Sn xut protein trn quy m ln

Nui cy cc vi sinh vt trn quy m ln l phng php kinh t nht do c cc u im sau:

- S dng cc mi trng n gin, r tin. - Cc chu k ln men ngn ngy. - C th kim sot trng thi sinh l ca vi sinh vt trong qu trnh ln men v m bo c tnh ng nht ca cc m ln men.
2

Nui cy cc vi sinh vt trn quy m ln l phng php kinh t nht do c cc u im sau:

- C th k hoch ha vic thu hoch protein mt cch hp l, ph hp vi cc qu trnh phn tch v tinh sch u ra. - Hiu sut protein c th c tng ln nhiu ln bng cch ci thin cc chng truyn thng v pht trin qu trnh ln men, cng vi vic s dng thm cng ngh DNA ti t hp.
3

Nui cy cc vi sinh vt trn quy m ln l phng php kinh t nht do c cc u im sau:

- Cc k thut ti t hp DNA cng m ra cc c hi cho vic sn xut sinh khi enzyme t vic nui cy nhng loi vi sinh vt i hi iu kin sinh trng khc khe nh: mi trng dinh dng t tin hoc phi b sung cc nhn t cm ng (inducers)...
4

Nui cy cc vi sinh vt trn quy m ln l phng php kinh t nht do c cc u im sau:

- Cc enzyme ca cc dng vi sinh vt sng iu kin khc nghit (sinh trng cc iu kin cc oan ca nhit , mui, p lc thm thu, kim) nh k thut ti t hp DNA nay c th sinh trng thun li trong cc nui cy mesophilic (a nhit trung bnh 20-40oC), v c th sn xut cc enzyme vi cc c im chu nhit hoc chu mui nng cao.
5

Ln men E. coli ti t hp
Cc enzyme c ngun gc t cc sinh vt prokaryote c th c sn xut d dng trn quy m ln vi hiu sut cao trong cc vt ch E. coli ti t hp. Cc qu trnh ln men ny c th c tin hnh quy m t 3.000-6.000 L, v khng i hi cc bc cy gy (inoculum cuture) phc tp. Mt cu trc vt ch thch hp cho qu trnh ln men c trng c th nh sau:
6

Ln men E. coli ti t hp
Vt ch E. coli mang (1) plasmid vector c gen m ha cho enzyme cn thit, (2) cng vi gen ch th khng khng sinh thch hp nh ampicillin hoc neomycin, v (3) mt nhn t cm ng nh l TAC (b ba c trng cm ng lactose hoc isopropylthiogalactose).

Ln men E. coli ti t hp
Hiu qu sn xut cao nh mong mun c th t c bng cch ln men m c cung cp dinh dng, trong mi trng nui cy m cha cc thnh phn cho s sinh trng ban u ca vi khun, v d: glucose 2%, dch chit nm men (yeast extract) 1%, phosphate 1% v cc loi mui khc cng vi khng sinh c chn. Sau khi s sinh trng ban u c thit lp, cc cht dinh dng b sung c cung cp cc t l thch hp m bo ngun carbon v nitrogen.
8

Ln men E. coli ti t hp
Ngun carbon thun li l glucose, v nitrogen c th l mt phc hp t nhin (chng hn nh dch chit nm men, dch thy phn casein), sir ng (corn sirup), mt loi mui ammonium n hoc urea. Nui cy E. coli trong mi trng c b sung cc hn hp amino acid cho kh nng sinh trng v sn xut protein nhanh hn, tuy nhin vic s dng cc ngun dinh dng n gin hn (chng hn ngun nitrogen), mc d c th i hi thi gian ln men lu hn, vn c th to ra s sinh trng ca t bo v hiu sut protein cao tng t.

Ln men E. coli ti t hp
Sn xut enzyme c cm ng thun li bng cch b sung isopropylthiogalactoside t 30-300 mg/L, hoc lactose t 1-10 g/L. Vic quyt nh thi gian v tn s b sung cht cm ng l rt quan trng thu c hiu sut enzyme cc i. Mt ti u trn 200 ca OD600nm (50 g khi lng kh ca t bo/L) v s biu hin protein khong 10 g/L (30% ca protein tng s) l mt kt qu ln men mong mun. Qu trnh ln men thng thc hin trong hai ngy nhit thp hn nhit sinh trng ti u ca c th, ngha l t 24-28oC.
10

Ln men nm
Cc loi nm vt ch nh Aspergillus v Fusarium, v nm men hng methyl (Pichia) thch hp cho sn xut cc protein glycosyl ha t cc ngun ng vt hoc nm. DNA ca protein c hp nht trc tip trong DNA nhim sc th cng vi h thng promoter iu ha biu hin gen.
11

Ln men nm
Cc vt ch ti t hp c th c ln men trong mt kiu tng t nh cc nui cy khng ti t hp v ln men mt cao ca t bo c th d dng thu c bng cch dng cc phng php ca k thut sinh hc truyn thng.

12

Ln men nm
Cc vt ch Aspergillus hoc Fusarium c th c sinh trng trn cc nguyn liu th r tin nh bt u tng c b sung thm cht dinh dng l sir ng. Nm men, nh Saccharomyces v Pichia, c th sinh trng trn dch chit nm men hoc cc dch thy phn protein v sir ng.
13

Ln men nm
Qu trnh nui cy c th cho sinh khi t bo cao bng cch s dng cc mi trng c xc nh y c b sung xen k thm mt vi cht dinh dng khc. Ln men c trng ko di t 4-8 ngy. S biu hin protein vt qu 10 g/L thu c quy m cng nghip

14

Cc enzyme vi sinh vt thay th cc enzyme ng-thc vt

Mt s enzyme cng nghip vn c tch chit t cc ngun ng vt nh b tim n nguy c ca s nhim bn bnh no dng xp ca b (bovine spongiorm encephalopathy). Chng hn renin, sau khi thu t d dy ca b con mi sinh s c s dng lm phomt. N vn khng c m bo liu c hin din bt k ri ro no cho sc kho ca ngi tiu dng hay khng, tuy nhin renin ti t hp ca b con hin nay c th c sn xut nh ln men vi sinh vt l hon ton an ton.
15

Cc enzyme vi sinh vt thay th cc enzyme ng-thc vt

Mt cch khc, bng phng thc tch chit enzyme truyn thng t cc ngun vi sinh vt v vic s dng k thut sng lc thch hp, nhiu enzyme mi ging nh enzyme ng-thc vt hin nay c sn xut

16

Cc enzyme vi sinh vt thay th cc enzyme ng-thc vt

Enzyme protease ca Mucor trong nhiu trng hp thay th renin t d dy ca b. - Protease ca Aspergillus v Bacillus thay th cc enzyme dng trong thuc da. - Rt nhiu loi amylase vi sinh vt c pht trin thay th hoc b sung cho cc amylase thc vt trong to malt ca la mch hoc la m.
17

Cc enzyme vi sinh vt thay th cc enzyme ng-thc vt

Hn na, mt s enzyme c ngun gc ng vt (nh hot t plasminogen ca m) cng c sn xut trong cc vi sinh vt ti t hp v c th cung cp chui protein c ti cun xon. Cc enzyme glycosyl ha ca ng vt c v c th c biu hin trong cc c th eukaryote nh Saccharomyces v Aspergillus. y, vt ch s glycosyl ha enzyme cung cp hot tnh y cho d cc ng glycosyl ha y c th khc ng vt c v.
18

Cc enzyme vi sinh vt thay th cc enzyme ng-thc vt

Hn na, mt s enzyme c ngun gc ng vt (nh hot t plasminogen ca m) cng c sn xut trong cc vi sinh vt ti t hp v c th cung cp chui protein c ti cun xon. Cc enzyme glycosyl ha ca ng vt c v c th c biu hin trong cc c th eukaryote nh Saccharomyces v Aspergillus. y, vt ch s glycosyl ha enzyme cung cp hot tnh y cho d cc ng glycosyl ha y c th khc ng vt c v.
19