© asian Network for Scientific information 2000, Production Physiology of Cellulases and B-glucosidase Enzymes of Aspergillus niger Grown under S 0 State Fermentation Conditions M, Fadel Microbial Chemisty Department, National Research Center, Dokki, Cairo, Egypt (ec a 2 1/31) and fe gheandace O42 1 ‘anate eb insin aubatte, Cull 70%. twa itil pH 4S, inoculum ase 10% Substrate) aranced.snayme ectvtles, Shlry, witeat bien st 20% canbe suceertuly repented for four attra eye fer eptaning nigh eye vil Producton whan the. oct PH 4.5 55 and 45. The fect of tontmert wih sStlernt concentrations of regu hysroude on thos erhoncing the dstigiiewion ot whew! straw _sibrtate and endorsing moist eve, “Te fUngue expresses hgh neve producton fein} 9t'32°C ater T2h which yilded 91.5, 45.0 snd 218.2 1 Uig ‘Sipplmaniaton ofthe cuture. medurn wath, wrmaiom supe fat ive ot 20° my NG To posses snasgucsrase ECS TIN wrogucsnan Iheubeten” peed, intel pH, edbeton (wind wen the mest autabla eaten sours Ws I Ug. sbstate of Pare, CMs, ond Pati petition ith ‘aes ‘nevnaie hydrant ie socymesracion end mauldy autetate were comp ered with crude zyme aruact to hydrshae the allalrasted uhest stan The highest hyarsyes #20. “me” enyraie hydwelyate contaring. 69% gicees of toa niga Celiore isthe most abundant cabohyate palmer Wwostes tem forest prodet ture, rts and Vegete ies Thi ceulose palmer cull be converted In imple suger by snsymic"degradston These. sages con smanufacure of organic chericle (yu "ane. Mandel, the probian of fading new food sours for ou expanding Dopuleten by oneg mcterpenar en euch mati ot Economen wtimton ot rich igreeluone mene hindered beens of the high conte of procuce that ate, wich aelgnceluone mates, ‘There have Sgnocetutonce (Cowling, 1075). The development of ISGF) procese that requces reste fantan endo 14 Be gicen 1148.0: EC 3.21), oxo TAP gem (140 Ds collbiohyttace” EC 9.2.1.81); and Bi guider (EC 3.2121 BO-gheohyecles) or colonise. Thue the hydrelvle ef ealloee ie completed by he eyreciee ‘The ceulese ystems of ‘ypecelulce producing mutante of Techegarma reese’ are” gereatyeticnt Slucosiéne (Outt at a!, 1986) Ourng the hvdrohale Eeluore it the ype ot enmyme eter elton 2 celulice system hevng © roto of (> gucosare acuity lose to. 1.0 ie neceesary to cbtan the Fighe and highest gucone content in the the roducionphysnopy of the grou of enoymes by © fected sean of Aspeplur niger under candiion ot SSF Materials ané Methods Mict-organame: A. niger F-118 wo lctty lated trom decayed vest stan and. identied by lent Pathology potato destose agar POA) mudum Saccharomyces ‘erevinie AFZ. 990 ve inlted om sug cme, lee ‘htsined fom Mirobil Gnetc Lab. ©. Baye. “eqyot. The substte vat died at 108°C to constant Canight ad ground to 20 math we,of fungal inseam: oculum we prepared by ‘0.08 M ciate butfer pH 8, then shaked for 10 Iminutee at 200 tor before using for iocueen of the Cukure Condkions: A expeiments wae condicted 250m! conlet Haake contained 10° radia except in the cave of studying eect of te medium depth on the encyme ‘evlindrical botles, OTM ettete butter pH 48 wor used & here ured to aap the bud phase fo vous fovsen 9.07.0, when the fect. of pH on anevmes fof the fingus, The afect of dilret sonemtatons of firogen scuoee a wel ae the, addon of insu ‘Successive tame of femented subwurte: After enaymes etn, te ei nae an gid an ted mi O1 Molva buffer pHA8. and then shaking for 90 Iminutes at 180 rpm et 32°C, felowed by fikteton though cording to Mandel method dererbed by Habit 11981) ot a 974, Protein determination: Tote proten nthe tupematint wat rmessued by the method desebed by Loony eta 11981 ‘Ammonium sulphate procpkaion of enrymes: To aliquots ‘nother attarot the precbitaion wn conied ot in four ‘thoy ons note. caperted by contugetin ond wire Went sem dlteetin: What try as ground an ‘were tented with set concentrations of NaOH [1.1.53 Tevame and Osene, 1072: Reo eal, 1560). in another wes and glucosidase enzymes 20-40meeh naymatic ydicis of wheat stew by the fongel enzymes: Mystayes of aa ested whest srs ne Carried aut by hesbatrg 39 edbevate te form of 5% cxperimente did fermented mouey aubstete for abe rented heat senor tyarivele one chad out in 100. apacltyconleu ‘Res contained 6 akalh toned wheat fucose. The percent hydlvos was cakated from the hydra Weight of wiativested wheat eee (@) according to Sacro o (198 Ethanol podicton by & cores gronn on ontrmatc sith died fermented subaate (250 LFPaca) in 2. total Volume of one bt of O.08M cate butter. pH 48, at SO%C tor 48h on tomy shar 130 "om alter hyarave for dh by actualy growing yeast ld type Saccharomycer ‘vows and ribo bende glcore tor ethane! proavetion Results and Diseussion Production of ealdaer and §- gueosiare under varius otsure tvs: Modture content in sold sbstete ec eth sven and mutants eliity and euteity to be snaymee lal produced by the Angie, at wel ae the lwo) moiture levee, some ameunt of protein nae ‘60% twin) moltre more spectie 10 exoglucaaee and ‘oglucnnae. stiven, On the other hand. at 70%. ooh Imoisture the specie actvides were meteued by cbeut 5.9, 118 and 115% for FPaue, CMCare and ivconesee respactvaly The miture level demande fod state fermentation ttre cording to enyine tobe Produced, sunetut, mictcrgaier at wall at the porte fine of the subrtate ee wet os the confiqution of the Pertices (Mariaweran and Chrys, 1994 Nendakumer et {Ubeptinal gro, lee subevate sneling ane high witer Inia! msetsre content might be due to the stare (Gants and Lonsane, 1984), In gener) cur cbtoned nthe present eth agree th thse for ellese production In SSF by Shuma ot aFadel: Production physiology of celllases and D-lucosid poets Sige tee rene se 2: oct of iaitason pod en the Roninhene of cases and B guscadem arses by niger F118 sated on afte caces 1995 and Srite et a. (1986. Gupte and Medarwar for enzemet_producion offer the potential for inexpensie 11997) ‘ans Ginek ‘nd Garvie (1987) raportad tat an production a enzymes (Sony et a, 1995). incur Incrsae iit lets content of the subtle fom time _rnecesary for opti! produclon varied. Detwewt So-74% peody anhinced the fit paper sett, liferat ayes produced from one tubule Site tiect of incubation paid on dhe calases and Bs ghcoroce ven deteced_ after 24h, herons eigiicant ‘Guceslace eneymes” Monymbers: Short heubaton"pelod icteue war noted after Bh.” The ow ectvty” for fFadel: Production physiology of celllases and [-glicosdase enzymes Table: Stour of iaryaiannogen eases co ha Diccyehas of cluaice ad 8. guesses ensyres by niger AID uns eels Treg ser Poermaa Cares a Tasty ra ea mmuipo, 2% abe 208 see ta ° Te? hmrintin same aon erat wth adn cn te Henan of exces ard Be dscns by A. er sine wou Foon Pgimontern fom cows urease losin dutng fat 24h may be doe to the fect tat time needed for aneyme prediction see found to be shorter Srigial subavele contained "no carpaunde erlang in aold state fermentation thar the ewomerges eure Sensyme producton. The sctan of bot endogucenase Machin tat, 1980; Manes at af, 1992) afe ot al, (eitcare) ana ogluconae (Paes) cteng the ewty stges 1093). Endo ghemaze war cbtamec afer 58h by ‘ot growth tenited In celobces production thet can indice Trichoderma herianum Oxachamps etal, 1986). The B- lucerdece bognmene at leer stogee (Gotten at al, highest Miter wet” actity and eaborymetnicetula 1903). The seubaton me ropired forthe forason of sctviies of 427 ane 1208 Wig rapectvaly were Imcroorganam under shidy esciavoe et al, 1905, celloblawe acy that could obtned ater 8 cave Guoeui tnd Fide, 18091. On the other hand. meubatin {Muniewecan and” Charly, T8961 ‘On the sontay” Ohilonef aL (1996) btsned the masinur yield from CMC, QM 9414 nae catvated on soe stam The fact tat futures of Aepegiton app. potuce mere bet licence war repeded by many autre Mamet a tfect of pH vartion on the binymbese of cetlane ant B- uconilere eneymer:” The hydrogen ion concentation the stabity of exact eneynes at Wi parila find the wold denetuaton ot lower or higher pH wakes ellicees and Go gucondese The enzymes Monyntest Swe inhibited at both low pH under 4.0 and a high Shove 88. Great emancemant in enzyme. bleyntesle. wae Fecorded between pH tange 40 t0 5.5 and the otinum PH In SSF procemes a not uavely eftempte, probaly ome medum contuene i epioyed to. senate the fact of temporaure of inten on the hiceyeie of SMiaees and B- ghuceske enzymes: The election of Ineubtion tengereture ls guided by the optimum growt ‘synthesis of both endoscaace and D> glucosidase vas more inibited by low and hgh tempers (24 and ne recored for the production of the tee anne = the present study by AL aigerF=119, The vasa Lempereture mamtained In SSF ayeere, ie uty femployed for he. fementatan purpores Lancane ef a from thermostable Aaperiue ap vat prosuced at 37°C Maximum crises producion "wee cheered wen the SSF wae petfomed at 38 and 48°C "utng Pelli chrysogenum (Sheime, et al.” 1995). Tenpeetre for ‘ubstrates under SSF ware sided in the Reratire by many Invewigetre Woneane tal, 1986) Abdulih ot a, 1968). {ChiCuow sore romatheiy tect by Inoculum siete ww) Roca abe contls and. shortens the ap ents edd! ition! bariar together wath Hk wes and glucosidase enzymes decrease in groveh and ensyme production Muniwaran @ Iaximal enzyme prosocton in the present sudy agiee wif Ia onsitve to the nivogen scurce ard ntogen vel In the ced Invbtion was seen sth amon chloe, soluble proten 'p auberate vee reered wer” 250 Me foo of anmoriom suphste wae ineoduced wth the fe aouring to reed 21.9, 21.8 and 20% foivay cf Peco” CMCare end + ghicosdam (ucericese formation were inhtied “by the ah fulphate oar sown to fellate tre cette and. ‘Jucetdece production ia Pau)” Yunieuowum Mrothecium SP. Chectenums colldlyicum, Tichederra raesas A niger and A. tareus (ema ane Murphey. 1900 Effect of supplementation with adorn! caton sources Tatle 7 shove the afunce. of enrymes Seep, by Tevale namnly 10, 20 and. 20% tw Date show thet Ponder adden the highest P- glucosace was prose! ‘hen 20% com bran er vast bran vat wdded long ith adie Catenymethyelulcee (CoC) reeled in shout fwive) aad. recuted in 154 19.8% tncraaoe on CMCaae ation supplemented in 10 ard. 20%% (wi rerpectvly a Well ae the high ewete ln fh gluconante wee “16.7% hen” CMC supplemerted level wee, 20%1 fwin) Com bean Sed wheat bran at inv! 20% (win) ware found f9 be the most elective rutsent for promoting sty of hott Colllasse and f- gucosdare oe tw ieratee, were 408, 289 and 310% for fam, OMCs and pe guuconda bran wae supplemented. ‘Supplementtion of rutionts for Inducing entmenheeben tudes by mony vers. Te suttort amcunt of ‘nutients ané "nae. found to roman Toots sven in the mace rate, thereby provdng. Inge Sr al, 1906). A mitre of wheat etaw 60% and whest bran 20% awe ued for cobitee, production by T (Deschamps et al.” 1905). Yabo er al 1996), rented 105 fiw) wheat bran vith corn stover for T harsonum 912, .Fadel: Production physiology of celllases and [-glicosdase enzymes bisa ess ours ugtoncssn ut cole sugae She snes Pong Eas sey a coucaee Prpeatcnse Taleo: cftect of ai deh of matum op aha caluaat and P> gusslse ayes Nenwahess by ger F119 uns cohd xe 7 sure fpamn cane Bquccdone spare ue ‘a femision sisters ips Gomes Pgusssiney fae fiect of supplomeniation with sohble suger: Thee lve 1 2 ed 3% of fw supe namely, gone, celebione, isscer and ps guconese by A niger F118. Reaute = Table 8 chow ‘at the beeythene responded deer to the ensymer eidtond of ger ae well ae te rracallier pectin released. General, the extocelalar Drow averted inthe medium was mgher than con, ind the Dionne of el tented zyme wee nites SiigorsFPase production ves Induced by luce, raltore Sind wuroce, whorene, Chicane ves Inind by ghcore ne matoce. fo pucosdace war exited gh level of ctivity by addon 186 sucrose then ober levle ond other eed ‘eugre. Ting im eonidertion the specie scevty me Epectbealy promstng for benynthea of beth salons and B» aicolsee, Many investigators used doer ouble suger for inducing. cebloser and B= gucondae Bone and Munoz {1084} showed thet addon of gicess at levels Sane. 10% of raw vaight eimulned Ian ‘the other hand Sanya! et a. 11960) showed that low lve fof glucose (13) dil not raprace the symiesa of CUCaze, ut when ahecote wan adaed in the grant) median higher sonemurtont (Om ther utawion of CMC mise‘or promoting scivty ef the slicanace encyrne ae wet at ‘ilerone wan the mort ineicng ewan eautee for eeotee sucrose induced “calobiace beter than glcore, whereas ‘cov euerd Fare better than sucrose In ‘horse celebinee bgrymils. wos nies by rato ‘When. P.chnsogenum wee ctured on Sgroculaes fect of the medio depth: Doth it» potenti contig actor M1 SSF Haven ef wl, 1899), th hey festr & determing. te amount of rat tml awh oe find Ghery, 1886). Depth moy ase be portrt inthe Shaloner saevate. fle mare pronounced at” seater spine. Mestre gradients may out in deap tubeeater fon the growth of the metostganim wich In tum woud Colldores “and. ff cosdere | production hive” beat decrensed viare the enzyme yield had been reduced to t apectic cepih te move stale to prelong the phase = Wahish these enayrs bre bcnythesa ad rlesed. Te Fupenite, and pause see The eect of the pth of the {ubatrat an annyne production for SSF har not been wal GfHects of depth of the subset. In the evarinaon by 11960) the portance of depth Sacunted. were intly perormes on thn ede IO cml to. elm ‘maximum xylene hen elated 7. leghmehiten on Suocershe sukivetan on faented substi Eslatiog products not yet" saved. ‘The browse socesth ‘Sith reeyding the rendue of fomented, red afer wes and glucosidase enzymes promising quantson of FPare, CMCare and Piglucenden the fou successive cutvaien, Sagal incrace Pubetrate utienlon ary epht) wee noted rng each ‘stration to improve the callase ad - guceldae In SSF mised cuties of exllciie tng vet not le @ get erhercea eneyne yield (Sharale and” Steckontah, tte ct of exacting agent on the sneymer recovery tom the eed ewe eae: Table 11 shows the. vorout scent ‘enzyme tom microurgniam substete may depend an Iany tector e. 9 type of eneime. source of enzymes and Phosphate and scrote butfora [ph 5.9} were found mare ultatia for abha = ayiaeaeacton (Fenkor et a! 47960; Ondeer ot a, 1980), Fenanaezanore at al 1998 found thet O8M sodlim chide war mor subi for asd Srectansah 11067) soaked the mouley substate in water th for recovery of celulmee and Pltosidee. Inthe Present study irate bufler acetate buffer and. radu horde canbe repertag for celular and gicondase tram mouldy = redile wheat trav then dated water, ter Perel purifeaion with smmentn: slucowaes tom cre entct of AL niger F119. from sniphate: The step ana 8 for FPaco CMCose and fs gucondace rezpectily with Protein of Pace, CMCate end B ghcosdaee reapecvey setiviier wor cbtaned by using two stapes The fe recitation win sxturaton 20%. wv) ammonium fuiphate to gets of rome of unwanted. pices and (1 fs ueonaace wat ference in apes aches from 103 and 63-4 Wing protean In crude extect to 19.7 128 and T2465 Wing. protein Geioled in 01 M techn vate bulls pH 48 fer Fate, CMCure and f- gucesame Teapectvely. On te ath band hgh recoveien were tehieved ving 20-40%" ammeium —slghatesxtrston Chicase and fe slecaiace reopectly. One sep nee opine for parsl —puriowion of cablanes and ‘Jucendece by many workers. Eahty percent emontim St aL 199%) ‘and the resovey wae 70S for Care_ fe Bigucuiee tes cee 2 Perial puntemien fo clans ans Paucencees sututes producee by Ange F119 on tale mastm SSF ner cama sss sauce soos spss pga ch alias nat 5 ages CCharastaiaton of parity puifed ceubeer and tect of ineubation tompuatre on enymse actin fone scvty of Fase, CMC and fe ghemidese nee Setivity of glicostnce eshed radial incrnce from 440. “Cand. the maori acvity es eshibhed “wt 65°C fetivity wat studled by “many” investigors, Gag end 'S65C wan reperted for Strptormene ap CMCate,whatee05 and [- glucosid 3975 Gh 95 3003 Wo Fe a5 0 ‘temperature C F118 ron on rite under SSF 3o5 1 45 555 8 657 HH Fo. 2etfect of pH on pretty punted entonte and Ettact of hs The dats on he aac of diferent oH on the FPece, CMCate end §- gSucoridece we given in Fig 2 ChICoce wee. noted at pH 8:5. Patucciane expeced ie pH 45:55 (Gag nd Neslatatan, 1982, Shama et 199, Wheat stew byt: In deveosing = technalogy fx elldoce hyiralysie by ersyme, one of the mar fetes # cnsymes.entadt fcutis mpamatant} won te hysrolyee of 5% (wh) abat vested wheat saw it different skal concentatons ae rantioned in materale ‘tnd: methods Data we gen in Table 13. showed tht the Straw at 485%. sogum hydrovide ae the hysotele nae fhout BOS twin Uoing law at high concantton of Sodium hyerside wu used to prevent giocebilsie to frmore the exiles or hemicamione for hyéroveng Cneymes Chabal etal, 1996). The hyrcytic. potential (Wo%N of celuacer produced the present stody maybe ue 12 Meh fv _ghloncnee contents when prevented the ‘ccumulaon of enlbiens Inthe hyrcvnteconeequany {Chala 1996; Abavarn and Kur, 1987, Comparative rudy on_whnat stow hydiolyis with cre feraymes, paciaby pied” ensyme prpartion and wath mouldy.subsuate: Tale 14 shows the hydro ot ‘eligntig wheat evawr 148% NAOH!” vl crode enymoe fend: patialy purfed ammonium suphete presptated (20 440%) fracten se well se with moldy subetate es 2 couee ff the eraymer Renate show that emia hydchie coud fehieved by appeation of crude enzymes extct rmeniim euphatepreiitted —neymee nd mou Pfaniculnum for “hydching alll fewed suger cane egeese and” cbtamed — cmiaryerayaing poner from S8F for calles production with T. rene! on wheat tra war uiee for hyerovee of 55S (wi) degre Face ig. debqiied whet stam and the hyiroyc easel and. B-ghicosaaee fom A. nigar were used” eignfeston weriment the ate ot hyatiieracned 454% Paro oo. 1988), Under submerged feramnteton condons, the eonvantiond Strvmen se proiuced in dined soklons A ewe Teauemente te enw ‘serandng prcrses IB shoner re and coneequety tay Felp in reducing the ont ofprodvetin of thee eneyn[Ethanol production fem srxymatis hydtovate of fof araymate.hyarsate of aol Wanted whem ra we aiven im Table 16. Ethane! production fromm the Avérelyzmtes by tee prepaon of annrmee tom A. niger The tower carverson of sugar to acohal (24:4 to 93.2% 5! cerevsive SL cute due to the wt of the fate corevane BL yous to conver igure other then gucore & Sgnocelulnce to aleahol ao et af 1905) ould hydarse {or aeohal production” by. cerevisiae. They cbtaied 40: Acknowedgment The auton wish to sxprat his deep gratiudes to Prof. Or. A" Ek Roti Prot of mircilog and Neteenlogy end reveuone of the ate Reterances Abdutn. AL, ALP Tengeedy and VG. Murphy, 1965. AAbrahom, M. aed M4. 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