Ch :

THU NHN ENZYME CELLULASE T NM MC TRICHODERMA HARZIANUM

GVHD: TS. V Ngc Bi Nhm 5: Phm Th Sng Thi Vit Hiu Nguyn Th Tm Ha Ng Th Giang

PHN 2: THU NHN ENZYME

CELLULASE T NM MC TRICHODERMA HARZIANUM

Nui nm si thu enzyme. Thu ch phm enzyme th. Tinh sch enzyme bng sc k lc gel.

Phng php sn xut enzyme t vi sinh vt

Ngun thu enzyme: nm si Trichoderma Harzianum c im sinh l, sinh ho v sinh thi - Khun lc Trichoderma Harzianum trn mi trng PGA c dng bng, khi cn non c mu trng, khi gi c mu vng xanh do mu ca bo t, mt tri ca Petri c mu vng nht n vng ngh.

- c tnh sinh l: sinh trng nhit ti thiu 00C, ti u 20- 300C, ti a 34- 380C . - c t: khng sinh c t - Sinh thi: chim u th vng kh hu nhit i.

Phng php nui:

thu nhn enzyme cellulase, ngi ta thng nui cy theo phng php b mt bng mi trng xp (mi trng bn rn). - m : 55%. - C cht : cellulose: Cm m : b ma (6:4).

Pha khong cho mi trng NaNO3 3.5 g/l. K2HPO4 1,5g/l. MgSO4.7H2O 0,5g/l. KCl 0.5 g/l. FeSO4.7H2O 0.01g/l.
- pH mi trng ban u cho nm si 4,5 5,5. - Trong qu trnh nui, cn thng xuyn cung cp oxy. - Xp: khng kh c th lu thng t bn ngoi t mi trng vo trong khi mi trng.

Lng nc cn b sung vo t m cn thit( m 55%):

A(B%-C%)

F=

100%

F: lng nc b sung vo mi trng c m cn thit(ml) A: khi lng c cht cn b sung B: m cn t c C: m c sn trong c cht 100%: m 100%

1. S quy trnh thu nhn

Mi Trng B ma,cm m, khong,nc

Hp kh trng 121 C trong 20

X l mi trng

Thu canh trng Ging Nhn ging

Nghin
Khuy trong 30 Nc Trch ly Lc B

ng ho

Thu ch phm enzim th Ta

Trch ly

Li tm Ho vo m Na acetat Sc k lc gel Thu ta

Tinh sch

Enzyme tinh khit

Thu nhn enzim bng sc k lc gel

i vi cc sn phm ta bng cn lnh v acetone, qu trnh tinh sch ch c thc hin trn Biogel P-100

i vi cc sn phm ta bng mui , qu trnh tinh sch xy ra qua hai bc: loi mui bng gel Sephadex G- 25 v tinh sch trn Biogel p- 100

 Ha cht: Gel Biogel P- 100 c c tnh : ht mn , kch thc ht 45- 90m, kh nng ngm nc: 12ml/g gel kh, phm vi phn tch 5000- 150000 Dal. Gel Sephadex G- 25 c c tnh phm vi phn tch:1000-5000 Dal, kh nng ngm nc :2,5 ml/g gel kh, th tch nn 6 ml/g gel kh. m Na-acetat 50mm, pH = 5 kh bt kh trc khi dng

Biogel-P100 Huyn ph ho ht gel Trong 12 h 20oC hoc l 4h 100oC Hydrat ho Gn lp b mt 5-10 pht

Thm dd m vi V gp 2ln

Kh kh ca dung dch

4 ln

Gn lp b mt
n nh gel

Ht mn

Np gel vo ct Mu enzim

Dung dch gel

Tin hnh chy sc k

 Thu v xc nh mu tch c Dch ra khi ct c o bc sng 280nm bng detector trong h sc k v c th hin hp th di dng sc k Tin hnh thu cc phn on cui cng v phn tch hm lng protein. Enzyme sau khi lm sch v xc nh trng lng phn t bng k thut in di trn gel polyacrylamide.