công nghệ sinh học và giống cây trồng

CễNG NGH SINH HC V GING CY TRNG 330 THIT K DềNG T BO CM NG N NH S DNG TRONG H THNG BIU HIN PROTEIN Nguyn Th Thu Hng1 ABSTRACT The ecdysone inducible system was generated for checking the the toxicity of target gene which was transfected into the cells The retinoid X receptor (RXR) was transfected into HEK293FT to generate the stable cell line pVgRXR-HEK293FT For checking the function, the cells transfected with pIND-GFP and induced by 1àM ponasteron A for GFP expression The fluorescence signal was detected in the transfected and induced cells informed the success of expriment TểM TT H thng biu hin gen cm ng ecdysone c thit k vi mc ớch kim tra nh hng gõy c ca gen ớch Yu t phiờn mó (RXR) c chuyn vo t bo HEK293FT to dũng t bo cm ng n nh cú tờn gi pVgRXR HEK293FT nh vo k thut chuyn gen bng húa cht kim tra hot tớnh, plasmid pIND-GFP cha protein ch th GFP c chuyn vo cỏc t bo ny v s dng ponasteron A cm ng biu hin gen ớch Tớn hiu fluorescence cỏc t bo c chuyn gen v cm ng bi ponasteron A ch s biu hin ca gen GFP T VN Vi mc ớch to nhng protein mong mun, cỏc h thng biu hin c s dng mt cỏch rng rói Tuy nhiờn, thnh cụng ca vic biu hin protein thng b gii hn bi vỡ cỏc nh hng gõy c ca protein ớch khc phc c nhng nhng gii hn ny, cỏc h thng cm ng c thit k nh h thng ph thuc tetracycline (Tet), promoter/receptor tng t Drosophila cm ng ecdysone, virus gõy u chut cm ng glucocorticoid (Ficca et al, 2004) H thng cm ng ecdysone cú hot tớnh c s thp, tớnh cm ng cao, di hot ng cao, ỏp ng nhanh, cú th tt khi loi b cht cm ng (Padidam et al, 2003) Bờn cnh ú, ecdysone l cht a lipid t nhiờn nờn cú th xuyờn qua tt c cỏc mụ v cú mt na thi gian sng thp nờn cho phộp cm ng chớnh xỏc v cú hiu lc (No et al, 1995; Oehme et al, 2005) Thờm vo ú, ecdysteroids khụng c v khụng cú nh hng ti cỏc chc nng sinh lý ca ng vt cú vỳ (No et al, 1996) Vỡ nhng lý ny, h thng promoter cm ng ecdysone c thit k cm ng biu hin cỏc tiu n v khỏc ca AchR nghiờn cu ny Thuc h thng ecdysteroid, ecdysone l hormoe steroid nhng cụn trựng thay lụng nh Drosophila melanogaster Khi nng ecdysone tng lờn, gen mó húa cho cỏc protein m u trựng cn v gõy s phng ca chromosome c biu hin.Trong phũng thớ nghim, ecdysone hoc cỏc cht tng hp tng t (munisterone A, ponasterone A), c s dng liờn kt vi th th v thỳc y quỏ trỡnh nh trựng hp n v phiờn mó th nht, th th X retinoid (RXR) v n v phiờn mó th 2, th th ecdyson ci bin (VgEcR), c thit k t mt promoter shock nhit ti thiu kt hp vi yu t ỏp ng ecdysone/ glucocorticoid (Lueers et al, 2000) Cu trỳc ny hot ng nh mt phc hp yu t phiờn mó, cm ng cho s biu hin gen ớch Trng i hc Hng c 331 Tuy nhiờn, vic chuyn c hai vector vo cựng mt t bo l mt yu t tr ngi ỏng k ca h thng ny Sau kt hp vo h gen, hot ng phiờn mó ca c hai yu t ny cú th b nh hng t trỡnh t genome vựng cỏnh, kt qu ca mt h thng khụng cú chc nng (Luers et al, 2000) NGUYấN LIU V PHNG PHP 2.1 Nguyờn liu Dũng t bo HEK293FT (Life Technology); Plasmid pIND-GFP (Add gene); Plasmid pVG-RXR (Invitrogen) 2.2 Húa cht Cỏc húa cht c s dng nghiờn cu ny u l nhng húa cht cú tiờu chun cht lng cao ca cỏc hóng cung cp ln nh Merck (c), Sigma Aldrich (c), Invitrogen (M), Carl Roth (c), Fermentas (c), VWR (c) 2.3 Phng phỏp 2.3.1 Nuụi cy t bo Cỏc t bo HEK 293FT c nuụi cy mụi trng Dulbecco's Modified Eagle (DMEM) Mụi trng nuụi cy c b sung thờm 10% huyt bo thai bũ (FBS), 2mM LAlanyl L- Glutamine, 1% cỏc amino acid khụng thit yu, 2.25 mM Na2CO3 v mM sodium pyruvate Phng phỏp nuụi cy v tỏch chit t bo c tin hnh theo hng dn ca Kỹpper- 2009 2.3.2 Thit k dũng t bo cm ng n nh pVgRXR HEK293FT Plasmid pVgRXR c chuyn vo t bo HEK293FT nh LipofectaminTM 2000 cỏc a 24 ging Mt ngy sau chuyn gen, cỏc t bo s c thu hoch bng trypsin, v chuyn vo cỏc a nuụi cy t bo 10cm vi s lng t bo khỏc (104, 5x104, 105TB/a) Hai ngy sau chuyn gen, Zeocin (200àg/ml) c s dng phõn lp cỏc t bo c chuyn gen Cỏc t bo c tip tc nuụi cy tun hỡnh thnh cm t bo v mụi trng nuụi cy t bo HEK293FT cha Zeocin (200 àg/ml) c thay ln/ 1tun Loi b cỏc a cha nhiu hn 50 cm t bo kim tra chc nng, plasmid pINDGFP c chuyn vo cỏc t bo mi c chuyn gen s dng cỏc a 96 ging bng phng phỏp s dng LipofectaminTM 2000 24h sau chuyn gen, cỏc t bo c b sung ponasteron A 1àM cm ng biu hin gen ớch (GFP) Quỏ trỡnh cm ng ny c thc hin hng ngy sut ngy Hai mu i chng c s dng: 1) cỏc t bo HEK293FT khụng chuyn gen; 2) cỏc t bo pVgRXR-HEK293FT c chuyn gen vi pIND-GFP nhng khụng c cm ng bi ponasteron A KT QU Sau tun nuụi cy, 21 cm t bo c hỡnh thnh trờn b mt nuụi cy (s liu khụng ch ra) Sỏu s 21 cm t bo ny cú tớn hiu mnh sau c chuyn gen vi pINDGFP v c cm ng vi ponasteron A 1àM Kt qu ca cm t bo s 11 v 16 c s dng lm kt qu i din 332 Khụng cú tớn hiu fluorescence no c tỡm thy mu i chng s 1-t bo HEK293FT khụng c chuyn gen (IC) Tuy nhiờn, tớn hiu fluorescence yu c tỡm thy cỏc t bo HEK293FT c chuyn gen pIND-GFP cm ng hoc khụng cm ng vi ponasteron A (IA, IB) Trong trng hp cỏc t bo pVgRXR-HEK293FT mi c hỡnh thnh (cm t bo 11 v 16 c s dng lm i din), tớn hiu fluorescence rt rừ c phỏt hin cỏc t bo chuyn gen vi pIND-GFP v cm ng bi ponasteron A 1àM (IIA, IIIA) Tuy nhiờn, ch hai cm t bo 11 (II) v 17 (s liu khụng ch ra) khụng tỡm thy tớn hiu fluorescence sau c chuyn vi pIND-GFP v khụng cm ng bi ponasteron A Ngc li, mt vi tớn hiu yu c tỡm thy cm t bo cũn li (cm t bo 16- IIIB) Thờm vo ú, cỏc tớn hiu fluorescence yu cng c tỡm thy cỏc t bo pVgRXR-HEK293FT khụng c chuyn gen pIND-GFP nhng c cm ng ponasteron A 1àM (IIC, IIIC) Figure 2: Kim tra chc nng ca dũng t bo cm ng n nh pVGRXR HEK293FT I: t bo HEK 293FT cell, II: cm t bo pVgRXR HEK293 FT 11, III: t bo pVgRXR HEK293FT 16 A: cỏc t bo c chuyn gen pIND-GFP v cm ng bi ponasteron A; B: cỏc t bo chuyn gen pIND-GFP nhng khụng c cm ng ponasteron A; C: Cỏc t bo khụng c chuyn gen nhng c cm ng bi ponasteron A S biu hin GFP yu c tỡm thy cỏc t bo HEK293FT c cm ng (IA) v khụng c cm ng (IB) ponasteron A Khụng tớn hiu fluorescence no c phỏt hin cỏc t bo khụng chuyn gen pIND-GFP nhng c cm ng bi ponasteron A (IC) Tớn hiu fluorescence mnh c tỡm thy cỏc t bo pVGRXR HEK293FT cm 11(IIA) v cm (IIIA) sau c chuyn gen pIND-GFP v cm ng bi ponasteron A Cỏc t bo chuyn gen khụng c cm ng ponasteron A, s biu hin gen GFP khụng c tỡm thy cm t bo 11(IIB) nhng th hin yu cm t bo 16 (IIIB) cỏc t bo pVgRXR HEK293FT khụng c chuyn gen pIND-GFP nhng c cm ng ponasteron A mt vi t bo biu hin gen GFP (IIC, IIIC) Hỡnh nh c chp t kớnh hin vi fluorescence CKX41 Scale bar: 100 àm; Expose time: 2.0s THO LUN chn lc dũng t bo n nh pVgRXR-HEK293FT, cỏc t bo c x lý Zeocin 200àg/ml cỏc cm t bo c phõn lp, tớn hiu fluorescence th hin s biu hin ca gen ớch GFP cỏc t bo c chuyn gen vi pIND-GFP v cm ng bi ponasteron A 1àM Kt qu ny phự hp vi bỏo cỏo ca Lueers v cng s nghiờn cu ny, sau chuyn pVG-RXR v pER-EGFP vo cỏc t bo CHO (Chinese hamster ovary) v nuụi cy 333 hỡnh thnh cỏc cm t bo v s dng Zeocin 100àg/ml phõn lp Cỏc tớn hiu fluorescence ch s biu hin ca gen ớch GFP cỏc t bo chuyn gen v s dng ponasteron A 1àM cm ng (Lueers et al, 2000) S xut hin ca tớn hiu fluorescence cỏc t bo HEK293FT chuyn gen pIND-GFP k c cm ng v khụng cm ng ponasteron A Kt qu ny cho thy s biu hin ca gen ớch GFP cú th thc hin iu kin bỡnh thng Thờm vo ú, s cm t bo thu thp c cú tớn hiu fluorescence yu sau chuyn gen vi pIND-GFP nhng khụng c cm ng ponasteron A Kt qu cho thy s rũ r ca h thng biu hin protein cm ng Ngay c ch mt s ớt gen ớch c biu hin, kt qu ny s tng theo cp s nhõn theo chu k phõn chia t bo Do ú, cm t bo ny b loi b v cm t bo khụng biu hin bt k tớn hiu fluorescence no sau chuyn gen vi pIND-GFP nhng khụng c cm ng ponasteron A, c gi li cho cỏc nghiờn cu sõu hn Kt qu ny chng t h thng biu hin protein cm ng ecdysone l mt cụng c hu hiu vic kim soỏt s biu hin ca gen ớch KT LUN Thit k thnh cụng dũng t bo cm ng n nh pVGRXR HEK293FT tip tc hng nghiờn cu ny, hai cm t bo thu thp c s c kim tra li chc nng v s dng biu hin cỏc gen mong mun Li cm n Nghiờn cu c thc hin ti Phũng Thớ nghim Sinh hc Phõn t, Trng i hc K thut Brandenburgische, Cottbus- Senftenberg, c TI LIU THAM KHO David No, Tso Pang Yao, and Ronald M Evan (1995), Ecdysone inducible gene expression in mammalian cells and transgenic mice, Proc Natl Acad Sci USA, Volume 93, pp: 3346-3351 Georg Hermann Lueers, Nicole Jess, Thomas Franz (2000), Reporter linked monitoring of transgene expression in living cells using the ecdysone inducible promoter system, European Journal of Cell Biology, Volume 79, pp: 653-657 I Oehme, S Boăsser, and M Zoărnig (2006), Agonists of an ecdysone-inducible mammalian expression system inhibit Fas Ligand- and TRAIL-induced apoptosis in the human colon carcinoma cell line RKO, Cell Death and Differentiation, Volume 13, pp:189201 Invitrogen (2006), LipofectaminTM 2000 user guider Kỹpper JH (2009): Lab Manual and SOPs Molecular Cell Biology Senftenberg Mirella L Meyer- Ficca, Ralph G Meyer Heike Kaiser, Alexandra R Brack, Reinhard Kandolf, Jan-Hainer Kuepper (2004), Comparative analysis of inducible expression systems in transient transfection studies, Analytical Biochemistry, Volume 334, pp: 919 334 PHN LP V TUYN CHN DềNG NM CHO SN XUT MEN RU BNG PHNG PHP SY THNG HOA Trnh Lan Hng1 ABSTRACT In this research, Five dominant mold and three dominant yeast strains were isolated and purified By screening test of saccharification efficiency, mold isolates were selected and designed as TB-M2, THM1, and DL-M By screening test of alcohol production, yeast strains were selected and designed as TBY, TH-Y and DL-Y The potential of mold and yeast strain were TB-M2 and DL-Y, identified as Mucor spp HLT01 and Saccharomyces cerevisiae, respectively for freeze dried starter culture Keywords: Rice wine, Saccharomyces cerevisiae, Mucor, freeze-dried starter TểM TT Kt qu nghiờn cu ó phõn lp c dũng nm mc v dũng nm men in hỡnh S tuyn c dũng nm mc cho kh nng ng húa tinh bt cao nht l TB-M2, TH-M1, v DL-M, v dũng nm men cho kh nng ru húa cao nht l TB-Y, YH-Y v DL-Y Dũng nm mc TB-M2 v dũng nm men DL-Y c nh danh s b tng ng vi Mucor spp HLT01 and Saccharomyces cerevisiae v l hai dũng c tuyn chn cho sn xut men ru theo phng phỏp sy thng hoa T khúa: Ru, Saccharomyces cerevisiae, Mucor, men ru ụng khụ, sy thng hoa T VN Vit Nam, ru np c sn xut t ngun ging vi sinh vt di cỏc dng bỏnh men lm ru Cỏc ngun ging chng ny nh hng ln n nng sut v cht lng ru Mt s bỏo cỏo iu tra cho thy cú rt nhiu loi nm men v nm mc tn ti cỏc bỏnh men ru S a dng v chng loi ph thuc vo vựng min, nguyờn vt liu, kinh nghim v mụi trng lm bỏnh men Bờn cnh cỏc h vi sinh vt cú ớch cho quỏ trỡnh lờn men ru, bỏnh men truyn thng cũn cú rt nhiu cỏc cht nhim bn v cỏc chng vi sinh vt gõy hi n sc khe ngi cng nh lm gim cht lng ru thnh phm Nguyờn nhõn ch yu ca bỏnh men khụng an ton l ti cỏc a phng, ngi dõn ch yu sn xut bỏnh men theo phng phỏp truyn thng vi hn hp cỏc dũng vi sinh vt khụng thun chng t cỏc loi lỏ v bỏnh men sn cú T ú, nghiờn cu ny thc hin nhm gii quyt cũn hn ch v cht lng kộm, khụng n nh v tui th ngn ca bỏnh men ru truyn thng, bng cỏch phõn lp v tuyn chn cỏc dũng vi sinh vt l nm men, nm mc hu ớch cho sn xut ru t cỏc mu bỏnh men thu thp Bc Trung Nam ng dng sn xut men ru theo phng phỏp sy thng hoa NGUYấN VT LIU V PHNG PHP NGHIấN CU 2.1 Nguyờn vt liu, thi gian, a im Cỏc mu bỏnh men ru truyn thng thu thp t Bc Trung Nam Nghiờn cu c tin hnh nm 2014 ti Phũng thớ nghim vi sinh thc phm, Trng i hc Los Banos, Philippines Trng i hc Hng c 335 2.2 Phng phỏp nghiờn cu 2.2.1 Phõn lp cỏc dũng nm thun chng in hỡnh Ly 10g mu men ru ó nghin nh cho vo 90ml nc mui sinh lý vụ trựng 0.85%, ng nht mu, pha loóng theo dóy s thp phõn v nuụi cy mụi trng thch khoai tõy PDA 30oC 48 gi Chn cỏc khun lc nm mc v nm men in hỡnh em cy chuyn trờn mụi trng thch nghiờng v nuụi cy 30oC 48 gi nhiu ln n thu c cỏc dũng thun 2.2.2 S tuyn cỏc dũng nm mc cú kh nng ng húa tinh bt Dch huyn phự ca cỏc dũng nm mc c cy vo 50g cm np ó lm ngui n nhit khong 40-45oC bỡnh tam giỏc v iu kin hiu khớ ngy Cỏc mu c em phõn tớch lng ng tng s v ng kh Dũng nm mc cho ch s ny cao c chn cho sn xut men ụng khụ 2.2.3 ỏnh giỏ hiu sut ng húa ca men nm mc ụng khụ Quy trỡnh sn xut men ru ụng khụ bng phng phỏp sy thng hoa c tin hnh theo quy trỡnh ca Dizon v cng s (2009) Cỏc mu men ụng khụ vi dũng nm mc n v hn hp sau nghin nh ln lt c cy u vo cỏc phn cm np 100g riờng r theo t l 1% Sau ngy iu kin hiu khớ, tt c cỏc mu c phõn tớch t l ng tng s v ng kh Mu men cho t l cao nht tng ng vi chng nm mc c la chn v ng dng sn xut ru 2.2.4 ỏnh giỏ kh nng lờn men ru ca cỏc dũng nm men Cỏc dũng nm men c cy riờng bit dng n v hn hp vo cỏc bỡnh lờn men c dng trờn c cht l cm vi chng nm mc c chn Hn hp c lờn men ym khớ 10 ngy, lc vo bỡnh vụ trựng, lng v xỏc nh hm lng cn 2.2.5 nh danh s b dũng nm cho sn xut men ụng khụ Cỏc dũng nm men v nm mc la chn c nh danh s b da trờn cỏc thớ nghim c trng v mụi trng, hỡnh thỏi, sinh lý v sinh húa theo phng phỏp ca Lodder (1970), Alexopoulus v cng s (1996), Kurtzman v Fell (1998), Cappuccino v Sherman (2001), Watanabe (2002) KT QU V THO LUN 3.1 Phõn lp cỏc dũng nm thun chng in hỡnh Kt qu phõn lp v chn la c dũng nm mc v dũng nm men in hỡnh Cỏc mu c dỏn nhón da trờn loi dũng nm v khu vc thu thp mu 3.2 S tuyn cỏc dũng nm mc cú kh nng ng húa tinh bt ỏnh giỏ kh nng ng húa ca cỏc dũng nm mc da trờn t l hm lng ng tng s v ng kh Kt qu s tuyn c dũng cú ký hiu TB-M2, TH-M1, v DL-M l cho kh nng ng húa tinh bt cao nht 336 3.3 ỏnh giỏ hiu sut ng húa ca men nm mc ụng khụ T dũng nm mc tim nng c la chn, to c mu men theo phng phỏp sy thng hoa cú cha dũng nm n v nm hn hp Sau thớ nghim, hai mu men tng ng cha dũng nm TB-M2 v TH-M1 c ỏnh giỏ l tim nng nht mu men cho sn xut ru Bng Hm lng ng tng s v ng kh ca cỏc dũng nm mc sau ngy NG TNG S NG KH DềNG NM MC (% cht khụ) (% cht khụ) a Dũng nm n TB-M2 44.13 0.03 33.01 0.03b b TH-M1 38.70 0.02 20.23 0.04c c DL-M 17.35 0.04 9.28 0.03d Hn hp cỏc TH-TB - M TH-DL M dũng TB-DL - M TB-TH-DL M 22.24 0.04d 21.82 0,03e 21.47 0.01f 16.99 0.02g 16.24 0.01a 17.45 0.03e 15.76 0.06a 11.00 0.04f Giỏ tr trung bỡnh cựng mt ct cú cựng ch cỏi khụng khỏc bit ỏng k ti mc ý ngha 0.05 3.4 ỏnh giỏ kh nng lờn men ru ca cỏc dũng nm men Hm lng cn to bi ba dũng nm men riờng r trờn c cht t nm mc TH-M1 tng i thp ch t 11%, t l ny thp hn so vi t l c to trờn c cht t nm mc TB-M2 Hm lng cn to nhiu nht sau quỏ trỡnh lờn men l 13% t mu ng dng dũng nm men DL-Y trờn c cht ca dũng nm mc TB-M2 Do ú dũng nm mc TB-M2 v dũng nm men DL-Y l hai dũng c tuyn chn cui cựng ng dng sn xut men ru theo phng phỏp sy thng hoa v quy trỡnh sn xut ru Bng Hm lng cn to bi cỏc dũng nm men khỏc MU NM HM LNG CN (%) Nm mc Nm men TH M1 TB Y 11.0ac TH Y 9.0b DL Y 11.0ac TB M2 TB Y 12.0ac TH Y 10.0ac DL Y 13.0d Giỏ tr trung bỡnh cú cựng ch cỏi khụng khỏc ỏng k ti mc ý ngha 0.05 3.5 nh danh s b dũng nm cho sn xut men ụng khụ 3.5.1 nh danh s b nm mc ký hiu TB-M2 Khun lc ca TB-M2 cú mu trng mi xut hin sau ú chuyn nõu, dng bụng, phỏt trin rt nhanh, ng kớnh sau ngy nuụi cy a petri nhit phũng lờn ti 68-81 cm Si nm khụng cú vỏch ngn v r gi Giỏ nang phõn nhỏnh, sut, nhn, cú ch phỡnh ra, kớch thc giỏ nang l 57,91 x 3,28 àm (26,67-106,68 x 1,14-7,62 àm, n = 10) 337 Tỳi bo t hỡnh bu dc n hỡnh cu, cú tr bo t, sut, nhn, kớch thc 20.00 àm x 18.59 àm (11,43-38,10 àm x 11,43-34,29 àm, n = 10) (Hỡnh 1) ú l cỏc c trng v hỡnh thỏi ca dũng nm mc Mucor sp v c t tờn l Mucor sp HLT01 3.5.2 nh danh s b nm men ký hiu DL-Y Dũng nm men DL-Y mang nhng c tớnh thuc h Saccharomycetoideae, chi Saccharomyces v loi cerevisiae V hỡnh thỏi, nú hỡnh thnh bo t tỳi cú cha 1-4 bo t, khụng hỡnh thnh si nm õy l loi nm cú kh nng ng húa ng succrose, maltose, melibiose nhng khụng ng húa raffinose Hỡnh (1A) c tớnh hỡnh thỏi ca dũng nm mc Mucor sp.HLT01; (1B) S xỏc nh loi nm men thuc chi Sacchromyces 1A 1B KT LUN Phõn lp v s tuyn bc u ó chn c dũng nm mc v dũng nm men in hỡnh da trờn kh nng ng húa tinh bt v kh nng chuyn húa to cn Trong ú cú dũng nm mc tim nng ng dng lờn men ru l TB-M2 v TH-M1 Kt qu cui cựng tỡm dũng nm phự hp nht ng dng vo sn xut men ru ụng khụ bng phng phỏp sy thng hoa, gm mt dũng nm mc ký hiu TB-M2 cho hm lng ng tng s v ng kh cao nht tng ng t 44.13% v 33.01% c nh danh s b l Mucor sp HLT01 v mt dũng nm men ký hiu DL-Y cho t l cn cao nht t 13% c nh danh l Saccharomyces cerevisiae 338 TI LIU THAM KHO Alexopoulos CJ, Mims CW, Blackwell M (1996) Introductory Mycology 4th ed John Wiley and Sons, Inc., N.Y., USA 869 pp Cappuccino JG, Sherman N (2001) Microbiology: A laboratory manual 6th Ed Pearson Education Inc., San Francisco Boston New York, USA Dizon EI, Del Rosario OM, Romero MV, Bandonill EH, Morales AV (2009) Standardization of Starter Culture for Rice Wine (Tapuy) Processing Annual Report PHILRICE/UPLB Dizon EI, Del Rosario OM, Lizardo RCM, Tabion MJ, Ibanez VIC (2013) Establishment of Microbial Succession of Starter Culture for Rice Wine (Tapuy) Processing Terminal Report TECHNICOM-DOST and UP Los Banos Dung NTP, Rombouts FM, Nou MJR (2005) Development of defined mixed-culture fungal fermentation starter granulate for controlled production of rice wine Innov Food Sci Emerg Technol., 6, 429-441 Kurtzman CP, Fell JW (eds) (1998) The Yeasts, a Taxonomic Study 4th ed Elsevier Science B.V., Amsterdam, The Netherlands 1035 pp LEE AC, FUJIO Y 1999 Microora of banh men, a fermentation starter from Vietnam World Journal of Microbiology and Biotechnology 15: 57 62 Lodder J 1970 The Yeasts, A Taxonomic Study 2nd ed North-Holland Publishing Co., Amsterdam, The Netherlands 339 PHN LP V TUYN CHN CC CHNG VI KHUN NI SINH Cể KH NNG C NH M, PHN GII LN, TNG HP IAA T CY LA Phm Th Hi1, Nguyn Th Sn1, Nguyn Quang Thch1 ABSTRACT Currently, application of endophytic bacteria which has capable of nitrogen fixing, phosphate solubilizing, IAA synthesizing is one of effective solutions in agricultural production Using this method, chemical fertilizers are reduced; the environment is fresh and production cost is saved Besides, the yield and quality of crops are also increased In this research, 30 endophytic bacterial strains were isolated on RMR medium from stems, leaves and roots of rice in Gialam, Hanoi The results indicated that three endophytic bacteria strains N6, N15, N26 with the best ability of nitrogen fixing, phosphate solubilizing and IAA synthesizing were selected All three strains showed the highest activity at pH=7 after days inoculation At both 32oC and 37oC, the ability of nitrogen fixing is the best, while the optimum temperature for phosphate solubilizing is at 32 oC and IAA synthesizing is at 37oC Keyword: nitrogen fixing, phosphate solubilizing, IAA synthesizing, rice endophytic bacteria TểM TT S dng cỏc chng vi khun ni sinh cú kh nng c nh m, phõn gii lõn v tng hp indole-3-acetic acid (IAA) l mt nhng bin phỏp cú hiu qu sn xut nụng nghip hin Bin phỏp ny gim ti vic s dng quỏ nhiu phõn húa hc, khụng gõy ụ nhim mụi trng, tit kim chi phớ sn xut nhng m bo cht lng ng thi tng nng sut cõy trng Trong nghiờn cu ny, 30 chng vi khun ó c phõn lp trờn mụi trng RMR (Rennie medium supplemented with rice extract and malate) t thõn, lỏ, r ca cỏc ging lỳa trng ti Gia Lõm H Ni Kt qu cho thy, ba chng N6, N15, N26 cú cỏc kh nng c nh m, phõn gii lõn v tng hp IAA tt nht C chng ny u th hin cỏc hot tớnh núi trờn cao nht sau ngy nuụi cy v ti pH =7 nhit 32oC v 37oC, cỏc chng ny u cú kh nng c nh m mnh nht Tuy nhiờn, s phõn gii lõn mnh nht din 32oC v tng hp IAA mnh nht 37oC T khúa: vi khun ni sinh lỳa, c nh m, phõn gii lõn, tng hp IAA T VN gim ti vic s dng quỏ nhiu phõn húa hc sn xut lỳa, vic s dng cỏc chng vi khun ni sinh cú cỏc kh nng c nh m, phõn gii lõn v tng hp IAA l mt nhng bin phỏp cú hiu qu m khụng gõy ụ nhim mụi trng, tit kim chi phớ sn xut nhng m bo cht lng ng thi tng nng sut cõy trng Vi khun ni sinh l vi khun tri qua phn ln vũng i cõy trng (Quispel, 1992) Vi khun ni sinh cú th c xem nh l vi khun trung mụ thc vt m khụng cú nhng du hiu nhim bnh hay hu qu tiờu cc i vi cõy ch (Schuluz v Boyle, 2005) Trờn th gii, nhiu vi khun ni sinh c tỡm thy r, vựng r v thõn cõy lỳa nh Azospirillum sp., Burkholderia sp., Enterobacter sp., Herbaspirillium sp., Klebsiella sp(Koomnok et al., 2007; Mano v Morisaki, 2008) Vit Nam, c bit vựng ng bng sụng Cu Long ó cú nhiu nghiờn cu v ng dng vi khun ni sinh cõy lỳa nh phõn lp c Burkholderia vietnamiensi, Azospirillum lipoferum t cõy lỳa trng Nam Vit Nam (Van et al., Hc vin Nụng nghip Vit Nam 497 1994; Cao Ngc ip et al., 2007) Tuy nhiờn, vic nghiờn cu v cỏc loi vi khun ni sinh hu ớch trờn cõy lỳa trng ng bng sụng Hng cha c trung nghiờn cu VT LIU V PHNG PHP 2.1.Vt liu Thõn, r lỏ mu lỳa ti Gia Lõm Húa cht: Cn, HgCl2, H2O2, Mg(SO4)2, KH2PO4, K2HPO4, CaCl2, NaOH, Na2SO4, (NH4)2SO4, Ca3(PO4)3, 2.2 Phng phỏp nghiờn cu 2.2.1 Phõn lp cỏc chng vi khun Chn cỏc mu lỳa ang giai on tng trng mnh Sau ú cõy lỳa di vũi nc chy cho sch t bỏm thõn v r, bo qun 40C Sau ú ct ri thõn, lỏ v r thnh tng on nh, kh trựng b mt thõn, lỏ, r ln lt bng cn 70% phỳt, sch bng nc ct vụ trựng Tip n cho HgCl2 0.1%, lc nh phỳt, sch bng nc ct vụ trựng Lm tng t vi hydrogen peroxide (H2O2) 3%, sau ú sch ln bng nc ct vụ trựng kim tra vi sinh vt cũn sút li trờn b mt thõn, lỏ, r cõy lỳa sau kh trựng: 200 àl nc ct vụ trựng ó mu ln cui cy lờn cỏc a cha mụi trng Tryptone Yeast extract glucose agar v 300C 24 gi Nu sau 24 gi, cỏc a mụi trng ny khụng xut hin cỏc khun lc thỡ cỏc mu thõn lỏ r ó kh trựng t yờu cu Mu thõn, lỏ v r sau ó kh trựng cho vo ci vụ trựng, gió nhuyn, thờm 1ml nc ct vụ trựng vo ci, trn u v hỳt dch trớch mu cho vo tube 1,5ml vụ trựng Hỳt 50àl dch trớch mu cho vo a mụi trng RMR sau ú dựng que chang chang u lờn mt thch, 300C khong 1-2 ngy Sau 1-2 ngy, chn cỏc khun lc (khỏc v hỡnh dng, mu sc, kớch thc) t a mụi trng trờn cy chuyn nhiu ln n cỏc khun lc ri Cy cỏc chng vi khun ó thun vo ng nghim cha mụi trng RMR c v gi 40C (Cao Ngc ip, 2011) 2.2.2 Xỏc inh kh nng c nh m bng phng phỏp Indophenol blue Hỳt 0,5ml phn dch sau ly tõm dch nuụi vi khun cho vo cỏc ng nghim ó cha sn 2ml nc ct kh trựng cng vi 0,5 ml EDTA Thờm 1ml dung dch Phenol nitroprusside v 2ml dung dch Sodium hypocloride vo mi ng, trn u dung dch bng mỏy Vortex n nh nhit phũng khong 30 phỳt Sau ú tin hnh o OD bc súng 636nm (OD636nm) Kt qu o OD ca cỏc dũng vi khun c thay vo phng trỡnh th ng chun, t ú suy c hm lng ammonium sinh dung dch (Page et al., 1982) 2.2.3 Xỏc nh kh nng hũa tan lõn bng phng phỏp acid ascorbi Hỳt 0,5ml phn dch sau ly tõm dch, nuụi vi khun cho vo cỏc ng nghim ó cha sn ml nc ct kh trựng Sau ú nh 4ml hn hp thuc th (H2SO4 5N, K(SbO)C4H4O6.1/2H2O, (NH4)6Mo7O24.4H2O,Acid Ascorbic 0,1M), trn u dung dch bng mỏy Vortex n nh nhit phũng khong 30 phỳt Sau ú tin hnh o OD bc súng 880 nm (OD880nm) Kt qu o OD ca cỏc dũng vi khun c thay vo phng trỡnh 498 th ng chun, t ú suy c hm lng PO43- dung dch (Murphy and Riley, 1977) (Cao Ngc ip, 2011) 2.2.4 Xỏc nh kh nng tng hp IAA ca vi khun bng phng phỏp Salkowski Hỳt 1ml phn dch sau ly tõm lnh (40C) dch nuụi vi khun cho vo cỏc ng nghim cha sn 2ml thuc th Salkowski (H2SO4 10,8M v FeCl3), trn u bng mỏy vortex hn hp trờn ti 10-15 phỳt phn ng xy hon ton Tin hnh o OD bc súng 530nm (OD530nm) Kt qu o OD ca cỏc dũng vi khun c thay vo phng trỡnh th ng chun IAA, t ú suy c hm lng IAA sinh dung dch (Glickmann v Dessaux, 1995) 2.2.5 Kho sỏt nh hng ca thi gian nuụi cy, nhit , pH n hot tớnh c nh am, phõn gii lõn, tng hp IAA ca cỏc chng vi khun tuyn chn Cỏc chng vi khun c nuụi cy trờn cỏc mụi trng Burk khụng m, NBRIP, King B 1; 2; 3; 4; 5; ngy, cỏc di nhit 25; 32; 37; 450C, cỏc di pH, 5; 6; 7; 8; lc 120 vũng/phỳt Sau ú tin hnh kim tra cỏc hot tớnh c nh m, phõn gii lõn, tng hp IAA ca cỏc chng vi khun tuyn chn 2.2.6 Kho sỏt nh hng ca mụi trng, nhit , pH n kh nng sinh trng, phỏt trin ca cỏc chng cú kh nng c nh m, phõn gii lõn, tng hp IAA cao Cỏc chng vi khun c nuụi cy trờn loi mụi trng khỏc Nfb, King B, Fallik cỏc di nhit 25; 32; 37; 450C, cỏc di pH 5; 6; 7; 8; 9, lc 120 vũng/phỳt Tin hnh o OD 660nm sau 48h nuụi cy kim tra kh nng sinh trng ca cỏc chng vi khun tuyn chn KT QU 3.1 Phõn lp cỏc chng vi khun T thõn, lỏ r ca cỏc mu lỳa ó phõn lp c 30 chng vi khun trờn mụi trng RMR gm 13 chng t mu r, 10 chng t thõn v chng t mu lỏ c im khun lc v t bo ca cỏc chng c th hin di bng sau Bng 1: c im khun lc v t bo ca cỏc chng vi khun phõn lp c TT 10 Chng N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 Ngun phõn lp Lỏ Lỏ Lỏ Lỏ Lỏ Lỏ Lỏ Thõn Thõn Thõn c im t bo c im khun lc Kớch thc Trũn u 1,5 Trũn u 1,5 Trũn u Trũn u Khụng u 1,5 Trũn u Trũn u Trũn u Trũn u Khụng u Hỡnh dng B mt khun lc Trng trong, cú nhõn Trng trong, nhy Trng c,cú nhõn Trng c Trng c Trng trong, nhy Trng c, cú nhõn Trng Trng c Trng 499 Mộp khun lc Bng phng Bng phng Bng phng Bng phng Nhn Bng phng Bng phng Nhn Bng phng Bng phng Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 N22 N23 N24 N25 N26 N27 N28 N29 N30 Thõn Thõn Thõn Thõn Thõn Thõn Thõn R R R R R R R R R R R R R Trũn u Khụng u Trũn u Khụng u Trũn u Trũn u Trũn u Trũn u Trũn u Trũn u Trũn u Trũn u Trũn Trũn u Trũn u Trũn u Trũn u Trũn Trũn trũn u 1,5 2,5 1,5 1,5 1,5 1 1,5 1,5 1,5 2 1,5 1,5 Trng c Trng Trng Trng c Trng c Trng c,cú nhõn Trng trong, nhy Trng c Trng c Trng c,cú nhõn Trng Trng trong, lan Trng Trng c Trng c, lan Trng c Trng trong, cú nhõn Trng trong, nhy Trng c,cú nhõn Trng c Nhn Bng phng Nhn Bng phng Bng phng Nhn Bng phng Nhn Bng phng Nhn Nhn Bng phng Bng phng Bng phng Bng phng Nhn Bng phng Bng phng Nhn Nhn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Que ngn Hỡnh 1: Hỡnh nh khun lc ca chng N6, N26 3.2 Kh nng c nh m ca cỏc chng vi khun T 30 chng vi khun phõn lp c ó tuyn chn c 19 chng cú kh nng c nh m Cú 5/19 chng cú kh nng tng hp NH4+ mnh l chng N15 (1,797mg/l), N26 (1,526mg/l), N9 (1,429mg/l), N28 (1,243mg/l), N2 (1,187mg/l)/l Hỡnh 2: th biu hin hm lng NH4+tng hp ca cỏc chng vi khun Hỡnh 3: Kh nng tng hp NH4+ca cỏc chng vi khun 500 3.3 Kh nng phõn gii lõn ca cỏc chng vi khun Kt qu kho sỏt kh nng phõn gii lõn ca 30 chng vi khun cho thy 19 chng cú kh nng phõn gii lõn 19 chng vi khun u cú kh nng phõn gii lõn ú cú cỏc chng N6, N26, N23, N21 cú kh nng phõn gii lõn mnh nht tng ng l 7,784mg/l; 6,992 mg/l; 6,014mg/l; 4,197mg/l Kt qu ny thp hn rt nhiu so vi nghiờn cu ca Lng Ngc Du (2006) v cỏc chng vi khun ni sinh phõn lp t thõn v r cõy lỳa cao sn trờn mụi trng Nfb vi lng lõn hũa tan cao nht t ti 1334,51mg/l Hỡnh 4: Hm lng PO43-phõn gii ca cỏc chng vi khun Hỡnh 5: Kh nng phõn gii lõn ca cỏc chng vi khun 3.4 Kh nng tng hp IAA Nhn thy, cỏc chng vi khun u cú kh nng sinh tng hp IAA, hm lng IAA sinh dao ng t 3,75 n 14,67mg/l Cỏc chng N15, N26, N6 cú hm lng IAA mnh nht tng ng vi 14,67mg/l;13,52mg/l; 12,92mg/l Kh nng sinh tng hp IAA ca nhng chng vi khun phõn lp c thp hn cỏc chng vi khun phõn lp c t r v thõn cõy lỳa phõn lp trờn mụi trng Nfb ca Lng Ngc Du (2006) (15,61mg/l) Hỡnh 6: Hm lng IAA tng hp ca cỏc chng vi khun 3.5 nh hng ca thi gian nuụi cy, nhit , pH n kh nng c nh m, phõn gii lõn v tng hp IAA ca cỏc chng vi khun tuyn chn 3.5.1 nh hng ca thi gian nuụi cy n kh nng c nh m, phõn gii lõn v tng hp IAA ca cỏc chng vi khun tuyn chn 501 Thi gian nuụi cy cú nh hng ln n kh nng sinh c nh m, phõn gii lõn v tng hp IAA ca chng vi khun Hot tớnh ca cỏc chng vi khun tng theo thi gian t ngy th hai n ngy th sau ú li gim dn v t giỏ tr cao nht ngy th Hm lng NH4+ tng hp ca chng N15 t 1,807mg/l v chng N26 t 1,529mg/l Hm lng PO43-phõn gii ca chng N6 t 7,907 mg/l, chng N26 t 7,050 mg/l Hm lng IAA ca chng N6 t 13,58 mg/l, chng N15 t 15,42, chng N26 t 14,50mg/l Hỡnh 7: nh hng ca thi gian n kh nng tng hp NH4 + ca cỏc chng vi khun tuyn chn Hỡnh 8: nh hng ca thi gian n kh nng phõn gii lõn ca cỏc chng vi khun tuyn chn Hỡnh 9: nh hng ca thi gian n kh nng tng hp IAA ca cỏc chng vi khun tuyn chn 3.5.2 nh hng ca nhit n kh nng c nh m, phõn gii lõn, tng hp IAA ca cỏc chng vi khun tuyn chn Kt qu nghiờn cu cho thy nhit cú nh hng ti kh nng c nh m, phõn gii lõn, tng hp IAA ca cỏc chng vi khun Nhit i thớch hp cho kh nng c nh m ca chng N15 l 320C, chng N26 l 370C Cỏc chng cú kh nng phõn gii lõn mnh nht ti 320C Ba chng vi khun tng hp IAA cao nht 370C Hỡnh 10: nh hng ca nhit n kh nng c nh m ca cỏc chng vi khun tuyn chn Hỡnh 11: nh hng ca nhit n nng phõn gii lõn ca cỏc chng tuyn chn 502 Hỡnh 12: nh hng ca nhit n kh nng tng hp IAA ca cỏc chng vi khun tuyn chn 3.5.3 nh hng ca pH n kh nng c nh m, phõn gii lõn v tng hp IAA ca cỏc chng vi khun tuyn chn T th ta nhn thy c ba chng vi khun u cú kh nng nng c nh m, phõn gii lõn v tng hp IAA mnh nht ti pH =7, nng NH4+ ca chng N15 t 1,714mg/l, chng N26 t 1,524mg/l Nng PO43- phõn gii ca chng N6 t 5,456mg/l, chng N26 t 6,973mg/l Nng IAA tng hp ca cỏc chng t c ln lt l N6: 12,42mg/l, N15: 14,92mg/l, N26: 13,83mg/l Hỡnh 13: nh hng ca pH n kh nng c nh m ca cỏc chng vi khun tuyn chn Hỡnh 14: nh hng ca pH n kh nng phõn gii lõn ca cỏc chng vi khun tuyn chn Hỡnh 15: nh hng ca pH n kh nng tng hp IAA ca cỏc chngvi khun tuyn chn 503 KT LUN Trờn mụi trng RMR, t cỏc b phn r, thõn, lỏ lỳa ó phõn lp c 30 chng vi khun ni sinh gm 13 chng t mu r, 10 chng t thõn v chng t mu lỏ ó ỏnh giỏ c kh nng c nh m, phõn gii lõn, sinh IAA ca cỏc chng phõn lp c v tuyn chn c chng cú cỏc hot tớnh c nh m, phõn gii lõn, sinh IAA cao nht: N6, N15, N26 C chng u cho hot tớnh c nh m, phõn gii lõn, tng hp IAA cao nht sau ngy nuụi cy, pH =7 Cỏc chng u c nh m mnh nht tai 320C v 370C, phõn gii lõn mnh nht ti 320C, tng hp IAA mnh nht ti 370C LI CM N Chỳng tụi xin chõn thnh cm n GS.TS Cao Ngc ip ó h tr phng phỏp nghiờn cu cụng trỡnh c hon thnh TI LIU THAM KHO Cao Ngc ip, Phm Th Khỏnh Võn v Lng Ngc Du, (2007) Phỏt hin vi khun Azospirillum lipoferum ni sinh cõy lỳa cao sn (Oryza sativa L.) trng ng bng sụng Cu Long Bỏo cỏo khoa hc Hi ngh ton quc Nghiờn cu c bn khoa hc s sng, Quy Nhn 10-08-2007, NXB KH-KT, trang 456-459 Cao Ngc ip, (2011) Vi khun ni sinh thc vt (Endophytic bacteria) NXB i hc Cn Th, trang 1-48 Lng Ngc Du, (2006) ỏnh giỏ kh nng c nh m, hũa tan lõn v tng hp IAA ca vi khun sng r v thõn lỳa mỳa c sn ng bng Sụng Cu Long Lun tt nghip thc s ngnh Cụng ngh sinh hc, Trng i hc Cn Th Glickmann, E and Y Dessaux, (1995) A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria Appl Environ Microbiol, 61(2):793-796 Murphy, J.;Riley, J R.Amodified, (1977) Single solutionmethod for determination of phosphate in natural waters Anal Chem.,27,31-36 the Page, L., R.H Miller and R.D Keeney, (1982) Methods for Soils Analysis, Part 2: Chemical and Microbial properties, 2nd edition American Society of Agronomy Incorporation USA Quispel A, (1992) A search of signal in endophytic microorganisms, In: Verma, DPS (Eds.) Molecular Signail in Plant-Microbe Communications, CRS Press, Boca Raton, FL p.475-491 Schuluz B v Boyle C, (2005) The endophytic continuum Mycol Res 109: 661-686 Van, T.V., O Berge, S Ngo Ke, J Balandreau and T Heulin (2000), Repeated beneficial effects of rice inoculation with a strain of Burkholderia vietnamiensis on early and late yield component in low fertility sulphate acid soils of Viet Nam Plant Soil, 218:273-284 504 PHN LP V TUYN CHN CC CHNG NM MC ASPERGILLUS SPP SINH TNG HP ENZYME INVERTASE Phm Thựy Trang1, Nguyn Th Thu1, Nguyn c Thỏi1, Thỏi Th H Phng1, ng Quang Cnh1 ABSTRACT Invertase (-fructofuranosidase E.C.3.2.1.26) catalyzes the hydrolysis of sucrose to glucose and fructose so it is mainly used in the food industry This study was conducted to isolate and screen fungal strains Aspergillus spp which have invertase activity and study some biochemical characterization of invertase enzyme From different samples, we isolated 16 fungal strains which have the ability synthesis invertase enzyme with O1, L1.2 expressing highest activity Strains O1 and L1.2 produced high levels of invertase enzyme under optimized culture conditions at an optimum temperature 35oC, pH 6,5 with ure as nitrogen source and sucrose as carbon source Enzyme invertase of strain O1 exhibited optimum at temperature 50oC, pH 5,5; strain L1.2 exhibited optimum at temperature 40 oC, pH This research showed that the activity of this enzyme increases as additional Mn2+ Key word: Invertase enzyme, Aspergillus spp., , enzyme activity, TểM TT Invertase (-fructofuranosidase E.C.3.2.1.26) l enzyme cú kh nng xỳc tỏc phn ng thy phõn sucrose thnh glucose v fructose nờn c ng dng nhiu cụng nghip thc phm Nghiờn cu ny nhm mc ớch tuyn chn cỏc chng nm mc Aspergillus spp cú kh nng sinh enzyme invertase v kho sỏt mt s c tớnh ca enzyme ny T cỏc ngun mu khỏc chỳng tụi ó phõn lp c 16 chng cú kh nng tng hp enzyme invertase ú chng O1, L1.2 biu hin hot tớnh invertase cao nht Hai chng O1 v L1.2 cú kh nng sinh tng hp enzyme tt nht 35oC v pH 6,5 vi ngun nitrogen l urea i vi chng L1.2 v NaNO3 i vi chng O1, ngun carbon l sucrose i vi chng L1.2 cũn vi O1 l fructose Enzyme invertase ca chng O1 hot ng ti u 50oC, pH 5,5; i vi chng L1.2 enzyme ny hot ng tt nht 40oC, pH Kt qu nghiờn cu cho thy hot tớnh ca enzyme ny tng lờn b sung Mn2+ T khúa: Enzyme invertase, Aspergillus spp., hot tớnh enzyme, T VN Invertase (EC3.2.1.26) l mt enzyme thuc h GH 32 ca glycoside hydrolase (Alberto et al 2004), nú cũn c bit n vi cỏc tờn gi khỏc nh: -fructofuranosidase, saccharase, glucosucrase, sucrase, fructosidase Invertase cú kh nng xỳc tỏc phn ng thy phõn sucrose thnh glucose v fructose (t l 1:1) gi l ng nghch o Invertase c s dng rng rói cụng nghip thc phm c bit l ngnh sn xut bỏnh ko v nc gii khỏt ng nghch o ngt hn v ớt kt tinh hn so vi ng sucrose Ngoi ra, invertase cũn c ng dng sn xut ng fructooligosaccharide (FOS) l loi ng cú chc nng lm gim cholesterol, phospholipid v triglycerides mỏu, cng nh lm gim huyt ỏp tõm trng (Nguyen et al, 2005) Invertase l mt enzyme c thu nhn t nhiu ngun khỏc nh thc vt, vi khun, nm men Saccharomyces v mt s vi nm nh Aspergillus niger (Rubio v Navarro, 2006) Tuy nhiờn nghiờn cu nc v cỏc chng Aspergillus sinh tng hp invertase cũn hnch nờn chỳng tụi thc hin nghiờn cu ny nhm Hc vin Nụng nghip Vit Nam 505 mc ớch phõn lp, tuyn chn cỏc chng nm mc Aspergillus spp cú kh nng sinh enzyme invertase v kho sỏt mt s c tớnh ca enzyme ny VT LIU V PHNG PHP NGHIấN CU 2.1 Vt liu thớ nghim Vt liu phõn lp nm mc: cỏc chng nm mc c phõn lp t cỏc ngun vt liu khỏc nhau: mu lỏ mc, lc, go, t ong mc, v chanh c ly t cỏc khu vc v a im khỏc nhau: H Ni, Nam nh 2.2 Phng phỏp nghiờn cu Cỏc chng nm mc Aspergillus c phõn lp theo phng phỏp Ulster (Bựi Xuõn ng, Nguyn Huy Vn, 2000) trờn loi mụi trng phõn lp l PDA v Czapek-Dox Sau ú lm thun bng cỏch cy truyn trờn mụi trng PDA Cỏc chng nm mc Aspergillus c nuụi lc mụi trng Czapek-Dox lng pH 5.5 (Sigma- M), tc lc 150 rpm, nhit 35oC, mt tip ging 106 bo t cho 100ml mụi trng lờn men Sau ngy nuụi cy thu dch nuụi em li tõm 10000 rpm 4oC 20 phỳt Phn dch lng thu c sau li tõm s c a i th hot tớnh enzyme invertase Da vo nguyờn lý enzyme invertase cú kh nng thy phõn liờn kt -1,2 glucoside ca sucrose sn phm l glucose v fructose t l 1:1 Hot tớnh tng i ca enzyme invertase c xỏc nh bng phng phỏp khuch tỏn a thch: phng phỏp ny s dng thuc th TTC 0.1% (2,3,5- triphenyl tetrazolium chloride NaOH 0,5M) ng kh (glucose) sinh t quỏ trỡnh thy phõn sucrose ca enzyme invertase s kh TTC thnh triphenyformazon cú mu hng, lng triphenyformazon t l thun vi lng ng kh sinh Ging thch c c mụi trng c cht sucrose 3% m natri acetate 50mM pH 5,0 Cho 100àl dch enzyme thụ vo vo ging qua ờm nhit phũng Thuc th TTC c thờm vo bng cỏch phun lờn mt thch v búng ti 20 phỳt, m acetate 0,1M pH 5,0 Enzyme invertase khuch tỏn vo a thch sn phm thy phõn ca nú c khng nh bng s xut hin vũng quanh ging Phng phỏp so mu DNS: hn hp phn ng gm 200àl enzyme thụ, 500àl dung dch sucrose 5% v 300àl m acetate 50mM pH 5,0 500C 30 phỳt (Bernfeld et al.,1955) 1ml DNS (3, 5-dinitrosalicylic acid) c cho thờm vo hn hp trờn v kt thỳc phn ng bng cỏch ngõm ng nghim b nc sụi phỳt Sau lm lnh bng nc ti nhit phũng dung dch phn ng c dựng xỏc nh lng ng kh glucose sinh bng cỏch o OD540 KT QU V THO LUN 3.1 Phõn lp v tuyn chn chng vi sinh vt cú kh nng sinh invertase T cỏc ngun vt liu mu chỳng tụi phõn lp c 16 chng nm mc cú c im ging vi Aspergillus ú cú chng khun lc cú mu en chng khun lc cú mu xanh rờu, vng lc Cỏc chng nm mc c lm thun v nuụi lng lc mụi trng Czapek-Dox ngy 35oC vi mt 106 bo t/ 100ml mụi trng Sau ngy nuụi thu dch enzyme thụ tin hnh xỏc nh hot tớnh bng khuch tỏn a thch vi c cht sucrose v thuc th TTC 0.1% Kt qu c trỡnh by ti bng 506 Bng Kt qu kh nng tng hp enzyme invertase ca 16 chng nm mc trờn mụi trng Czapek-Dox Chng O1 O2 O3 O4 L1 LB G2.2 G2.1 ng kớnh vũng hot tớnh (mm) 23.670.47 9.000.82 10.670.47 140.82 14.330.47 90.82 21.330.47 14.330.94 Chng G0.2 L1.1 L1.2 L1.3 L1.4 PL2.1 PL2.2 243 ng kớnh vũng hot tớnh (mm) 18.330.47 12.670.47 22.330.47 6.000.82 13.670.47 13.670.94 12.670.47 4.330.47 Chỳng tụi nhn thy cỏc chng nm mc biu hin kh nng thy phõn sucrose khỏc nhau, s 16 chng nm mc tham gia kho sỏt, cú chng biu hin hot tớnh invertase mnh nht l O1 v L1.2 vi ng kớnh vũng phõn gii sucrose tng ng l 23,67 mm v 22,33mm 3.2 nh hng ca mt s yu t n kh nng tng hp invertase 3.2.1 nh hng ca pH mụi trng Cỏc chng Aspergillus c nuụi trờn mụi trng Czapek-Dox lng vi cỏc pH thay i t 10 Sau ngy nuụi, dch nuụi c ly tõm v th hot tớnh phõn gii sucrose pH mụi trng ó nh hng ti kh nng sinh trng v tng hp enzyme invertase ca cỏc chng nm mc Aspergillus Cỏc chng Aspergillus cú kh nng sinh trng v tng hp enzyme invertae di pH khỏ rng t 3-9, ú vựng pH t 6.5 l thớch hp nht i vi c hai chng nm mc thỡ pH tt nht tng hp invertase l 6.5 (hỡnh 1) Hỡnh nh hng ca pH mụi trng n kh nng tng hp invertase 3.2.2 nh hng ca nhit Cỏc chng nm mc Aspergillus c nuụi trờn mụi trng Czapek Dox lng vi pH 6.5 cỏc nhit : 25, 30, 35, 40, 45oC Sau nuụi ngy thu dch nuụi em i ly tõm th hot tớnh phõn gii sucrose ca cỏc chng Cỏc chng Aspergillus u cú kh nng tng hp invertase di nhit t 25 - 40oC, ú chng O1 v L1.2 cú kh nng tng hp enzyme cao nht 35oC Kt qu ny phự hp vi kt qu nghiờn cu ca 507 Madhanasundareswari v cng s (2015) nghiờn cu sn xut v ti u húa cỏc iu kin phỏt trin ca chng nm mc sinh enzyme invertase Ti nhit 45oC thỡ c hai chng u khụng cũn kh nng tng hp invertase Hỡnh nh hng ca nhit nuụi cy n kh nng tng hp invertase ca cỏc chng Aspergillus 3.2.3 nh hng ca mt ngun carbon Enzyme invertase l enzyme cm ng mun tng hp enzyme cm ng cn cú iu kin: cú gen tng ng nhim sc th ca t bo, cú y c cht tng ng xõy dng cỏc phn t enzyme, cú nng lng cn thit cho s tng hp v phi cú cht cm ng Nu khụng cú cht cm ng thỡ cú iu kin trờn cng khụng th tng hp enzyme Tng hp enzyme chu nh hng ln ca cỏc loi ng s dng nh ngun carbon v c cht cm ng sinh enzyme (Ottoni et al., 2012) Cỏc loi ng khỏc nh glucose, frucose, sucrose v raffinose vi nng 1% ó c la chn thu invertase theo nh mụ t ca Uma et al., (2010) Chng L1.2 sn xut invertase nhiu nht mụi trng cú ng sucrose l ngun carbon Kt qu ny phự hp vi kt qu nghiờn cu ca Uma et al., (2010) nghiờn cu sn xut, tinh sch v kh sỏt c tớnh enzyme invertase t Aspergillus flavus v kt qu ca Ottoni et al (2012) nghiờn cu ti u húa iu kin sn xut invertase t Aspergillus oryzea (hỡnh 3) Trong ú vi chng O1 thỡ ngun carbon tt nht li l fructose nhiờn vi ngun carbon l sucrose thỡ ng kớnh vũng hot tớnh cng tng ng chng L1.2 Hỡnh nh hng ca ngun carbon khỏc ti kh nng tng hp enzyme invertase Hỡnh nh hng ca ngun nitrogen ti kh nng tng hp invertase 508 3.2.4 nh hng ca ngun nitrogen Ngun nitrogen v ngun carbon l hai yu t nh hng n s tng trng ca t bo v sn xut enzyme ca cỏc chng vi sinh vt (Wen-Chang Chen, Chihsien Liu., 1996) Hai chng O1 v L1.2 c nuụi lc trờn mụi trng Czapek-Dox lng pH 6.5, nhit 35oC, 180 vũng/phỳt, ngun carbon l sucrose v thay th ngun nitrogen bng cỏc ngun nitrogen khỏc nhau: urea, NaNO3, pepton v cao nm men Cỏc ngun nitrogen kho nghim ó nh hng ti s tng hp enzyme invertase ca chng O1 v L1.2 (hỡnh 4) Urea v NaNO3 l ngun nitrogen tt nht cho s tng hp enzyme invertase ca cỏc chng Aspergillus thớ nghim, ú cao nm nem li cho kt qu tng hp invertase ngoi bo khỏ thp mc dự sinh nm tng trng mnh Kt qu ny phự hp vi nghiờn cu ca Ottoni et al 2012 nghiờn cu ti u húa iu kin sn xut invertase t Aspergillus oryzea Ngc li Wang v Zhou (2006) cho thy urea hn ch c cỏc hot ng sn xut invertase v tng trng sinh ca chng Aspergillus sp.JN19 v cao nm men c chn lm ngun nitrogen tt nht Mt khỏc, urea cng c dựng ci thin sn lng invertase sn xut t Saccharomyces cerevisiae (Ikram v Sikander 2007), Rajoka v Yasmeen (2005) thy rng NaNO3 l ngun nitrogen nghốo nht cho s tng hp -fructofuranosidase bi A.niger 3.3 Mt s yu t nh hng n hot tớnh ca invertase 3.3.1 nh hng ca pH m Enzyme invertase cỏc chng Aspergillus tng hp cú gii pH hot ng khỏ rng (Nguyen Q D et al, 2005) Chng O1 hot ng mnh nht pH 5.5 Kt qu ny phự hp vi nghiờn cu ca Nguyen.Q.D v cng s (2005) tin hnh tinh sch v kh sỏt mt s thuc tớnh ca of -fructofuranosidase t Aspergillus niger IMI303386 Trong ú chng L1.2 hot ng mnh pH (hỡnh 5) Hỡnh nh hng ca pH m n s phõn gii sucrose ca enzyme invertase Hỡnh nh hng ca nhit lờn hot tớnh ca invertase 3.3.2 nh hng ca nhit Nhit nh hng ti cu trỳc v hot ng ca enzyme, chớnh vỡ vy bit c nhit hot ng ti u ca enzyme l yờu cu cn thit Dch chit enzyme thụ ca cỏc chng O1 v L1.2 c x lớ cỏc nhit t 30oC n 80oC v kim tra hot tớnh bng phng phỏp khuch tỏn a thch Kt qu (hỡnh 6) cho thy vi nhit ti u cho hot tớnh ca enzyme invertase thụ ca chng O1 v L1.2 tng ng l 50oC v 40oC Sanjay v Sugunan (2006) 509 cho rng enzyme ny hot ng ti u 50oC, pH 5,0 Mt s tỏc gi khỏc cho rng invertases hot ng ti u phm vi nhit rng 35-75C tựy thuc vo ngun gc ca chỳng v thi gian (Kern et al.,1992) Nguyen et al., 2005 bỏo cỏo nhit ti u ca invertase t A niger l 50C Giỏ tr nhit ti u ca invertase cao hn c bỏo cỏo bi nhiu tỏc gi (Rubio et al.,2002; Guimaraes et al.,2007; Hussain et al.,2009) giỏ tr thp hn (30C) ó c bỏo cỏo bi (Rashad et al.,2006) 3.3.3 nh hng ca mt s ion kim loi Cỏc ion kim loi (nng mM) ó c thờm vo hn hp phn ng mt cỏch riờng bit S thy phõn ca enzyme c o v so sỏnh vi mu i chng khụng cú ion kim loi (Rahul Kumar v Balakrishnan Kesavapillai., 2012) Kt qu cho thy cỏc ion kim loi cú nh hng khỏ mnh ti hot tớnh ca invertase, hot tớnh c biu hin mnh hn thờm vo cỏc ion Mn2+, Na+, hay Sn2+ cng cú tỏc ng n hot tớnh invertase Khi b sung ion Na+ hot tớnh invertase t chng O1 gim ỏng k so vi b sung cỏc ion Cu2+, Ca2+ Ion Zn2+ cng gõy c ch hot tớnh inertase t hai chng Cỏc nghiờn cu khỏc cng cho thy cỏc ion kim loi tỏc ng rt khỏc ti hot tớnh invertase sn xut t cỏc ngun khỏc Shankar, Thangamathi, Rama, Sivakumar (2014) cho kt qu rng enzyme invertase t chng nm men Saccharomyces cerevisiae MK hot ng ti u cú Ca2+, b c ch mnh bi K+ Rahul Kumar v Balakrishnan Kesavapillai (2012) cho thy enzyme ny c tng hot tớnh vi ion Fe3+ , Mn2+ , nhng ion Cu2+ c ch hot tớnh ca enzyme ny Hỡnh nh hng ca ion kim loi lờn hot tớnh ca enzyme invertase KT LUN T cỏc ngun mu thu thp t nhiu a im khỏc chỳng tụi phõn lp ó c 16 chng cú hot tớnh enzyme invertase, ú chng O1 v L1.2 cú hot tớnh cao nht iu kin mụi trng thớch hp cho kh nng sinh tng hp enzyme invertase cao nht ca chng ny cng ó c xỏc nh: Ngun carbon v nitrogen tt nht vi chng L1.2 v O1 tng ng l : sucrose v NaNO3, fructose v urea; pH v nhit thớch hp cho hai chng ny sinh trng, phỏt trin l 6,5; 350 C Hot tớnh enzyme invertase biu hin tt nht ti nhit 40oC- 50oC v pH 5,5- Ion Mg2+ ó tng hot tớnh thy phõn ca enzyme invertase TI LIU THAM KHO Alberto, F., C Bignon, G Sulzenbacher, B Henrissat and M Czjzek, 2004 The three dimensional structure of invertase (-fructosidase) from Thermotoga maritime reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases Journal of Biological Chemistry, 279:18903-18910 510 Bernfeld, P., S.P Colowick and N.O Kaplan, 1955 Amylases a and , pp Method in Enzymol Vol I, Academic Press Inc., New York, pp: 149-158 Guimaraes LHS, Terenzi HF, Polizeli MLT, Jorge JA (2007) Enzyme MicrobTechnol 42:52-57 Hussain A, Rashid MH, Perveen R, Ashraf M (2009) Purification, kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccarumofficinarum) Plant PhysiolBiochem 47:188-194 Ikram-Ul-Haq, Sikander A., Kinetics of invertase production by Saccharomyces cerevisiae in batch culture Pak J Bot., 39,907-12(2007) Kern G, Schỹlke N, Schmid FX, Jaenicke R (1992) Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast Protein Sci 1:120-131 Nguyen QD, Rezessy-Szabo JM, Bhat MK, Hoschke A (2005) Purification and some properties of -fructofuranosidase from Aspergillus niger IMI303386 Process Biochem 40:2461-2466 Ottoni C A.; R Cuervo-FernỏndezII; R M PiccoliI; R MoreiraI; B GuilarteMaresmaII; E Sabino da SilvaI; M F A RodriguesI; A E Maiorano Media optimization for -Fructofuranosidase production by Aspergillus oryzae Braz J Chem Eng vol.29 no.1 Sóo Paulo Jan./Mar 2012 Rahul Kumar and Balakrishnan Kesavapillai.(2012) Stimulation of extracellular invertase production from spent yeast when sugarcane pressmud used as substrate through solid state fermentation 10 Rajoka, M I., Yasmeen, A (2005) Improved productivity of -fructofuranosidase by a derepressed mutant of Aspergillus niger from conventional and non-conventional substrates Word J Microbiol Biotechnol., 21,471-78 11 Rashad MM, Mahmoud AEE, Desouky MA, Nooman MU (2006) Purification and characterization of extra and intracellular -fructofuranosidase from Saccharomyces cerevisiae growing on Eichhorniacrassipes leaf extract Deutsche Lebensmittel Rundschau 102:157-166 12 Rubio MC, Runco R, Navarro AR (2002) Invertase from a strain of Rhodotorula glutinis Phytochemistry 61:605-609 13 Sanjay G, Sugunan S (2006) Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10 Food Chem 94:573-579 14 Shankar T., P Thangamathi, R Rama, T Sivakumar (2014) Characterization of Invertase from Saccharomyces cerevisiae MK obtained from toddy sample 15 Uma C., D Gomathi, C Muthulakshmi and V.K Gopalakrishnan Production, Purification and Characterization of Invertase by Aspergillus flavus Using Fruit Peel Waste as Substrate Advances in Biological Research (1): 31-36, 2010 16 Wang, L M., Zhou, H M (2006) Isolation and identification of a novel Aspergillus japonicus JN19 producing -fructofuranosidase and characterization of the enzyme J Food Biochem., 30,641-58 17 Wen-chang Chen and Chi-hsien Liu 1996 Production of P-fructofuranosidase by Aspergillus japonicus Enzyme and Microbial Technology 18:153-160, 1996 511 ... 0,1720 0,0057 0,0076 0,0696 0,2558 3.3 Kt qu ng dng levan sn xut sa chua Ch phm levan sau sy (hỡnh 1A) t ch tiờu vi sinh, húa lý i vi thc phm (bng 3.4 ) Do levan l polymer sinh hc cú nhiu hot tớnh... xut levan trờn quy mụ cụng nghip Trong s ngun carbon, B subtilis natto D s dng tt nht sucrose sinh tng hp levan tng t nh cụng b ca Shin I.L.v cs (2005), t ngun glucose khụng thu c sn phm ny 3.2 ... tng hp levan Mc ớch ca nghiờn cu l tỡm s tng tỏc ng thi ca cỏc yu t nh hng ti sinh tng hp levan, nhm xỏc nh mụi trng ti u cho levan cao nht Vi 17 thớ nghim c thit k bng phn mm ti u (bng 3.2 ), hm