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TP CH KHOA HC & CNG NGH CC TRNG I HC K THUT S 70 - 2009

96

NG DNG VI KHUN CORYNEBACTERIUM SP. C NHTRONG LN MEN THU NHN LYSINE

IMMOBILIZING CORYNEBACTERIUM SP. CELL AND APPLICATION FOR PRODUCING LYSINE

Nguyn Thu Hng, Trn Th Minh TmTrng i hc Bch khoa -HQG Tp. H Ch Minh

TM TT

Bi bo nghin cu vic ng dng cc ch phm t bo vi khun Corynebacterium sp. c nhtrn mt s cht mang alginate, Bacterial Cellulose (BC) v phc Bacterial Cellulose - Alginate (BC-A) ng dng ln men thu nhn lysine. Kt qu thu c nh sau:

- Cht mang BC c nhiu u th hn cc cht mang truyn thng alginate trong k thut cnh t bo vi khun Corynebacterium sp.

- BC hon ton ph hp cc yu cu ca cht mang trong k thut c nh t bo vi khun.

- Phc cht mang Bacterial Cellulose- Alginate (BC-A) pht huy c nhng u th ca chtmang Bacterial Cellulose v Alginate.

- Hiu qu s dng ch phm Corynebacterium sp. c nh trn phc cht mang BC-A trong lnmen thu nhn lysine kh cao: c th ti s dng ch phm 12 ln vn m bo v mt thi gian lnmen v sn lng lysine so vi i chng.

ABSTRACT

In this paper, we investigate the immobilization of Corynebacterium sp. in some carriers(alginate, bacterial cellulose -BC and bacterial cellulose- alginate BC-A) and apply in producing lysine.The results are given as follows:

- The bacterial cellulose (BC) showed many advantages compared with traditional carrier(alginate) in immobilization Corynebacterium sp.

- BC entirely satisfied the conditions required for immobilization Corynebacterium sp.

- The effect of using Corynebacterium sp., immobilized in BC-A, for producing lysine was high: itcould be reused about 12 times, the quantity of lysine remained rather good against controlexperiments.

I. M U

L-lysine l mt trong 9 acid amin thityu v l acid amin khng thay th. C thngi v ng vt khng t tng hp lysine.Hai lnh vc ln ng dng lysine l thc phm

v dc phm.Lysine c thu nhn t dch thy phn

protein ng vt, thc vt v phng php lnmen vi sinh vt, trong u th thuc v

phng php ln men. Do nhu cu ln vlysine, vic sn xut lysine cc quc gia M,

Nht, Nga, tin hnh quy m cng nghip. Vit Nam, lysine phi nhp khu hon tonv cha c mt c s no sn xut lysine quym ln. Hn na, do bt hp l thnh phn thc

n nn ngi Vit Nam rt d b thiu htlysine [1,3].

K thut c nh t bo ang c cpv quan tm nhiu, c bit trong cc lnh vcln men sn xut cc sn phm trao i cht. T

bo c nh c nhiu u im so vi vic s

dng t bo t do nh: t bo sau khi c cnh c th s dng c nhiu ln, khng lnvo sn phm v c th ch ng ngng phnng theo mun [4,5].

Trong gii hn bi bo ny, nhm nngcao hiu ququ trnh ln men thu nhn lysine,chng ti nghin cu vic ng dng cc ch

phm t bo vi khun Corynebacterium sp. cnh trn mt s cht mang ln men thu nhnlysine.

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II. VT LIU V PHNG PHP

2.1 Vt liu

- Chng vi khun: Corynebacterium sp. cngun gc t b su tp ging trng i hcBch khoa TP.HCM.

- Cc cht mang: Na-alginate (Kanta, NhtBn), Cellulose vi khun (Bacterial Cellulose-BC) [6].

2.2 Cc phng php c nh t bo

C nh trn cht mang alginate: T boCorynebacterium sp. c c nh trn chtmang alginate bng phng php nht: iukin c nh l nng alginate 3%, nng CaCl2 2% v nng t bo vi khun a voc nh l 1010 t bo/ml. [4,5,6]. Ht alginate

to thnh dng trn c ng knh khong 3,5-4 mm.

C nh trn cht mang BC:Trn cht mangBC (kch thc 2cm x 2cm x 1 cm), t boCorynebacterium sp. c c nh bng

phng php by- hp ph (gm 2 giai on:hp ph vi khun trn b mt BC v by tngsinh trn v trong cht mang BC). C th gmcc bc nh sau: Hot ho chng vi khun B sung cht mang BC v trng vo dch t bo hot ho Qu trnh c nh trn cht

mang BC s tri qua 2 giai on lin tip:Giai on hp ph: trong khong 30 pht

u tin sau khi ngm BC trongdch t bo, BCs trng n v trng thi ban u, ng thi shp ph t bo vo h thng si bn trong cngnh trn b mt bn ngoi.

Giai on by tng sinh: k tip giaion hp ph. Sau giai on hp ph, chtmang BC c tch khi huyn ph vi sinh vtv cc t bo c hp ph giai on trntip tc pht trin v tng sinh trn gi BC,

khi tip tc iu kin ti u ca ging visinh vt c nh.

Cc iu kin ti u c nhCorynebacterium sp. trn cht mang BC: hp

ph bng my lc, thi gian 3 ngy, nhit 300C, lng t bo vi khun c nh trncht mang t c nh sau:

- Mt trung bnh l: 8,8 x109/g .

- Mt vi khun mt ngoi cht mang l:4,9x10

4t bo/cm2.

- Mt vi khun bn trong cht mang l:4,3x10

4t bo/cm2 [6,7].

C nh trn phc cht mang BC-A:

Trn phc cht mang BC-A, chng tikt hp c 2 phng php nht v by hp pht bo Corynebacterium sp..

u th ca phng php nht trong gelalginate l t bo b nht s khng khuch tn rami trng xung quanh, nhng vn cho c chtc phn t lng nh vo v cc sn phm traoi cht ca t bo ra khi mng li gel.

u th ca phng php by hp phtrn cht mang BC l tnh cht c l bn vng,

d ph hp vi hnh dng thit b ln men. Tnhcht c l bn vng ca BC gip ch phm t

bo c nh chu c cc iu kin khuy otrong qu trnh ln men v gp phn gia tng sln ti s dng.

Cc iu kin ti u c nhCorynebacterium sp. trn phc cht mang BC-A chnh l tng hp cc iu kin ti u trncht mang alginate v BC, gm cc bc nhsau:

-Dch alginate 3% v t bo Corynebacteriumsp. ng nht vi mt khong 1010t bo/ml.

- BC (kch thc 2cm x 2cm x 1cm) hp phdch ging- alginate bng my lc trong 30

pht.

- Khi cho cht mang BC vo huyn ph vikhun c cha 3% alginate, trnh s nhhng nht ca huyn ph n s phn bca vi sinh vt v cht mang trong dung dch,cn thc hin trn my lc (200 vng/pht, thigian 30 pht). Ch ny c ghi nhn lti u cho s phn b vi sinh vt v qu trnhhp ph [2,6].

- B sung cht h tr to gel CaCl2 2%.

- Thi gian 3 ngy, nhit 300C.

Vi cc iu kin trn, lng t bo vikhun c nh trn phc cht mang BC-A tc:

- Mt trung bnh l: 8,9 x109/g .

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- Mt vi khun mt ngoi cht mang l: 5,0 x10

4t bo/cm2.

- Mt vi khun bn trong cht mang l: 4,6x10

4t bo/cm2 [6,7].

2.3 Phng php nghin cu

- nh lng vi khun c nh trn b mt chtmang BC: Mu i din Dp mu v trng X l bng cellulase nh lng bng

phng php m gin tip.

- Xc nh t l ra tri t bo (T) sau mi chuk ln men:

T= B

x100%A

(vi A: mt t bo ban u ca ch phm, B:

mt t bo trong dch ln men ti chu kkho st).

- Ln men bi ch phm t bo c nh quym phng th nghim trong fermenter (5 lt)

thu nhn lysine. Lng ch phm c tnhton sao cho t l ging l nh nhau c 4nghim thc: s dng t bo t do v s dng 3ch phm vi khun c nh trn alginate, BC vBC-A. iu kin ln men thu nhn lysine c

b tr nh nhau [1,3,6].

- nh tnh L-lysine bng phng php sc kgiy. nh lng L-lysine bng phng phpquang ph [1,3].

- Phng php x l s liu: S liu c x lbng chng trnh Excel, Stagraphic Version7.0.

III. KT QU V THO LUN

3.1 Cc c im sinh hc ca chngCorynebacterium sp. kho st.

Cc bin ng v tng trng, pH mitrng v sn lng lysine ca chngCorynebacterium sp. c th hin qua th1.

0

5

10

15

20

25

30

35

Ban u 1 2 3 4 5Ngy ln men

Mt t bo (triu/ml) pH Lysine (g/l)

th 1. Bin ng v tng trng, pH mi trng v sn lng lysine ca chng Corynebacteriumsp. theo thi gian ln men

Nh vy, vi chng kho st, thi gianln men cho sn lng lysine cao nht l 3 ngyt khong 32 g/l v mt t bo t c caonht sau 3-4 ngy nui cy.Xt v thi gian v

hiu sut qu trnh ln men sinh tng hplysine, sn lng lysine ca chng kho st nm mc 70-80% so vi cc bo co ngoi nc[1,3] v tng ng so vi bo co trong nc[9]. Vi mc ch nng cao hiu qu qu trnhln men thu nhn lysine, chng ti s dngchng ny cho cc nghin cu tip theo v cnh t bo.

3.2 ng dng vikhun Corynebacterium sp.c nh trong ln men thu nhn lysine

S dng 3 ch phm Corynebacteriumsp. (c c nh bng 3 cht mang alginate,BC v phc BC-A gii thiu trong phn

phng php) ln men thu nhn lysine nhm

so snh hiu qu ca cc ch phm c nh.Tiu ch nh gi xc nh s ln ti s dngl sn lng lysine ca cc ln ti s dng lnmen bng cc ch phm vi khun c nh trongcng mt thi gian ln men l 72 gi. Thi gian72 gi c xc nh qua th 1.

Cc ch phm vi khun Corynebacteriumsp. c nh trn alginate di tc ng khuyo trong ln men, b trng v v rasau 5ln ti s dng. T l cht mang alginateb v

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l 50%. Trong mi trng ln men c mtlng Na+ v gc phosphat l nhng tc nhnph v cu trc h gel ca alginate. Trong khi, mng BC gi nguyn cu trc rt chc sau10 ln theo di ti s dng( th 2).

Da vo cc yu cu ca cht mang trongk thut c nh t bo, u im ni bt cacht mang BC th hin qua 4 im: tnh cht cl bn vng, d ph hp vi hnh dng thit bln men, gi thnh thp v s ln ti s dngcao. Nhng kiu c nh t bo bng phng

php by-hp ph dn n hin tng ra tri tbo sau mi ln ti s dng. V vy, khi c nht bo bng phng php by hp ph trn BCch c kh nng ti s dng khong 9 ln ( th2). Khi kt hp vi nht t bo bng alginatetrn phc BC-A khc phc c hin tng

ra tri t bo. Sau 10 ln ti s dng ch phmvi khun c nh trn BC, t l ra tri ln n32%, tng ng l sn lng lysine gim 40%so vi i chng. i vi cht mang BC-A, sau13 ln ti s dng t l ra tri l 27%, tngng sn lng lysine gim 25% so vi ichng. Kt qu kho st t l ra tri t bo thhin qua bng 1.

nh gi hiu qu c nh ca cc chphm Corynebacterium sp. c nh trn cccht mang thng qua sn lng lysine trong qutrnh ln men v x l s liu, cho kt qu nhsau:

- i vi cht mang alginate: c th ti s dng3 ln cho hiu qu ln men khng khc bit sovi i chng. Ln ln men ti s dng th 4,sn lng lysine gim khong 24%. Ln ti sdng th 5, sn lng gim 32%. Di tc ngkhuy o trong qu trnh ln men, ch phmCorynebacterium sp. c nh trn alginate btrng v v ra sau 5 ln ti s dng. T l chtmang alginate b v l 50%.

- i vi cht mang BC: c th ti s dng 8ln cho hiu qu ln men khng khc bit so

vi i chng. Ln ln men ti s dng th 9,sn lng lysine gim khong 24%. Ln ti sdng th 10, sn lng lysine gim 40%. Chnhv s khc bit ca sn lng lysine qua cc lnln men ti s dng, nn i vi cht mang BC,s ln ti s dng c xc nh l 8 ln.

0

5

10

15

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25

30

35

C [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

Ln ti s dng ln men bng ch phm vi khun c nh

Lysin

(g/l)

Cht mang Alginat Cht mang BC Phc cht mang BC-A

th 2. Sn lng L-lysine ca cc ln ti s dng ln men bng cc ch phm vi khun c nh(C: i chng ln men bng t bo t do)

Bng 1. T l ra tri t bo sau ti s dng ln men ch phm t bo c nh(%)

Chtmang

Ln ti s dng

[2] [4] [6] [8] [10] [12] [13]

Alginate 8 35 x x x x x

BC 12 15 19 25 32 x x

BC-A 9 12 17 19 22 23 27

(x): khng tip tc ti s dng

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- i vi phc cht mang BC-A: c th ti sdng 12 ln cho hiu qu ln men khng khc

bit so vi i chng. Ln ln men ti s dngth 13, sn lng lysine gim khong 25%. Lntis dng th 14, sn lng lysine gim 38%.

Do s khc bit ca sn lng lysine qua ccln ln men ti s dng, nn i vi phc chtmang BC-A, s ln ti s dng c xc nh l12 ln.

- Cht mang BC v phc BC-A, sau s ln theodi ti s dng ln men vn gi nguyntrng thi cu trc ban u. y cng l mtuim, th hin qua tnh cht vt l ca BC [2]v cng l mt yu cu quan trng ca chtmang trong k thut c nh t bo vi sinh vt[2-6]. Vi phc BC-A, cc t bo c nht bi

gel alginate trong mng li BC, gip gim tl ra tri t bo trong qu trnh ti s dng ln men (bng 1), v vys lnti s dnggiatng ( th 2).

- Ccs liu th 2 c x l thng k chothy s khc bit v hm lng lysine trong ccchu k t 1-8 (i vi cht mang BC) v chu k

t 1-12 (i vi phc cht mang BC-A) khngc ngha tin cy 95%. Vi chu k 8-9(i vi cht mang BC) v chu k 12 -13 (ivi cht mang BC-A), s khc bit c ngha tin cy95% .

IV. KT LUN- Cht mang BC c nhiu u th hn cc chtmang truyn thng alginate trong k thut cnh t bo vi khunCorynebacterium sp.

- BC hon ton ph hp cc yu cu ca chtmang trong k thut c nh t bo vi khun.

- Phc cht mang Bacterial Cellulose- Alginate(BC-A) pht huy c nhng u th ca chtmang Bacterial Cellulose v Alginate.

- Hiu qu s dng ch phm Corynebacterium

sp. c nh trn phc cht mang BC-A trong lnmen thu nhn lysine kh cao: c th ti s dngch phm 12 ln m vn mbo v mt thigian ln men v sn lng lysine so vi ichng.

TI LIU THAM KHO1. Gunji Y., Yasueda H.; Enhancement of L-lysine production in methylotroph Methylophilus

methylotrophusby introducing a mutant LysE exporter; Biotechnology 6, 2006, 126-132.

2. Krystynowicz A., Czaja W.; Factors affecting the yield and properties of bacterial cellulose;

Industrial Microbiology and Biotechnology29, 2002, 189-195.3. Kiefer, Elmar Heinzle, Oskar Zelder, and Christoph Wittmann; Comparative metabolic flux

analysis of lysinee-producing Corynebacterium glutamicum cultured on glucose or fructose;Applied and Environmental Microbiology 1, 2004, 229-239.

4. Lee K. Y.; Survival of Bifidobacterium longum immobilized in calcium alginatee beads in

simulated gastric juices and bile salt solution; Applied and Environmental Microbiology 66,2000, 869-873.

5. Sheu T.Y.; Microentrapment ofLactobaciliin calcium alginatee gel; Food science 54, 1999, 557-561.

6. Nguyn Thy Hng; Tuyn chn v ci thin cc chng Acetobacter xylinum to cellulose vikhun sn xut v ng dng quy m pilot ; Lun n Tin s Sinh hc,Trng i hc Khoa

hc T nhin TP.HCM, 2006.7. Nguyn Thy Hng; Mt s ng dng ca cellulose vi khun trong lnh vc thc phm ; Tp ch

Sinh hc,tp 30, s 1, 2008, 62-69.8. Nguyn Thy Hng, TrnThTngAn;C nh vi khunLactobacillus lactis ng dng ln

men thu nhn bacteriocine, Tp ch Khoa hc v Cng nghcctrngi hc k thut, s 63,2008, 82-86.

9. V Kim Thoa;Hi ngh Cng ngh Sinh hc ton quc, trang 381-383,H ni-2003: .

a ch lin h: Nguyn Thu Hng- Tel: 0955.019.067, e-mail: [email protected] mn Cng ngh Sinh hc- Trng i hc Bch khoa - HQG Tp.HCM
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Gunji%20Y%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Yasueda%20H%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Yasueda%20H%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Gunji%20Y%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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