PHN I TNG QUAN TI LIU 1.1 i cng v h enzyme chitinase 1.1.1 Khi qut v enzyme 1.1.1.

.1 nh ngha [1, 23] Enzyme l mt loi phn t protein c sinh vt tng hp nn v tham gia xc tc cho cc phn ng sinh hc. Enzyme c phn t lng t 20.000 n 1.000.000 dalton, c cu to t cc L-acid amin lin kt nhau bi lin kt peptid. B phn c hiu tham gia phn ng gi l trung tm hot ng ca enzyme. Enzyme gm hai nhm: nhm enzyme mt cu t gm nhng enzyme c thnh phn ha hc duy nht l protein; nhm enzyme hai cu t gm nhng enzyme c hai thnh phn: phn protein thun gi l apoenzyme c vai tr xc tc, phn th hai phi protein l coenzyme l nhng cht hu c c hiu c vai tr thc y qu trnh xc tc. Ngoi ra c mt s kim loi nh Zn, Cu, Mn, Fe ... ng vai tr lin kt enzyme v c cht trong qu trnh xc tc phn ng, lin kt gia apoenzyme v coenzyme, tham gia trc tip vo qu trnh vn chuyn in t.
1.1.1.2 C ch hot ng [16, 23]

Trung tm hot ng ca enzyme (E) c cu trc khng gian tng ng vi c cht m chng xc tc, phn ng hnh thnh trong qu trnh enzyme tip xc vi c cht nh cha kha- kha to phc hp enzyme-c cht. Qu trnh tc ng ca enzyme vo c cht to sn phm tri qua ba giai on: Giai on 1: Enzyme (E) tng tc vi c cht (S) nh nhng lin kt to phc E-S Giai on 2: Khi c cht (S) to phc vi enzyme (E), c cht s b thay i cu hnh khng gian v mc bn vng cc lin kt, lin kt b ph v to sn phm. Giai on 3: Enzyme tch ra, c gii phng nguyn vn. Sn phm (P) to thnh. S c ch tc ng enzyme: E + S E-S E + P 1.1.2 Enzyme chitinase 1.1.2.1 nh ngha Chitinase hay poly -1,4-(2-acetamido-2-deoxy)-D-glucosid, glucanohydrolase, thuc nhm enzyme thy phn (Hydrolase), l enzyme thy phn chitin thnh chitobiose qua vic xc tc s thy gii lin kt 1,4- -glucosid gia C1 v C4 ca 2 phn t Nacetylglucosamine lin kt nhau trong chitin. 1.1.2.2 Phn loi a. Da vo phn ng phn ct [67]

Enzyme phn gii chitin bao gm: endochitinase, chitin-1,4-chitobiosidse, N-acetyl- -Dglucosaminidase (exochitinase). - Endochitinase: (EC 3.2.1.14): l nhm enzyme phn ct ni mch chitin mt cch ngu nhin to cc on oligosaccharide. Cc enzyme ny c nghin cu t dch chit mi trng nui cy nm mc Trichoderma harzianum (e loi endochitinase: M1= 36kDa, pI1= 5,3 0,2 v M2= 40kDa, pI2=3,9), Gliocladium viens (M=41kDa, pI=7,8). - Chitin-1,4-chitobiosidse: l enzyme phn ct chitin to thnh cc sn phm chnh l cc dimer chitobiose. - N-acetyl- -D-glucosaminidase (exochitinase): l enzyme tip tc phn ct chitin t mt u cho snr phm chnh l cc monomer N-acetyl- -D-glucosamin.

Hnh: s phn ct chitin bi cc enzyme thuc nhm chitinase b. Da vo cu trc phn t [67] Enzyme chitinase c sp xp vo 2 h Glycohydrolase: - H Glycohydrolase 18: l h chitinase ln nht vi khong 180 chi, c cu trc xc nh gm 8 xon / cun trn, c tm thy hu ht cc loi thuc Eukaryote, Prokaryote v virus. H ny bao gm ch yu l enzyme chitinase, ngoi ra cn c cc enzyme khc nh chitodextrinase, chitobiase v N-acetyl- -D-glucosaminidase. Cc chitinase ny hot ng thng qua mt c ch kim sot m trong cc on - polymer b phn ct to ra sn phm -anomer. Cc chitinase thuc h Glycohydrolase 18 c tng hp t Aeromonas hydrophila, Bacillus circularis, Trichoderma harzianum, Aphanocladium album, Serratia marcscens - H Glycohydrolase 19: h ny gm hn 130 chi, thng thy ch yu thc vt nh c chua (Solanum tuberosum), ci (Arabidopsis thaliana), u H Lan (Pisum sativum), ngoi ra cn x khun Streptomyces griceus, vi khun Haemophillus influenzae Chng c cu trc hnh cu vi mt vng xon v hot ng thng qua c ch nghch chuyn.

c. Da vo trnh t amoni acid [75]

Da vo trnh t u amin (N), s nh v ca enzyme, im ng in, peptid nhn bit v vng cm ng, ngi ta phn loi enzyme chitinase thnh 5 nhm: - Nhm I: l nhng ng phn enzyme trong phn t c u N giu cystein ni vi tm xc tc thng qua mt on giu glycin hoc prolin u cacboxyl (C) (peptid nhn bit). Vng giu cystein c vai tr quan trng i vi s gn kt enzyme v c cht chitin nhng khng cn cho hot ng xc tc. - Nhm II: l nhng ng phn enzyme trong phn t ch c tm xc tc, thiu on giu cystein u N v peptid nhn bit u C, c trnh t amino acid tng t chitinase nhm I. Chitinase nhm II c thc vt, nm v vi khun; chng c cm ng bi cc tc nhn bn ngoi. - Nhm III: Trnh t amino acid hon ton khc vi chitinase nhm I v II. - Nhm IV: l nhng ng phn enzyme ch yu c l cy hai l mm, 41 47% trnh t amino acid tm xc tc ca chng tng t nh chitinase nhm I, phn t cng c on giu cystein nhng kch thc phn t nh hn ng k so vi chitinase nhm - Nhm V: Da trn nhng d liu v trnh t, ngi ta nhn thy vng gn chitin (vng giu cystein) c th gim i nhiu ln trong qu trnh tin ha thc vt bc cao. 1.1.2.3 Cu trc ca h enzyme chitinase a. Cu trc enzyme chitinase thuc h Glycohydrolase 18 Cu trc khng gian (cu trc bc 3) ca mt s enzyme chitinase huc h Glycohydrolase 18 c nghin cu, c th l chitinase vi khun (Serratia marcascens) v chitinase thc vt. Tm hot ng ca cc enzyme ny to thnh t 8 si xon / cun trn. Si s 8 ca phin cun vo bn trong cu trc hnh nhng vi vng xon th hin nh mt chic nhn hng ra ngoi (hnh v [92])

Hnh 3: M hnh cu trc khng gian ca enzyme chitinase Serratia marcescens b. Cu trc enzyme chitinase thuc h Glycohydrolase 19 Chitinase tch chit t la mch (Hodeum vulgare) c tinh th ha v phn tch cu trc bng tia X. u tin ngi ta tin hnh quan st phn gii 2,8 A0 v sau quan st phn gii 1,8 A0(hnh v). [93]

Hnh 4: M hnh cu trc khng gian ca chitinase Hodeum 1.1.2.4 Cc c tnh c bn ca h enzyme chitinase [11] a. Trng lng phn t Enzyme chitinase tm thy thc vt bc cao v to bin c trng lng phn t khong 30kDa (kilodalton). cc loi thn mm, chn t, ng vt c xng (c, lng c, th), mt s chitinase c trng lng phn t khong 40-90 kDa hoc cao hn c l khong 120kDa. Trng lng phn t ca enzyme chitinase thu nhn t nm v vi khun c khong bin i rng, t 30 n 120 kDa. b. im ng in, hng s Michaelis Enzyme chitinase c gi tr im ng in pI thay i rng, t 3- 10 thc vt bc cao v to; pI t 4,7-9,3 cn trng, gip xc, thn mm v c; pI t 3,5 8,8 vi sinh vt. Hng s Michaelis : 0,010 0,011 (g/100ml). c. nh hng ca nhit [32, 63]

Theo nhiu nghin cu, chitinase hot ng gii hn nhit t 20 500C (Frandberg v Schnure, 1994; Huang v cng s, 1996; Bhushan v Hoondal, 1998; Wiwat v cng s, 1999; Bendt v cng s, 2001). Nhn chung nhit ti u cho h enzyme chitinase vi sinh vt hot ng l 400C, ngoi tr chitinase ca Aspergillus niger hot ng trn c cht l glycol chitin c nhit ti thch l 50OC (Jeuniaux, 1963). Tuy nhin, ty theo ngun gc thu nhn m cc enzyme chitinase c th c nhng gi tr nhit ti thch khc nhau. Cc enzyme chitinase thc vt thuc nhm III v chitinase t Bacillus licheniformis phn lp sui nc nng cho thy kh nng chu ng nhit cao n 800C. Bendt v cng s (2001) pht hin hot tnh thy phn chitin mnh nht ca chitinase t Vibrio sp. T 30-450C v chitinase chu nhit t chng Bacillus sp. BG-11 hot tnh cao nht 40-600C. Lorito (1998) kho st hot tnh enzyme chitinase t chng Trichoderma harzianum Rifai nhn thy enzyme ny c kh nng hot ng trong khong nhit rng t 25-600C, nhit ti u l 400C. d. nh hng ca pH [32] Gi tr pH ti thch (pHop) ca h enzyme chitinase t 4-9 i vi cc enzyme chitinase thc vt bc cao v to; h enzyme chitinase ng vt l 4,8- 7,5 v vi sinh vt l 3,5- 8,0. Theo cc nh khoa hc, pHop ca enzyme chitinase c th c s ph thuc vo c cht c s dng. a s cc enzyme chitinase c nghin cu c pHop khong 5,0 khi c cht l glycol chitin nm trong khong pH kim yu. Cc nghin cu chng t rng chitinase hot ng c trong khong pH t 4,0-8,5 (Morrisey v cng s, 1976; Wiwat v cng s, 1999; Bendt v cng s, 2001). Chitinase ca nm hot tnh cao nht pH = 5, trong khi vi khun pH ti thch l 8,0. Theo Bhushan v Hoondal (1998), hot tnh ca chitinase t Bacillus sp. BG-11 cao nht pH = 8,5. e. Cht tng hot cht c ch * Allosamidin: allosamidin l cht c ch c nghin cu, c bit l vi chitinase cn trng. Allosamidin c ch cnh tranh vi enzyme chitinase, gi tr KI khong 0,1m. Cht ny c cu to tng t dng trung gian ca c cht: mt vng oxazoline; vng ny c th gia carbonyl oxygen ca nhm N acetyl v C 1 ca N-acetyl-D-glucosamin trong qu trnh thy gii. [67]

Hnh 5: Cu trc ha hc ca allosamidin v dn xut allosamidin [94] Allosamidin: R1=R2=CH3 Demethylallosamdin: R1=CH3,R2=H Didemethylallosamidin: R1=R2=H * Cc ion kim loi: Cc ion kim loi: Hg2+, Ag+ l nhng cht c ch, cn ion Cu+ th ty theo dng enzyme chitinase: dng chitinase b c ch hoc tng cng hot tnh (tm thy mt s loi c v vi sinh vt). Bn cnh albumin cng c vai tr lm tng hot ng ca enzyme chitinase, nhng s nh hng n ch r rng sau 2 3 gi u ca phn ng. f. n nh hot tnh [65] Enzyme chitinase th hoc tinh sch n nh trong trng thi ng lnh khong 2 nm. Chng b mt hot tnh nhanh chng 370C trong trng hp khng c mt c cht. Chu k bn hy 370C l 40 ngy v 50C l 230 ngy. S n nh ca enzyme chitinase s cao hn khi c mt ca c cht l chitin. Enzyme chitinase bt hot bi oxygen, hng s bt hot 200C l K = 0,145/h. 1.1.2.5 Cc loi c cht ca enzyme chitinase a. Chitin [67] C cht ch yu ca enzyme chitinase l chitin. Chitin c tm thy trong thnh t bo ca nm si v l cht hu c chim khi lng ln hnh thnh nn lp v ngoi ca ng vt khng xng khp (cn trng, gip xc, thn mm). Chitin l mt polymer mch thng c cu to dng chui, thnh phn ch yu l cc monomer N- acetyl D glucosmin ni vi nhau bng lin kt 1,4 glucosid. Mi on chitin c xc nh c di l 10,4 A0. V mt cu trc, chitin c cu trc tng t nh cellulose, im khc bit ha hc duy nht l nhm acetamido v tr s 2 trn khung carbon ca chitin c thay bng nhm hydroxyl (-OH) cellulose [1,76]. Ngoi ra, chitin cng c cu trc lin h vi murein cu trc polymer hin din vch t bo v khun. lp v cn trng v gip xc chitin c gn kt vi cc polysaccharid khc (cellulose, mannan, glucan...) hm lng chim ti a khong 3-5% sinh khi nm ti. (hnh v)

Hnh 5 : cu trc ha hc ca chitin [95] V mt cu trc lp th, chitin c 3 dng : , , ; S khc nhau ny biu hin s sp xp cc chui. - chitin cc chui xui v ngc xen k nhau, - chitin th cng hng v - chitin c 2 chui xui xen k vi 2 chui ngc. Dng chim nhiu nht l - chitin. [64] Trong gii ng vt, chitin l mt thnh phn cu trc quan trng trong lp v ca mt s ng vt khng xng sng nh cn trng, nhuyn th, gip xc v giun trn. Trong gii thc vt, chitin c thnh t bo ca nm v mt s to Chlorophiceae. Chitin tn ti trong t nhin dng tinh th, l cu trc gm nhiu phn t c ni vi nhau bng cc lin kt hydro to thnh mt h thng si. Trong t nhin, chitin him khi tn ti trng thi t do m gn nh lun lun lin kt di dng phc hp chitin- protein. iu ny dn n s khng vi cc ha cht v cc enzyme thy phn, gy nhiu kh khn cho vic chit tch, tinh ch chng. Ty thuc vo cc c tnh c th v s thay i tng giai on sinh l m trong cng mt loi c th thy s thay i v lng v cht ca chitin. Tnh cht ca chitin [27, 31] Chitin th rn, c cu trc bn vng nh cc lin kt hydro trong v gia cc mch. Chitin khng tan trong nc, trong dung dch acid v kim long, trong cn v trong cc dung mi thng thng. N ch tan c trong mt s acid v c c (HCl, H 2SO4, H3PO4). b. Cc dn xut ca chitin [64, 65] Enzyme chitinase c th tc ng ln mt s dn xut ca chitin nh glycol chitin, carboxymethylchitin, chitosan, chitinsulfat, 4- methylumbellferyl tri N acetyl chititrioside (MUC pht hunh quang). Enzyme chitinase khng tc ng trn cc c cht : chitin nitrat, cellulose, hyaluronic acid, alginic acid hoc mucin. 1.1.2.6 C ch cm ng ca h enzyme chitinase [57] tl tham kho Hin nay, c ch cm ng ca h chitinase ca Trichoderma l ch rt c nhiu nh khoa hc quan tm. S cm ng enzyme ngoi bo rt hiu qu khi nui cy

Trichoderma trong mi trng c ngun cacbon duy nht l chitin tinh sch, vch t bo nm hoc h si nm. Khng c hoc rt t c hin tng cm ng khi thnh phn mi trng c cha chitosan, cellulose, chitin cha tinh sch hoc laminarin. Cc enzyme khc nhau th c c ch cm ng khc nhau. V d : N-acetylglucosamine ch cm ng c trng cho vic to ra -N- acetyl-glucosamine ch cm ng c trng cho vic to ra -N- acetyl-hexosaminedase ( N-acetylglucosaminidase) m khng cm ng to ra endochitinase Trichoderma. Trong qu trnh k sinh ca Trichoderma trn nhng k ch khc nhau th mc cm ng v thnh phn cc sn phm enzyme to thnh khc nhau, ch yu l 1,4- N acetylglucosaminiase 102 kDa v 72 kDa (CHIT102, CHIT72) v mt vi enzyme endochitinase ca nhm II. Hn na vch t bo nm thu nhn t nhng loi nm m Basidiomycetes khc nhau, c s dng cm ng endochitinase t cc chng Trichoderma cng khc nhau. S hnh thnh h enzyme chitinase in vitri b c ch bi t l gia tng ca lng ng glucose, sucrose v snr phm cui, iu ny cho thy rng qu trnh sinh tng hp enzyme c iu chnh mt cch c hiu bi s c ch d ha. iu ny c nghin cu r hn endochitinase 42kDa (CHIT42) v gen m ha ThEn-42 ca T. Harzianum. Nhiu tc gi cho rng, glucose c ch s hnh thnh CHIT42 cng nh cp mRNA. S hnh thnh CHIT72, CHIT42 v CHIT33 c iu ha cp phin m. Inbar v Chet chng minh s to thnh enzyme chitinase ca chng T. Harzianum k sinh c bt ngun bi vic tit ra cht trung gian tng tc vi k ch l lectin. Hin tng ny xy ra khi c s cm ng ca chitooligomer. Lectin l h thng nhn bit k ch ca vi nm k sinh v rt c gi tr trong nghin cu pha sm ca qu trnh k sinh nm. S tht l trong iu kin in vitro h si nm hp kh trng c tc dng cm ng CHIT33 v CHIT42 mnh hn tc nhn cm ng l chitin tinh sch. Do , khi c s tn ti ca k ch nhng khng c chitin cng vt qua c s c ch ca glucose i vi CHIT42. [58] S cm ng cng b hn ch bi nh sng, qu trnh to bo t hay s tip xc vt l gia t bo v c cht. Cng c kin cho rng, hin tng b b i cng l tc nhn cm ng mnh m kh nng sinh tng hp CHIT42. S hot ng ca h enzyme chitinase b nh hng bi cc hot ng ca nhng hp cht cm ng khc nhau nh cc enzyme ph hy vch khc (protease v glucanase), cc lin kt protein trong vch t bo cht, permease v cht khng sinh. 1.1.2.7 C ch tc dng ca h enzyme chitinase (TLTK) Enzyme chitinase xc tc cho phn ng thy phn lin kt 1,4- -glucosid trong chitin. N phn ct dc theo mch carbon ca chitin v sn phm to thnh ch yu l chitobiose v chiotriose. Nhng cht ny sau tip tc b phn ct thnh cc monomer l cc N-acetyl D-glucosamine. [65] Qu trnh phn gii chitin c tm tt nh sau :

Chitin Citobiose, chitotriose N-acetyl D-glucosamine S thy phn chitin c th xy ra theo 2 c ch : c ch gi li cc cu t -anomer trong sn phm v c ch nghch chuyn t dng sang dng (hnh 29) V tr kt ni gia c cht chitin v enzyme chitin c gi nh bao gm ti thiu 6 tiu phn ng c k hiu t A n F (bt u t u khng kh). Ti v tr ny, hexasaccharid s c phn ct thnh hai trisaccharid (hnh 2.10) [86]

1.1.2.8. Cc ngun thu nhn enzyme chitinase [30, 57, 58]- LASH Chitin l mt polysaccharide ph bin trong t nhin, l mt polyme sinh hc c tng hp vi s lng ln t sinh vt. Lng chitin c sn xut hng nm trn th gii ch ng sau cellulose, chng c to ra trung bnh 20g trong 1 nm/1m2 b mt tri t. Trong t nhin chitin hin din hu ht cc gii vi sinh vt. n nay c nhiu nghin cu v enzyme chitinase ca cc vi sinh vt, thc vt, ng vt. [53] a. Chitinase vi khun [67] Enzyme chitinase c tm thy cc vi khun : Chromobacterium, Klebsiella, Pseudomonas, Clostridium, Vibrio v c bit l nhm Streptomyces. Enzyme chitinase c th l enzyme cu trc hoc enzyme cm ng. Tuy nhin trong cc mi trng nui cy vi sinh vt, ngi ta u cho thm chitin c cht ca enzyme chitinase lm tng kh nng tng hp enzyme chitinase, ng thi n nh hot tnh enzyme chitinase sau qu trnh chit tch. Vi khun tng hp enzyme chitinase nhm phn gii chitin trong mi trng to ngun cacbon cho vi khun sinh trng, pht trin. b. Chitin nm [67] Chitinase cng c to ra bi cc loi nm si. Cc chng nm mc cho enzyme chitinase cao nh : Trichoderma, Gliocladium, Calvatia,...c bit l cc loi nm ln nh Lycoperdon, Coprinus...cc nm phn hy chitin cng c tm thy trong cc thy vc nh loi nm Karlinggiomyces asterocystic thuc lp Phycomycetes. Tng t nh vi khun, enzyme chitinase ca nm ng vai tr quan trng v mt dinh dng, nhng khc l hot ng ca chng rt linh hot trong qu trnh pht trin v trong s pht sinh hnh thi ca nm bi v chitin l thnh phn chnh ca vch t bo nm. Chitinase cn gi vai tr chnh trong hot ng k sinh nm i khng li cc li nm gy bnh thc vt. c. Chitinase thc vt [67] Chitinase tham gia vo c ch t v ca thc vt chng li cc loi cn trng v nm k sinh gy bnh [54]. Ngi ta quan st thy chitinase tch chit t cy cn ty c kh nng c ch si nm pht trin. Tuy nhin cng c tc gi cho rng chitinase cn c vai tr khc nh tham gia vo qu trnh hnh thnh phi [41]. Cc thc vt bc cao c kh nng to enzyme chitinase nh : cao su (Hevea brasiliensis), thuc l (Nicotiana sp), la mch (Hordeum vulgare), c rt, ht u nnh ... v c bit mt s loi to bin cng l ngun cung cp enzyme chitinase [52]. d. Chitinase ng vt [65] T mt s ng vt nguyn sinh v t cc m, tuyn khc nhau trong h tiu ha ca nhiu loi ng vt khng xng : rut khoang, giun trn, thn mm, chn t (v d trong dch rut ca c sn Helix aspersa), ta c th thu nhn c enzyme chitinase. i vi ng vt c xng sng enzyme chitinase c tit ra t tuyn ty v dch d dy ca cc loi c, lng c, b st n su b ; trong dung dch d dy ca nhng loi chim, th n su b.

Trong ng vt thy sn, c bit l trong v tm, cua gh, mai mc, hm lng chitin chim kh cao t 14-35% so vi trng lng kh. Chitin c tm thy t nhiu ngun khc nhau vi hm lng khc nhau [45,51] B cnh cng 37% Nhn 38% B cp 30% Su 20-38% Nm 5-20% Tm 33% Cua 70% Mc 3-20% Mc d chng c ph bin rng ri nhng cho n nay ngun thu nhn chnh ca chitin l t v cua v tm. Trong cng ngh ch bin, do chitin tn ti dng phc hp vi mt s cht nh: CaCO3, protein, lipid, cc cht hu c nn vic tch chit cn kh khn v phi m bo c hai yu t cng mt lc l va loi ht tp cht ng thi khng lm bin i tnh cht ca chitin. Ngoi ra, enzyme chitinase cn c thu nhn t dch biu b ca giun trn trong sut qu trnh pht trin v dch tit biu b ca cc loi chn t vo thi im thay v, lt da. Enzyme chitinase gip cn trng tiu ha mng ngoi (cutincun) trong qu trnh bin thi hay lt xc. 1.2 ng dng ca chitinase trong nng nghip v y hc 1.2.1 Mt s ng dng h enzyme chitinase trong nng nghip Cc loi cn trng v vi sinh vt (ch yu l nm mc) phn ln u c hi cho ng thc vt, chng gy ra nhiu loi dch bnh cho cy trng v vt nui, nh hng trc tip n sn xut nng nghip. [85, 113] Hu ht cc nhn t c hot tnh khng nm (fungicid) hay dit cn trng (insecticid) kim sot dch bnh thng qua vic tiu dit hoc kim sot cc tc nhn gy bnh, cc k ch trung gian hoc cc vector mang bnh u s dng mt trong cc kiu tc ng sau : - Cc cht c gy ngt th hoc lm cht kh. - Cc cht c lm kt ta hoc bt hot cc enzyme, lm gim chc nng ca cc bo quan trong nguyn sinh cht. - Cc cht c gy bt hot cc enzyme trong chui h hp. - Cc cht c nh hng ln cc c quan nh h thn kinh, h tun hon, D nhin l mt loi thuc tt nht s gy hi cho vt ch ca cc loi k sinh m n tc ng. Tuy nhin, do s phc tp v s ph thuc qua li ln nhau trong cc qu trnh sng th khng lc no ngi ta t c mc ch trn v v th mt s fungicid v insecticid gy c chng mc no , mt s khc gy ra cc hiu ng ph khng mong mun,

V chitin khng phi l thnh phn ph bin thc vt v ng vt c xng nn ngi ta s dng cc tc nhn km hm s sinh tng hp chitin trong cc fungicif v insecticid nh nikkomycin, polyoxin D, Khi p dng trn cy cnh, cy lng thc, trn ng vt, nhng tc nhn trn chng t khng gy hi ng k cho thc vt hoc ng vt c xng. 1.2.1 C s khoa hc ca ng dng enzyme chitinase trong phng tr nm gy bnh thc vt Thnh t bo ca vi nm dy khong 0.2 m, c tnh phn quang rt mnh nn c th phn bit c r rng knh hin vi quang hc. Nhiu cng trnh nghin cu chng minh cu to thnh t bo vi nm c cu trc bn mng, va c cu trc si [15]

Cu to chnh ca thnh t bo cc nhm nm ch yu : [15] Nhm phn loi Nm nhy myxomyces Plasmodiphore Nm non oomycetes Nm c Hyphytridiomycetes Chytridiomycetes Nm tip hp Zygomycetes Nm nang Ascomycetes Nm m Basidiomycetes Nm bt ton Deuteromycetes Cu to chnh ca thnh t bo Cellulose Chitin Cellulose Glucan Cellulose Chitin Chitin Glucan Chitin chitosan Chitin Glucan

Theo Hirohi Ihui [80], enzyme chitinase lun c mt trong c th thc vt mc d trong cy khng cha chitin. Chitinase v 1,3 glucanase c to ra trong m thc vt khi t bo b kch thch bi nm gy bnh cha chitin, xc tc s thy phn vch t bo nm v ngn cn s pht trin ca bnh. [59] S kch thch hot tnh enzyme chitinase l du hiu tr li ca t bo i vi tc ng ca tc nhn gy bnh, i km vi s kch thch hot tnh phn gii amoniac, phenylalanin lm tin cho s tng hp lignin v phytoalexin thc vt...

Bn cnh cc nh khoa hc cng chng minh qu trnh chng li cc mm bnh thc vt c lin quan n vic sn xut ra enzyme chitinase. Tht vy, vi khun c kh nng chng li nm bnh bng cch sn xut ra chitinase [48]. Chitinase ca Streptomyces c kh nng c ch s pht trin ca nm bnh. [112] Chng Serratia marcescen hoang di c kh nng kim sot sinh hc i vi cc mm bnh thc vt. chng Serratia marcescens t bin c mang gen ChiA (gen m ha enzyme chitinase), khi gen b bt hot th chng ny mt hiu lc kim sot sinh hc. Khi ti t hp gen ChiA t Serratia marcescens vo E.coli, E.coli c kh nng lm gim cc bnh gy ra bi Sclerotium rolfsii v Rhizoctonia solani [87].[8] Trong chu k sng ca k sinh vi nm bnh, Trichoderma c tit ra enzyme chitin phn hy chitin, bao gm phc hp vi 6 enzyme khc bit nhau : hai enzyme -1,4-Nacetyl glucosaminidase c trng lng phn t ln lt l 102, 73, kDa v bn enzyme endochitinase c trng lng phn t ln lt l 52, 42, 33 v 31 kDa. Trong . Endochitinase (42 kDa) c kh nng thy phn vch t bo botrytis cinerea in vitro. Qu trnh khng nm bnh mun t c hiu qu cao nht cn c s phi hp hot ng b sung cho nhau ca 6 enzyme ny,[76] 1.2.2 Phi hp chitinase v chng vi khun Enterobacter cloecae khng nm y l s kt hp gia enzyme phn hy thnh t bo nm mc vi vi khun khng nm Enterobacter cloecae ( vi khun thng thy trn b mt ht v cc vng r ca thc vt ). S kt hp ny nhm mc ch km hm s sinh trng v pht trin ca c loi nm mc thng qua s tc ng kp : chitinase phn gii thnh t bo ca nm mc, to mi trng dinh dng cho Enterobacter cloecae tng sinh trng trong t nhin, ng thi tng kh nng kim sot sinh hc thng qua tng kh nng gn vo thnh phn khun ty ca nm mc. S tng tc gia chitinase v Enterobacter cloecae trong hn hp gia tng kh nng khng nm mc gy bnh nh chng vi khun c th bm vo thnh t bo, thm ch khi c s hin din D-glucose, saccharose, do c th tri rng phm vi s dng trn cc ht ging v thc vt c s tit r cc loi ng ny. Hin nay, ngi ta s dng hn hp ny bo v ht ging, l, r hoc qu, vng t xung quanh ht Hn hp ny c th dng khng cc loi nm mc gy bnh thc vt nh Fusarium, Gliocladium, Trichoderma, Saccharomyces, 1.2.3 Ch phm biocid s tc ng cng hng ca h enzyme chitinase, cc enzyme khc v cc tc nhn khng nm Cc nh khoa hc nghin cu s thy phn vch t bo vi nm bi cc enzyme chitinase, glucanase, cellulase,[107] v th nghim trn ng rung tc dng ca cc ch phm biocid. Khi s dng ring bit ch phm chitinase, nhn thy hiu qu tiu dit thp trn nm mc, giun trn, ch c kh nng km hm. Ch phm thy phn bi cc enzyme protease phi trn vi mt s tc nhn khc c hiu qu tiu dit nm mc, giun trn, v cn trng cao. [111]

Khi phi trn 2 ch phm ny theo t l 1 :1 th thu c hiu qu cao hn khi s dng ring l. y c th l do s cng hng hot tnh ca h sinh ha phc tp a nhn t. Ngoi ra, ngi ta cn tm cch tng cng hot tnh khng nm thng qua tc ng cng hng gia hn hp ch phm trn vi cc tc nhn khng nm : captan, manzate, chloroneb, [111] 1.2.4 B sung chitin vo t nhm to iu kin sinh trng tt cho cc chng vi sinh vt trong t tng hp chitinase khng nm Toyoda ngh cung cp vo t mt lng thch hp chitin nhm kch thch s pht trin ca cc chng vi khun sn sinh chitinase (nhng vi sinh vt ny chim u th vng r). X khun Streptomyces anulatus c c nh trong mt git gel alginat Ca v a vo t, chng x khun ny pht trin mnh trong t c cha chitin v c ch mt cch mnh m s pht trin ca nhng mm bnh t nm (v d kim sot bnh ho ca cy c chua gy ra bi nm Fusarium oxysporum). 1.2.5 Tuyn chn loi nm i khng cn trng gy hi cy trng [10, 53,87] Trong cng tc bo v thc vt, ngi ta t c nhiu kt qu tt trong vic dng mt s loi nm phng tr su bnh hi cy trng. C th nh nm Metarhizium anisopliae dng phng tr su xm, su c thn ng, su c nn da. nm Beauveria bassiana tr trn 80 loi su hi khc nhau, trong c su c thn ng, b xt, b hung,.. Phn ln cc loi nm xm nhp vo c th su bng si nm xuyn qua lp v cutincun (phc h protein chitin) vo trong. Cc vi nm tit ra enzyme chitinase phn gii chitin to thnh mt l xm nhp vo trong Cc nh khoa hc chng minh mi lin h gia kh nng tiu dit sy bnh ca vi nm i khng v s tng hp enzyme chitinase cc loi vi nm ny. Hin nay, cc nh khoa hc ch ng tuyn chn cc ging vi nm i khng su bnh hi cy trng da trn ch tiu hot tnh chitinase, s dng mi trng cm ng kch thch biu hin hot tnh enzyme chitinase v nhn sinh khi loi vi nm ny, ng thi thu nhn cc gel m ha chitinase, chuyn sang nhng loi vi nm khc khng c gel ny lm tng kh nng tiu dit su bnh ca chng. 1.2.2 Mt s ng dng trong Y hc 1.2.2.1 Tng hp chitooligosaccharid Hin nay hot tnh sinh hc ca cc chitooligosaccharis ngy cng c nghin cu su. Trong y hc ngi ta s dng cc oligomer chitohexaose v chitoheptaose lm tc nhn khng ung th. Enzyme chitinase ca Vibrio alginolyticus phn ct huyn ph chitin thnh chitopentaose v chitotriose. Enzyme N,N diacetylchtobiase c s dng rng ri lm nguyn lieuj khi u cho sinh tng hp cc hp cht c hot tnh sinh hc. Chitinase thu nhn t S. Griseus c kh nng thy phn chitin huyn ph thnh chitobiose tip tc c ci bin ha hc thnh mt dn xut disaccharid mi 2- acetamido- 2- deoxy- D-

allopyranose, y l cht trung gian tng hp nn cht c ch enzyme. Ngc li, Kobayashi v cng s cho bit c th s dng chitinase ca Bacillus tng hp chitobiose nh s kt ni N- acetyl- D- glucosamin vi dn xut ng oxazolin. [34] 1.2.2.2 Chn on cc bnh truyn nhim do vi nm bng enzyme chitinase [76] Nhiu phng php chn on bnh nm c xut nh ELISA, s ngng kt khng th, mu d phn t ..., pht hin c hiu cc nm gy bnh trong cc dch c th nhng gi thnh qu cao. Nhng bt li chung trong hu ht cc phng hin s dng l kh p dng i vi cc mu dch c th bi v kh c nh c cc mu ny. Cc phng php nhum nh GMC (Grocott methenamine AgNO3 staining), calcofluor/cellufour, India Ink, lectin label, rylus BSU c dng nhum c nh cc tiu bn nm nhng khng c tnh c hiu cao v cn s dng cc thit b t tin. Chitin hin din nhiu trong vch hu ht cc nm gy bnh, t nht l mt giai on trong chu trnh sng ca nm hay nm men th hin din trong nhng vt chi. Do cn mt phng php nhum chitin c hiu cho nm, to c s xy dng mt phng php chn on nhanh chng, hiu qu cc loi nm gy bnh. Hin nay, cc nh khoa hc xut mt phng php chn on mi cc bnh truyn nhim do nm bng cch s dng enzyme chitinase c phn lp to dng t Vibrio parahemolyticus (t tn l chitinase VP1), n kt hp cht ch vi chitin v c th s dng nh mt mu d trong vic chn on vi nhy cao, nhn din mt cch c hiu cc vch t bo nm hay nhng vt chi nm men trong nhng lt ct mu m bnh. 1.2.2.3 Ch phm thuc mi : enzyme chitinase v cc dc cht khng nm [5, 52] Hin nay, cc nh khoa hc ngh s dng chitinase vi cc tc nhn khng nm c th chp nhn khc nhm b tr cho hot ng ni sinh ca chitinase. Cc tc nhn bao gm : - Amphotericin B v nhng phc cht c cu trc tng t nystatin v Pyramycin. - 5-fluorocytosin v cc dn xut azol nh fluconazol, ketoconazol, itraconazol, miconazol... - Allylamines thiocarbamates nh olnaftat, terbinafin... - Griseofulvin ; acid undecylenic ; bezoic... Enzyme chitinase c th pht huy hiu qu cc tc nhn khng nm liu lng khng gy tc dng ph cho bnh nhn. Ngoi ra vic kt hp gia enzyme chitinase v laminarinase c ghi nhn l hu hiu hn trong vic tn cng vo vch t bo nm (so vi ch dng enzyme chitinase). Cc nh khoa hc th nghim hot tnh khng nm ca enzyme chitinase ti t hp trong c th chut v th b nhim cc loi nm khc nhau thuc nhm Aspergillus, Candida...Hiu qu ca s iu tr vi tc nhn khng nm c c lng trn 3 im : - Gim t l cht. - Gim s lng t bo nm c nui cy t cc c quan. - Gim mc lu thng khng nguyn nm.

1.3 c im sinh hc ca Trichoderma 1.3.1 Phn loi v c im hnh thi ca Trichoderma 1.3.1.1 Phn loi Trichoderma l mt trong nhng vi nm gy nhiu kh khn cho cng tc phn loi do cn nhiu c im cn thit cho vic phn loi vn cn cha c bit y . Persoon ex Gray (1801) phn loi Trichoderma nh sau: Gii: Fungi Ngnh: Ascomycota Lp: Euascomycetes B: Hypocreales H: Hypocreaceae Ging: Trichoderma Ainsworth v Sussman li cho rng Trichoderma thuc lp Deuteromyctes, b Moniliales, h Moniliaceae. Theo hai nh khoa hc Elisa Esposito v Manuela da Silva (1998), Trichoderma thuc h Hypocreaceae, lp Nm ti Ascomycetes; cc loi Trichoderma c phn thnh 5 nhm: Trichoderma, Longibrachiatum, Saturnisporum, Pachybasium v Hypocreanum. Trong , 3 nhm Trichoderma, Pachybasium, Longibrachiatum c giai on telemorph (hnh thi giai on sinh sn hu tnh ) l Hypocrea; nhm Hypocreanum him khi gp di dng teleomorph c lp; nhm Saturnisporum khng tm thy hnh thc teleomorph. 1.3.1.2 c im hnh thi ca Trichoderma Trichoderma l mt loi nm bt ton, sinh sn v tnh bng nh bo t t khun ty.[12] Khun ty ca nm khng mu, cung sinh bo t phn nhnh nhiu, cui nhnh pht trin thnh mt khi trn mang cc bo t trn khng c vch, khng mu, lin kt nhau thnh chum nh u cnh nh cht nhy. Bo t hnh cu, hnh elip hoc hnh thun. Khun lc nm c mu trng hoc t lc trng n lc vng, xanh, lc xn n lc m. Cc chng ca Trichoderma c tc pht trin nhanh ( c hnh) chng c th t c ng knh khun lc t 2 9 cm sau 4 ngy nui cy 200C. 1.3.2 c im sinh l, sinh ha Mi trng sng Trichoderma spp l nhm vi nm ph bin t nng nghip, ng c, rng, m mui v t sa mc. Hu ht chng l nhng vi sinh vt hoi sinh, nhng chng cng c kh nng tn cng cc loi nm khc. Trichoderma rt t tm thy trn thc vt sng v khng sng ni k sinh vi thc vt. Chng c th tn ti trong tt c cc vng kh hu t min cc Bc n nhng vng ni cao cng nh min nhit i. Tuy nhin, c mt s tng quan gia s phn b cc loi v cc iu kin mi trng. T.polysporum v T.viride c mt vng kh hu lnh, trong khi T.harzianum c cc vng kh hu nng. iu ny tng quan vi nhu cu cn nhit ti a cho tng loi.

Cc loi Trichoderma thng xut hin t acid, v Gochenaur (1970) cho rng c tng quan gia s hin din ca T.viride vi t acid trong vng kh hu rt lnh Peru, Trichoderma pht trin bt c pH no nh hn 7 v c th pht trin tt t kim nu nh c s tp trung mt lng CO2 v bicarbonat. Trichoderma c th s dng nhiu ngun thc n khc nhau t Carbonhydrat, amino acid n ammonia. Trichoderma l vi nm a m, chng c bit chim u th nhng ni m t, nhng khu rng khc nhau. T. hamatum v T. pseudokoningii c th chu iu kin m cao hn so vi nhng loi khc. Tuy nhin, Trichoderma spp thng khng chu c m thp v iu ny c cho l mt yu t gp phn lm cho s lng Trichoderma gim r rt trong nhng ni c m thp, song cc loi Trichoderma spp khc nhau th yu cu v nhit v m cng khc nhau. Trichoderma spp c th c pht hin trong t bi mi hng ca chng, hng da (6-pentyl--pyrone d bay hi) thng c to ra trong qu trnh sinh trng ca Trichoderma. Vi phng php pha long ngi ta c tnh Trichoderma c th t n 3% tng s vi nm hin din trong cc loi t rng v 1,5% s lng trong t trng c. Turner v cng s (1997) ch ra rng T.longibrachiatum v T.citrinoviride c nhiu s trng nhau v khu vc phn b a l. S phn b rng khp ny l do s pht tn hiu qu ( nh gi hoc cn trng) hoc biu hin mt qu trnh tin ha rt sm. Cht chuyn ha th cp v khng sinh Trichoderma spp sn xut nhiu loi khng sinh. Ngy nay, danh sch ca cc cht trn c ko di thm ra, bao gm a dng cc cht c hot tnh: glioviridin ( mt diketopiperazin), sesquiterpenoids, trichothecenes ( trichodermin), cyclic peptides, isocyanid bao gm cc cht chuyn ha (trichoiridin). Bn cnh kh nng c ch vi sinh vt khc, chc chn nhng cht chuyn ha ny lin quan n s tng trng yu km ca thc vt bc cao hn v cng l nguyn nhn gy ra bnh ci cu thng qua hot ng c ch vi sinh vt phn gii cellulose trong d c ca chng. Cc chng Trichoderma cng sinh ra nhiu loi hp cht c ch d bay hi c th tr gip cho s hnh thnh khun lc c chng trong t. Trichoderma v Gliocladium sn xut a dng cht chuyn ha th cp. Nhng cht ny bao gm sc t anthroqinon (pachybasin-[1,8-dihydroxy-3-methyl-,10-anthraquinon]; emodin- [1,6,8-trihydroxy-3-methyl-9,10-anthroquinon]), chc nng ca chng vn cha c bit, mt s cht khc nh benzoquinon (thermophyllin), cardinan (avocettin); dihydrocoumarins, polyacetyl mch nhnh (trichodermen) v dn xut cc acid bo (methyl2,4,6-triene-1-1 carboxylat). Nhng cht ny cng cha c bit r v hiu qu ca chng trong s hnh thnh khun lc. 1.3.2.1 Dinh dng v con ng trao i cht c bn ca Trichoderma a. Ngun Cacbon [56]

Trichoderma ni bt v kh nng tit ra enzyme phn hy nhiu loi polysaccharide ( nh cellulose, hemicellulose) v nhng polymer lin quan nh chitin. Nhng enzyme ny c cng nhn c gi tr thng mi. Manczinger v Pollner (1985) [74] s dng ngun cacbon l phng thc phn loi cc ging Trichoderma thnh tng nhm. Theo phn tch, nhng ngun cacbon sau c s dng bi tt c nhng chng nghin cu: D-glucose, D-galactose, D-fructose, Dmannose, D-cellobiose, trehalose, D-xylose, L-arabinose, d-mannitol, D-arabitol, glycerol, salacin, esculin, arbutin, glycerol-1-monoacetat, -methyl-D-glucosid v N-acetyl- -Dglucosamin. Ni chung ngun cacbon tt nht l glucose, fructose, mannose, galactose, xylose, rehalose v cellobiose. Ngc li Trichoderma spp thng khng c kh nng s dng -methyl-D-xylosid, -methyl-D-mannosid, methanol, ethanol, n-propanol, ethylamine, 5-ketogluconic acid, Ltartaric, propionic acid, butyric acid, oxalic acid, glyoxalic acid, DL-isocitric acid, adipic acid, DL-lactic acid, manolic acid, acetoin, maltitol, dertran, uracil, cytosine, cytidin, Llysin, L-histidin, L-methyonin, L-cystein, -DL-aminoadipic acid, -alanin, ethanolamine, nhiu loi D-amino acid, benzoic acid, ferrulic acid v anthanilic acid. Vic s dng mt vi ngun cacbon (nh inulin, tinh bt, xylan, pectin, lactose, sucrose, maltose, mt vi polyol, sugar acid, hu ht cc amino acid v mt vi pentoses) th ty thuc vo tng loi, v c th s dng cho mc ch phn loi ha hc. b. Ngun Nit [56] Trichoderma sppc kh nng s dng c nhng ngun nit kh phc tp hay n gin tng trng. Khi Trichoderma ang tng trng trn ngun cacbon l carbohydrate, ngun nit thng c s dng l ammonium hn l nitrat. Mt vi chng nh T.reesei hay T.koningii T-1 cng khng th s dng nitrat. l s thiu enzyme nitrate permease. Ngun nit hu c nh peptone thng c s dng trong mi trng hn ch s tng trng chm trn c cht polymeric nh l cellulose. Tuy nhin cn ch rng peptone c s dng nh l c ngun nit ln ngun cacbon, v c u tin s dng khi c cung cp ng thi vi polysaccharide. Trong s nhng amino acid, ngun nit hu c tt nht cho Trichoderma l alanin, aid aspartic v aicd glutamic. Ngoi amino acid th T.lignorum ( = T.viride) c bo co l c th s dng baz purin, purin nucleoside v nucleotide tng ng nh l ngun nit duy nht. c. Ngun dinh dng khc [56] Hu ht cc chng phn lp hoang di ca Trichoderma khng yu cu nhng nhn t tng trng phc tp hay vitamin. Thnh phn ion kim loi ca si nm T.reeei c phn tch bi Gaunt v cng s (1984) v c s dng tnh ton nhu cu v ion kim loi. Nhng ion kim loi nh st th cn thit ch s tng trng v c th c tm thy mt nng rt thp trong mi trng. Mt s lng ln nhng ion kim loi khc cng rt quan trng cho s tng trng nhng nng thp, ngc li nng cao li c ch tng trng. S thm vo ca Cd2+ v

Hg2+ nng 1 10mM dn n c ch tng trng T.viride v dn n kiu hnh bt thng ca nm. Tuy nhin tng t nh loi nm khc, Trichoderma cng bt cc ion kim loi trong mng t bo ca chng, pht hin ny c s dng trong vic loi b nhng ion kim loi nng ra khi nc thi cng nghip bng h si nm Trichoderma. d. Oxi v CO2 [56] Trichoderma l vi sinh vt hiu kh bt buc, mc d nhng chng phn lp u c thu nhn trn nhng mi trng sng c p sut tng phn oxi rt thp. S cung cp oxi v hot ng ca ti th cng c bo co l nhng nhn t iu ha s hnh thnh cellulase ca T.reesei [53], v nng O2 mc di cc thun dng nh l thch hp cho s tng hp enzyme. CO2 sn phm cui cng ca s oxi ha cacbon, s c tch ly mt mc no trong mi trng tng trng rn, ph thuc pH v nhit . V vy, vi loi Trichoderma spp b c ch bi CO2 theo phng thc ph thuc vo pH, s c ch mnh nht trong mi trng kim nh v trung tnh. Hutchinson v Cowan (1972) [61] bo co rng T.harzianum to ra hiu ng c ch CO2 v ethanol trn s tng trng v to bo t ca nhi nm khc (Aspetgillus niger, Pestalotia rhododendri), trn cy con ca Lactuca sativa. iu ny cho thy mt vi chng Trichoderma c th chp nhn s tch t CO2 nhiu hn mt s nm khc. 1.3.2.2 nh hng ca yu t bn ngoi ln s pht trin ca nm Trichoderma [56] a. Nc. Jackson v CS (1991) kho st trn T.virens, T.citrinovride v T.viride v thy rng mc pht trin ca si nm ca tt c cc loi nm gim khi tng in th nc vt qua ngng -0,7 n -14,0 Mpa. Trong khong ny tt c nhng chng phn lp ny d chp nhn in th thm lc (NaCl, glycerol) hn l in th cht nn (polyethyeneglycol) [32] S gim lng nc thc y s to bo t ca T.harzianujm,c im ny c th s dng trong mi trng nui cy lng to nh bo t cng vi vic tng mc sy kh rt c ch cho nhng mc ch iu khin sinh hc. S ny mm ca nh bo t ca Trichoderma c bit nhy cm vi vic tng p sut thm lc [32] b. Nng ion H+ Nng ion H+ c nh hng ln trn s tng trng ca nm, v nhiu cht dinh dng (v d nh ng v amino acid), u c thu nhn bng s ng vn chuyn vi H+. V vy nm thng ph hp vi mi trng c pH hi acid. S tng trng ti u thng trong khong pH= 4 6,5, v mt vi chng Trichoderma spp thch hp vi pH < 3. c. Nhit Nhit ti u cho s tng trng ca hu ht Trichoderma l trong khong 25 0 30 C.

S tng trng ph thuc nhit l mt hin tng c tnh thch nghi Trichoderma, l loi c ngun gc t vng kh hu m p, c nhit ti u cao. Nhng loi thuc Trichoderma nhm Longibrachium c nhit ti u cao nht t 38 44 0C. Bn cnh , T.polysporum v T.viride c quan st c mt cao nht nhit hi lnh 20 250C. Nhng chng phn lp ca T.viride thm ch c th tng trng 50C [32]. 1.3.3.3 Mi trng Vic phn lp v nh hng c tin hnh bng cch nui Trichoderma trn mi trng nui cy chn lc. Trong mi trng dng nhng ha cht v thuc nhum nh Bengal , tm kt tinh ( crystal violet), oxgall (2-deoxycholat) v pentachloronitrobenzen (PCNB) u c phi hp vi nhiu thuc dit nm. Mi trng phc hi c in l TSM (Trichoderma Selective Medium) sau dc ci tin hn ch s pht trin ca Fusarium bng cch phi hp vi benomyl cho nhng chng khng li thuc dit nm ny. [44, 45] 1.3.3.4 Cu trc mc t bo phn t a. Vch t bo [56] B mt t bo ca hu ht cc loi nm bao gm 3 lp lu thng: mng nhy, vch t bo v mng t bo. Cu trc vch t bo ca Trichoderma thuc dng chitin--glucan. Tuy nhin, chitin hin din h si li khng c bo t ca T.viride. S tng hp chitin rt cn thit cho s ny mm ca bo t nh. Vch t bo ca mt vi chng Trichoderma cng c pht hin l cha galactose v N-acetyl--D-galactosimin lectin. Lora v cng s (1994) [72] m t s hin din ca mt protein vch t bo c kh nng c ch chitinase trong S.cerevisiae. b. Nhn [56] Ging nh tt c Eukaryot cc t bo nm mang mt nhn in hnh c bao quanh bi mt mng i. Nhn ca nm thng nh hn (ng knh t 1-5m), v cha t hn 1pg DNA. Bo t nh thng cha nhiu hn mt nhn. Tuy nhin Trichoderma spp c s lng nhn khc nhau. Mt vi chng n nhn (Hammill, 1974 [83]) ngc li vi chng a nhn (nh T.atroviride ATCC 36042). T.reesei hnh thnh nhn nh sau khi x l colchicin do s phn chia nhn bt bnh thng. Hm lng DNA trung bnh ca nhng nhn nh ny bng 30% ca nhn bnh thng, gi l a bi lch. Nhng nhn ny c th hu ch cho vic vn chuyn nhng lng DNA nh vo trong th nguyn sinh [85] c. Bo quan v mng t bo [56] Nhng nghin cu trn T.reesei cho thy trong t bo ca chng hin din tt c cc bo quan ca Eukaryot, tng t vi cc chng Trichoderma khc. Cu trc ca mt s bo quan (nh th Golgi) th khc bit so vi Eukaryot, nhng vn cha bit r nhng c tnh v sinh ha c khc nhau hay khng. Mt vi loi Trichoderma spp cha mt lng li ni cht kh ln v nhiu nht l chng T.seesei t bin. 1.3.3.5 Hin tng ki sinh ca nm Trichoderma Hin tng k sinh ca nm Trichoderma spp bao gm mt s bc sau:

Bc 1: Tng tc u tin l t nm Trichoderma hng v t nm k ch [38] . Hin tng ny l c tnh hng ha ca Trichoderma spp, hng v ni c cht ha hc do t nm k ch tit ra (Chet vf Elad, 1983, [42]). Khi t nm Trichoderma n t nm k ch, chng c xu hng tip xc v cun xung quanh si nm k ch hnh thnh cu trc mc hoc p st si nm v pht trin song song vi nm k ch. Theo Chet v Baker (1981), s tip xc nhn bit ca nm Trichoderma vi k ch ca n rt c trng. V d: Trichoderma harzianum nhn bit t nm R.solani nh mt cht bm dnh (agglutinin), cht ny lin kt c hiu vi lectin c trong cu trc vch t bo ca nm Rhizoctonia slain [38] Bc 2: T nm k sinh thy phn vch nm k ch bng cch tng hp tit ra cc enzyme: glucanase, chitinase, cellulose (Elad, 1983 [42]) Enzyme -1,3-glucanase v chitinase ca Trichoderma harzianum khng ch tc ng kt hp vi nhau m cn kt hp vi cc hp cht khng nm. Tm li, cc chng Trichoderma c th c s dng lm tc nhn sinh hc l da trn kh nng k sinh nm ca chng (ch yu l k sinh trn cc loi nm gy bnh thc vt). Qu trnh k sinh nm ca Trichoderma bao gm cc bc sau: S ha dng ca Trichoderma S nhn bit k ch Tit enzyme ngoi bo v cht khng sinh Xm nhp vo t nm k ch Tiu dit k ch. c tnh k sinh ca loi Trichoderma trn cc loi nm gy bnh thc vt c nghin cu kh k [38,43]. Cc nh khoa hc a s tp rung tm hiu qu trnh k sinh nm trong kim sot bnh cy. Hnh v 1.3.3.6 Mi lin h gia kh nng i khng nm gy bnh thc vt v ht ng enzyme chitinase ca Trichoderma spp. [58] Trichoderma spp c kh nng phn gii vch t bo nm. Harran kho st c ch phn t ca enzyme phn gii lin quan n hot ng kim sot sinh hc ca T. harzianum. S thy phn vch t bo nm ch yu da vo hot tnh ca chitinas, glucanase v protease. Sau khi bm v qun quanh nm bnh, khun ty ca nm k sinh to ra l hng trn vch ca khun ty k ch. Mu sc ca khun ty k sinh thay i v pht hunh quang mnh bi s kt hp ca fluorescein isothiocyanat vi lectin gn vo chitotriose hoc vi calcofluor White. C phc hp ny c gn vi glucan v oligomer N acetyl D glucosamin. Hin tng ny c nhn nh do enzyme phn gii c tit ra ti v tr tip xc bi T. harzianum khi phn hy vch ca R. solani v S. rolfsii. Nu c s hin din ca cycloheximid, hin tng i khng b ngn chn v hot tnh ca enzyme b suy gim. Hot tnh ca enzyme chitinase cng c tm thy khi Trichoderma tn cng R. solani v S. rolfsii trong mi trng t.

T.harzianum c kh nng tn cng ln nm bnh th tit 1,3 glucanase v chitinase nhiu hn nhng chng khng co kh nng tn cng nm bnh, kh nng i khng ca S. rolfsii khi b T. harzianum tn cng cng c nghin cu. Khun ty T. harzianum khi xm chim vo hch ca S. rolfsii v t l trn b mt ca hch ny, cui cng lm thay i hnh dng v lm bin mt t bo cht ca n. chng Trichoderma khc nhau ty thuc cp enzyme phn gii to ra tn cng si nm ca cc loi nm bnh. tm ra s hnh thnh enzyme chitinase trong qu trnh tng tc k sinh, Harran thc hin th nghim nui cy ghp (dual - culture) trong T.harzianum ng u ring l vi hai loi nm bnh u cha chitin trong thnh phn cu to vch t bo l R. solani, nhng kh c th mc trn qua phn mi trng c cha khu ty ca S. rolfsii trong cng iu kin. S biu hin ca h enzyme chitinase T. harzianum trong tng tc ny c iu ha rt c bit bi ty loi t bo k ch. Hiu qu i khng chng li R. solani cao do s biu hin ca c 3 loi endochitinase l CHIT52, CHIT42 v CHI33 v N acetylglucosaminidase 102kDa. Ngc li, ic chng li S. rolfsii ca T. harzianum trong tng tc k sinh khng c hiu qu do ch c duy nht 2 loi exochitinase l Nacetylglucosaminidase (CHIT102, CHIT73) c pht hin. Nhng kt qu ny chng minh rng h enzyme chitinase ca T. harzianum khng phi c iu ha mt cch n gin theo c ch bt/tt phn ng li s hin din hay vng mt ca chitin. a. S i khng gia T. harzianum v nm bnh S. rolfsii. Inbar v Chet nghin cu s cm ng chitinase c th ca T. harzianum trong sut qu trnh k sinh vi S. rolfsii. Trc khi tip xc, c T. harzianum ln S. rolfsii u c cha -1,4-N-acetylgucosaminidase ca ring mnh. Nhng khi c s tng tc -1,4-Nacetylgucosaminidase ca S. rolfsii bin mt. Trong giai on u ca qu trnh tng tc th enzyme 1,4-N-acetylgucosaminidase 102 kDa (CHIT102) ca Trichoderma c cm ng u tin. Ngay sau ( khong 12 g sau khi tip xc) th hot ng ca CHIT102 b suy gim nhanh chng ng thi vi s cm ng to ra nhiu enzyme -1,4-Nacetylgucosaminidase kDa (CHIT73). Hin tng ny s khng xy ra nu si nm ca S. rolfsii kh trng trc khi b sung vo mi trng vi .T. harzianum. nhn bit hiu ng ca qu trnh k sinh, Inbar v Chet d s dng h thng biomimetic, gn lectin tinh sch t S.rolfsii ln si nylon. Khi Trichoderma pht trin trn nhng si nylon c ph lectin, hot tnh ca CHIT102 tng nhanh hn so vi nui trn mi trng si nylon khng gn lectin. iu lu tm y l h thng biomimetic tuyt i khng c s hin din ca chitin, kt qu ny cho thy cm ng CHIT102 Trichoderma trong qu trnh i khng xy ra rt sm. [62] b. S i khng gia T. harzianum v nm bnh R. solani. Xem xt s phc tp trong tng tc in vivo gia nm k sinh v k ch ca n trn vng r ca thc vt hoc trong t. Phng php phn tch tng tc kp (dual

interaction assay) l phng php kh thi nht nghin cu cc c tnh sinh ha ca nm k sinh. Flores s dng phng php ny nghin cu s biu hin ca gen protease (pbr1) T. harzianum trong qu trnh tng tc gia T. harzianum v R.solani. ng nhn thy rng, nng mRNA ca gen pbr1 tng khi s tng tc xy ra. Carsolio nghin cu s biu hin ca CHIT42 T. harzianum IMI 206040 trong sut th nghim nghin cu s k sinh trc tip ca n trn R.solani v ng nhn thy s biu hin ca CHIT42 c cm ng mnh m trong sut qu trnh tng tc i khng. Trong qu trnh i khng gia T. harzianum v R.solani; CHIT102 cng l enzyme c cm ng u tin. Tuy nhin khong 12 gi sau khi tip xc hot tnh ca CHIT102 khng h suy gim m cn tip c gia tng, ng thi 3 loi endochitinase l CHIT52, CHIT42 v CHIT33 cng c cm ng. Thi gian sau , hot tnh ca CHIT102 mi bt u gim dn trong khi hot tnh ca CHIT52, CHIT42, CHIT33 dn dn gia tng [37] 1.3.3.7 Nhng hp cht khng nm ca cc chng trichoderma. gliotoxin (C13H14N2S2O4): l cht khng sinh t nm T. viride Gliotoxin khng bn v d b phn hy nhanh chng trong nh sang, ph khng sinh ca gliotoxin bao gm vi khun (ch yu vi khun gram dng) v cc nm gy bnh hot tnh khng sinh ca gliotoxin lin quan n s c mt ca phn t lu hunh trong cu to ca chng. Viridin (C9H16O6): ngoi gliotoxin, ngi ta cn tch c cht khng sinh Viridin (sc t vng), chng c kh nng c ch mt s loi nm gy bnh nh Fusarium coeruleum, Botrytis alli, Penicillium notatum. Mt s cht khng sinh khc - Theo Dennis v Webster (1971) [40], T. Viride c kh nng tit trichodermin, T. hamatum to ra cc polypeptide c bn cht khng sinh. - Okuda (1982) cho rng nhiu loi Trichoderma spp tit isonitrile c bn cht khng sinh. - Trichozianine l khng sinh c hot tnh khng nm c pht hin nhiu loi T. harzianum. Trichozianine kt hp vi nhng enzyme thy phn vch trong qu trnh c ch s ny mm v ko di t nm trong qu trnh k sinh nm [79] - Trichothecene t T. harzianum cng c hot tnh khng nm (Corley et al.1994). - Tricholin l protein bt hot ribosome do T. viride tit ra, chng lm gim s hnh thnh chui polysome nm bnh Rhizoctonia solani [70] - Claydon (1987) [30] xc nh c cht alkylpyrons d bay hi do T. harzianum c ch nm R. solani gy bnh ho r trn ci trong iu kin in vitro. 1.3.3 ng dng ca vi nm Trichoderma 1.3.3.1 Trong nng nghip a. Bo v thc vt [12,28] Trichoderma spp hin din khp ni trong t v trn cc loi cy g va b n ng l mt bng chng th hin tnh cnh tranh mnh m ca chng, mt khc vi c tnh k sinh trn mt s nm gy bnh thc vt, cc nh khoa hc nghin cu v a ra nhng

kt qu thuyt phc v kh nng i khng nm ca Trichoderma spp thng qua hot ng k sinh nm. Mt s bnh do nm lin quan n cc b phn ca cy di mt t (r) rt kh tr bng phng php ha hc truyn thng, v khng th tc ng ton b h r bng thuc dit nm. Trichoderma spp l tc nhn i khng t nhin ca cc nm gy bnh trong t v c ng dng lm tc nhn kim sot sinh hc thnh cng trong nh knh v ngoi ng rung, chng l nhng k sinh rt hu hiu trn nhiu loi nm gy bnh khc nhau nh: Phytophthora spp, Rhizoctonia solani, Pythium spp, Sclerotium rolfsii. Nm 1993, Harman v Hayes th nghim dung hp t bo trn nhm to ra chng c kh nng kim sot bnh hu hiu. Mt s nh khoa hc khc tp trung vo ci bin mt s c tnh lin quan n cc hot ng i khng nh to chng t bin sinh enzyme mnh hn tng hiu qu i khng. Mt s loi Trichoderma c s dng ph bin trong kim sot sinh hc l T. koningii, T. harzianum; T. viride, T.hamatum. T. harzianum c th dng kt hp vi nhng chng Trichoderma khc hoc dng di dng phn bn vi sinh. Hin nay trn th gii c mt s ch phm c thng mi ha nh: [15]

TRICHODEX (tc nhn chnh c T. harzianum) BINAB-T (tc nhn chnh bao gm T. harzianum v T. polysporum) ca Thy in. TRI 002, TRI 003 (chng T. harzianum) ca H Lan. T22 Planter Box (y l chng dung hp protoblast T. harzianum) ca Hoa K.

Trichopel, Trichoject, Trichodowels, Trichoseal (tc nhn chnh l T. harzianum v T. viride) ca c. b. Ci thin nng sut cy trng Vi loi Trichoderma c kh nng kch thch s ny mm v s ra hoa. c nhiu cng trnh khoa hc chng minh rng T. harzianum v T. koningii kch thch s ny mm v tng trng ca cy. i vi cc loi c trng trong nh knh, T. harzianum y nhanh s ra hoa bng cch rt ngn ngy ra hoa hay tng s lng hoa. Trichoderma y mnh tc tng trng ca cy trng nh kh nng gip cy trng to ra h r cng cp hn. Mt nghin cu gn y cn cho bit nu bp T. harzianum T-22 h sinh r th cn lng phn m t hn 40% so vi r khng c T -22. Trichoderma ci thin cu trc v thnh phn ca t, y mnh s pht trin ca vi sinh vt nt sn c nh nito trong t, duy tr s can bng ca cc vi sinh vt hu ch trong t, bo ton v tng ph nhiu, dinh dng cho cy trng. Tng sc khng ca cy trng, mt s chng T. harzianum cn c th xm nhp vo m bo cy, lm tng tnh chng chu bnh ca cy trng. Nh vy, cc chng nm Trichoderma spp trong cc ch phm phn hu c vi sinh khng nhng cung cp mt ngun phn bn an ton, hiu qu m cn gip kim ch cc bnh gy hi cy trng v taoh c nhng sinh thi phng bnh lu di trong t nhin.

Nhng loi Trichoderma c dng ph bin trong kim sot sinh hc l T. harzianum, T. koningii, T. viride, T. hamatum v T. polysporum. 1.3.3.2 Trong lnh vc x l mi trng [47] T.harzianum c kh nng phn hy cc cht gy nhim trong t rng. T.harzianum chng t kh nng phn gii hiu qu ca chng trn ciliatin, glycophosphat v amino methylphosphat acid (3-methoxyphenyl). T.harzianum 2023 (Khoa sinh l hc vt Trng i hc California) c th phn gii DDT, endosulfan, pentachloronitrobenzen v pentachlorophenol. T.harzianum CCT-4790 phn gii 60% thuc dit c Duirion trong t trong 24 gi, y l mt tim nng tt x l sinh hc cc ha cht nhim trog t v trong m ly. 1.3.3.3 Trong cng ngh thc phm [11] Enzyme ca chng Trichoderma longibrachiatum c kho st v cho thy c th lm tng hng v ca men ru. ng thi, cc loi glucanase v glucosidase c tit ra bi chng ny c kh nng thy phn lin kt glycoside, do th gii cc lin kt glycoside-terpen phng thch terpen, to mi thm trng. Bt la m sn xut bnh m thng cha y hm lng amylase nhng li thiu amylase nht nh. amylase c th ly t la i mch ny mm (malt), vi khuaane hay nm. Trong nm l ngun amylase phong ph v tt nht. Ngi ta chuyn gen amylase nm Trichoderma vo nm men bnh m di s kim sot ca promoter ACT1. Bnh m to ra c kch thc ln hn trc v rut bnh m cng mm hn, chm cng hn lm tng thi gin bo qun. Ngoi ra trong cng nghip sn xut du olive, c s dng cellulose v hemicelulase chit t Trichoderma trn vi pectinase chit tch t Aspergillus, iu ny lm tng sn lng du sn xut ra mt cch r rt, ng thi cht lng du cng c ci thin so vi phng php sn xut du truyn thng. 1.3.3.4 B sung thc n cho gia sc Hin nay tren th trng c ch phm EconaseTM vi cc enzyme c ngun gc t Trichoderma reesei b sung vo thc n ca gia cm c cc u im sau: Thnh phn cht dinh dng linh ng hn. S dng ngun nguyn liu r tin hn D tiu ha hn Lm tng gi tr nng lng tht ca cc loi ng cc Cc gia cm tng trng ng dng Gip tng trng tt hn Trng gia cm sach s hn Tng mu lng trng g Gim lng rc thi ra mi trng

Bn cnh , ch phm AvizymeTM cng c nhng u im tng t nh Econase TM ph hp cho g trong giai on trng. Porzyme TM l thng hiu ca mt hn hp cc enzyme cellulose, hemicellulase, protease. Amylase b sung vo khu phn n ca ln con va thi b. Tt c cc enzyme ny u c ngun gc t Trichoderma. 1.3.3.5 Ngun gen s dng trong chuyn gen [12] Nhiu vi sinh vt kim sot sinh hc u c cha mt s lng ln gen m ha cc snr phm c hot tnh cn thit s dng trong kim sot sinh hc. Nhiu gen c ngun gc Trichoderma c to dng v c tim nng ng dng rt ln trong chuyn gen to ra cy c kh nng khng c nhiu bnh. Cha c mt gen no c thng mi ha tuy nhien hin c nhiu gen ca Trichoderma c nghin cu v pht trin. 1.1.11. 1.3.3.6 Tnh hnh nghin cu v kh nng ng dng Trichoderma spp Vit Nam nc ta, hin nay c mt s cng trnh nghin cu ng dng vi nm Trichodema: theo bo co ca Trn Th Thun ( B mn nghin cu bnh cy Vin bo v thc vt (1995)), ghi nhn bc u thnh cng khi s dng mt s chng Trichoderma phn lp c trong t, nhm hn ch bnh kh vn hi bp do nm Rhizoctonia gy ra. Gn y (2001), TS. Hong Quc Khnh (phng vi snh ng dng Vin sinh hc nhit i) th nghim thnh cng trn din rng ch phm BIOPROMOT (bao gm 2 chng ging T. harzianum v T. Koningii) trn hai loi cy c chua v ci, ch phm dng x l t nhm hn ch bnh v kch thch cy tng trng. Nm 2005, inh Minh Hip v cng s bc u hon tt vic kho st s phn b ca cc chng nm Trichoderma ti Thnh Ph H Ch Minh v cc tnh min ng Nam B. ng thi nhm nghin cu ny cng c nhng nghin cu su v hot tnh i khng ca vi nm Trichoderma phn lp i vi cc loi nm gy bnh cy trng ph bin nh Sclerotium rolfsii, Rhizoctonia solani, Phytophthora primulae v th nghim ng dng ch phm Trichoderma ti mt s ni nh x Bu Lm (huyn Xuyn Mc, tnh B Ra Vng Tu), trm bo v thc vt Long Khnh thu c mt s kt qu bc u kh tt. Cc kt qu nghin cu ca trng i hc cn th, Vin Nghin cu la ng bng Sng Cu Long, Cng ty Thuc sat trng Vit Nam, Vin Sinh hc nhit i cho thy hiu qu rt r rng ca nm Trichoderma trn mt s cay trng ph nam. Cc nghin cu cho thy nm Trichoderma c kh nng tiu dit nm Furasium solani (gy bnh thi r trn cam qut, bnh vng l cht chm trn tiu) hay mt s loi nm gy bnh khc nh Sclerotium rolfsii, Fusarium Oxysporum, Rhizoctonia solani. Cng dng th 2 ca nm Trichderma l kh nng phn hy cellulose, ha tan ln chm tan. Li dng c tnh ny ngi ta phi trn Trichoderma vo qu trnh sn xut phn hu c vi sinh, thc y qu trnh phn hy hu c c nhanh chng. Cc sn phm hu c sinh hc c ng dng kt qu nghin cu mi ny hin c trn th trng nh loi phn Cugasa ca Cng ty Anh

Vit (TP. H Ch Minh), phn VK ca Cng Ty Vin Khang (ng Nai) d c nng dn cc vng trng rau, cy n tri, cy tiu, cy iu, hoan nghnh v ng dng hiu qu.