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Ch :

THU NHN ENZYME CELLULASET NM MC TRICHODERMA

HARZIANUM

GVHD: TS. V Ngc Bi

Nhm 5:

Phm Th Sng

Thi Vit HiuNguyn Th Tm Ha

Ng Th Giang

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PHN 2: THU NHN ENZYMECELLULASE T NM MCTRICHODERMA HARZIANUM

Nui nm si thu enzyme.

Thu ch phm enzyme th.

Tinh sch enzymebngsc klc gel.

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Phng php sn xut enzyme t vi sinh vt

Ngun thu enzyme:

nm siTrichoderma Harzianum

c im sinh l, sinh ho v sinhthi

- Khun lc Trichoderma Harzianumtrn mi trng PGA c dng bng,khi cn non c mu trng, khi gi cmu vng xanh do mu ca bo t,

mt tri ca Petri c mu vng nhtn vng ngh.

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- 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.

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Phng php nui:

thu nhn enzyme cellulase, ngi ta thng nui cytheo phng php b mt bng mi trng xp (mitrng bn rn).

- m : 55%.

- C cht : cellulose: Cm m : b ma (6:4).

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- 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.

Pha khong cho mi trngNaNO3 3.5 g/l.

K2HPO4 1,5g/l.MgSO4.7H2O 0,5g/l.KCl 0.5 g/l.FeSO4.7H2O 0.01g/l.

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Lngnccnb sung vo tmcnthit(m 55%):

F=

F: lngncb sung vo mi trng c mcnthit(ml)A: khilngcchtcnb sungB: mcntcC: m c sn trong ccht

100%: m 100%

A(B%-C%)

100%

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1. S quy trnh thu nhn

B

ng ho

Mi TrngB ma,cm m,khong,nc

X l mitrng

Ging

Thu canh

trng

Nghin

Nc Trch ly

Thu ch phmenzim th

Ta

Tinh sch

Nhn ging

Lc

Li tm

Trch ly

Thu taHo vo m Na acetat

Sc k lc gel

Hp kh trng121 C trong 20

Khuy trong 30

Enzyme tinh khit

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Thu nhn enzim bngsc klc gel

i vi cc sn phm ta bng cn lnh vacetone, qu trnh tinh sch ch c thchin trn Biogel P-100

i vi cc sn phm ta bng mui , qutrnh tinh sch xy ra qua hai bc: loi

mui bng gel Sephadex G- 25 v tinh schtrn Biogel p- 100

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Ha cht:

Gel Biogel P- 100 c c tnh : htmn , kchthcht 45- 90m, khnngngmnc:12ml/g gel kh, phm vi phn tch 5000- 150000Dal.

Gel Sephadex G- 25 c c tnh phm vi phn

tch:1000-5000 Dal, khnngngmnc :2,5ml/g gel kh, th tch nn 6 ml/g gel kh.

m Na-acetat 50mm, pH = 5 khbt khtrckhi dng

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Biogel-P100

Huyn ph ho htgel

Hydrat ho

Gn lp b mt

Kh kh ca dungdch

5-10 pht

Gn lp b mt

Np gel vo ct

Mu enzim

Tin hnh chysc k

Thm dd m viV gp 2ln

Trong 12 h 20oC

hoc l 4h 100oC

n nh gel

Ht mn4 ln

Dung dch gel

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Thu v xc nhmu tch c

Dch ra khictcobc sng 280nm

bng detector trong h sc k v c thhinhpthdidngsc k

Tin hnh thu cc phn on cui cng v

phn tch hm lng protein.Enzyme sau khi lm sch v xc nhtrng

lng phn tbngkthutin di trn gel

polyacrylamide.