\RQXQXQ DUGÕQGDQ KHPLVHOOR] VHOOR] YH OLJQLQLQ D\UÕúWÕUÕOPDVÕ\OD GHOLJQLILNDV\RQJHUoHNOHúWLULOPHNWHGLU>21,33,51,52]. Sonuç olarak; etanol üretiminde KHNVR] YH SHQWR]GDQ PH\GDQD JHOHQ NDUÕúÕP IHUPHQWDV\RQ LoLQ NXOODQÕOPDNWDGÕU Lignoselülozik materyaller ikinci nesil biyo-yakÕWUHWLPGHLONNH]NXOODQÕOPÕúODUGÕU [33]. øODo YH NLP\DVDO X\JXODPDODU .VLODQD] YH NVLODQÕQ NoN ELU NÕVPÕ LODo HQGVWULVLQGHNXOODQÕOPDNWDGÕU.VLODQD]ODUED]HQELUEHVLQWDNYL\HVLRODUDNVLQGLULPL G]HQOHPHN DPDFÕ\OD KHPLVHOOD] SURWHD] YH GL÷HUOHUL\OH EHUDEHU NXOODQÕOPDNWDGÕU [31].. 1.3. Ksilanaz Üretimi. %L\RWHNQRORMLGHNL JHOLúPHOHUH SDUDOHO RODUDN HQGVWUL\HO HQ]LP NXOODQÕPÕ GD JLGHUHN DUWPDNWDGÕU (Q]LP UHWLPOHULQLQ E\N NÕVPÕ VÕYÕ NOWU IHUPHQWDV\RQ \|QWHPL\OH JHUoHNOHúWLULOLUNHQ JQP]GH NDWÕ NOtür fermentasyon yönteminin NXOODQÕPÕQGD GD ND\GD GH÷HU ELU DUWÕú V|] NRQXVXGXU %XQXQ EDúOÕFD QHGHQOHUL DUDVÕQGD\NVHNYHULPGúNPDOL\HWEDVLWDOHWYHHNLSPDQNXOODQÕPÕVDIODúWÕUPDGD NROD\OÕN\HUDOPDNWDGÕU $\UÕFD WDUÕPVDO PDWHU\DOOHULQ YH HQGVWUL\HO DWÕNODUÕQ NDWÕ RUWDP RODUDN NXOODQÕOPDVÕQD RODQDN VD÷ODQPDVÕ\OD RUJDQLN DVLWOHU SURWHLQOHU YH HQ]LPOHU JLEL \NVHNWLFDULGH÷HUHVDKLSUQOHULQUHWLOPHVLJHUoHNOHúPHNWHGLU[53].. 14.

(53) .VLODQD]ODU KHP .DWÕ .OWU )HUPDQWDV\RQ.DWÕ 6XEVWUDW )HUPHQWDV\RQ (KSF) hem de 6ÕYÕ.OWU)HUPDQWDV\RQ(SKF) ile üretilebilmektedirler [20].. 1.3.1 .DWÕ6XEVWUDW)HUPHQWDV\RQ .6)