1.

Gii thiu Cht thi v x l ca h tr thnh mt vn mi trng trn ton th gii c bit l khi nhng cht thi c kh nng phn hy cc hng ha v dch v hu ch (Shide et al, 2004). Lm gim lng du v pht trin bt n chnh tr trong sn xut du m cc quc gia t ra mt mi e da nng lng ln vi th gii cn phi c gii quyt bng cch gii thiu cc la chn thay th (Banerjee et al, 2010). N c c tnh rng sn xut hng nm ca sinh khi cellulose c th cung cp 10 ln nhu cu nng lng ca chng ti v 100 ln nhu cu thc phm ca chng ti trn quy m ton cu (Hall, 1979). Cht thi Cellulolytic t cc hot ng nng nghip c th c s dng sn xut cc hp cht quan trng nh ru qua gip trong vic kim sot nhim mi trng (Omojasola v Jilani, 2008). V cam thuc nhm cht thi sinh khi c gi tr (Mrudula v Anitharaj, 2011; Omojasola v Jilani, 2008). Trng cam c d on tip cn 66,4 triu tn vo nm 2010, i din cho mt s gia tng 14% trong vng 12 nm (Plessas et al, 2007). Khong 40-60% cam vt nc tri cy v phn cn li, c v, mng phn khc v cc sn phm ph c coi l x l cht thi cam qut (Grohmann v Baldwin, 1992). V cam ch yu bao gm cellulose (13,6%), Hemicellulose (10%) (Ververis et al. 2007), 25% -30% (trng lng kh) pectin (AravantinosZafiris et al., 1994), sc t dip lc v cc hp cht trng lng phn t thp khc nh limonene v nh vy (Nagy et al, 1977). Trong bioconversion cht thi cng nng nghip nh v cam, thy phn ca polyme l iu cn thit cho s c ca h monome m c th tip tc c ln men ethanol (nh l mt thay th cho ngun nng lng) v cc sn phm khc. Hai polyme chnh cn thit c thy phn v cam l nhng pectin v celluloses. Thy phn enzym cc cht pectin v cellulolytic i hi phi c hnh ng hip ng ca cc enzym pectin v cellulolytic (Grohmann v Baldwin, 1992). Cc men ny l thng mi t tin. Nm sn xut enzyme pectinase v cellulase ph v cellulose v pectin vo cc hnh thc n gin. Kh nng ca cc si nm tit ra xenlulaza v cc enzym thy phn khc vo cc phng tin truyn thng vn ha ca h dn n thu hoch ca cc enzym hu ch (Berry v Paterson, 1990; Chinedu et al, 2008.). Enzyme thng mi t tin, c bit l cc nc ang pht trin nh Nigeria, bi v chng c sn xut t cc cht nn tinh ch v cc sinh vt thng c cp bng sng ch. Do , bt buc m cht nn r hn t cc ngun a phng sn xut enzyme c iu tra v rng cc loi nm vi cng sut sn xut enzyme b c lp ti a phng. Bo co trong lnh vc nghin cu ny t i Nigeria, c l, do s ph thuc hn v d tr du th ca chng ti. Do , cng vic ny bo co vic s dng v cam m l c sn trong mi trng ca chng ti sn xut pectinase v phc tp enzyme cellulase ca ba phn lp nm. 2. Vt liu v phng php Mu B su tp V cam c thu thp t cc thng nhn tri cy ti Agbowo Ibadan th trong khu vc pha ty nam ca Nigeria. Mu t cng c thu thp t cc khu vn thc vt ca trng i hc Ibadan. V cam c s dng lm cht nn phn lp vi sinh vt, trng trt v sn xut enzym. Cc mu t cng c s dng phn lp vi khun. X l mu V cam cn li phn hy t nhin nhit phng trong hai tun vi mc ch vi sinh vt c lp. trng vi khun v sn xut enzyme, v cam cng phi kh, sau l sy kh, mt t v c lu tr trong khng kh cht ch ti polyethylene gi m. C lp ca Nm Mt gam mi v cam mc nt v mu t c cn v trng vo 9ml ca nc ct ring v lc k. T , pha long sau c thc hin ln n 10-4 v m 10-2, 10-3, v 10 -

4 c thc hin trong mi trng thch kh trng ng khoai ty (PDA). Streptomycin (100mg / l) c thm vo PDA sau khi kh trng ngn chn s pht trin ca vi khun. Tm l trong mt v tr o ngc 30oC trong 7 ngy. Sau khi , cc a c quan st i vi tng trng nm. Nn vn ha c tinh ch bng ph nui 2 - 4 ln c c nn vn ha tinh khit ca cc thuc a c sau duy tr trn nghing PDA 4oC. Tiu Chiu cho Pectinolysis v Cellulolysis Cc phng tin truyn thng c s dng sng lc l Czapek-DOX sa i Agar (Nwodo-Chinedu et al, 2010) vi sucrose thay th vi s lng bng nhau ca pectin cam qut mn hnh cho pectinolysis hoc carboxymethylcellulose (CMC) mn hnh cho cellulolysis. Streptomycin (100mg / l) cng c thm vo cho mi phng tin ngn nga nhim khun. Sng lc c thc hin pH 5.4. sng lc pectinase sn xut nm, tt c cc chng c cy trn a petri c cha pectin cy c mi nh ngun carbon duy nht v 37oC trong 24 gi. Vo cui thi gian bnh, tm c nhum mu vi it 50mm xem cc khu r rng xung quanh cc thuc a (Yogesh et al, 2009). i vi nm cellulase sn xut, phn lp c cy trn a petri c cha cc phng tin truyn thng vi cacboxymetylxenluloza (CMC) nh ngun carbon duy nht v 29 1oC trong 48 gi sau khi h c nhum mu vi congo cho 15minutes hnh dung khu vc gii phng mt bng theo phng php ca Omojasola v Jilani (2008). Trung Screening Cc chng nm cho thy khu vc thanh ton b tr ng k trong c hai pectin c cha v CMC thch c cha c la chn sng lc th cp bng cch c tnh polygalacturonaza v carboxymethylcellulase sn xut. Cc phng tin c s dng c sa i mi lng Czapek-DOX. Cc chng nm chn lc c cy vo c hai h cam qut c cha pectin v CMC c cha sa i Czapek-DOX phng tin truyn thng cht lng. S dng mt su c lie flamed v lm mt (5mm), a ca si nm t mp ngy cng tng ca tch cc vn ha ngy cng tng b ct trn a petri. Vi mt que cy flamed v lm lnh, hai a c a vo 100ml v trng i Czapek-DOX phng tin truyn thng cht lng trong bnh Erlenmeyer 250ml. Cc bnh c bao ph bng bng gn v trng v trong bng ti 29 1oC trong 7 ngy. Sau khi , cc nn vn ha c thu hoch bng cch lc qua giy lc Whatman s 1. Lc c lu tr 4oC v c s dng nh cc enzym th cho polygalacturonaza v xt nghim nh lng carboxymethylcellulase. Phn lp vi cc hot ng enzyme cao nht c la chn cho cc th nghim tip theo. Sn xut xenlulaza v Pectinaza trong Solid Nh nc ln men chun b ngun bnh, hai a ca si nm tng ng c trn vi 10ml nc ct v trng v h thng treo c thc hin. 5g trung bnh ln men c ca ch bin v cam trn vi 4mls ca Czapek-DOX khong sn gii php bn trong mt bnh Erlenmeyer 250ml v tit trng 121oC trong 30 pht. Trng trt c thc hin bng cch thm 1 ml inocula cc. Sau , cc bnh c 29o 1oC. Nhng nh hng ca thi gian bnh, pH, nhit v ngun nit c th nghim xc nh iu kin ti u cho sn xut cellulase v pectinase. Sau khi , cc b lc vn ha c thc hin tri qua polygalacturonaza v xt nghim endoglucanase. Mi trng ln men c phn tch cho polygalacturonaza v endoglucanase. Xt nghim Hot ng polygalacturonaza (PG): PG hot ng c xc nh bng cch o pht hnh gim nhm t pectin cam qut bng cch s dng 3, 5, dinitrosalicyclic axit tinh khit (DNSA) kho nghim (Miller, 1959). Hn hp phn ng c cha 0.8ml 1% citric pectin (Sigma) trong b m acetate 0.2m, pH 5,0 v 0.2ml dung dch enzyme th c 50oC

cho 10min (Silva et al, 2002). Mt n v (U) ca cc hot ng enzyme c nh ngha l lng enzyme pht hnh 1mol axit galacturonic mi pht. Endoglucanase Hot ng: Cc th tc theo kho nghim 0,5 ml m t bi Jeffries (Jeffries, 1996). Gii php enzyme th c pha long trong citrate 0.05M m, pH 4,8. Enzyme pha long trong b m v 1% CMC (0,5 ml) c trn u v 30 pht 50oC. 3ml DNSA c thm vo v ng c t trong cc nc si khong 5 pht. Cc ng c lm lnh v lng ng kh (glucose) c xc nh (Miller, 1959). iu ny c xc nh bng cch o hp th 540nm bng cch s dng Lambda 25 UV / Vis quang ph. Ph hp vi Hip Hi Quc T Ha sinh, mt enzyme n v (U) bng 1mol glucose c pht hnh mi pht. 5. Kt lun Cc enzyme s b hin ang c s dng trong mt s sn phm cng nghip khc nhau v cc qu trnh v c ngy cng tng nhu cu thay th cc qu trnh ha hc truyn thng vi cc qu trnh cng ngh sinh hc tin tin lin quan n vi sinh vt v cc enzym. Do , cc khu vc mi ca ng dng c lin tc c nghin cu. Tht th v, mt lng ln cc vi sinh vt c kh nng sn xut cc enzym trong t bo v extracellularly. Mt s trong nhng yu t m lm cho cc men vi khun mnh m hp dn trong nhiu qu trnh cng nghip bao gm thn thin vi mi trng thin nhin, gim nhu cu nng lng v d dng c cc nguyn liu cho sn xut ca h. Vic s dng cc ph liu trong mi trng ca chng ti l cht sn xut cc enzyme do vi sinh vt l mt bc tin y ha hn gii quyt cc vn nhim mi trng m thng c gy ra bi cc cht thi. Lm gim lng du v pht trin bt n chnh tr ti cc quc gia sn xut du t ra mt mi e da nng lng ln cho th gii m cn phi c gii quyt bng cch gii thiu cc la chn thay th. Cc quc gia nh M v Brazil ang dn u cc nh sn xut ethanol sinh hc t cy trng. Chuyn hng cy trng mc ch ny t ra mt nguy c thiu lng thc cho ton b dn s th gii ang gia tng mt t l hnh hc. Khi nim v sn xut nhin liu sinh hc m l mt ngun nng lng ti to t cc vt liu ph thi nhanh chng t c s ch ca nhiu nh nghin cu trn ton th gii. Cc cht thi ny l majorly nng nghip v cc ph liu rng c ca h thnh phn cellulose, tinh bt, lignin, xylan v pectin. S dng cc cht thi xenlul sn xut nhin liu sinh hc bao gm mt bc thy phn vi s tr gip ca enzym. Enzyme thng mi c s dng cho mc ch ny l kh t tin. Cc tin tt l mt s vi khun c kh nng chuyn ha ny cht nh carbon v cc ngun nng lng bng cch sn xut mt lot cc enzym. Trong tc phm ny, nm cellulolytic v pectin c phn lp t t v mu da cam ang phn hy v v tt nht ba phn lp nm c xem nh l Penicillium atrovenetum, Aspergillus flavus v Aspergillus oryzae c la chn sn xut cellulase v pectinase trong mt qu trnh ln men trng thi rn ca v cam. Polygalacturonaza v endoglucanase xt nghim c thc hin c tnh tit ra cellulase v pectinase bi cc loi nm v iu kin vn ha, h tr sn xut cao nht ca cc enzym c nghin cu. Cng trnh nghin cu ny do cho thy vic s dng mu cam v c sn trong mi trng ca chng ti cho vic sn xut pectinase v cellulase ca ba phn lp nm nghin cu trong cng vic ny. Tuy nhin, nghin cu su hn s c thc hin kim tra cc tnh cht l ha ca cc enzym sn xut v hiu qu trong qu trnh thy phn v cc loi ng ln men sn xut ethanol. 4. Tho lun Citrus pectin v carboxymethylcellulose c s dng mn hnh cho pectin v cellulolytic nm. Carboxymethylcellulose l mt ngun carbon thun li hn cho vic kim tra cc nm cellulolytic (Shahriarinour et al, 2011). Hai th tc kim tra lm vic chn chng vi kh nng cao nht sn xut cc enzym. Samira et al. (2011) cng lm nh vy cho bit

cellulase hot ng ca mt s mu vi khun. Vic kim tra chnh l mt cht lng hnh dung qu trnh thy phn ca ngun carbon trn a. ng knh ca khu r rng xung quanh cc thuc a c o v t l ca ng knh vng r rng i vi ng knh thuc a c tnh ton cho mt so snh nh gi cc hot ng ca cc phn lp. C lp khc nhau trong phn ng ca h th nghim ny. Vic kim tra th cp l mt phng php nh lng c s dng xc nh cc hot ng enzyme ca phn lp. Ba phn lp, S2, S4, P1, so vi nhng ngi khc, cho thy tnh nht qun trong sn xut cao ca hai loi men gan v cc hot ng ca h endoglucanse 15.31U/ml, 13.11U/ml v 13.02U/ml trong khi hot ng polygalacturonaza 11.58U/ml, 12.12U/ml v 10.17U/ml tng ng. Ba phn lp ny sau c thit k da trn hnh thi hc nh Penicillium atrovenetum, Aspergillus flavus v Aspergillus oryzae. Penicillium v Aspergillus l mt trong nm cellulolytic nghin cu nhiu nht v pectin (Sukumaran et al, 2005; Favela-Torres et al. 2006). Mc d cc chng Penicillium sp. c l c bit n nhiu nht cho vic s dng trong ngnh cng nghip dc phm cho s tng hp ca penicillin v griseofulvin, Fawole v Odunfa (1992) trc y cho thy, Aspergillus, Fusarium, Penicillium v Rhizopus cho thy hot ng cao pectolytic. Chng Penicillium chrysogenum cng c bo co to ra lng ng k ca cc enzym cellulolytic (Nwodo-Chinedu et al, 2007). Hn hp cam b ma v cm la m l cht nn tt nht cho sn xut ca pectinase trong qu trnh ln men trng thi rn bng cch s dng mt sp Penicillium. (Silva et al, 2002). Cng nghip, s dng ca v cam bt c th c tnh kinh t cao. Dhillon et al. (2004) bo co vic s dng v cam chanh trong na rn qu trnh ln men sn xut pectinase. Cc thng s khc nhau trong qu trnh ln men trng thi hin ti vng chc ca v cam bao gm thi gian bnh, pH ca mi trng c bn, nhit bnh v ngun nit. Ba phn lp sn xut polygalacturonaza ti u vo ngy th 5 trong khi endoglucanase c sn xut ti u vo ngy mng 7. Cao nht sn xut c quan st polygalacturonaza v endoglucanase bi Penicillium atrovenetum ti 5 pH, 40oC v persulfate amoni 0,2%. Sn xut ti a polygalacturonaza v endoglucanase Aspergillus flavus c quan st thy pH 5,5, 40oC v persulfate amoni 0,25% trong khi Aspergillus oryzae sn xut hai enzyme ti a pH 5.5, 35oC v persulfate amoni 0,2%. ng ch l s so snh v mc enzyme sn xut bi cc sinh vt khc nhau trong thi gian th nghim khc nhau ca reseachers l khng n gin v iu kin vn ha khc bit v xc nh hot ng ca enzyme c s dng. Tuy nhin, Banu et al. (2010) trnh by quan st tng t cho sn xut polygalacturonaza Penicillium viridicatum, Trichoderma longibrachiatum sn xut s lng cao nht ca glucose vo ngy 7, pH 5 nhng glucose 45oC ti u, s dng Aspergillus niger c sn xut pH 4.5 vo ngy th 5 v 45oC trong khi s dng nm men Saccharomyces cerevisiae, ti u sn xut glucose c sn xut ti pH 4,5 vo ngy th 3 v 45oC (Omojasola v Jilani, 2008). Mrudula v Anitharaj (2011) bo co sn xut ti u ca pectinase v cam bi Aspergillus niger 50oC, pH 5, 96 gi, 1:2 (v / w) m, kch thc inoculums 2.5ml v trong s hin din ca sucrose v Triton- X-100. Thi gian khc nhau ca thi gian bnh ti u c th c bin minh bng vic np Patil v Dayanand (2006) rng thi gian ca qu trnh ln men ph thuc vo bn cht ca mi trng, sinh vt ln men, nng cc cht dinh dng v iu kin qu trnh sinh l. Hu ht cc loi nm iu tra cho sn xut pectinase cho thy s tng trng ti u trong khong 45 n 60oC (Freitas et al, 2006;. Rubinder et al, 2002). PH ca mi trng cng s gii hn s pht trin ca nn vn ha hay to nh hng khi hot ng xc tc ca enzyme. Sn xut polygalacturonaza ti a c quan st trong mi trng vi cc gi tr pH ban u c tnh axit trong mt phm vi ca 4 n 6 (Aminzadeh et al, 2007). Quan st trong nghin cu ny cho thy pH ti u cho cc enzym sn xut trong vng 5 n 5,5.

Ngun gc ca nit trong mi trng pht trin c mt vai tr rt quan trng trong tng trng vi khun v sn xut enzyme (Mrudula v Anitharaj, 2011). Ammonium persulfate h tr sn xut cc enzyme ca vi sinh vt cao nht trong nghin cu ny. iu ny l tng t nh bo co ca Banu et al, (2010). Penicillium chrysogenum. Cc nh nghin cu khc bo co nhiu ngun nit khc l tt nht, v d nh amoni sulfat (Patil v Dayanand, 2006), s kt hp ca chit xut nm men v amoni sulfat (Mrudula v Anitharaj, 2011), Amoni oxalate cho nit th hai tt nht persulfate ngun amoni sau y trong cng vic ny. 3. Kt qu Sng lc cho Cellulase v pectinase Sn xut Nm flavus c c vo ngy th 5 pH 5.5 ti 40oC vi persulfate amoni (0,25%) nh l ngun nit tt nht trong khi sn lng ti a ca endoglucanase thu c vo ngy th 7 pH 5.5 ti 40oC vi 0,25% persulfate amoni (hnh 3- 7). Aspergillus oryzae sn xut polygalacturonaza ti a vo ngy th 5 pH 5.5 35oC vi 0,2% persulfate amoni kt hp trong mi trng c bn trong khi sn xut endoglucanase ti a thu c vo ngy th 7 pH 5.5 35oC v cng vi amoni sulfat (0,2%) (Hnh 3-7). Trong nhng ngun nit th nghim, amoni oxalat cho kt qu tt nht th hai sau persulfate amoni. Mi ba phn lp nm thu c c th nghim ln u tin cho endoglucanase v sn xut polygalacturonaza trn a thch s dng carboxymethylcellulose v pectin cam qut tng ng nh cc ngun carbon duy nht. Cc chng c ch nh l O1, O2, O3, O4, O5, P1, P4, P5, S1, S2, S3, S4 v S5. C lp O3 khng pht trin trn c hai phng tin truyn thng sau khi 48 gi, trong khi c lp S1 ch tng trung bnh c cha cellulose. Colony v khu vc gii phng mt bng ng knh khng th no o cho c lp P5 do ch ca n tng trng. Gii phng mt bng khu vc ch s cho mi c lp c tnh bng cch chia ng knh ca gii phng mt bng khu vc bng ng knh ca thuc a (Hnh 1). By phn lp vi ch s khu vc kh cao gii phng mt bng cho c hai enzyme c sng lc trong mt ngp nc ln men cho c tnh nh ca endoglucanase polygalacturonaza tng hp v. Ba phn lp, S2, S4, P1, so vi nhng ngi khc, cho thy s nht qun cao ca h sn xut ca 2 enzyme v cc hot ng ca h endoglucanse 15.31U/ml, 13.11U/ml v 13.02U/ml trong khi hot ng polygalacturonaza 11.58U/ml, 12.12U/ml v 10.17U/ml (hnh 2). Ba phn lp c xc nh l Penicillium atrovenetum, Aspergillus flavus v Aspergillus oryzae theo hnh thi sau c la chn cho cc th nghim tip theo. Sn xut v Kho nghim Enzymes Thu c t Solid State th nghim ln men. Penicilium atrovenetum by t cc hot ng polygalacturonaza ti a vo ngy th 5 pH 5 40oC vi persulfate amoni (0.2%) nh l ngun nit tt nht. Hot ng endoglucanase ti a thu c vo ngy th 7 pH 5 40oC vi ammonium persulfate (0,2%) (Hnh 3-7). Sn xut ti a polygalacturonaza bi Aspergillus flavus c c vo ngy th 5 pH 5.5 ti 40oC vi persulfate amoni (0,25%) nh l ngun nit tt nht trong khi nng sut ti a ca endoglucanase thu c vo ngy th 7 pH 5.5 ti 40oC vi 0,25% ca amoni persulfate (hnh 3-7). Aspergillus oryzae sn xut polygalacturonaza ti a vo ngy th 5 pH 5.5 35oC vi 0,2% persulfate amoni kt hp trong mi trng c bn trong khi sn xut endoglucanase ti a thu c vo ngy th 7 pH 5.5 35oC v cng vi amoni sulfat (0,2%) (Hnh 3-7). Trong nhng ngun nit th nghim, amoni oxalat cho kt qu tt nht th hai sau persulfate amoni.