Tgp chi Khoa hoc - Cong nghe Thuy sdn SS1/201^
Phong Gilng vi sinh vgt thupe Trung tam Cdng nghfi sinh hpe, Dai hpe Quoc gia H i Ndi. Chung dope ciy truyen v i nudi d 30°C ttong moi ttifdng thgch nghieng YS.
- Rong mipt Porphyra Vietnamensis, nang bien ty nhien, thu mua tgi Nha Trgng v i o thing 11/2011.
Rong cd m i u tim, tool, sgeh, cpng rong dai, mem.
Cd mill dgc trung. khdng lin vdi eic loii rong khic, khfing ed min Ig, mau la, khdng ed dau hieu ho hdng.
- Mdi tnfdng null ely ISP-4 eai ttIn: K^HPO, 0,1%. MgS0,.7H,0 0,1%. CaCO30,2%, NaCI 0.1%.
Da thay doi nguon cartxin v i nitnDgen ban d i u l i bpt e i v i ba mia. cd bo sung chit cam dng CMC [6].
2. N$i dung nghien ciru Nghien edu eac npi dung chinh sau.
- Lya chpn chiing xg khuin Micromonospora VTCC thich hpp va nghifin edu inh hodng ciia mpt s l dieu kifin nufii ely den khi ning sinh tong hpp cellulase cd hoat tinh cao.
- Nghien cdu phoang phip thich hop d l thu nhfin djeh chilt v i c ^ l phim enzyme ky thuat d quy mfi phdng thi nghifim.
- Bode d i u dng dyng c h l pham enzyme tren thuy phin rong mirt san xuit bpt rong thye pham v i dinh gii chit topng eiia bfit rong nay 3. Phuvng phip nghifin cdu
-Cichhlnhinhttifnghifim: Chung Mcromonospora
VTCC-A-1787 - * Hogt hda -» Null ciy -> Lytam-, Djch chda cellulase —>• Ket tiia -* C-CPE rellulase
—»Thuy phan rong —* S l y —* Bfit rong [7].
- Phoang phip xae djnh dodng cong siiti trodng: Do dp h i p thy quang hpe d bode s6ng 620 nm [4].
- Phoang phip x i e dinh hogt tinh cellulase, Dya v i o djnh lopng dodng khd theo phoang phsp so mau (phoang phip Hagedom Jensen), m dan vj hogt tinh cua enzyme cellulase dooc djnti nghla la loang enzyme cd khg nang ttiiiy phjn ca ehit CMC tao thinh dodng khd hjong dmn^
1 pg glucose trong thdi gian 24 gid d nhiet do phbi^
{30f>C) [1].
- Phoang phip xac djnh dodng kinh phan gi^
ciia enzyme bang phoang phip dye 16 thgch [3|, IM. KtT QUA NGHlfeN Cl>U VA THAO LUAN 1. Ket qua lira chpn chiing xg khuin Mkmmonospm VTCC va mdi trodng null cly thich hpp
Khio sat 3 chung xg khuin dope nudi cay sinh enzyme trong ba mdi trodng khae nhau 1^ YS, ISP-4, Gause-I cd bo sung ea chit cam dng CMC 0,5%, pH 7,0, lac 180 v/phiit d nhifit do 30°C. Sau 72 gid, chpn chiing Micromonospora VTCC-A-t787 tilp tye nghien cdu do k h i ning sinh ting h?p cellulase cao trong mfii tnjdng ISP-4 vdi dodng kinh thiy phan ban d i u l i 15mm, hogt tfnh 92,0 ± 0,011 UN
Hinb 1. Chung Micromonospora trong cic md Chung Micromonospora VTCC-A-1787 ed khi nang len men v i sd dyng 4 nguon dodng l i glucose, fructose, lactose v i sucrose, khdng cd kha nang ding hda manitol, saccharose. Chiu dope ning dp muli 4%, cd khi nang sinh tong hpp protease v i amylase. Dya vio mfit dp te bio \^^^^ cho thiy ehung Micromonospora VTCC-A-1787 smh trodng v i phit trien cd sinh khoi dgt gia tri cao nhat d mdi tardng ISP-4 sau 72 gid nudi ely (OD^^^, = 1,554), do dd chpn thdi diem niy d l tilp gilng nudi sinh enzyme vdi ty le 10%.
VTCC-A-1787 v^ kha nang sinh enzyme trtrfrng nuoi cay
2. Ket qui xie djnh ning dp co chat cim iJug, nguin va nong dp carbon, nitrogen
Chpn c h l dfi nudi ely lac 180v/phiit tgo ra wy hoa tan, giup cac chit dinh dodng phin bfi dw trong mdi trodng. lim ting trao dli ehit dinti dif&ng l i dilu kien can thilt de chiing Micromonospors VTCC-A-1787 sinh trodng v i sinh ting h9P enzyme tot.
Theo k i t qua nghien cdu (bing 1a) cho thiy nong dp chit c i m dng CMC cd anh hodng rit ton d i n kha nang sinh ting hpp cellulase. Vdi di^'''^
1 1 0 - TRUONG OAI HOC NHA TRANG
ip chi Khoa hoc - Cong nghe Thuy sdn So 1/2014
nh thodng enzyme dope tong hpp vdi mdt s lpng ca ban toang d l i it, nhilu khi khd phit hifin, long khi tang mfit loong CMC nhit djnh v i o mdi Lfdng nudi ely thi ham lopng cellulase dope ting 3'p se tang len. Tuy nhifin neu cd tiep tyc tang bng dfi CMC, sfi lam cho do nhdt ciia mdi trodng mg gay anh hodng d i n be mgt tt-ao doi chat ciia sinh vat, hgn che trao d l i chit, l i m tang i p suit llm thau gay ra hifin toang co nguyen sinh ehit.
lim node t y do trong te bao ra ngoai. K i t qua nghien cdu chpn nong dfi CMC thieh hpp bo sung vao mdi trodng null eay la 1,5% cho hogt tinh cao nhatla141,3UI/ml
Tilp tyc nghifin edu i n h hodng eiia nguon cartaon v i nitrogen (bang l b va le) cho thay ehung Micromonospora VTCC-A-1787 thieh hop vdi earison phdc tap d dgng thd nho phe phim ba mia.
vdt nguon nitrogen hdu ea la bfit e i . B i n g 1. Anh hodng cua t h i n h p h i n moi tnrdng den hogt tinh enzyme
Nong d$ CMC, (b) Nguon carbon, (c) Nguon nitrogen N i n g d i CMC
0 0.5
1 1 5
2 2.5
3
Hofttinh (Ul/ml) 20,8 ± 0,025 110,310,02 117,7 ±0,03 141,3 ±0,025 118,5 ±0,04 73,7 ± 0,01 45,2 ± 0,034
Ngudn nitrogen (Nong lip 0,2%)
BOtcS Bpt d#u tirang
Pepton NaNO, (NHJ,SO,
Urea
Ho^t tinli cei- iulase (Ui/ml) 164,1± 0,045 112,0 ±0,056 151,8 ±0,026 84,3 ± 0,040 125,8 ± 0,035 26,5 ± 0,030
Ngu6n carbon (Ndrtg do 1%) Ba mia Baram Cao nam men
Mfit n dodng Mungd Tinh bdt b i p Tinh bpt gao Tinh bdt sin Tinh bdt tan Glu(X)se Fnjctose Sucrose Lactose
Hoat ti'nh cellulase (Ul/ml) 148,6 ± 0,035
76,1 ± 0,02 95,7 ± 002 10,2 ±0,01 11,9 ±0,015 77,8 ±0,011 100.1 ± 0,01 126,6 ±0,02 119.3 ±0,02 49,3 ± 0,02 58,2 ± 0,025 51,7 ±0,01 102,2 ±0.025
(b)
K i t qua cho thiy khi bo sung ba mia vdi ning dfi 2% va bpt c i 0,4% thi khi nang sinh tong hpp cellulase ao nhit vdi hogt tinh l i n lopt l i 148,6UI/ml v i 164,1 Ul/ml
. Toi UHJ hda d i l u Icien nuoi c i y quy hoach Box - Behnken
Sau khi x i e djnh cic khoing dilu kifin nudi ciy thfch hpp: nhiet dp (30-^40''C). pH (6,0-^7,0), thdi gian I20+I4h). Chiing tdi tien hinh t l i ou hda cic dilu kifin niy. nhim xie dinh moi quan he toang l i c ciia 3 yeu t l
&y, ham myc tieu ciia q u i trinh nufii ely la dat hogt tinh enzyme cao nhat. K i t qua dope trinh biy Irong hinh 2.
• b c Iflnh 2. B4 mit dip ung ho^t tinh enzyme v cic khoing yeu th kbic nhau
XJ, PH'6,S; (b) X,, ThM gian 133 gifr; (c) X,, Nhift dp 32,5'<;
TRUONG OAI HOC NHA TRANG - 1 1 1
Tgp chi Khoa hoc - Cong nghe Thuy sdn
Phoang ttinh h l i quy dope chip nhgn vdi R^ = 0,9815, p < 0,05:Y= 188.2 + 6.0125X,+ 3 6625X,+ O.725X3 +3.875X,Xj-10.6X,X3+1.45X^X3-16.7625X,^
- 28.7625X^2-15.1375X3^
K i t qua dio thay nhifil dp l i yeu t l ed inh hodng ttve ttep va ro ring nhii din so sinh trodng v i phit trien, kha nang ting hpp cellulase cua ehiing Micromonospora VTCC-A-1787. Tiiy theo dieu klfin sin xuit, ed the CO ^nh mfit y l u t l va thay doi thing s l khic ttong khoing ttjang dng ttiich hpp Xae djnh dilu klfin nudi ciy t l i ou d l thu dope cellulase ed hogt dfi cao: Mdi ttirdng eii tien ISP-4 vdi pH 6,5;
d nhifil dO 33°C ± 1, tie dp lie 180v/phiit. ttidl gign nufii ciy 132 gid, cellulase thu dope ed hogt dfi eao 188,93UI/ml. Vific tang ning dfi CMC 1,5%, thay the nguin carbon l i ba mia 2%, nguon nitnsgen l i bfit e i 0,4% v i o mdi ttudng giup ting hogt tfnh enzyme.
4. K i t q u i x i e dinh t i c nhan ket tiia Mil enzyme se cd logi l i c nhan kit tOa thfch hpp, khi dd C-CPE thu dope sfi cd hogt dp cao nhit. Tnang qui trinh thu nhfin v i lim sgeh enzyme thi phoang phip tiia bing ethanol 96° cho hifiu qui v i hogt tinh cao ban so vdi aceton 99,5% d ning d0 thich hop l i 75% (216,2UI/g) v i dung djch (NHJ^SO^ bao hda khfing ed hifin topng kit Ida.
Bing 2. Anh hirdng t i c nhan k i t tiia den hogt d$ enzyme
So t/2[
Dung mdi hihi c v
Aceton 99,5%
Ethanol 96°
Ammonium sulfete K h i i lu-g-ng kit tiia trung binh (g)
1,61 ±0.035
2.57 ± 0,047 Hl^u suit thu nh^n
(%)
4.03
6,43 CPE (Ul/g)
152,7 ± 0,023 216,2 ±
0,01 Khdng cd hi0n tuwig kit tOa Khi ting ning dp ethanol td 60% len 75% ttii hogt dfi cellulase tang Ifin v i dgt gia tri cyc dgi d ning dp 75%. Tuy nhifin nlu tilp tuc tang nong dp kit tiia den 80% thi hogt dfi enzyme se giim vi giy biln tinh enzyme. Oilu niy ed t h i giii Ihich khi d ning dfi Ihip Ihi choa du gay k i l tOa hoan toin h i l enzyme ed trong djch I h l , nlu ting nong dp vopt ngodng cho phep Ihi se giy biln linh enzyme.
5. K i t qua thiJy phan rong mirt Porph vietnamensis sir d^mg che p h i m enzyme t h u i t tip Micromonospora VTCC-A-i787
Bode diu dng dyng enzyme C-CPE cellul td Micromonospora VTCC-A-1787 thiiy phin r mirt vdi ty Ifi C-CPE vdi nguyen lifiu t i 2%, nhie 45'C, pH 6,0, lopng node l i 1:1 (v/w) so vdl ngu lifiu, lac 180 v/phut. Sau 48 gid eho ttiiy enzj niy d i ed tac dfing, lam giam lopng cellulose rong va lim ting him lopng dodng khd ban da Bang 3. Thinh p h i n dinh dirdng b^t rong
tru>dv v i sau khi thuy p h i n
STT
1 2 3 4 5 6
Tin chi tliu Protein Lipid Ceiiuiose Duang khir Tro Dolm
Hdm tir?ng (%) Trwic
38 1,5 39,2 3,7 2,6 13,1
Sau 42,4 0,8 13,5 14,12 2,1 12,3 Ve thinh phin dinh doang: bfit rong da dng dope nhu c i u dinh dodng v i dim bio chi VI sinh vfit dinh eho thye pham yfiu c l u gia n tnj'dc khi sd dyng. Tuy nhifin. dinh gii chit lui cam quan eiia bfit rong thi choa dgt yfiu clu, khi nang hda tan trong node cdn thip, dfi tan dgt 68%
IV. K £ T L U A N V A K i f N NGHj 1. Ket lugn
Theo c i c k i t qua nghien cdu, di Micromonospora VTCC-A-1787 dope chpn d l 1 ciy sinh long hpp cellulase trong mdi tnidng ISI pH 6,5, lie 180 v/phut d 33°C sau 132 gid. Nlni ehit c i m dng CMC t l i ou l i 1,5%, nguin car va nitrogen Ihich hpp l i ba mia vdi tj l# 2% vd ea 0,4%. Da thu dope C-CPE cellulase ed hog cao nhit l i 216,2UI/g, sd dung t i c nhin kit ti ethanol 96° d ning dp 75%, thdi gian k i t tua la gid d 4<<;. Nhifit dp thfch hpp cho hogt dfing C-CPE cellulase eiia chiing Micromonosi VTCC-A-1787 l i 45=C. pH 6,0.
Cfng dyng c h l phim enzyme niy thiiy f rong thu dope bfit rong cho chit lopng dgt yfiu
112 • TRUONG OAI HOC NHA TRANG
Tgp chi Khoa hoc - Cong nghe Thdy sdn
Kit qua nghifin cdu cho thiy danh gia cam quan sin phim rong biln k h i t i t v l chit lopng miu, miii v i vj, ham lopng protein v i dodng tang len, ham lopng lipid v i cellulose giim, dilu niy cd nghla cellulase da cd t i c dpng d i n hp cellulose trong rong.giup thCiy phin cellulose v i tao thinh mpt lopng dodng khd toang dng
2. Kien nghj
Tiep tye nghifin cdu phoang phap v i c h l dp siy d l s i n phim bfit rong dope tot han.
TAI LIEU THAM KHAO Fling Vi^t
1 Nguyin Trpng cAn, Nguyen Th| HiSn, D6 Thi Giang„ Tran Thi Luyen, 1998. Cong nghg enzyme. NXB Nong nghi?p.
TP HCM.
2. Nguyen Lan Dung, Nguyen Dang Diic, Djing H6ng Mien, Nguyin Vinh Phuac, Nguyin Dinh QuySn, Nguyen Phiing Tiln, Pham Van Ty, 1976. lM$t s6 phuong phip nghien cuu vi smh vat - T?p 2 va 3, NXB Khoa hoc va Ky thuat. Ha Npi.
3. Pham Thi Anh Hfing, 2003. Ky thu|it Sinh hoa. Truimg Dai hoc Khoa hoc T\t nhien. NXB Dai hgc Quic gia TP. HCIVl 4. Tran Thi Luyen, Do Minh Phung, Nguyen Anh Tuan, Ngo Dang Nghla, 2004. Che biSn rong bien. fJXB Nong nghipp.
TP. HCM.
5 Nguyin Diic Lupng, 2003. Thi nghiem vi sinh vat hpe - Tap II. NXB D91 hpe Qu6c gia TP. HCM.
6 Tran Linh Thudc, 2007. Phirong phap phan tich vi sinh vat trong nude, thye pham va my phim NXB Giao due.
lUngAnh
1 Teruo Nakakuki (Ed), 1993. Oligosaccharides: Produclion, Properties and Applications, Japanese, Technology Reviews, Vol. 3, No. 2, Gordon and Breach, Switzerland.
TRLTONG DAi HOC NHA TI5ANG " I B
