Giao Trinh Vi Sinh Vat Hoc Cong Nghiep

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MC LCChng1: M U 1. i tng, nhim v v ni dung ca vi sinh vt hc cng nghip 2. Lc s pht trin ca vi sinh vt hc cng nghip 3. V tr v yu cu mn hc Cu hi n tp Chng2: C S KHOA HC CA VI SINH VT HC CNG NGHIP 1. c im cu to v hot ng sng ca vi sinh vt 2. C s ha sinh ca vi sinh vt hc cng nghip 3. C s di truyn vi sinh vt Cu hi n tp Chng 3: S PHN LOI SN PHM 1. Phn loi theo tnh cht thng mi 2. Phn loi theo sinh l trao i cht Cu hi n tp Chng 4: CC PHNG PHP V K THUT LN MEN 1. Qu trnh ln men 2. Vi sinh vt 3. C cht dinh dng 4. Nhu cu v oxy v s thng kh trong qu trnh ln men 5. Kh trng 6. Phng php nui 7. Ni ln men Cu hi n tp Chng 5: S THU NHN SINH KHI T BO 1. Tiu chun v chng 2. Mi quan h vi sinh trng 3. Cht lng sn phm 4. Ging khi ng 5. Protein n bo (SCP) Cu hi n tp Chng 6: CC SN PHM LN MEN 1. Ln men ethanol

Trang 4 5 7 3

8 13 21 32 33 34 47

48 52 55 61 62 66 70 74 75 76 80 82 92 98 99

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2. Ln men lactic 3.Ln men 2,3 butadiol 4. Ln men butanol -aceton Cu hi n tp Chng 7: CC CHT TRAO I BC MT 1. Nguyn l ca s tng hp tha 2. Cc phng php to ra th t bin tng hp tha 3. Aminoacid 4. Sn xut cc purin nucleotit 5. Vitamin Cu hi n tp Chng 8: CC CHT TRAO I BC HAI 1. Cc cht khng sinh 2. Cc c t nm Cu hi n tp Chng 9: CC SN PHM CHUYN HA 1. S chuyn ha cc steroit 2. S to thnh phenyl-axetylcacbinol 3. Sn phm t vi khun axetic 4. Sn xut vitamin C 5.Sn xut destran Cu hi n tp Chng 10 : X L NC THI BNG BIN PHP SINH HC 1. Vi sinh vt hc ca cc ngun nc ung 2. X l nc thi 3.Ln men methane Cu hi n tp Chng11 : S TUYN KHONG NH VI SINH VT 1. Cc vi khun ngm chit 2. C ch tc ng ca vi khun 3. Mt s qu trnh thy luyn kim sinh hc 4. S tch ly kim loi nh vi khun v to Cu hi n tp Chng 12: CC BI TP C S V NNG CAO 1. Phn cu hi 2. Tr li mt s cu hi

114 120 125 127 129 134 138 146 150 151 153 172 180 182 187 187 190 198 202 204 208 210 218 220 221 223 233 234 236 245

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NHNG CH VIT TTADP AMP APG A-1,3-DPG ATP A-6PA CoA CKS DNA R-1,5-DP R-5-P RNA VSV F-6-P FAD G-6-P GAP KDPG N NAD NADH NADP NADPH PP X-5-P Adenosine diphosphate Adenosine monophosphate Acid 3-phosphoglyceric Acid 1,3 diphosphoglyceric Adenosine triphosphate Acid 6-penicillanic Coenzyme A Cht khng sinh Deoxiribonucleic acid Ribulose-1,5-diphosphate Ribulose-5-diphosphate Ribonucleic acid Vi sinh vt Fructose-6-phosphate Flavin adenine dinucleotide Glucose-6-phosphate Glyceraldehyde phosphate 2-Keto-3-deoxi-6-phosphogluconate Nitrogen Nicotinamid adenine dinucleotide dng oxi ha Nicotinamid adenine dinucleotide dng khNicotinamid adenine dinucleotide phosphat dng oxi ha

Nicotinamid adenine dinucleotide phosphat dng kh Pentose phosphate Xylulose-5-phosphate

Ngi bin son 1. PGS.TS. Kiu Hu nh 2. TS. Bin Vn Minh (Ch bin) 3. TS. Phm Ngc Lan 4. ThS. Bch Thy

Bin son cc chng 7, 8, 9, 10 1, 2, 3, 11 v 12 4, 5 6

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Chng 1

M u1. i tng, nhim v, ni dung ca vi sinh vt hc cng nghipVi sinh vt hc cng nghip (Industrial Microbiology) l mt ngnh ca Vi sinh hc, trong vi sinh vt (VSV) c xem xt s dng trong cng nghip v nhng lnh vc khc nhau ca k thut. Vi sinh vt hc cng nghip (VSVHCN) gii quyt hai vn chnh tri ngc nhau: Mt mt, n dn ti lm r hon ton nhng tnh cht sinh hc v sinh ho ca nhng c th sng l nguyn nhn c bn v trc tip ca s chuyn ho ho hc, nhng cht c c cht ny hay c cht kia. Trong trng hp ny, VSVHCN s dng nhng VSV thu nhng sn phm quan trng v c gi tr thc t bng con ng ln men. Phng php sinh ho thu nhiu sn phm l phng php duy nht c li v kinh t. Mt khc, chng ta cng bit s ln men do VSV gy ra khng lun lun din ra theo mt hng nh mong mun. S ph hu mt qu trnh ln men thng xy ra do s hot ng ca nhng VSV l. Trong trng hp ny, iu rt quan trng l khng nhng phi bit nhng VSV gy ra qu trnh cn thit m cn phi bit c nhng VSV c hi gy tn tht cho sn xut. Nh VSVHCN c kinh nghim phi khm ph ra chng, lm r tnh cht c hi do chng gy ra v tm ra nhng phng php u tranh vi chng. 1.1. Mc tiu mn hc Sau khi hc xong hc phn ny, sinh vin cn hiu c cc ng dng cng nghip quan trng ca vi sinh vt, s khc bit gia cng ngh sinh hc vi sinh vt hin i v vi sinh vt hc truyn thng, phn bit c cc nhm sn phm v qu trnh cng nghip, vai tr ca vi sinh vt trong tuyn khong v trong x l nc thi bng con ng sinh hc. 1.2. M t mn hc VSVHCN l mt b phn quan trng trong cng ngh sinh hc, l mn khoa hc nghin cu nhng hot ng sng ca vi sinh vt p dng n mt cch tt nht vo cc quy trnh sn xut quy m cng nghip v cc lnh vc khc nhau ca k thut. VSVHCN l mt ngnh khoa hc mi pht trin, nhng do ngha quan trng ca n v mt l thuyt cng nh thc tin nn pht trin ht sc nhanh chng.

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2. Lc s pht trin ca vi sinh vt hc cng nghipS pht trin ca VSVHCN c chia thnh 3 giai on chnh: Giai on u, tnh n na sau th k 19. Vic ng dng tim nng ca VSV c t bui u ca nn vn minh nhn loi nh sn xut ru vang, bia, dm. Vit Nam ngh nu ru, lm dm, lm tng cng c t rt xa xa. Tuy mt s qu trnh c thc hin quy m rng ri, nhng nhng s thnh cng cn ph thuc vo s ngu nhin hay kinh nghim ca nhng ngi th gii truyn cho cc th h sau. Vai tr ca VSV trong s chuyn ho cc cht hu c c con ngi bit n khong hn 100 nm trc y. Nhng cng trnh nghin cu VSVHCN bt u t Pasteur (1878). Nh ta thy Pasteur nghin cu nhiu qu trnh ln men p dng trong sn xut v hc thuyt v mm bnh. Pasteur cng ra phng php thanh trng Pasteur tit trng ru nho, bia m khng lm hng phm cht. Phng php ny hin nay c ng dng rt ln. Bi vy Pasteur c coi l ngi sng lp ra VSVHCN. Vic nghin cu v s dng cc chng nm men thun khit trong sn xut bia (Hansen, 1886) c th xem l bc m u cho cng nghip ln men da trn c s khoa hc. Nm 1898 Buchner cng nghin cu tc dng ln men ca nhiu nm men, vch ra mi lin h gia nm men v ho hc v men, v ng dng hot ng ca nm men vo sn xut tip ging ngoi. ng nghin nm men ly ra dung dch c men zymase v cho ln men ru. Nh vy giai on th nht l giai on s dng cc hot tnh ca VSV- giai on ny c nh du bng vic t c s khoa hc cho qu trnh sn xut ung cha ru.

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Hnh 1.1: Cc nh vi sinh vt hc cng nghip tin bi 1. Louis Pasteur (1822-1895); 2. Gerhard H. A. Hansen (1841-1912); 3. Eduard Buchner (1860- 1917)

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Giai on th hai ca VSVHCN c tnh n gia th k XX, bao gm s xut hin ca cc cht khng sinh, nhng tin b v di truyn hc trong vic chn lc cc th t bin vi khun, s nghin cu cc iu kin ln men ti u, k thut hc ln men, vic tch v tinh ch sn phm... Giai on th ba c c trng bi s pht trin ca mt nn cng nghip VSV c lp. Ngi ta iu khin c cc qu trnh siu tng hp VSV v to ra c hng lot cc chng t bin VSV. Nh cc thnh tu ny m ngi ta sn xut quy m ln m chnh, lysine v nhiu loi aminoacid khc.

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Hnh 1.2: 1. Alexander Fleming(1881-1955); 2. Francis Crick (1916-2004); 3. James Dewey Watson (1928-); 4. Joshua Lederberg(1925-)

Giai on th t (giai on hin nay) c nh du bng s pht hin ra cc enzyme ct gii hn restrictase v cc plasmid vi s gn cc gene l mang cc thng tin tng hp cc protein c bit vo mt c th tr thnh mt phng php thng dng v s kim sot ngy cng tt hn s biu hin ca cc gene ny.

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Hnh 1.3: 1.Daniel Nathans (1928-1999); 2.Werner Arber(1929-); 3.Hamilton Othanel Smith (1931-); 4.Herbert Boyer (1936-).

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3. V tr v yu cu mn hcMn VSVCN nhm cung cp cho ngi hc nhng kin thc v k nng ng dng VSV trong mt s quy trnh cng ngh phc v khoa hc v i sng con ngi, ngoi ra cn gip cho sinh vin phng php nm bt c mt s quy trnh k thut v gii thch c qu trnh sn xut trn c s khoa hc, tin ti c th ch ng hng dn gup mt s c s sn xut trong nhng trng hp cn thit, ng thi cung cp thm nhng kin thc su, rng v VSV hc ng dng, gp phn y mnh sn xut, tng thm ca ci vt cht, ci thin i sng cho nhn loi.

Cu hi n tp1. i tng nghin cu ca vi sinh vt hc cng nghip l g ? 2. Cc giai on pht trin ca vi sinh vt hc cng nghip ? 3. Giai on pht trin u tin ca vi sinh vt hc cng nghip c nh du bng cng trnh ca Pasteur (1878) chng minh vi sinh vt l tc nhn ca s ln men, v sau l cc cng trnh ca:.............(1886) dng cc chng nm men thun khit trong sn xut bia, v ...............(1898) pht hin ra dch chit nm men c kh nng gy ra qu trnh ln men ru ( chng minh ln men thc cht l mt qu trnh enzyme). 4. Ti sao c ngi ni vi sinh vt va l ngi bn thn thit, va l k th nguy him ca con ngi.

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Chng 2

C s khoa hc ca vi sinh vt hc cng nghipI. c im cu to v hot ng sng ca vi sinh vtVi sinh vt (Microogranisms) l tn gi chung ch tt c cc loi sinh vt nh b, ch c th nhn r di knh hin vi quang hc hoc knh hin vi in t. Vi sinh vt bao gm nhiu nhm khc nhau: Cc virus (nhm cha c cu to t bo), cc vi khun v vi khun lam (nhm sinh vt nhn s), cc vi nm (nhm sinh vt nhn chun) v c mt s ng vt nguyn sinh cng nh to n bo cng thuc nhm ny. Gia cc nhm trn khng c mi lin h cht ch v mt hnh thi hay phn loi, nhng ngi ta gp chng li v chng cng c mt s phng php nui dng, nghin cu v hot ng sinh l gn ging nhau v u c cc c im chung. 1. c im chung ca cc vi sinh vt 1.1. Kch thc nh b

Hnh 2.1: Cc phng php quan st th gii sng (t nguyn t n t bo)

Cc Vi sinh vt c kch thc rt b, o bng n v nanomt (1nm = 10-9 m) nh cc virus hoc micromet (1m = 10-6 m) nh cc vi khun, vi nm. Chng hn:

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- Cc th thc khun (hay phage) T2, T4, T6 c kch thc bin thin trong khong: (65 - 95) x (25 - 100) nm. - Cc vi khun c kch thc thay i trong khong (0,2 - 2) x (2,0 - 8,0) m; trong vi khun Escherichia coli rt nh: 0,5 x 2,0m. - Cc t bo nm men Saccharomyces cerevisiae c ng knh 5 10m. Kch thc cng b th din tch b mt ca vi sinh vt trong 1 n v th tch cng ln. Chng hn ng knh ca 1 cu khun (Coccus) ch c 1mm, nhng nu xp y chng thnh 1 khi lp nhng c th lch l 1cm3 th chng c din tch b mt rng ti ...6 m2 ! 1.2. Hp thu nhiu, chuyn ha nhanh Tuy vi sinh vt c kch thc rt nh b nhng chng li c nng lc hp thu v chuyn ho vt xa cc sinh vt khc. Chng hn 1 vi khun lctic (Lactobacillus) trong 1 gi c th phn gii c mt lng ng lactose ln hn 100-10 000 ln so vi khi lng ca chng, tc tng hp protein ca nm men cao gp 1000 ln so vi u tng v gp 100 000 ln so vi tru b. Nng lc chuyn ha sinh ha mnh m ca VSV dn n cc tc dng v cng to ln ca chng trong thin nhin cng nh trong hot ng sng ca con ngi. 1.3. Kh nng sinh sn nhanh Chng hn, 1 trc khun i trng (Escherichia coli ) trong cc iu kin thch hp ch sau 12-20 pht li phn ct mt ln. Nu ly thi gian th h l 20 pht th mi gi phn ct 3 ln, sau 24 gi phn ct 72 ln v to ra (4 722 366. 1017) t bo- tng ng vi 1 khi lng ... 4722 tn. Tt nhin trong t nhin khng c c cc iu kin ti u nh vy ( v thiu thc n, thiu oxy, d tha cc sn phm trao i cht c hi...). Trong ni ln men vi cc iu kin nui cy thch hp t 1 t bo c th to ra sau 24 gi khong 100 000 000- 1 000 000 000 t bo. Thi gian th h ca nm men di hn, v d vi men ru (Saccharomyces cerevisiae) l 120 pht. Vi nhiu vi sinh vt khc cn di hn na, v d vi to Tiu cu ( Chlorella ) l 7 gi, vi vi khun lam Nostoc l 23 gi...C th ni khng c sinh vt no c tc sinh si ny n nhanh nh vi sinh vt. y l c im quan trng c con ngi li dng sn xut nhiu sn phm hu ch nh ru, bia, tng chao, m chnh, cc cht khng sinh...

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Vi kun Escherichia coli

Nm men Saccharomyces cerevisiae

Nm si Alternaria

Vi to Chlorella

Hnh 2.3: Mt s vi sinh vt c s dng trong VSVHCN

1.4. Kh nng thch ng rt cao v pht sinh bin d mnh Trong qu trnh tin ho lu di vi sinh vt to cho mnh nhng c ch iu ho trao i cht thch ng c vi nhng iu kin sng rt khc nhau, k c nhng iu kin ht sc bt li m cc sinh vt khc tgng khng th tn ti c. C vi sinh vt sng c mi trng nng n 1300C, lnh n 0-50C, mn n nng 32% mui n, ngt n nng mt ong, pH thp n 0,5 hoc cao n 10,7, p sut cao n trn 1103 at. hay c phng x cao n 750 000 rad. Nhiu vi sinh vt c th pht trin tt trong iu kin tuyt i k kh, c loi nm si c th pht trin dy c trong b ngm t thi vi nng Formol rt cao... Vi sinh vt a s l n bo, n bi, sinh sn nhanh, s lng nhiu, tip xc trc tip vi mi trng sng ... do rt d dng pht sinh bin d. Tn s bin d thng mc 10-5-10-10. Ch sau mt thi gian ngn c th to ra mt s lng rt ln cc c th bin d cc h h sau. Nhng bin d c ch s a li hiu qu rt ln trong sn xut. Nu nh khi mi pht hin ra penicillin hot tnh ch t 20 n v/ml dch ln men (1943) th nay c th t trn 100 000 n v/ml. Khi mi pht hin ra acid glutamic ch t 1-2g/l th nay t n 150g/ml dch ln men (VEDAN-Vit Nam). 1.5. Phn b rng, chng loi nhiu Vi sinh vt c mt khp mi ni trn Tri t, trong khng kh, trong t, trn ni cao, di bin su, trn c th, ngi, ng vt, thc vt, trong thc phm, trn mi vt... Vi sinh vt tham gia tch cc vo vic thc hin cc vng tun hon sinh-a-ho hc (biogeochemical cycles) nh vng tun hon C, vng tun hon N, vng tun hon P, vng tun hon S, vng tun hon Fe...

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Trong nc vi sinh vt c nhiu vng duyn hi (littoral zone), vng nc nng (limnetic zone) v ngay c vng nc su (profundal zone), vng y ao h (benthic zone)... Trong khng kh th cng ln cao s lng vi sinh vt cng t. S lng vi sinh vt trong khng kh cc khu dn c ng c cao hn rt nhiu so vi khng kh trn mt bin v nht l trong khng kh Bc cc, Nam cc... Hu nh khng c hp cht carbon no (tr kim cng, grapht...) m khng l thc n ca nhng nhm vi sinh vt no (k c du m, kh thin nhin, formol. dioxin...). Vi sinh vt c rt phong ph cc kiu dinh dng khc nhau : quang t dng (photoautotrophy), quang d dng (photoheterotrophy), ho t dng (chemoautotrophy), ho d dng (chemoheterotrophy), t dng cht sinh trng (auxoautotroph), d dng cht sinh trng (auxoheterotroph)... 1.6. L sinh vt xut hin u tin trn tri t Tri t hnh thnh cch y 4,6 t nm nhng cho n nay mi ch tm thy du vt ca s sng t cch y khong 3,5 t nm. l cc vi sinh vt ho thch cn li vt tch trong cc tng c. Vi sinh vt ho thch c xa nht c pht hin l nhng dng rt ging vi Vi khun lam ngy nay. Chng c J.William Schopf tm thy ti cc tng c min Ty Australia. Chng c dng a bo n gin, ni thnh si di n vi chc mm vi ng knh khong 1-2 mm v c thnh t bo kh dy. Trc cc nh khoa hc cng tm thy vt tch ca chi Gloeodiniopsis c nin i cch y 1,5 t nm v vt tch ca chi Palaeolyngbya c nin i cch y 950 triu nm.

Vt tch vi khun lam Vt tch Vt tch Gloeodiniopsis Palaeolyngbya Cyanobacteria cch y 3,5 t nm cch y 1,5 t nm cch y 950 triu nm Hnh 1.2: Cc vi sinh vt ho thch c xa

(cn li vt tch trong cc tng c min Ty Australia)

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2. Nhng im khc bit gia cc t bo sinh vt nhn s v nhn chun Cc sinh vt nhn s v nhn chun c mt s c im ging nhau: u c t bo l n v cu to, chc nng l n v di truyn; vt cht di truyn trong cc t bo l DNA xon kp cng cc sn phm ca n l RNA, protein... Tuy nhin gia chng c nhiu nt sai khc rt r. Thm ch ngay c cc t bo nhn chun nh nht cng khc bit mt cch cn bn so vi cc t bo nhn s trong cu to, cch t chc thng tin di truyn cng nh trong cc kiu tng hp RNA v protein ca chng (bng 2.1).Bng 2.1: Nhng sai khc gia cc t bo sinh vt nhn s v nhn chun c im Sinh vt nhn s Sinh vt nhn chun 1. T chc di truyn - Mng nhn Khng c C - S nhim sc th khc nhau 1 >1 - Cc nhim sc th cha Khng c C histon - Hch nhn Khng c C - Trao i di truyn Mt chiu, qua plasmid Bng s kt hp giao t 2. Cc cu trc ca t bo - Li ni cht Khng c C - B my Golgi Khng c C - Cc lysosome Khng c C - Cc ty th Khng c C - Cc lp th Khng c C thc vt - Kch thc ribosome 70 S 80 S - Si thoi v sc Khng c C -Vch t bo cha C, ngoi tr Khng c peptidoglycan Mycoplasma v vi khun c 3. Mt s c tnh chc nng - Thc bo Khng c i khi c - m bo (ung bo) Khng c i khi c - V tr vn chuyn in t Mng t bo Mng bo quan - Dng t bo cht Khng c C (Ngun: Stanier et al, 1976; dn theo Watson et al, 1987. p. 97)

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[A]

[B] Hnh 2.2: Cu trc t bo vi khun [A] ; t bo ng vt [B]

II. C s ha sinh ca vi sinh vt hc cng nghip1. ng phn. ng phn l qu trnh phn hu phn t glucose (C6H12O6) to thnh acid pyruvic v NADH+ H+. im c bit ca ng phn l khng phi phn t glucose t do b phn hu m phn t ng glucose c hot ho bi vic gn gc P vo to dng ng phosphate. Qu trnh ng phn gm 2 giai on vi nhiu phn ng phc tp: - Phn ct phn t glucose thnh 2 phn t triose l AlPG v PDA.

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- Bin i 2 phn t triose thnh 2 phn t acid pyruvic. Qu trnh ng phn c th tm tt theo s sau:

Hnh 2.3. S ng phn

Kt qu ca ng phn c th tm tt l: C6H12O6 + 2NAD+ + 2ADP + 2H3PO4 2CH3COCOOH + 2NADH+H+ + 2ATP Trong h hp hiu kh, acid pyruvic tip tc phn hu qua chu trnh Krebs, cn 2NADH+H thc hin chui h hp to H2O. 2NADH+H+ + O2 2NAD+ + 2H2O Phn ng ny km theo vic tng hp c 6ATP qua qu trnh phosphoryl ho.

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Vy kt qu ca ng phn trong h hp hiu kh l: C6H12O6 + O2 2CH3COCOOH + 2H2O ng thi to ra c 8 ATP Trong h hp k kh, 2NADH+H+ s c dng kh CH3COCOOH (trong ln men lactic) hay kh CH3CHO (trong ln men ru) nn khng thc hin chui h hp. Phn ng ln men lactic nh sau: 2CH3COCOOH + 2NADH+H+ 2CH3CHOHCOOH + 2NAD+ Vy kt qu ca ng phn trong h hp k kh l C6H12O6 2CH3CHOHCOOH Qu trnh ny ch to ra c 2ATP 2. Chu trnh Krebs. Sn phm ca ng phn l acid pyruvic s c decarboxyl ha to acetyl-CoA v mt phn t CO2. Acetyl-CoA tip tc phn hu qua chu trnh Krebs trong h hp k kh. Qu trnh phn hu acid pyruvic qua chu trnh Krebs c thc hin ti ty th do nhiu h enzyme xc tc. Chu trnh xy ra qua 2 giai on: - Phn hu acid pyruvic. Trong qu trnh ny s to ra nhiu coenzyme kh (NADH+H+, FADH2). - Thc hin chui h hp qua cc coenzyme kh c to ra do phn hu acid pyruvic. Chu trnh Krebs c tm tt qua s sau:

(a) (b) Hnh 2.4: a) Krebs, Sir Hans Adolf (1900-1981); b) S minh ho chu trnh Krebs

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T phn t acid pyruvic qua chu trnh to ra CH3COCOOH + 3H2O 3CO2 + 5H2 (4NADPH +H+ + 1FADH2) Cc coenzyme (NADH + H+, FADH2) thc hin chui h hp: 5H2 + 5/2O2 5H2O Vy kt qu chu trnh s l: CH3COCOOH + 5/2O2 3CO2 + 2H2O T 1 phn t glucose qua ng phn to ra 2 phn t acid pyruvic vi kt qu phn tch trn. T 2 acid pyruvic qua chu trnh Krebs to ra: 2CH3COCOOH + 5O2 6CO2 + 4H2O Kt hp vi giai on ng phn, ta c kt qu tng qut ca qu trnh phn hu glucose qua h hp hiu kh l: C6H12O6 + 6CO2 6CO2 + 6H2O Trong qu trnh phn hu acid pyruvic qua chu trnh Krebs s to c 4 NADH+H+ v 1 FADH2. Cc coenzyme kh ny qua chui h hp s tng hp ATP vi kt qu: -4NADFH+H+ to ra 12 ATP -1FADH2 to ra 2ATP -Trong chu trnh to ra 1ATP. Nh vy, phn hu 1 phn t acid pyruvic qua chu trnh Krebs to ra 15ATP. Nu phn hu 2 acid pyruvic s to ra 30ATP, kt hp vi giai on ng phn th phn hu 1 phn t glucose to ra c 38ATP. 3. Chui h hp v phosphoryl ho 3.1. Chui h hp Trong t bo s trao i nng lng lun gn vi phn ng oxi hokh. Trong h thng oxi ho kh hai phn ng oxi ho v kh lun i km nhau. AH2 + B A + BH2 Trong t bo phn ng trn xy ra thng cn h thng cc cht truyn in t v H+ trung gian, l h enzyme oxi ho-kh. Cc enzyme ny cng vi c cht hot ng trong mt chui phn ng cht ch chuyn H2 t c cht n O2 to nn chui h hp. Khi u ca chui l c cht dng kh AH2. AH2 lm nhim v l cht cho H2. H2 tch ra t c cht c h thng cc coenzyme ca h enzymee oxi ho-kh vn chuyn n khu cui cng ca chui l O2 kh O2 to phn t H2O.

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Trong chui h hp in t c chuyn t c cht l cht c nng lng cao nht n oxi c nng lng thp nht, th oxi ho cao nht (+0,81V). Gia hai thnh phn trn l cc coenzyme c th oxi ho-kh trung gian, th kh gim dn t c cht n O2. Bi vy chui h hp l qu trnh gii phng nng lng. Nng lng thi ra trong chui h hp c xc nh theo phng trnh: G = -nF. Eo (kCal/mol) Trong : G : mc bin i nng lng ca phn ng oxi ho-kh n: s in t trao i trong phn ng. F: s Faraday (23,06) (hng s Faraday). Eo: chnh lch th oxi ho-kh ca 2 cht tham gia phn ng. Vi phng trnh trn c th xc nh c nng lng thi ra ca tng phn ng trong chui trn c s th oxi ho-kh ca cc h xc nh. 3.2. Phosphoryl ho Qu trnh tng hp ATP trong t bo l qu trnh phosphoryl ho: ADP + H3PO4 ATP + H2O Phn ng ny i hi nng lng tng ng nng lng ca lin kt cao nng th nht (7,3 Kcalo/mol - trong iu kin chun). Tu ngun nng lng cung cp m c cc hnh thc phosphoryl ho quang ha (xy ra trong quang hp) v phosphoryl ha oxi ha (xy ra trong h hp). Trong h hp c hai hnh thc tng hp ATP - Phosphoryl ho mc c cht: l qu trnh tng hp ATP nh nng lng thi ra ca phn ng oxi ho trc tip c cht.

- Phosphoryl ho qua chui h hp: l qu trnh tng hp ATP nh nng lng thi ra ca cc phn ng trong chui h hp. Chui h hp xy ra nhiu phn ng, phn ng no tho mn cc iu kin ca qu trnh phosphoryl ho th qu trnh tng hp ATP xy ra . Trong chui h hp c 3 v tr iu kin tng hp ATP. Nh vy, c vn chuyn c H2 t c cht n O2 s to ra c 3 ATP cho t bo.

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4. S trao i protein, lipid, acid nucleic trong t bo vi sinh vt 4.1. S trao i protein 4.1.1. S tng hp protein Tng hp protein l qu trnh rt phc tp nhng c vai tr rt quan trng trong hot ng sng ca VSV. Con ng tng hp protein ch yu l tng hp theo c ch khun, tc l qu trnh gii m. Phn t protein c m ho trong gene theo m b ba: c 3 nucleotide trn mch khun ca gene m ho mt amino acids trn cu trc bc I ca protein. B ba m gc trn gene c phin m sang b m ho trn mRNA thng qua qu trnh phin m - tng hp mRNA. T mRNA l khun chui polypeptid-protein bc I s c tng hp ti ribosome. Qu trnh tng hp protein xy ra qua nhiu giai on: - Hot ho amino acids nh ATP v gn amino acids vo tRNA; - Tng hp chui polypeptid ti ribosome; - Hon thin phn t protein. 4.1.2. Phn hu protein Protein trong t bo VSV lun lun c i mi. Bi vy qu trnh phn hu protein xy ra thng xuyn cng vi qu trnh tng hp. S phn hu protein xy ra qua 2 giai on - Thy phn protein thnh cc amino acids nh enzyme thy phn protease. - Cc amino acids khng cn thit s b phn hu tip hay chuyn ho thnh cc amino acids cn thit khc. S phn hu amino acids xy ra bng nhiu con ng: kh amin, kh cacboxyl, chuyn v amin. 4.2. Trao i acid nucleic.

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4.2.1. Tng hp acid nucleic Tng hp acid nucleic l qu trnh quan trng trong c ch truyn t thng tin di truyn. Tng hp acid nucleic gm tng hp DNA v qu trnh tng hp RNA. * Sao chp DNA: T 1 phn t DNA gc qua sao chp to ra 2 phn t DNA mi hon ton ging nhau v ging DNA gc. Qu trnh sao chp xy ra bng nhiu c ch trong c ch bn bo th l ph bin v quan trng nht. Trn DNA khun, hai chui c chiu ngc nhau do vy qu trnh sao chp xy ra trn hai chui khc nhau. - Trn chui c chiu 3-5 ca DNA gc: sau khi enzymee helicase tho xon, tch 2 mch ca phn t DNA gc, trn mch 3-5 tin hnh tng hp mt on RNA mi ngn nh primase xc tc. T RNA mi cc nucleotide mi tip tc n ko di chui theo chiu 5-3 nh DNA-polymerase III xc tc. Qu trnh ni cc nucleotide t do vo mch mi theo nguyn tc b sung vi mch gc. Sau khi tng hp b sung xong c mch, on RNA mi b ct b v thay vo bng mch DNA tng ng nh DNA-polymerase I xc tc. - Trn mch c chiu 5-3 ca DNA gc: do trn mch ny chiu tng hp mch mi ngc chiu tho xon nn din ra phc tp hn. Qu trnh tng hp mch b sung xy ra theo tng on ngn. C m xon mt on vi trm nucleotide qu trnh tng hp xy ra ngc chiu tho xon theo tun t tng hp on RNA mi ri tng hp tip on DNA. Tng hp xong on ny, tho xon tip v li tng hp nh on trc . C nh vy trn mch ny DNA c tng hp theo tng on ngn-on Okazaki. Sau cc RNA mi c ct b, thay vo cc on DNA tng ng, nh ligase ni cc on Okazaki li. * Phin m: xy ra qua 2 giai on. Giai on u phin m t gene thnh pro RNA. Sau t pro-RNA s bin i thnh mRNA tng ng. RNA tng hp trn DNA khun hay trn RNA khun (vi virus cha RNA). DNA khun nh RNA polymerase tho xon cc b to nn bng phin m. Bng phin m c chiu di 30 cp nucleotide. Nh yu t sigma () ca RNA-polymerase nhn bit im m u v chui DNA dng lm khun. Nh li enzyme polymerase tin hnh tng hp chui RNA b sung vi chui khun trn DNA. Qu trnh tip din cho n khi yu t rho () nhn bit im kt thc trn DNA v kt thc qu trnh

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tng hp chui RNA b sung. RNA tch khi DNA khun to ra proRNA. T proRNA qua nhiu bin i phc tp to mRNA trng thnh. - Ni thm m; - Ni thm ui; - Nh enzyme ct ghp tin hnh ct b cc on khng m ho intron (I) v ni cc on m ho exon (E) li vi nhau s to ra mRNA. Kt qu l t 1 on DNA (gene) to nn mt phn t mRNA. Thnh phn trt t cc nucleotide trn cc on exon ca DNA qui nh thnh phn, trt t cc ribonucleotide trn mRNA. 4.2.2. Phn hu acid nucleic Trong t bo acid nucleic lun bin i, nht l cc phn t RNA. i sng cc phn t mRNA, tRNA rt ngn. Chng thng xuyn b phn hu thay th cc loi mi. S phn hu acid nucleic xy ra qua 3 giai on: - Thy phn acid nucleic thnh nucleotide nh cc nuclease tng ng xc tc; - Thu phn nucleotidee thnh cc n phn (base-nitrogene, ng pentose v H3PO4) nh enzyme thu phn nucleotidease xc tc; - Bin i tip base-nitrogene, ng pentose thnh cc sn phm khc nhau. 4.3. Trao i lipid 4.3.1. Tng hp lipid Trong t bo lipid ph bin nht l cht bo. Cht bo c tng hp t glycerin v cc acid bo qua cc bc: - Glycerin + ATP glycero-P + ADP - Glycero-P + acid bo 1 monoglyceric. - Monoglyceric + acid bo 2 diglyceric. - Diglyceric + acid bo 3 Triglyceric + H3PO4. 4.3.2. Phn hu lipid. Qu trnh phn hu cht bo xy ra qua 2 giai on - Thu phn acid bo thnh glycerin v cc acid bo. - Phn hu tip glycerin v cc acid bo. + Glycerin + ATP glycero-P + ADP

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+ Glycero-P AlGP ng phn. Cc acid bo phn hu bng nhiu con ng trong ph bin nht l phn hu theo con ng -oxi ho. Cc acid bo phn hu theo con ng -oxi ho to nn cc acetyl-CoA. T acetyl-CoA phn hu tip bng chu trnh Krebs. S phn hu acid bo to ra nng lng ATP kh ln cho t bo VSV. V d khi phn hu acid palmitic to ra c 130 ATP. III. C s di truyn vi sinh vt Nh chng ta u bit, cho n u thp nin 1940 cc VSV mi bt u c s dng trong nghin cu di truyn hc. T y hnh thnh nn di truyn hc VSV v di truyn sinh-ha, m ng cho s ra i ca di truyn hc phn t (1953) v cng ngh DNA ti t hp sau ny (1972). 1. Di truyn hc virus Theo R.M. Atlas (1994) th Virus l mt thc th v bo c cha mt lng ti thiu protein v acid nucleic m ch c th sao chp sau khi xm nhp vo nhng t bo sng chuyn bit. Chng khng c qu trnh trao i cht ni ti, s sao chp da vo vic iu khin trao i cht t bo nh h gene ca virus. Trong t bo ch cc thnh phn ca virus c tng hp ring r, ri lp rp thnh dng thnh thc. 1.1. c im v cu to ca virus - Virus l cc th sng cha c cu to t bo, k sinh ni bo bt buc. Mi kiu virus c mt phm vi vt ch nht nh. Chng nhn din t bo ch theo nguyn tc kha v cha kha gia cc protein v ca n vi cc im nhn trn b mt mng t bo. Cc virus ca vi khun gi l bacteriophage (th thc khun), gi tt l phage. - Virus khng c h thng sinh nng lng, khng c ribosome, khng c h thng bin dng ring v do vy cc virus khng tng trng. - Virus khng to mng lipid ring; - Virus khng c khung sn t bo; - Virus khng b tc ng bi cc cht khng sinh; - Mi ht virus thng gm 1 phn t acid nucleic trong, gi l li, v lp v (capsid) bao bn ngoi l cc protein (t hp theo cc n v gi l capsomere), c mang cc thnh phn khng nguyn. Kt cu c bn gi l nucleocapsid.

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Hnh 2.5. Virus trn v virus c v ngoi

H gene ca virus rt c bit (bng 2.2), mi loi virus ch cha mt loi acid nucleic, hoc l DNA hoc l RNA, chui n hay chui kp, dng si thng hay dng vng. Virus b nht khong 4 gene v ln nht khong vi trm gene. Mt s virus ngoi v protein cn c thm vi lp mng bao khc gm (protein + phospholipid) bt ngun t mng sinh cht t bo ch, hoc c (protein + glycoprotein) t virus HIV, virus SARS, H5N1 l mt v d.(HIVgy hi chng suy gim min dch mc phi (HCSGMDMP); Virus SARS gy hi chng vim ng h hp cp (VHHC); H5N1 gy bnh cm g) Bng 2.2. Phn bit qu trnh truyn bnh AIDS, SARS, CM G Bnh Acid nucleic RNA RNA RNA Tn virus Vt ch Phng thc lan truyn -Qua mu -Quan h tnh dc -M sang con -H hp -H hp, tiu ha

AIDS SARS CM G

HIV1, HIV2 SARS H5N1

Ngi, kh Cy hng, ngi Gia cm

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Hnh 2.6: Virus v ng truyn bnh AIDS, SARS, CM G Bng 2.3: Dng acid nucleic v kch thc h gene mt s virus Virus Phage MS2, f2, R17 Virus TMV Cc Reovirus Phage X174 Virus SV40 Baculovirus Adenovirus Ad5 Phage (Lambda) Phage T2, T4 Vt ch E. coli Thuc l Th E. coli Th Cn trng Ngi E. coli E. coli Dng acid nucleic RNA si nthng RNA si kpthng DNA si nvng DNA si kpvng DNA si kpthng Kch thc h gene ( s cp base) 3.569 ~ 6.000 ~ 50.000 5.375 5.243 (100-150)x103 ~ 36.000 48.502 ~ 200.000

Ghi ch: Cc virus gy bnh ngi v ng vt c h gene ch yu l DNA si kp-thng, nh: cc virus thuc h Adenoviridae gy bnh ng h hp, h Herpesviridae gy mn rp herpes ngi, bnh u g, h Poxviridae gy u ma, u b ... Hoc l RNA si n-thng, nh cc virus thuc h Retroviridae

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gy ung th, AIDS, bch cu ..., h Paramyxoviridae gy si, quai b, bnh g toi, h Rhabdoviridae vi bnh di, h Orthomyxoviridae vi bnh cm, h Picornaviridae vi cc bnh vim ty xm, vim gan A do virus, bnh l mm long mng tru b...

1.2. Phng thc sinh sn v vng i ca virus y chng ta s tm hiu nhng nt chung nht trong chu trnh sng-sinh sn ca virus qua i din l cc phage k sinh E. coli. Thng thng c chia lm 5 giai on: Hp th Xm nhp Sao chp Thnh thc Phng thch, hoc 3 giai on nh sau: (1) Phage bm bng phn ui trn b mt t bo vi khun v tit enzyme tiu hy vch t bo; (2) Phage dng phn ui tim li acid nucleic (DNA) vo trong t bo ch; (3) Trong t bo ch, ty loi DNA cc phage khc nhau m c th din ra mt trong hai trng thi: gy tan hoc tim tan. + i vi cc phage c nh T2 v T4, lc ny DNA ca chng s sao chp nhiu ln v tng hp cc protein cn cho to v v ui, sau chng lp rp vi nhau to ra cc phage th h con. Sau , di tc dng ca lysozyme lm tan t bo ch v phng thch chng 100-200 phage con.

Hnh 2.7: Chu trnh hot ng gy tan (lytic cycle) v tim tan (lysogeneic cycle) ca phage E. coli.

+ i vi phage n ha nh lambda (), c th gy tan hoc khng gy tan. Trng hp tim tan (lysogeney) xy ra l do: sau khi chui vo t bo ch, DNA ca phage t ng vng ( nh c cc u dnh v xc tc

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ca ligase), ri xen vo nhim sc th chnh ca vi khun bng s ti t hp v tr c hiu v n tim n trng thi , gi l tin virus (prophage). Tuy nhin, cc prophage ny c th hot ng tr li gy tan. Chng hn, nu c tc ng ca tia UV gy tn thng nhim sc th vt ch th mt trao i cho ngc tr li s xy ra, nh vy DNA phage c tch ra khi nhim sc th vi khun ch v bt u chu trnh sinh sn gy tan. + i vi virus thuc nhm retrovirus (virus gy ung th, gy bnh AIDS, gy bnh bch cu t bo T...) c chu trnh sng in hnh (hnh 2.6).

Hnh 2.8: Chu trnh sng ca virus nhm retrovirus

y cn hai im quan trng lin quan cng ngh gene: + Ti t hp: Khi c nhiu virus nhim ng thi t bo ch th gia chng xy ra s trao i gene, to ra cc th ti t hp. Nh vy c th lp bn di truyn virus. Dng ti t hp im c hiu gn DNA phage vo nhim sc th vi khun ch v tch ra ( khi b cm ng tia UV) c xc tc bi cp enzyme tng ng l integrase v excisionase. Li dng c im ny, ngi ta s dng phage lm vector trong k thut gene. + Sao chp: Do vt liu di truyn ca cc virus rt a dng, nn s sao chp/sao chp ca chng cng khc nhau, theo 3 con ng:

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DNA DNA; RNA RNA; v RNA cDNA kp RNA. Trong , cc retrovirus c h gene RNA gy ung th, bnh AIDS (HIV), bnh bch cu t bo T (HTLV-I)...u c cha enzyme phin m ngc (reverse transcriptase) tng hp cDNA si n trn khun RNA ca n, ri sau l cDNA si kp c th xen vo nhim sc th vt ch trng thi provirus n nh. Li dng enzyme ny tng hp v to dng cDNA. + Phin m:

Hnh 2.8: S minh ha qu trnh phin m mt s virus

2. Di truyn hc vi khun 2.1. Vt liu di truyn ca vi khun Vi khun l mt nhm ln ca Prokaryote, c cu trc t bo nhng cha c nhn in hnh. B my di truyn ca vi khun phc tp hn virus nhiu, thng gm 1 phn t DNA si kp-vng kch thc ln (v d E. coli l 4,6 x106 cp base). N lin kt vi cc protein tng t histon to thnh cu trc siu xon tp trung mt vng gi l vng nhn (nucleoid). V trong dch bo c rt nhiu phn t DNA trn si kp, dng vng, kch thc b bng khong 0,05-10% kch thc nhim sc th vi khun v c kh nng sao chp c lp, gi l cc plasmid. E. coli c nhiu loi plasmid vi tnh cht v s bn sao c mt khc nhau trong t bo, y cp 2 loi: - Cc plasmid khng thuc, gi l plasmid R (R-resistance), thng c mang nhiu gene m ha cc enzyme c kh nng phn hy cht khng

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sinh tng ng c mt trong mi trng. V d, plasmid pBR322 (4362 cp base) c 2 gene khng ampicilline (AmpR) v tetracyline (TetR). Loi plasmid ny thuc loi sao chp mnh, c th to ra khong 50 bn sao (copies) trong mt t bo. l nhng c im chnh m ngi ta li dng n lm cng c c lc (vector) trong k thut di truyn. - Cc plasmid gii tnh, tc plasmid F (F-fertility) c cha cc gene truyn v bt buc tham gia vo qu trnh tip hp E. coli. Cc t bo vi khun c mang plasmid F, k hiu F+ v t bo khng c F, k hiu F-. Nhn t F c kch thc ln (khong 100.000 cp nucleotide), c sao chp ch 1 ln trong chu k t bo (nh xen vo trong nhim sc th vi khun) v phn ly v c 2 t bo con. Cho nn trong 1 t bo F+ in hnh thng c 1-2 bn sao ca plasmid F. 2.2. Ba phng thc trao i vt liu di truyn vi khun Ln u tin, nm 1946 Lederberg v Tatum pht hin ra s ti t hp vi khun. Cho n nay, ta bit rng vi khun c cc qu trnh sinh sn tng ng sinh sn hu tnh, gi l cn hu tnh (parasexuality), l: tip hp, bin np v ti np. Cc qu trnh ny c cc c im sau: (1) Truyn thng tin 1 chiu t t bo cho (donor) sang t bo nhn (recipient); (2) To ra hp t tng phn (merozygote), v th cho ch truyn sang th nhn mt phn b gene ca n; (3) V b gene ch l mt phn t DNA trn nn ch c mt nhm lin kt v ti t hp v thc cht l lai phn t. Nhng hiu bit su sc v cc qu trnh sinh sn cn hu tnh vi khun gp phn quan trng trong s pht trin ca di truyn hc phn t cng nh ca k thut gene sau ny. a) Tip hp (Conjugation) Tip hp l s tip xc trc tip gia hai t bo vi khun, trong din ra s truyn i mt phn vt liu di truyn t th cho sang th nhn. y, nhn t F c truyn t t bo F+ sang F- trong qu trnh tip hp, nh kiu sao chp vng ln (rolling circle) hay cn gi l sao chp sigma (). Kt qu ca s tip xc l t bo F- cng tr thnh F+, ngha l c s thay i gii tnh (ci c). Tuy nhin, tn s lai F+ x Frt thp, khong 10-6. V sau, ngi ta cn pht hin mt dng vi khun, trong plasmid F c xen ci vo trong nhim sc th vi khun, dng ny c kh nng lai vi t bo F- vi tn s cao hn khong 104 ln, gi l t bo Hfr (High

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Frequency of recombination). Khc vi cc t bo F+, cc t bo Hfr cn c th truyn i mt phn nhim sc th vi khun qua ng tip hp. Li dng c tnh ny ngi ta lp bn di truyn bng tip hp cho hn 700 gene ca nhim sc th E. coli v cho thy n c dng vng. b) Bin np ( Transformation) Bin np l hin tng xm nhp ca DNA ngoi bo vo t bo th nhn v gy bin i mt s c tnh ca th nhn bi nh hng ca DNA th cho. Hin tng bin np c Frederick Griffith pht hin ln u tin nm 1928 v v sau c Oswald T. Avery, Colin MacLeod v Maclyn McCarty cng pht hin li nm 1944.

1

2

3

4

Hnh 2.9: 1. Frederick Griffith; 2. Oswald T. Avery (1877-1955); 3. Colin MacLeod (1909-1972); 4. Maclyn McCarty (1911-)

Khc vi tip hp v ti np, bin np l qu trnh chuyn DNA trc tip tch ra t t bo th cho sang t bo th nhn. c trng ca bin np l ch DNA th cho phi dng xon kp. Hiu qu ca n ph thuc vo kh nng dung np ca t bo nhn, kch thc on DNA v nng DNA. T bo c kh nng tip nhn on DNA ngoi lai gi l t bo kh bin, v c th xy ra lng bi ha mt phn b gene (hp t tng phn). S gn kt on DNA ngoi lai vo DNA t bo nhn c thc hin nh c ch ti t hp tng ng. V sau khi chui qua mng t bo nhn, mt si ca on DNA ngoi lai b phn hy, cho nn nu nh on si n cn li khng c gn vo th s b phn hy lun. Ni chung, tn s xut hin cc t bo kh bin l rt thp (ngay c cc loi vi khun c kh nng bin np) v tn s bin np (i vi cc t bo kh bin) cng ch khong 10-3. V vy, bin np ch c dng lm

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k thut lp bn gene cho 1 s loi. Tuy nhin, ngy nay bin np c ng dng rt rng ri nh l mt khu c bn trong k thut di truyn: cy-chuyn gene, tim lp-ghp gene,... E. coli, nm men bia, thc vt, ng vt v c ngi. c) Ti np (Transduction) Ti np l qu trnh truyn DNA t vi khun cho sang vi khun nhn nh phage. C 2 kiu ti np: ti np chung v ti np c hiu. * Ti np chung l trng hp phage truyn bt k gene no ca vi khun cho sang vi khun nhn E. coli, phage P1 l phage ti np chung c nghin cu k. Trong khi nhim vo vi khun, phage P1 sn ra nuclease ct DNA vi khun ch thnh tng on mt cch ngu nhin. V sau, trong qu trnh lp rp v protein vi DNA phage, c khong 10-3 10-2 ht phage con mang on DNA vt ch. Khi cc phage ti np ny xm nhp vi khun khc, s xy ra s ti t hp tng ng vi NST vt ch mi. on gene ti np thng cha khong 50 gene v ti np c th c dng lp bn gene. * Ti np c hiu l trng hp phage ch truyn i nhng gene nht nh t vi khun cho sang vi khun nhn. Kiu ti np ny c mt s c im sau: c thc hin bi prophage lambda (); Nhng gene c chuyn nm st ch prophage xen vo; Do kt qu ca s ct sai ca phage khi tch khi NST vt ch; Cc vi khun ti t hp c th lng bi mt phn. Tuy nhin, do s ct sai ca phage lambda rt him nn ti np c hiu c tn s thp. 3. Di truyn hc vi nm Sau y l mt vi nt s lc v nm men. Hin nay, S. cerevisiae v Schizosaccharomyses pombe ang c nghin cu rng ri mc phn t. Chng c chu trnh sng n gin c lin quan ti tt c pha n bi (n) v pha lng bi (2n). i vi cc t bo (2n) din ra nhng phn chia gim nhim thng thng to ra cc bo t (n). Ton b 4 sn phm ca gim phn c bc bn trong mt ci nang dy c chia . iu cho php kim tra cc on NST trao i cho thun nghch ring bit. Cc t bo nm men S. cerevisiae phn chia bng qu trnh ny chi lm tch cc t bo cha m ra khi cc t bo con (chi) ca chng. Ngc li, S. pombe l nm men phn i (fission), cc t bo cha m sinh trng v sau ngt i to ra 2 t bo ging nhau v hnh thi.

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Trung bnh h gene n bi ca nm men S. cerevisiae c cha khong 14 x 106 cp base, ngha l c hm lng DNA ln hn E. coli khong 3,5 ln, trong khi rui gim Drosophila ln gp 25 ln v cc t bo thc vt bc cao cha nhiu gp t nht l 1.000 ln. Nh vy, dng nm men trong nghin cu di truyn nhn chun qu l n gin v r hn. S. cerevisiae DNA c phn phi trong 16 NST vi khong 5 - 6 ngn gene, trong khi S. pombe ch c 3 NST khc nhau. Tnh n 1996, c 6000 gene ca S.cerevisiae c lp bn . iu ng ni l nhiu t bo nm men cng c cc bn sao phc ca cc DNA-plasmid ni sinh dng vng 2 m (6300 cp base) c kh nng sao chp c lp, thng c 50 - 200 bn sao trn mt t bo. Bnh thng chng khng mang mt gene thit yu no. y l loi vector quan trng trong k thut di truyn. Trong s rt nhiu loi nm men c ng dng rng ri trong cng nghip sn xut bia, ru, nc gii kht, to sinh khi ..., loi S. cerevisiae hin ang c dng nh mt cng c c lc mang cc DNA ti t hp c cc gene quan trng ca ng vt v ngi, nhm sn xut cc protein c hot tnh sinh hc, nh: cc interferon, insulin, hormon sinh trng, hirudin, cc khng nguyn virus v.v.. dng trong chn nui, phng v cha bnh. 4. Cc sinh vt mang gene ti t hp 4.1. Cc vi sinh vt ti t hp Mt trong nhng ng dng u tin ca k thut di truyn l to ra mt chng Pseudomonas syringae. Chng hoang di ca vi khun ny thng thng cha mt gene to bng, gene ny kch thch s to bng trn cc b mt lnh v m. S bin i gene ny to ra mt th ti t hp c kh nng ngn nga s to thnh bng. c to ra di tn gi l Frostban, vi khun ny mang li mt thnh cng khim tn trong vic chng li vic to thnh bng trn cc cy du ty v khoai ty ngoi ng rung. Pseudomonas fluorescens c ti t hp vi cc gene sn sinh cht dit cn trng sinh hc ca Bacillus thuringiensis. Cc vi kun ny c th vo t, chng s bm vo r v gip tiu dit cc cn trng ang tn cng. Nm 1979 xut hin thng bo u tin v s biu hin gene insulin ngi sau khi c gp vo vi khun E. coli, nm 1982 insulin c sn xut bng k thut ny c bn ra trn th trng vi tn thng phm Humulin (Human insulin). Hin nay ngi ta dn thay th vi khun E. coli mang gene ti t hp bng cc VSV khc nh nm men thuc chi Saccharomyces, x khun- Streptomyces....

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4.2. Cc thc vt ti t hp Tham gia vo hu ht cc thao tc ca k thut di truyn thc vt c mt loi vi khun hp dn, l Agrobacterium tumefacien. Vi khun ny l mt tc nhn gy bnh t nhin thc vt, n xm nhp vo r v to nn mt khi u c gi l bnh mn tn. A. tumefacien cha mt plasmid ln (Ti), n c gn vo gene nom ca cc t bo r b nhim khun v lm bin i cc t bo ny. Ngay c khi cc vi khun b cht trong khi u th khi u vn tip tc pht trin. Plasmid ny l mt vector hon ho a cc gene l vo gene nom thc vt. Trong a s trng hp, Plasmit Ti c ly ra, ghp vo mt gene c la chn ri li a tr li vo Agrobacterium.

Hnh 2.10: Khi u thc vt cha A.tumefacien cha Ti plasmid

Khi thc vt b nhim khun, cc vi khun ti t hp s t ng chuyn gene mi vo cc t bo r ca cy. Vic gn gene theo cch ny lc u c tin hnh cc cy hai l mm nh khoai ty, c chua, thuc l v bng. Cho n nay, cc thc vt ti t hp, hay chuyn gene, ch yu l cc cy bng hoc thuc l khng vi cht dit c, cc thc vt c kh nng dit cn trng, nhiu loi khng vi virus hoc vi nm v mt loi c chua khng chn qu sm v khng b v khi vn chuyn. 4.3. Cc ng vt ti t hp Khng ch virus, vi khun, nm v thc vt m c ng vt cng c th c thit k v mt di truyn. Trong mt s iu kin ng vt cng c th biu hin cc gene c gn nhn to bi chuyn nhim. Cc ng vt u tin c chuyn gene l nhng con rui gim m nhng khuyt

32

ht v mu mt ca chng c sa cha nh vic a cc gene bnh thng vo trng khim khuyt. Chut l nhng ng vt c bit d bo trong vic chp nhn gene theo cch ny. Phi ca chut th tinh c cy cc gene quy nh cc hocmon sinh trng ca ngi sinh ra cc con chut khng l ln gp hai so vi chut bnh thng. Trong mt th nghim khc, cc hp t ca chut do mang mt khim khuyt di truyn thng lm cho chng b bnh run, c sa cha nh cha gene bnh thng mi c a vo. Ngnh chn nui nhanh chng tip thu mt s trong s k thut ny. Nm 1987, phi ln c cy bng gene hormon sinh trng ca b trong mt th nghim nhm tng tc sinh trng ca ln v ct gim lng m to thnh trong khu phn tht. Nhng con ln u tin c t m hn, song tht khng may, chng cng c nhng vn v sc kho nghim trng: vim khp, bnh tim v bnh thn. Khuynh hng gn y nht trong cc th nghim chuyn gene l thit k trng th tinh ca cc ng vt nui nh ln, cu vi cc gene ca ngi. Cc ng vt ti t hp ny s c dng lm cc h thng sng sn sinh ra mt lng ln cc protein c gi tr ca ngi nh hemoglobin, yu t ng mu v hormon.

Cu hi n tp 1. Vi sinh vt c nhng c im chung no ? 2. Hy gii thch ti sao trong thin nhin vi sinh vt tng lnkhng tng ng vi tc sinh sn v cng nhanh chn ca n. 3. Vt liu di truyn ca virus ? Mi quan h di truyn gia virus v t bo vt ch ? 4. Vt liu di truyn sinh vt nhn s khc vi sinh vt nhn chun nhng c im no ? 5. Th no l tip hp, bin np v ti np ? Ti sao ni cc qu trnh ny tng ng vi sinh sn hu tnh sinh vt bc cao ? ngha ca vic pht hin ra hin tng bin np vi khun.

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Chng 3

S phn loi sn phmI.Phn loi theo tnh cht thng miTheo Thomas D. Brock (1995), cc sn phm vi sinh vt c ngha cng nghip c phn thnh 5 loi chnh: - Bn thn cc t bo vi sinh vt (sinh khi) l cc sn phm mong mun. - Cc enzyme do vi sinh vt to nn: amylase, protease, lipase... - Cc dc phm: cc cht khng sinh v cc alcaloit. - Cc ho cht c bit v cc cht iu v thc phm: bt ngt nhn to aspartam l mt dipeptide gia aspartic v phenylalanin; acid glutamic, lysine v triptophan, mt s vitamin. - Cc ho cht thng dng c sn xut bng con ng vi sinh vt bao gm ethanol, acid acetic, acidlactic v glycerine 1. Sinh khi t bo l cc trng hp ca nm men dng cho mc ch dinh dng v lm n bt m, trng hp cc nm n dng lm thc n. Cc vi khun v vi to cng c nui cho mc ch dinh dng, song cho n nay cha c ngha ln v mt thng mi. y cn phn bit hai trng hp: Thut ng ''protein n bo'' (SCP) thng c dng ch cc t bo cc vi sinh vt nh l cc sn phm cng nghip vi l do l hm lng protein trong chng cao v c quan tm v mt thng mi. Cn ''ging khi ng'' (starter culture) l cc sn phm cng nghip trong bn thn cc t bo vi sinh vt c dng lm nguyn liu cy. Chng hn cc ging vi khun lactic c bn ra di dng cc nguyn liu cy (inoculants) sn xut cc sn phm sa ln men v xc xch. 2. Cc enzyme do vi sinh vt to nn Nhiu enzyme quan trng c ngha thng mi c sn xut quy m cng nghip nh vi sinh vt, bao gm cc enzyme phn gii tinh bt (cc amylase), cc enzyme phn gii protein- protease, cc enzyme phn gii cht bo (cc lipase) Mt enzyme quan trng v mt cng nghip l gluco-isomerase c s dng vi s lng ln sn xut fructose c ngt cao hn glucose. Mt enzyme vi sinh vt quan trng khc l penicillin -acilase c s dng trong cng nghp sn xut cc penicillin tng hp.

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3. Cc dc phm Cc cht khng sinh v cc alcaloid nn trong nhm cc sn phm bc 2. l cc hp cht khng c to thnh trong pha sinh trng u tin m vo lc sinh trng bc vo pha cn bng. Vic hiu bit bn cht ca s trao i cht bc hai c tm quan trng trong trong vic pht trin cc qu trnh sn xut mi. Cng ty Cng nghip ho cht Takeda (Nht Bn) sn xut v cung cp loi thuc khng sinh validacina dng phng tr nhiu loi bnh hi cy trng do nm gy ra. Validacina c bit c hiu lc vi bnh kh vn la, trc tip bo v, lm tng nng sut v cht lng la. Thuc khng gy bt c hin tng ng c no cho cy trng, rt an ton cho ngi, vt nui, c... v nhng ng vt c ch khc; khng tn d trong t v nng sn sau khi thu hoch. Validacina c th hn hp vi hu ht cc loi thuc khc phun. Thuc validacina c cc dng thng phm: Validacin 3L, Validacin 5L, Validacin 5SP. 4. Cc ha cht c bit v cc cht iu v thc phm Cht iu v thc phm: bt ngt nhn to aspartam l mt dipeptide gia acid aspartic v phenylalanin, c hai amino acid ny u c sn xut bng con ng ln men vi sinh vt. cc amino acids khc cng c sn xut bng con ng ny l: acid glutamic, lysine v tryptophan. Mt s vitamin cng c sn xut bng con ng vi sinh vt, l riboflavin, vitamin B12 v acid ascorbic (vitamin C). 5. Cc ha cht thng dng l nhng cht c gi thnh thp v do vy c bn ra vi s lng ln. Cc ha cht thng c dng lm nguyn liu khi u tng hp ha hc cc phn t phc tp hn. Cc ha cht thng dng c sn xut bng con ng vi sinh vt bao gm ethanol, acid acetic, acid lactic v glycerol. Trong s ny ethanol l sn phm quan trng nht. Ethanol c sn xut nh nm men, bn ny c th to ra mt sn lng ethanol v CO2 t ng. Hin nay ethanol c dng lm nguyn liu tng hp ha hc v lm nhin liu chy cc ng c (gazohol).

II. S phn loi sn phm theo sinh l trao i chtKhc vi s phn chia sn phm mang tnh thng mi (Fritsche, 1978) chia cc sn phm ca vi sinh cng nghip thnh 3 loi. 1. Vt cht t bo (sinh khi) bao gm cc loi protein n bo v cc loi ging khi ng nh men bnh m cc vi khun lactic.

35

Bng 3.1: Mt s enzyme cng nghp v ng dng Enzyme Amylase Catalase Cellulase Glucooxydase Pectinase Penicillinase Protease Streptokinase Aspergillus, Rhizopus Ngun ng dng chnh

Bacillus, B sung vo bt, cng nghp dt, tr gip tiu ha Phng oxy ha thc phm, sn xut phomat Thy phn g, tr gip tiu ha Loi glucose hoc oxy trong thc phm, xc nh glucose lm sng Lm trong vang, dm, bia, dch qu Loi penicillin trong nghin cu Bacillus, Lm trong sake, ch bin tht, loi gelatin Lm lnh cc vt thng vt bng

Micrococcus, Aspergillus Aspergillus,Trichoderma Aspergillus Aspergillus, Sclerotinia Aspergillus Aspergillus, Streptomyces Streptococcus

2. Cc sn phm trao i cht bao gm: - Cc sn phm ln men. V d ethanol, acid lactic, methane, acetolbutanol... - Cc cht trao i bc 1: V d amino acids, nucleotide, vitamins, ng,.... - Cc cht trao i bc 2: V d cht khng sinh, alcaloid, gibberellin, IAA... - Cc loi enzyme: V d cc enzyme ngoi bo nh protease, amylase; cc enzyme ni bo nh asparaginase, penicillinase. 3. Cc sn phm chuyn ho Cc sn phm chuyn ho bao gm steroids v cc sn phm ca s oxi ho khng hon ton nh s to thnh acid acetic v socbose.

II. Sinh trng v s to thnh sn phmTrong iu kin nui cy tnh, qu trnh sinh trng ca mt qun th VSV tri qua 4 giai on (Tim pht logarit cn bng ng suy vong). Ton b qu trnh nui gn lin vi s thay i ko di ca cc

36

iu kin nui. Cht dinh dng gim i, s lng t bo tng ln ri gim dn, ng thi hot tnh trao i cht cng thay i. S lin quan ca cc sn phm trao i cht vi t bo c th phn bit thnh 2 nhm: cc sn phm bc mt v cc sn phm bc hai. Mt cht trao i bc mt l mt cht c to thnh trong pha sinh trng u tin ca VSV trong khi mt cht trao i bc hai l mt cht c to thnh gn vo lc kt thc ca pha sinh trng, thng l vo gn chnh pha cn bng.

Hnh 3.1: ng hc ca s sinh trng v to thnh sn phm VSV a. Sinh trng v to sn phm din ra ng thi b. S to thnh sn phm bt u sau khi sinh trng kt thc

*c im ca sn phm trao i bc hai: - Cc sn phm bc 2 ch c to thnh bi mt s tng i t c th v dng nh khng cn tht cho sinh trng v sinh sn. - S to thnh cc sn phm bc hai ph thuc vo cc iu kin sinh trng, c bit l thnh phn ca mi trng. - Chng thng c to thnh di dng mt nhm cc cht c cu trc gn gi. Chng hn, mt chng Streptomyces sn sinh ra 32 cht khng sinh antracyclin khc nhau nhng c cu trc gn ging nhau. - Thng c th nhn c s tng hp tha t ngt mnh cc sn phm bc hai, iu ny thng khng gp cc sn phm bc mt l loi sn phm c lin quan cht ch n pha sinh trng. Trong trao i cht bc hai c hai pha trao i cht khc bit nhau c gi l pha sinh trng (trophophase) v pha sn xut (idiophase). Nu chng ta nh sn xut cc sn phm bc hai th chng ta phi m bo rng cc iu kin thch hp cn c to ra trong pha u sinh

37

trng din ra ti u v cc iu kin phi c thay i ng lc s to thnh sn phm c th din ra ti u. Trong trao i cht bc hai, sn phm mong mun c th khng bt ngun t c cht sinh trng ban u m t mt sn phm c to thnh t c cht sinh trng ban u. Nh vy, ni chung sn phm bc hai c sinh ra t mt s sn phm trung gian tch lu trong mi trng nui cy hoc trong t bo trong qu trnh trao i cht bc mt. Mt trong cc c im c trng ca cc cht trao i bc hai l cc enzyme tham gia vo s to thnh chng c iu ho mt cch c lp vi cc enzyme ca trao i cht bc mt. 3. Sinh tng hp tha Qu trnh ln men cng nghp i hi s to thnh sn phm mong mun vi lng cao nht. VSV tn ti trong t nhin sinh ra cc sn phm trao i cht v cc thnh phn t bo ch mc cn thit cho s sinh sn ti u v cho s duy tr loi. C ngha l, trong t nhin khng c s sn sinh d tha cc sn phm trao i cht bc 1, bc 2. Bi vy nhng th nghim nhm phn lp cc chng c kh nng sinh tng hp d tha t ni sng t nhin thng khng em li kt qu mong mun. Ch khi no nhng c th thch hp thu c do x l bng cc tc nhn gy t bin nh hng, c chn lc trong iu kin phng th nghim v nhng kh nng nht nh, th ta mi c th thu c cc chng tng hp tha b sai hng trong c ch iu ho. Nhng chng ny c coi l nhng chng c nng sut cao dng trong cng nghip. 3.1. Nhng nguyn tc iu ho trao i cht - iu ho hot tnh enzyme nh c ch bng sn phm cui cng hay cn gi l s km hm do lin h ngc; - S cm ng v c ch qu trnh tng hp enzyme; - iu ho tng hp enzyme nh s kim ch bng sn phm cui cng v s gii kim ch; - iu ho tng hp enzyme nh s kim ch d ho. 3.2. Nhng sai hng di truyn ca iu ho trao i cht v hin tng tng hp tha Cc c ch iu ho trao i cht c th b thay i do nhng t bin dn n s sinh tng hp tha cc cht trao i. Ton b nhng sai hng c th biu hin s cm ng enzyme. Nh nhng sai hng m cc enzyme cm ng tr thnh cc enzyme cu trc, ngha l chng tn ti trong t bo khng ph thuc vo c cht.

38

Nhiu hot tnh ca cc chng sn xut l do sai hng di truyn. Vic la chn cc chng sn xut trc ht l theo kinh nghim. Nhng thnh tu ca sinh hc phn t nh ch ng gy t bin nh hng, chuyn gp gen... to iu kin cho vic la chn nh hng hn v do c hiu qu cao hn. 4. Cng ngh DNA ti t hp Nh bit, hai s kin ni bt nht trong giai on u ca Sinh hc phn t l s khm ph ra cu trc phn t DNA nm 1953 bi James Watson v Francis Crick (nhn gii Nobel-1962) v s gii m thnh cng h thng mt m di truyn nm1966 bi Marsall Nirenberg v Har Gobind Khorana nhn gii Nobel-1968 cng vi nhiu nh khoa hc khc. Giai on tip theo ca nh cao pht trin sinh hc phn t l s ra i ca Cng ngh DNA ti t hp (recombinant DNA technology), vo gia thp nin 1970, vi cc tn gi khc nh: cng ngh gen, k thut di truyn (genetic engineering). Cng ngh DNA ti t hp bt u nh mt khoa hc t 7/1972 khi nhm nghin cu ca Paul Berg (Vin hn lm khoa hc quc gia M) hon thnh vic ch to b gen lai gia virus SV40 ca kh vi phage lambda. Ln u tin kt ni hai virus khc nhau hon ton v mt di truyn thnh mt chnh th v c nhn ln di dng plasmid trong t bo vi khun. Phn t DNA lai cha cc thng tin di truyn ca virus SV40, thng tin iu khin qu trnh t sao ca DNA phage lambda cng nh ton b chc nng ca operon galactosidase ca vi khun E. coli.

1

2

3

4

Hnh 3.2: 1. Har Gobind Khorana (1922-); 2. Temin, Howard Martin (1934-1994); 3. Daniel Nathans (1928-1999); 4. Paul Berg (1926-)

4.1. Khi nim cng ngh DNA ti t hp

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l cng ngh bao gm mt tp hp cc k thut nh phn ct v lai ghp cc phn t nucleic acid nh cc enzyme gii hn, ligase...nhn dng DNA trong cc plasmid hoc virus c tng cng vi khun hoc t bo nm men; xc nh trnh t nucleotide DNA on c nhn dng; biu hin ca gen c nhn dng di dng sn phm protein... S ra i ca cng ngh DNA ti t hp v gn y l cng ngh chp DNA, sinh tin hc, nui cy t bo gc, cng ngh nano gn lin vi cc s kin khoa hc sau: - 1965, Xc lp c cc gen khng thuc vi khun nm trong cc plasmid. - 1967, SB Zimmerman v cng s tch chit thnh cng DNAligase. - 1970, Hamilton Smith v cc cng s phn lp c enzyme gii hn u tin t vi khun Haemophylus influenzae c gi l Hind II. -1970, Har Gobind Khorana tng hp c mt gen nhn to u tin l gen m ha vic tng hp tRNAala vn chuyn amino acid alanine nm men. Gene ny di 77 cp nucleotide, khng c cc trnh t iu ha v v th khng c hot tnh, n 1973 mt gen nhn to khc c th hot ng bnh thng trong t bo sng, l gene m ha tRNAtyr vn chuyn cu tyrosin E. coli c chiu di khong 200 cp nucleotide. - 1972, Paul Berg kin to c phn t DNA ti t hp u tin trong ng nghim. - 1973, H. Boyer, S. Cohen, A. Chang v R. Helling - ln u s dng plasmid to dng DNA E. coli. -1975, Temin, Howard Martin, pht hin ra enzyme phin m ngc (reversetranscriptase) m ra s tng hp gene nhn to.

- 1977, xut cc phng php ha hc (Maxam v Gilbert) v phng php enzyme hc (Frederick Sanger v cng s) xc nh trnh t nucleotide ca nucleic acid. - 1977, Thnh lp hng cng ngh gen u tin M (Genetech) sn xut cc ch phm y hc bng phng php DNA ti t hp.

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- 1978, Gii thng Nobel u tin trong lnh vc khm ph v s dng enzyme gii hn c trao cho Daniel Nathans; Werner Arber;Hamilton Othanel Smith.

- 1983, Gii thng Nobel c trao cho b Barbara McClintock pht hin ra yu t di truyn vn ng hay gen nhy. - 1986, Binnig, Gerd Karl (Nh khoa hc c, gii Nobel 1986) khm ph ra loi knh hin vi hot ng khng theo nguyn tc quang hc, m u cho cng ngh nano. - Thng 4 nm 1996, men n bt m S.cerevisiae gii m xong gm 6.000 gene - Thng 12/ 2002, tp on Genome Sequencing Chut quc t hon thnh mt bn tho genome chut v so snh vi genome ngi c khong 3.000 gene chung. - Nm 2003, gii Nobel c trao cho hai nh bc hc Paul C. Lauterbur v Peter Mansfield c cng pht minh nhng ng dng ca hin tng cng hng t (magnetic resonance). - Nm 2005, gii Nobel Y hc 2005 c trao cho hai nh khoa hc Australia - Barry J. Marshall v J. Robin Warren - do khm ph ra vi khun Helicobacter pylori v vai tr ca chng trong bnh vim lot h tiu ho (chng vim d dy, lot d dy hoc t trng

1

2

3

4

Hnh 3.3: 1. Frederick Sanger (1918-); 2. Barbara McClintock (1902-1992); 3. Binnig, Gerd Karl (1947- ); 4. Paul C. Lauterbur (1929-)

4.2. Cc enzyme thng dng trong k thut di truyn Nm 1962, Werner Arber ln u tin chng minh rng c nhng enzyme c bit hot ng trong t bo vi khun, chng c kh nng phn bit DNA "ca mnh" v DNA "l" ca phage. Cc enzyme ny c kh nng hn ch s nhn ln ca phage l trong t bo vi khun bng cch phn hy chng mt cch c hiu, do c gi l "restriction

41

enzyme". Ch restrion (hn ch) c ngun gc lch s t v c dng n nay. Thng thng, khi cp n cc enzyme c s dng trong k thut di truyn, ch yu l ni n cc enzym gii hn (restriction endonuclease, hay gi tt l restrictase), tc cc enzyme t vi khun c kh nng nhn bit v ct DNA ti nhng v tr xc nh v ngoi ra l nhm hn hp cc polymerase, ligase v nuclease. Nhn chung, c hai kiu ct: ct thng v ct so le. + Kiu ct thng to ra cc u bng (blunt ends): Mt s enzyme gii hn ct hai mch DNA ti cng mt im, v d: AluI, SmaI (Bng 3.2). + Kiu ct so le to ra cc u dnh (cohesive ends): Mt s enzyme gii hn ct on DNA nhn bit ti v tr so le trn hai mch. Kt qu l to ra cc u si n cha vi base b sung c th dnh chp tr li. Cc enzyme ny c s dng rng ri trong k thut ti t hp DNA. V d: BamHI, EcoRI, XmaI (Bng 3.2).Bng 3.2 : Mt s enzyme gii hn v on nhn bit ca chng (mi tn ch v tr ct) Vi sinh vt Enzym gii hn on 5/3/ nhn bit 3/5/ AluI Arthrobacter luteus AGCT TCGA BamHI Bacillus GGATCC amylolyquefaciens H CGTAGG EcoRI Escherichia coli RY13 GAATTC CTTAAG SmaI Serratia marcescens Sb CCCGGG GGGCCC XmaI Xanthomonas CCCGGG malvaccarum GGGCCC

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* Cc enzyme thng dng khc Trong k thut di truyn, bn cnh s dng cc enzyme gii hn nh con dao m tinh vi, cn phi dng ti nhm enzyme sau: - Cc DNA- v RNA-polymerase: xc tc cc phn ng tng hp DNA v RNA. Chng c dng khi cn thit to ra mt s lng ln cc bn sao acid nucleic ( to cc mu d, xc nh trnh t nucleotide...). Trong phi k n DNA polymerase I ca E. coli, enzyme phin m ngc t cc retrovirus, enzyme transferase u cng. - Cc DNA- v RNA-ligase xc tc phn ng ni hai u ca cc on DNA hoc RNA thng dng trong to dng. - Cc nuclease. y l nhm enzyme phn ct DNA (DNase) v RNA(RNase) khng mang tnh chuyn bit cao nh cc enzyme gii hn. 4.3. DNA ti t hp v cc vector 4.3.1. Khi nim DNA ti t hp DNA ti t hp l phn t DNA c ti to trong ng nghim bng cch kt hp DNA t cc ngun khc nhau theo mt qui trnh k thut nht nh. Thng thng, mt phn t DNA ti t hp gm mt DNA ca plasmid hoc phage nguyn vn (gi l vector) c mang mt on DNA cn cho nghin cu (gi l DNA ngoi lai). 4.3.2. Khi nim vector: Vector (th ti, th truyn) l mt phn t DNA ng vai tr l vt trung gian mang mt on DNA cn nhn dng c kh nng xm nhp vo t bo ch v mn b my t bo ch to ra nhiu bn sao ca vector. Cc ng dng ch yu ca vector l: - To dng nhm khuych i s bn sao ca mt on DNA xc nh (nhiu bn sao ging nhau). - Nghin cu s biu hin ca mt on trnh t ca DNA. - a gen vo t bo hay c th (nhm bin i c tnh di truyn mong mun sinh vt c truyn gen). - Sn xut RNA vi s lng ln t DNA c to dng. - Sn xut protein vi s lng ln t DNA c to dng.4.3.3. Cc c tnh ca mt vector - C kh nng xm nhp vo t bo ch v tn ti c qua nhiu th h. - C kh nng t sao chp mnh v c lp vi s ti bn ca b gen t bo ch.

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- Phi c cc c tnh (c m ha bi cc gen chn lc) cho php d dng pht hin t bo ch c cha chng. Chng hn, nu vector l plasmid th t bo ch c th c pht hin theo kiu hnh ca plasmid c mt trong t bo ch, tc c tnh khng thuc gip vi khun sng c trn mi trng c cht khng sinh tng ng. 4.3.4. Cc loi vector chuyn gen

Hnh 3.4: Bn enzyme hn ch ca plasmid pBR322 ci bin, mang hai gen khng thuc (tetR khng tetracycline v ampR khng ampicillin) v mt lot v tr ct ca enzyme hn ch trong mi enzyme ny ch c mt v tr phn ct, nh vy plasmid vector ny p dng c vi nhiu DNA khc nhau.

C nhiu loi vector c s dng trong k thut di truyn: plasmid, phage M13, cosmid, vector l virus ca eukaryotes, YAC (nhim sc th nhn to ca nm men). * Cosmid l vector c cu to nn t plasmid c gn thm gene cos ca phage . Gene cos gip cho DNA ca phage t dng thng ni u li thnh dng vng. Cosmid c kh nng cha on gene l di n 45kb hin c dng lp th vin gene rui dm, chut, ngi. * Phage M13 l phage th si c DNA mch n dng lm vector nhm: + Xc nh trnh t nucleotide ca gene; + Sn xut cc mu th (hay mu d); + Thc hin t bin im nh hng.

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* Phagemid l vector c cu to t plamid gn thm mt on DNA ca phage M13. Vic la chn loi vector ty thuc vo kch thc on DNA cn to dng v mc ch to dng, y ch cp hai loi vector thng dng: plasmid v phage. - Cc plasmid ni chung c kch thc 2-5kb, nn ch cha rt t gene chn lc v ch c th nhn 8-9 kb DNA cn to dng. - Cc phage l nhng virus xm nhim v lm tan vi khun ch. Vic s dng phage lm vector c nhiu u im hn vector plasmid ch hiu qu xm nhim mnh v kh nng sinh si ny n nhanh; kch thc on DNA m vector c th tip nhn c l ln hn rt nhiu. Tuy nhin, thao tc trn phage th phc tp hn trn plasmid. Trong s cc phage c s dng lm vector, phn ln bt ngun t phage lambda (h gene cha 48.502 cp base, c gii xong nm 1983; n c hai u dnh t nhin khong 12 cp base, cho php DNA- ng vng sau khi xm nhp vo t bo ch). 4.4. Cc phng php thnh lp phn t DNA ti t hp 4.4.1. Phng php s dng cc u dnh Bt k on DNA no nu c ct bi cng mt loi enzyme gii hn (v d EcoRI) to cc u dnh th c th dnh lp li vi nhau v c ni bi DNA ligase (hnh 3.4). Nm 1973, H. Boyer v tp th ca S. Cohen to ra c phn t DNA ti t hp u tin gm vector l plasmid nh pSC101 ca E. coli v DNA ngoi lai l mt plasmid khc. Chnh s kin ny t ra trin vng to ln cho k thut DNA ti t hp sau ny. 4.4.2. Phng php ni trc tip hoc tng hp cc u b sung i vi cc on DNA c to ra bng vic s l enzyme gii hn ct thng, nh HindII chng hn, th vic ni cc on DNA u bng c thc hin theo mt trong hai cch sau: (1) Ni trc tip nh DNA-ligase ca phage T4 (2) Tng hp b sung cc u dnh vo cc u 3 mt s nucleotid, v d: poly (dA) vo u 3 ca on ny v poly (dT) vo u 3 ca on kia, nh enzyme transferase u mt. Sau cc on ny c ni li nh xc tc ca DNA-ligase vi khun. 4.5. To dng DNA ti t hp Mc ch ca vic to dng l nhm thu c mt s lng ln bn sao ca mt on DNA xc nh.

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V nguyn tc, mt quy trnh k thut DNA ti t hp (to dng) gm cc bc c bn sau : Bc 1: Tch DNA v plasmid ra khi t bo, chn lc v x l DNA thuc cc ngun khc nhau, mt lm vector v mt cn cho to dng. i vi vector, vic chn lc ty thuc nhiu yu t: kch thc on cn to dng, mc ch to dng... i vi DNA cn to dng, cn chn s khi cc on c kch thc gn nhau v tng ng vi loi vector. Sau , x l c hai loi DNA trn bi cng mt loi enzyme gii hn (v d: EcoRI) to ra cc u (dnh) so le. Bc 2: Kin to phn t DNA ti t hp trong ng nghim. Trn chung vector v DNA cn to dng c x l theo mt t l nht nh vi s c mt ca ligase. Nh s xc tc ca DNA-ligase m vector ti t hp s c to thnh; sau c tinh sch qua tch chit v ta.

Hnh 3.4: S minh ha cc bc c bn ca k thut di truyn

Bc 3: a vector ti t hp vo t bo ch. C hai loi t bo ch thng dng nht l: (1) T bo vi khun, thng l E. coli; (2) T bo sinh vt nhn chun, v d nm men S. cerevisiae hoc t bo ng-thc vt nui cy. Ty loi t bo ch m s dng k thut bin np thch hp. Ni chung, mc ch ca bc ny l li dng c tnh sinh si ny n nhanh ca t bo ch v s dng b my t bo ch ti bn vector ti t hp thnh mt s lng ln bn sao hoc c th cho biu hin gen (protein) mong mun.

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Bc 4: Pht hin dng DNA ti t hp c hiu. pht hin dng cn tm, ngi ta c th s dng cng c l mu d (probe). Mu d c th l mt khng th c trng cho protein c m ha bi gen cn tm hoc mt on DNA hoc RNA b sung cho gen cn tm. 4.6. ng dng ca cng ngh DNA ti t hp Trong sut hai thp k qua, cng ngh DNA ti t hp mang li nhng tin b to ln cho sinh hc. Nhng ng dng ch yu ca n l: - Sn xut cc protein, enzyme, vaccine... hu ch; to ra cc vi khun, nm men c kh nng tng hp cc cht c ngha v mt kinh t. - Gy ra nhng bin i kiu gen cc c th sinh vt lin quan n nhng c tnh mong mun. To ngun DNA v RNA cho cc nghin cu. - To ra kh nng sa cha cc khuyt tt di truyn ng vt v ngi (liu php gen hnh 3.5) - Chn gene kho vo trong ribonucleic acid (RNA)ca Retroviruses;- Cho restrovirus tip cn t bo b bnh; - Thm gene kho vo deoxyribonucleic acid ( DNA) t bo b bnh; - Tim vo bnh nhn. Cng ngh DNA ti t hp m ra mt trin vng to ln trong tng lai gn gip con ngi khm ph b n ca c th vi sinh vt v con ngi, trn c s c th nng cao nng sut vt nui, cy trng v bo v sc khe con ngi.

Hnh 3.5: Phng php cha bnh bng gen

Cu hi n tp

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1. Cc kiu phn loi sn phm hin nay ? Theo anh (chi) kiu phn loi no hp l nht ? V sao ? 2. c im ca sn phm bc hai ? 3. ngha thc tin ca vic nghin cu sinh tng hp tha ? 4. Th no l enzyme hn ch ? Chng c cc vai tr v tnh cht no c coi l quan trng i vi k thut DNA ti t hp ? 5. Bng hai v d v enzyme ct hn ch, hy chng t rng t nht c hai phng php thnh lp cc phn t DNA ti t hp trong ng nghim (in vitro). 6. Th no l phn t DNA ti t hp, vector tch dng ? Hy nu cc bc ca quy trnh to dng v cho s minh ha. 7. Anh (ch) hy nu mt s ng dng khc nhau ca cng ngh DNA ti t hp.

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Chng 4

Cc phng php v k thut ln menI. Qu trnh ln men1. Quy trnh ln men (hnh 4.1)Ging ng kh Ch to mi trng dinh dng Nguyn liu (C,N,P,K) du ph bt, mui khong, nc Kh trng thit b, h thng ng ng, h lc kh

Ging thch nghing

Kh trng mi trng

Ging nui trn my lc Ln men Nhn ging trong ni nhn ging Cung cp khng kh v trng

Thu hoch tinh ch to thnh phm Hnh 4.1 S qu trnh ln men

2. Chng ging chn c ging vi sinh vt thun chng, bc u tin phi phn lp chng t cc ngun t nhin nh nc, khng kh, t, cc vt liu hu c, v c b phn hy... T nhng k thut vi sinh vt hc c in t thi L. Pasteur v R. Koch ra, nhiu phng php c bit dng phn lp chng ging thun khit dng cho cng nghip c pht trin, nht l trong vic tm chng sn xut nhng cht khng sinh mi. T nhng sinh thi t nhin s phn lp c cc chng hoang di. Cc chng ny c mt s hot tnh sinh enzyme, tch t cc cht trao i bc 1, bc 2 ... nhng thng cho nng sut thp. Theo k thut vi sinh vt c in vic phn lp cc chng thun khit mt nhiu cng sc v chm. Ngy nay, ngi ta dng phng php sng lc va nhanh va c hiu qu. Phng php sng lc ch yu c s dng trong vic tm chng sn cc cht khng sinh. T nguyn l c bn

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phng php c ci tin ngy mt phong ph. chn cc chng sn sinh aminoacidngi ta s dng kiu chn lc theo k thut penicillin. Trong phng php ny cc iu kin c la chn sao cho cc t bo hoang di c th pht trin trong mi trng dinh dng thiu mt aminoacidno v b git cht bng penicillin. Cc t bo cn aminoacid(t dng acid amin) khng sinh trng c nn s sng st. i vi vi khun khng mn cm vi penicillin th dng cht khng sinh khc, nh canamycin hay xycloserin, i vi nm men th dng nystatin l thch hp. phn lp nhng chng c tnh cht c bit, v d nh chuyn ha steroid, ngi ta dng hn hp ca nhiu chng vi sinh vt em nui cy trong cng mt mi trng c cht m ta mun thc hin s bin i. Sau khi nui, chit xut v tch sn phm phn tch theo phng php sc k. 3. Nguyn liu cy Ging sn xut thng c bo qun trnh gim hot tnh. Do , vic cy ging trn mi trng thch nghing trc khi nhn ging l vic lm rt cn thit. C th coi y l vic nh thc chng ging ng thi kim tra hot tnh ca ging sau mt thi gian bo qun nhit thp. T nhng nhng t bo hoc bo t ring r ca chng bo qun, cy ra mt s culture, nhng culture ny c nhn ging trong phng th nghim v c kim tra hot tnh. Nu c s khc nhau th dng culture c hot tnh mnh nht gy nguyn liu cy v to thnh chng mi. 4. Nhn ging Cng ging nh trong phng th nghim v qui m pilot, mun thc hin mt qu trnh ln men qui m cng nghip phi tin hnh nhn ging, m bo s lng t bo vi tui sinh l ang thi k hot ng mnh nht cy vo mi trng ln men. Nhn ging y c th phi qua 2-3 bc, ta thng gi l nhn ging cp 1, cp 2, cp 3 v.v... tu thuc vo quy m sn xut. Vic nhn ging thng din ra bng cch nui chm. Cc iu kin nui c la chn sao cho ch xy ra s sinh trng ch khng xy ra s to thnh sn phm. Khi s dng nm sinh bo t v x khun, trc khi nui chm, ngi ta thc hin nhn bo t trn mi trng c nh mi trng cm, bt ng... Nhn ging cp 1 c tin hnh trn my lc vi nhit v thi gian tu thuc vo ni nhn ging (tng t ni ln men) c sc kh v n nhit. T l nhn ging t ng nghim vo bnh tam gic c th ch mt vng que cy hoc c dch huyn ph ca mt ng ging. T nhn ging cp 1 sang cp 2 (hoc t cp 2 sang cp 3) t l ging thng l 1- 10%

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th tch dch ln men hoc l cao hn, t dch nhn ging cui cng vo ni ln men khong 0,5-10% tu thuc vo c tnh tng chng vi sinh vt.

Hnh 4.2: My lc n nhit (Personal-11 , TAITEC)

Thnh phn mi trng nhn ging v mi trng ln men gn ging nhau. Thng thng th hm lng carbon mi trng nhn ging thp hn mi trng ln men, nhng cc thnh phn khc th giu hn, c bit l cc cht sinh trng phc v cho sinh sn v pht trin ca ging vi sinh vt. Ch nui c bit l nhit gia nhn ging v ln men cng khc nhau (nu cng ch nhit th khng cn phi quan tm lm). Nhng nu nhit ln men thp (nh ln men bia) th cn phi nhn ging vi nhit gim dn khi vo ln men, ging khng b chong sc. Ch sc kh cc cng on ny cng khc nhau, thng th trong thi gian nhn ging nhu cu v xy cao nh trong pha sinh trng ca ln men hoc cao hn. Ni chung mt chng vi sinh vt nhn ging a vo ln men m bo cc yu cu cng ngh nh sau: - Dch ging khng c tp nhim, c bit l thc khun th (Bacteriophage). - Cc t bo m bo tui sinh l thi gian sinh trng tt nht, c hot tnh cao nht, thng l na sau ca pha ch s. - Cc thng s k thut nh OD, pH, mu sc, mi v... ng nh quy nh ca tng dy chuyn cng ngh.

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5. Ni ln men Cc ni ln men c thit k v ch to sao cho c th to c nhng iu kin ti u cho tng qu trnh ln men. Nhng yu cu c th t c hot tnh ti a ca vi sinh vt c thc hin thng qua mt s nguyn tc k thut. Ni ln men cha mi trng nui c kh nng to thnh sn phm vi nng sut cao. Trong qa trnh ln men cn theo di lin tc s to thnh sn phm v trng thi v trng dng qu trnh ng vo thi im thu hoch tt nht.

Hnh 4.3: Ni ln men 75 lt Trung tm CNSH-HQGHN

6. Thu nhn sn phm v x l sau thu hoch Vic thu nhn sn phm c bt u bng cch tch ring t bo ra khi mi trng dinh dng. Nu l nhng c th c dng h si th ngi ta thng lc, cn i vi vi khun v nm men th li tm. Vic x l tip theo l tu theo sn phm c tit ra mi trng dinh dng hay tn ti trong t bo. Bn cht ho hc ca sn phm quy nh cc bin php x l tip theo. Cc bin php c s dng l chit rt, hp ph, sng phn t v kt ta. Cc bc tinh ch tip theo c tin hnh k tip ngay sau bc tch sn phm thng phi qua nhiu cp, trc khi sn phm cui cng c ng gi. Vic thu nhn sn phm vi hiu sut cao c ngha quyt nh i vi tnh kinh t ca mt phng php. Bi vy, vn tch v c lp sn phm phi c ch ngay t khi chn chng v chn dch dinh dng. Vic ti u ho phng php c lin quan n tt c cc bc. Vic loi

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b v s dng cc ph v ph phm cng cn c ch trnh gy nhim mi trng.

Hnh 4.4: My li tm ( centrifuge)

II. Vi sinh vt1. Sinh vt nhn chun 1.1. Nm men Nm men (Yeast, Levure) thng tn ti dng n bo, a s sinh sn theo li ny chi, cng c khi theo hnh thc phn ct t bo, nhiu loi c kh nng ln men ng v thch nghi vi mi trng cha ng cao, c tnh acid cao. Nm men phn b rng ri trong t nhin, nht l trong mi trng c cha ng, c pH thp, chng hn nh trong hoa qu, rau da, r ng, trong t trng cc loi cy n qu, trong t c nhim du m. Nhiu loi nm men c ng dng cao trong sn xut cng nghip nh ln men bia ru, glycerine, sn xut nm men bnh m, thc n gia sc... 1.2. Nm si Nm si (Microfilamentous fungi) l tt c cc nm khng phi nm men v cng khng sinh m nm. Nm si cn gi l nm mc, c dng si phn nhnh, khng hoc c vch ngn, li sng hiu kh, ch yu l hoi sinh. Nm si phn b rng ri trong t nhin, tham gia tch cc vo cc vng tun hon vt cht, nht l qu trnh phn gii cht hu c v hnh thnh cht mn. Rt nhiu loi nm c s dng rng ri trong cng nghip ch bin thc phm, cng nghip enzyme, cng nghip dc phm, sn xut thuc tr su sinh hc, kch thch t sinh trng thc vt... Nhiu nm si k sinh trn ngi, ng vt v thc vt gy ra cc bnh nm nguy him. Mt s nm si pht trin nhanh trn cc cht hu c gy h hng lng thc, thc phm, nguyn vt liu...

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1.3. To (Algae) Vi to (Microalgae) gm cc i din c kh nng quang hp, c dng n bo sng thnh tp on, phn b ch yu mi trng nc ngt, nc mn v t m. Vi to c th sinh sn theo hnh thc dinh dng, v tnh v hu tnh. Nhiu loi vi to c ng dng trong sn xut v i sng nh thu sinh khi giu protein lm thc n cho ngi v gia sc (Chlorella), nui to silic (Skeletonema costatum) lm thc n cho u trng tm, tch acid bo khng no. S dng vi to cho x l mi trng (Scenedesmus) hoc lm sinh vt ch th trong mi trng ngho calcium (calcium) (to lc Desmid). 1.4. Nm qu th Nhiu loi nm c qu th c s dng lm thc phm, do nm giu protein, cht khong, cc vitamin A, B1, B2, C, D, E. Ngoi ra chng cn c nhiu c tnh ca bit dc, c kh nng phng v cha bnh h huyt p, chng bo ph, ng rut, h tr cha ung th. a s nm n thuc ngnh nm m (Basidiomycota), thng gp nm n thuc b Agaricales nh nm rm Volvariella volvaceae, Agaricus bisporus... Ngoi gi tr ti nguyn, thc phm v dc phm, nhiu loi nm c ngha trong cng ngh sinh hc v i sng do chng c kh nng sn sinh ra nhiu cht c ch nh eter, acid acetic, acid tanic, cc cht khng sinh... Nhiu loi nm c kh nng hp th v o thi cc cht phng x, mt s loi nm c s dng phn gii cc cht thi c hi v cc ngun ph liu gy nhim mi trng. 2. Sinh vt nhn s 2.1.Vi khun Vi khun (Bacteria) c nhiu hnh thi v cch sp xp khc nhau, kch thc kh nh so vi nm si v nm men. Phn ln vi khun thuc nhm d dng, i sng c th hiu kh, k kh hoc l dng sng tu nghi. Nhiu vi khun c ng dng trong sn xut cng nghip. in hnh nh cc loi vi khun ln men cc acid hu c (lactic, propionic...), sn sinh enzyme, acid acetic, acid glutamic, lysine, vitamin, ln men methane, sn xut phn bn, thuc tr su sinh hc... 2.2. X khun X khun (Actinomycetes) thuc nhm vi khun tht (Eubacteria) phn b rng ri trong t nhin. Phn ln x khun l hiu kh, hoi sinh, c cu to dng si phn nhnh (khun ti). X khun l mt trong nhng nhm vi sinh vt ng vai tr quan trng trong t nhin. Chng tham gia vo cc qu trnh chuyn ha nhiu hp cht trong t nhin. Trn 80%

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cht khng sinh c pht hin l do x khun sinh ra. X khun cn c dng sn xut nhiu loi enzyme, vitamin, acid hu c... 2.3. Vi khun lam Trc y vi khun lam (Cyanobacteria) c gi l to lam (Cyanophyta) hay to lam lc. Thc ra y l mt nhm vi sinh vt nhn nguyn thy thuc vi khun tht. Vi khun lam c kh nng t dng quang nng nh c cha sc t quang hp. Vi khun lam phn b rng ri trong t nhin, nhiu loi c ngha trong sn xut sinh khi giu protein, c nh m hay s dng trong cng nghip x l nc thi... 3. Cc tiu chun ca mt vi sinh vt dng trong cng nghip 3.1. Kh nng s dng nguyn liu Tnh kinh t ca mt qu trnh sn xut i hi phi s dng cc ngun nguyn liu r tin, n gin v cc chng vi sinh vt khng i hi qu cao v nhu cu dinh dng. Thng thng, ngun carbon v nit (nitrogen) dng cho sn xut l nhng ngun thng dng nh cc loi r ng, tinh bt, dch kim sulfid, ngun nit k thut nh cao ng, bt u tng. 3.2. Tnh cht sn phm ph Yu cu cc chng vi sinh vt dng trong sn xut l khng to thnh cc sn phm ph khng mong mun. Thc ra th trong qu trnh sng vi sinh vt lun to thnh nhiu sn phm trao i cht tch lu trong mi trng, mt s sn phm trao i cht c th khng c li cho chnh trao i cht ca t bo v c th gy c ch t bo. S tch lu nhiu cc sn phm ph trong mi trng mt mt lm gim hiu sut to thnh sn phm chnh, mt khc gy nhiu kh khn cho qu trnh thu nhn v tinh khit sn phm. 3.3. Mc mn cm vi s ly tp v kh nng tch sn phm khi mi trng Trong qu trnh ln men thng b nhim cc vi sinh vt l. Ngun ly nhim c th t nguyn liu, khng kh hay t thit b ln men. Nu cc vi sinh vt l cng thch nghi vi iu kin sng trong mi trng ln men th chng s cnh tranh vi chng sn xut ng thi to ra cc cht c tc dng c ch hoc gy cht i vi chng sn xut. Do , cc chng sn xut phi c la chn sao cho khng mn cm vi s tp nhim do cc vi sinh vt l v c bit l vi bacteriophage. Trong trng hp b nhim bacteriophage lng ging trong mi trng ln men c th gim rt nhanh. V d trong mi trng b nhim vi khun butiric c th

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nh hng mnh n sc sng ca ging bi v mt lng nh ca acid butiric to thnh gy c i vi t bo. Ngoi cc ch tiu trn, kh nng tch sn phm ra khi mi trng ln men cng l mt tiu chun chn chng vi sinh vt cho sn xut, bi v c nhiu qu trnh sn xut cho sn phm nhiu nhng vic tch sn phm kh thc hin v hiu qu kinh t thp.

III. C cht dinh dng1. Nguyn t i lng C, O, N, H, P, S, Mg, Ca, Fe Mi trng dinh dng phi cha tt c cc nguyn t cn thit cho sinh trng v to thnh sn phm. Cc mi trng nui cy vi sinh vt cn thit b sung cc nguyn t khong. Nhng nguyn t khong m vi sinh vt i hi phi c cung cp vi liu lng ln c gi l cc nguyn t i lng, bao gm cc nguyn t nh C, O, N, H, P, S, Mg, Ca, Fe. Nhu cu v cht khong ca vi sinh vt khc nhau ph thuc loi, giai on sinh trng pht trin. Nng cn thit v mui khong ca cc nm, vi khun v x khun thng thay i trong cc phm vi sau (bng 4.1). 2. Nguyn t vi lng Mn, Na, B, Mo, Zn, Cu, Ni, Va, Cl, SiBng 4.1: Nng cn thit v mui khong ca nm, vi khun v x khun Nng cn thit (g/l) Mui khong i vi vi khun i vi nm v x khun

K2HPO4.3H2O KH2PO4 MgSO4.7H2O MnSO4.4H2O FeSO4.7H2O Na2MoO4 ZnSO4.7H2O CoCl2 CaCl2 CaSO4.5H2O

0,2 - 0,5 0,2 - 0,5 0,1 - 0,2 0,005 - 0,01 0,005 - 0,01 0,001 -0,005 ti 0,03 0,01 - 0,03 0,001 - 0,005

1-2 1-2 0,2 - 0,5 0,02 - 0,1 0,05 - 0,2 0,01 - 0,02 0,02 - 0,1 ti 0,06 0,02 - 0,1 0,01 - 0,05

Nhng nguyn t khong m vi sinh vt ch i hi vi liu lng rt nh c gi l cc nguyn t vi lng, bao gm cc nguyn t nh Mn, Na, B, Mo, Zn, Cu, Ni, Va, Cl, Si... Nng cn thit ca tng nguyn t vi lng trong mi trng thng ch vo khong 10-6 - 10-8 M.

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Thng thng, khi ch to mi trng nui cy vi sinh vt khng cn b sung cc nguyn t vi lng v trong cc thnh phn khong i lng hoc trong nguyn liu hay nc pha ch mi trng c thnh phn vi lng. Ngoi tr mt s trng hp nh b sung Zn vo mi trng nui cy nm mc, Co vo mi trng nui cy vi khun sinh tng hp vitamin B12 nhng vi hm lng thp (khong 3-5 g/l). 3. Dch dinh dng cho cc vi sinh vt d dng Trong qu trnh ln men, mt mi trng nui cy tt nht phi l mi trng m bo cho sn xut vi hiu sut cao trong thi gian ngn nht v gi thnh thp nht i vi chng ging vi sinh vt. Nhng chng vi sinh vt dng cho cng nghip u l cc ging d dng, tr cc chng to thuc ging t dng. Cc chng ca to thng c nui cy trong cc qu trnh kh bn cho nc thi hoc nui to thu sinh khi. Nhng vi sinh vt d dng ch sn sinh nng lng trong ATP dng cho sinh trng nh qu trnh xy ho nhng hp cht hu c. ATP l thnh phn quan trng nht m t bo dng vn chuyn nng lng. Trong nhng phn ng khng thun li v phng din nhit ng, ATP cho php thc hin nhng phn ng vi tc thch hp. Trong cng ngh vi sinh, mi trng nui cy nhng chng d dng thng c cc thnh phn nh sau: 3.1. Ngun carbon v nng lng Ngun carbon v nng lng thng c s dng l tinh bt, mt r, saccharose, glucose, dch ng thu phn t bt hoc g v.v... Mt s loi vi sinh vt c kh nng s dng cellulose, hemicellulose t bit l carbuahydro (alkan, methane). Ch yu ngun carbon s dng l carbohydrate. Lng carbon c b sung vo mi trng tu thuc chng ging vi sinh vt. Mt s chng hiu kh s dng khong 50% c cht, cn cc chng k kh tu tin ch dng ti 10% cho sinh trng. 3.2. Ngun nit Ngun nit ch yu trong cng ngh ln men l nc amoniac v mui ammon. Dng vo mc ch ny cn c cc ngun nit hu c nh cao ng, dch thu phn nm men, thu phn kh lc, u tng, ht bng, cc b thi ca cng ngh bia (dch ngm malt hoc r mm malt), b thi rau qu, khoai ty, sa loi b m, ph phm khi ch bin pho mt, tht c... Cc ngun nit hu c vi vai tr lm ngun nit v c ngun carbon, ng thi cn cung cp cc cht sinh trng. V vy, khi s dng cc ngun nit hu c, vi sinh vt thng pht trin mnh hn. 3.3. Ngun phospho (phosphorus)

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Ngun phospho cung cp cho mi trng ln men dng mui phosphate hoc acid phosphoric. Phospho hu c (phytin) cn c trong cao ng, bt u tng, trong cc loi cm.... 3.4. Cc ngun khong khc - Ngun lu hunh b sung vo dch ln men dng mui sulfate. - Ngun Mg v K thng c a vo di dng cation ca mui phosphate v sulfate. Trong mt s qu trnh ln men, calcium c a vo mi trng dng mui carbonate duy tr pH vng trung tnh hoc gn trung tnh khi acid c to thnh (v d nh ln men to cc acid hu c) - Ngun Fe: thng thng trong cc nguyn liu s dng c st, nn t khi phi b sung. 3.5. Vitamin v cc yu t sinh trng khc Trong cc mi trng dinh dng dng cho cng nghip, vitamin v cc yu t sinh trng thng c b sung dng nguyn liu lm giu vitamin nh cao ng, r ng, cao nm men. Chng cha hn hp cc acid amin, vitamin v c mt s yu t khc cha bit r, v d nh cc dch chit ng vt hay thc vt. Lng c ch ca cc yu t t bit i khi ch cn rt t. V d - alanine c hiu lc nng 1/100.000.000, cn acid pantotenic c hiu lc nng 1/50.000. Nhng yu t cha bit r gi chung l cc yu t sinh trng. Cao ng, cao nm men, dch thy phn t ngun protein thc vt, ng vt va ng vai tr l ngun nit hu c va l ngun cung cp cc cht sinh trng cn thit. Ngoi ra, trong cng ngh vi sinh cn dng nc chit cm, dch p khoai ty, dch p gi u, dch p c chua, bp ci hoc mt s rau qu khc cng cha nhiu vitamin v dng lm ngun kch thch sinh trng trong nui cy vi sinh vt. Dch dinh dng cha cc cht dinh dng mt nng m bo sut qu trnh nui. Nh vy, ngun carbon v nng lng c a vo phm vi 10-100g/l. nhiu c th, nng cn thit duy tr tc sinh trng cc i l rt nh; i vi ng th khong 1-10mg/l. Vi aminoacid v vitamin th t bo ch cn nng 1-100 g/l. Cc tr s c tnh c hiu c th v c hiu qu trnh. Dch dinh dng dng trong ln men cha cc thnh phn cn thit thng khng mt t l cn i m sinh trng i hi. Nh nhng iu kin nh t l C : N cc tr hoc s thiu phosphate m trao i cht c th c iu khin theo hng c ngha cho vic tng hp sn phm mong mun. iu ng vi cc nguyn t i lng cng nh vi lng. Chng hn bng cch a Co vo

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m t c thu hoch cao v vitamin B12, hay s thiu st kch thch qu trnh tng hp acid citric. 4. Ngun carbon k thut v ngun nit k thut 4.1. R ng Trong cng nghip ng thng thu c mt r l dch ng sau khi kt tinh. Cc loi mt r gm c: - Mt r hydrol: dch thu c sau khi kt tinh glucose cc x nghip thy phn bt bng acid sn xut glucose. Trong hydrol c ti 40 - 50% glucose v c mt hm lng ng k NaCl. - R ng: cc nh my ng sn xut saccharose c mt ph phm vi t l kh ln, l r ng. y l mt loi nc ct c tch ra sau khi kt tinh ng. C hai loi r ng l r ng c ci v r ng ma (ty thuc vo ngun nguyn liu ca nh my ng). C hai loi r ny u c mu nu sm do c nu v c nhiu ln nn c nhiu caramen v melanoid to thnh. Trong r ng c ti 70 - 80% cht kh, trong ch yu l ng saccharose 46 - 54%, ng kh 6 - 9%, rafinose 1- 2%, N tng 0,45 - 2,88% v cht khong 3 - 4%. Cc cht hu c c trong r ng l cc acid, ru, acid amin, purine v cc vitamin. Hm lng cc mui phosphate trong r ng thng rt thp. Phn ln cc hp cht phospho nm phn cn lng. Do , khi dng r ng x l v loi b cn th nht thit phi b sung ngun phospho vo mi trng dinh dng. Trong r ng c cha cc hp cht c tc dng kch thch sinh trng vi sinh vt, nhng nu dng r ng vi nng cao th sinh trng ca cc chng sn xut s b km hm, v trong r ng c cha cc cht c tc dng c ch nh SO2, hydroxy-methyl-fucfural hay kalium - imido - disulfonate, cht ny c th sinh ra t sulfate v nitrate do vi khun to thnh. Trong r ng c cha hm lng ln biotine (vitamin H) l cht sinh trng rt cn thit i vi nhiu loi vi sinh vt v l cht iu ha trong qu trnh sinh tng hp acid amin, hm lng khong 20 - 120 mg/kg trong r ng ma v 0,01 - 0,13 mg/kg trong r ng c ci. Cc vitamin trong r ng ngoi biotine cn c vitamin B1, B2, PP, acid pantotenic... 4.2. Dch kim sulfid Trong cng nghip men cn s dng dung dch thy phn t g dch kim sulfid l ph thi ca cng nghip giy. Thnh phn chnh ca dch kim sulfid l linhosunfonate v cc ng pentose. Thnh phn ca dch kim sulfid t g ca cy l bn v cy l kim l khc nhau. Ngoi ra,

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thnh phn ny cng thay i nhiu tu theo mc khai thc. Dch kim sulfid ca g cy l bn chim t l cao cc ng pentose (khong 80% ng) thng s dng nui cy thu sinh khi nm men. Cn dch kim sulfid g cy l kim th cc hexose li chim u th (khong 70% ng) dng ln men thu ru. Vic tin x l cht thi ny trc ln men l ti thiu. Bm hi nc hoc thng kh pH 1,5 - 3,0 l cn thit loi SO2 l cht vn km hm sinh trng ca vi sinh vt. Sau pH s c iu chnh ti ti u (pH khong 5) v mi trng c b sung cc cht dinh dng cha nit v phosphate. 4.3. Cc nguyn liu thy phn tinh bt cung cp ngun carbon ch yu l glucose th bt sn c l l ngun nguyn liu tt nht. Trong bt sn cha ch yu l tinh bt, hm lng N hu c, cht khong, vitamin c vi lng rt nh. Thy phn cc loi tinh bt thng thc hin theo hai cch: - Thy phn bng acid vi p lc d. Dch thy phn thu c qua trung ha bng Na2CO3 hoc NaOH, nu dng H2SO4 lm tc nhn thy phn th c th dng CaCO3 hoc nc vi trung ha, sau em lc qua lc p khung bn vi than hot tnh kh mu. Dch thy phn ny cha ch yu l ng glucose, mt lng nh cc acid amin, c mt cc cht bn, khong c dng chun b mi trng nui cy hoc em c c ti 60 - 70% cht kh s dng dn. - Thy phn bng enzyme: Cc ch phm enzyme ch yu l t nm mc c nui cy b mt hoc b su, dng vi t cch l phc h amylase gm c , - amylase v glucoamylase. Sn phm thu c l hn hp maltose v glucose. Cng c trng hp dng phi hp ch phm enzyme t mc v ch phm enzyme t vi khun nui b su ( - amylase chu nhit) nn hiu qu ca qu trnh s cao hn. Phng php thy phn cc loi bt (bt sn, go, ng, bt m, cao lng, khoai ty) bng cc ch phm enzyme ny c dng trong cng nghip sn xut ru cn. 4.4. Ht v bt ng cc Cc loi bt ng cc thng c dng l bt go, bt ng c tch phi, bt m, bt i mch... Ngoi thnh phn ch yu l tinh bt, cc loi bt ny cn cha khong vi phn trm cc hp cht protein, cc cht x (ch yu l cellulose) v cc cht khong. Bt sn l loi nguyn liu kh r tin so vi cc loi bt khc hin nay ang c s dng nhiu cho cng ngh ln men c bit l ln men cn.

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Cc nguyn liu tinh bt trong ln men c th dng trc tip lm thnh phn ca mi trng dinh dng cho cc chng sinh ra enzyme amylase ngoi bo, c bit l trong phng php nui cy b mt. Ngoi ra, ngun nguyn liu ny cn qua mt giai on thy phn thnh dung dch cc loi ng ri mi dng chun b mi trng dinh dng. Ngoi ra, cc parafin c mch t C8 - C18, kh methane... c th c s dng lm ngun carbon nui cy vi sinh vt thu cn hoc protein n bo cho sn xut thc n gia sc. 4.5. Ngun nit k thut - Bt u tng c dng nh l mt ngun nit k thut tng i ph bin trong nhiu mi trng dinh dng. Trong bt u tng c ti gn 40% l protein v khong 19% cht bo, c cc acid amin. i vi sinh tng hp nhiu cht khng sinh khng nhng ch c cc hp cht protein mi c tc dng m cn phi k n cc cht bo c trong u tng. Cng vi bt u tng hoc kh du u tng ngi ta cn dng bt kh lc (lc sau khi p du), bt hoc kh du cc loi ht bng, ht hng dng... vo trong mc ch ny. - Nc chit ng v cao ng l sn phm ph trong cng nghip ch bin bt ng. Trc khi xay, ng c ngm vi dung dch natrium sulfid. Trong khi ngm, cc acid amin, vitamin c chit ra v ha tan vo dung dch. Nc chit ng c th dng trc tip pha ch mi trng dinh dng, nhng thng thng ngi ta c trong iu kin chn khng ti 50% cht kh dng st gi l cao ng. Trong cao ng c 6,4 - 8% N tng s, khong mt phn na l cc acid amin, phn cn li l peptide v protein, hm lng carbohydrate dao ng trong phm vi kh rng do c lin quan n ln men lactic, hm lng tro 15 - 20%, nhiu vitamin c bit l vitamin nhm B v biotine. - Nc chit nm men v cao nm men: sinh khi men bia hoc men ru c ra sch v cho t phn 48 520C trong 2 - 3 ngy, lc b b, thu c dch thy phn gi l nc chit nm men, c c c cao nm men. Cc sn phm ny giu acid amin, peptid cng nhiu vitamin nhm B v cc cht khong. Trong cao nm men c 40 - 50% cht kh, 0,6 - 1,5 N tng s, 0,3 - 0,5 N - amin (trong c mt 18 loi acid amin). - Dch thy phn cc loi kh du (u tng, lc, bng, hng dng) v tht, c giu nit hu c, nhiu acid amin. Nu thy phn bng acid th nhiu vitamin v mt hay hai aminoacidb ph hy (nh triptophan v mt phn cysteine), cn thy phn bng enzyme th cc thnh phn ny y hn. Dng cc dch thy phn ny lm ngun nit

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hu c trong mi trng nui cy vi sinh vt v mt phn l ngun cht dinh dng.

IV. Nhu cu v xy v s thng kh trong qu trnh ln men1. ho tan ca xy trong nc T bo s dng xy h hp v lm gim lng xy trong mi trng. V th trong nui cy hiu kh phi cung cp xy mt cch u n. Thiu xy nht thi ti mt thi im no trong mi trng s dn n s ph v qu trnh trao i cht ca t bo. Vi sinh vt s dng xy trong mi trng lng. Lng xy ho tan trong nc thng l rt t. Phi cung cp xy sao cho tc ho tan ca n bng tc tiu th xy ca vi sinh vt. Tc ho tan ca xy vo mi trng lng c tnh theo cng thc: dc R= = K. (C - C1) dt Trong : R- tc ho tan xy, C - nng xy bo ho p sut ring bit, C1- nng xy ho tan thi im la chn, K - hng s t l, t - thi gian. ho tan xy cn ph thuc vo nhit khi nui cy, vo nng cc cht hp phn v nht ca mi trng. Khi nhit tng ln th ho tan ca xy gim. ho tan ca xy trong mi trng gim i 2 ln khi nhit tng t 30 - 370C. iu ny c th khc phc bng cch cho sc kh mnh hn trong qu trnh ln men. Nng xy ho tan cng s gim khi dng cc cht hot ng b mt, cc cht ph bt v hm lng sinh khi vi sinh vt tng. Trong qu trnh nui cy khng kh nn c thi vo thng ln men c h thng cnh khuy. Tc sc kh mnh s tng tc ho tan xy v trn u c cht dinh dng trong mi trng. Nhng khng nn khuy qu mnh v c th dn n s h hng c hc cc t bo v dn n hin tng t phn. 2. Nng xy gii hn xy rt cn i vi i sng ca vi sinh vt hiu kh. Tng thng kh n gii hn nht nh th s pht trin ca vi sinh vt cng tng ln theo. i vi nhiu vi sinh vt, thng kh s lm tng tc sinh trng, rt ngn pha tim pht, nng cao lng sinh khi. Khi tng tc ho tan xy t 0 - 5 milimol O2 /l.pht, lng sinh khi cui cng ca Serratia marsescens s tng mt cch ng k; sinh khi cc i t c khi

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cng thng kh khong 5 milimol O2/l.pht. Nu tip tc tng thng kh hn na th lng sinh khi cui cng s gim. Hin tng ny cn gp rt nhiu ging vi sinh vt. duy tr vic cung cp xy ti thch cho t bo khng cn thit phi lm bo ho mi trng bng xy ho tan. Ch cn mt nng xy nh hn rt nhiu cng cung cp cho cc enzyme phn ng vi c cht . Nng xy gy ra h hp ti a c gi l nng xy ti hn (hoc p sut ring phn ca xy). khng phi l mt i lng c nh m l mt hm s ca tc sinh trng hoc ca tc h hp c lin quan vi n. Tr s ny vo khong 10 mol/l. Khi s vn chuyn xy b cn tr bi nhng tp hp t bo (cc cc si nm) hoc bi lp cht nhy bao quanh cc t bo th nng xy gii hn c tr s cao hn. 3. S cung cp xy cho cc t bo chm S cung cp xy cho cc t bo chm l mt qu trnh chuyn dch cht, trong xy c chuyn t bng khng kh vo mi trng dinh dng v t vo t bo. Qu trnh xy ra nh dng chy v s khuch tn. Lc y l s chnh lch nng xy. S chuyn dch cht t tng kh sang tng lng c quy nh bi b mt gii hn gia hai tng v do vy bi s lng v kch thc cc bng khng kh. Ch mt phn nh ca khng kh c cung cp i vo dung dch. Bi vy trong thc tin, ngi ta thng s dng t s ca th tch khng kh/th tch ni ln men/pht. i vi mi qu trnh ln men cn phi nghin cu nh hng ca cng thng kh i vi hiu sut to thnh sn phm. Trong sn xut cng nghip khng kh c nn qua my nn, qua mt h thng lm ngui, qua h thng lc loi ht tp khun ri thi vo cc thng ln men. Trong cc thng ln men v cc thng nui cy nhn ging u c h thng khuy tu thuc vo yu cu ca tng loi vi sinh vt, vo tng iu kin nui cy nhm thu c hiu sut ti a.

V. Kh trng1. Ln men khng v trng cho qa trnh ln men din ra c kt qu th cn phi ngn cn s pht trin ca cc c th l. Trong nhiu qa trnh ln men (sn xut acid amin, enzyme, cht khng sinh...) iu c thc hin thng qua vic kh trng mi trng dinh dng, khng kh v cc thit b ln men (ln men v trng). Tri li trong vic sn xut sinh khi nh sinh khi nm men, sinh khi vi khun, to thng tin hnh ln men khng v trng. S pht trin ca cc c th l b ngn cn mnh m bng cch to ra nhng

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iu kin nui sao cho chng sn xut c th sinh trng tri hn, v d nh c cht c hiu hay pH mi trng. 2. Ln men v trng Nhim v thanh trng l dit ht vi sinh vt c mt trong mi trng do c sn t trong thnh phn nh nc, nguyn liu, khng kh v trn b mt cc thit b tip xc vi mi trng. Nhng vi sinh vt ny cn sng st s pht trin cnh tranh vi chng sn xut, lm hng qu trnh ln men. Cc vi sinh vt thng c sc bn vi nhit, mt s c mt trong nguyn liu di dng bo t, mun dit c chng phi gia nhit ti 120 1210C trong vi chc pht. 3. Kh trng bng ni hp

Hnh 4.5:Ni hp p lc cho ln men xp ( Autoclaver)

Hnh 4.6:Thit b kh trng Pasteur

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Hin nay phng php thanh trng trong cng ngh vi sinh ph bin l dng hi nc qu nhit do ni hi cung cp. Trong ln men tng m cc thit b sau khi lm v sinh c kh trng bng hi nng ti 120 1300C, sau mi cho mi trng lng vo cc ni ln men kh trng mi trng cng vi c h thng khuy v cc on ng ng, van tip cn. Vic gia nhit cao c th dn n s ph hu cc thnh phn dinh dng mn cm vi nhit v caramen ho cc ngun ng cng nh xy ra cc phn ng melanoid gia ng vi aminoacidhoc cc vitamin b ph hng. Nhng thnh phn mn cm vi nhit c khi phi kh trng ring, sau mi trn ln hoc kh trng theo phng php khc nh lc qua phin v trng. Ngoi cch kh trng theo phng php hi nc gin on ny, ngi ta cn dng phng php kh trng bng hi lin tc bng cch cho mi trng chy qua thit b kh trng chuyn dng nhit 1400C trong vi pht. * Kh trng mi trng nui cy b mt Mi trng nui cy b mt thng l nhng hp cht rn, gm c cm, bt v cc cht dinh dng. Trong sn xut cng nghip mi trng rn c kh trng bng hi nng trong thit b chuyn dng vi p sut d 0,05 Mpa t nhit 104 - 1100C. iu chnh pH mi trng thch hp bng acid clohydric, sulfuric hoc lactic. Thanh trng bng hi nng c th qua 2 giai on. Giai on u nng nhit ti 1000C v o khi mi trng lin tc trong 15 - 20 pht. Giai on sau nng nhit ti 1100C khong 60 - 90 pht v c sau 15 pht li o mi trng 3 - 5 pht. * Kh trng mi trng lng ln men b su Kh trng mi trng lng c th chn mt trong nhng phng php gin on hoc lin tc. Phng php gin on thng dng trong trng hp khi dch khng ln, th d dch dng trong cc bnh ln men th nghim, cc ni nhn ging v cc ni ln men khng qu ln. Tin hnh ln men tng m ngay trong ni theo nguyn l ni hp p lc qua mt s giai on: - Kh trng ni ln men v h thng ng ng tip xc vi mi trng bng hi nng trc tip hoc gin tip. - Cho dch mi trng pha ch vo ni (lng dch bng 3/4 th tch ca ni v phi tnh thm phn nc ngng khi cho hi trc tip vo mi trng).

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- Gia nhit ti nhit thanh trng. C 2 bc gia nhit: cho hi vo v ni hoc ng xon trao i nhit khi dch ti gn 1000C th cho hi vo ni trc tip vi dch nng nhit n nhit ti hn. - Gi nhit ny trong khong thi gian thanh trng cn thit. - Lm ngui dch ngay trong ni bng cch cho nc vo v hoc ng xon trao i nhit cng vi h thng khuy lm vic. Phng php kh trng ny tng i lu v trnh bin i trong thnh phn dinh dng ca mi trng nn ch tin hnh p sut 0,05 0,1Mpa vi nhit 110 - 1200C trong khong 1 - 1,5 gi t lc t c nhit ti hn. Ngoi ra, c th tin hnh thanh trng lin tc nhit cao hn (140 - 1450C) v gi thi gian ngn hn (5 - 15 pht) nhit ny. 4. Kh trng bng ho cht i vi nhng cht km bn nhit d b phn hu nhit cao th vic kh trng c th bng cch lc qua phin lc hoc bng cc ho cht dit khun. Nhng vic s dng cc ho cht dit khun cn phi cn nhc k cng v tnh gy c cho vi sinh vt nui cy, cho ngi s dng sn phm cng nh v v sinh an ton lao ng. Mt s ho cht c dng kh trng trong mt s trng hp ngoi l, nh etylenoxyl, propiolacton rt thch hp c