PROTEASE

Protease c mt ng dng nh trong quy trnh sn xut ru vang. vai tr ca protease l phn gii protein trnh hin tng protein kt ta gy lng cn trong sn xut ru vang. Protease phn b thc vt, ng vt, vi sinh vt. Tuy nhin, ngun enzyme phong ph nht l ngun t vi sinh vt, c hu ht cc vi sinh vt nh vi khun, nm mc v x khun, C th ni vi sinh vt l ngun nguyn liu thch hp nht sn xut enzyme quy m ln dng trong cng nghip v i sng. Nhm enzyme protease xc tc qu trnh thy phn lin kt peptid ( -CO-NH)n trong phn t protein, polypeptid n sn phm cui cng l cc acid amin. Ngoi ra, nhiu protease cng c kh nng thy phn lin kt este v vn chuyn acid amin.
1.1. Cc enzyme th y phn protein Protease thuc enzyme nhm 3 (enzyme phn gii) v phn nhm 3.4 (phn ct cc lin kt peptide). Da vo v tr ct t lin kt trong chui lin kt peptide, ngi ta chia protease lm 2 nhm: Endopeptidase v Exopeptidase [67]. Protease xc tc cho s thy phn lin kt peptide (-CO-NH2-) trong phn t protein v c cht tng t. Nhiu protease cng c kh nng thy phn lin kt este v vn chuyn axit amin [3]. 1.1.1. Exopeptidase

Exopeptidase ch tc dng gn im kt thc ca chui polypeptide, ti u N hoc u C. Da vo v tr tc ng trn mch polypeptide, exopeptidase c phn chia thanh hai loai:

a/ Aminopeptidase xc tc thy phn lin kt peptide

u N t do ca chui polypeptide gii phng ra mt amino acid, mt dipeptide hoc mt tripeptide. Trong qu trnh aminopeptidase xc tc thy phn lin kt peptide, nu gii phng mt amino acid gi l aminopeptidase, gii phng mt dipeptide c gi l dipeptidyl-peptidase v gii phng mt tripeptide c gi l tripeptidyl-peptidase.

b/ Carboxypeptidase xc tc thy phn lin kt peptide

u C ca chui polypeptide v gii phng ra mt amino acid hoc mt dipeptide. Trong qu trnh exopeptidase tc dng ti u C, nu gii phng mt amino acid gi l cacboxyl-peptidase hoc gii phng mt dipeptide gi l peptidyl-dipeptidase. 1.1.2. Endopeptidase Endopeptidase tc ng vng bn trong ca chui lin kt peptide, cch xa 2 u C v N. Da vo c ch tc ng ca endopeptidase, ngi ta chia chng thnh 4 nhm: serine protease, cysteine protease, aspartic protease v metallo protease.

a/ Serine protease (EC.3.4.21) l nhm peptidase ln nht

v c pht hin mi gii sinh vt nh eukaryote, prokaryote, archaea v virus. Nhng enzyme ny u c chung mt c ch xc tc phn ng thy phn thng qua hai bc chnh [25]:

 Bc 1, acyl ha: hnh thnh lin kt cng ha tr gia nhm -OH ca serine vi nguyn t cc bon trong nhm cacboxyl ca phn t c cht nh c h tr ca nhm imidazole t histidine. Kt qu phn ng ny l to ra mt hp cht trung gian v mt ion imidazolium (phn ng cng). Hp cht trung gian khng bn ny nhanh chng b thy phn thnh mt acyl-enzyme, vng imidazole v mt amin (phn ng kh). Bc 2, kh acyl ha: phc h acyl - enzyme b thy phn bi phn t H2O theo chiu ngc li ca bc mt. Trong , nhm imidazole chuyn proton ca gc -OH t serine cho nhm amin ti sinh li enzyme. Serine protease c 1 serine trung tm hot ng, nhm ny bao gm hai nhm nh: chymotrypsin v subtilisin. Nhm chymotrypsin bao gm cc enzyme ng vt nh chymotrypsin, trypsin, elastase. Nhm subtilisin bao gm hai loi enzyme vi khun nh subtilisin Carlsberg, subtilisin BPN. Serine protease c Cc fluorid mt trong ny rt b hoc nhiu c ch loi b i

ca Aspergillus v Bacillus. Phenylmethylsulgonyl

enzyme (PMSF)

diisopropyl

fluorophosphat (DFP). Phn ln enzyme ny hot ng ti thch trong khong pH t 7 11 v c trng lng phn t khong 20.000 35.000 dalton.

b/ Cysteine protease (EC.3.4.22) c 1 cysteine trung tm

hot ng. Cysteine protease bao gm cc protease thc vt nh

papayin, bromelin, mt vi protease ng vt v k sinh trng v t thy nm. Phn ln cc enzyme ny hot ng ti thch pH t 5 8, c tnh c hiu c cht rng. Chng mn cm vi cc cht cha sulphydryl nh l P-chloromercuri benzoate.

c/ Aspartic protease (EC.3.4.23) c 2 axit aspartic trung

tm hot ng, l enzyme quan trng th 2 trong s cc protease cng nghip sau serine protease. Chng ch yu l cc protease axit vi mt axit aspartic c coi nh cha kha phn gii trong vng tc ng. Enzyme ny hot ng tt nht khong pH t 3 4, vi trng lng phn t t 30.000 45.000 dalton. Aspartic protease rt ph bin nm v khng n nh pH 7,0.

d/ Metallo protease (EC.3.4.24) s dng ion kim loi trong

trung tm hot ng, enzyme ny hot ng tt nht pH 5 9, mn cm vi cc EDTA nhng khng b nh hng bi cc cht c ch ca serine protease hoc thuc th sulphydryl. Km, Coban hoc Canxi, Mangan hot ha tr li khi b EDTA lm bt hot [85]. Phn ln cc metallo protease l enzyme c cha Km, cu trc phn t protein lun n nh bi s c mt ca Canxi. ng ch cc enzyme ny l phn ln a nhit v rt ph bin Bacillus

thermoproteolyticus[58].
Endopeptidase l mt trong nhng thnh phn quan trng nht ca nhm enzyme cng nghip, chim khong 60% tng s enzyme c s dng rng ri [61], [28], [34]. Trong s cc loi protease t vi sinh vt, protease vi khun l ng k nht. Trong s

cc vi khun th Bacillus l sinh vt c trng sinh protease ngoi bo. Protease c mt s c im rt l tng i vi cng ngh sinh hc, v vy chng tr thnh mt trong nhng enzyme quan trng nht trong sn xut cng nghip. Nhng protease ny c ng dng rng ri trong cc nghnh cng nghip nh dc phm, thuc da, ty ra, thc phm v cng nghip x l rc thi [82]. 1.2. Protease t vi sinh v t Protease c tm thy nhiu nhm vi sinh vt nh ng vt nguyn sinh, vi khun, nm mc, nm men v virus. ng vt 8%, thc vt 4%, nm mc v nm men 50%, vi khun 30%. Protein b phn gii bi nhiu vi sinh vt. Nhng vi sinh vt ny s dng c cht protein lm ngun cung cp nit, cc bon v nng lng cho sinh trng v pht trin. S phn gii protein c bt u bi protease c tit ra t vi sinh vt tip sau l peptidase bn trong hay bn ngoi t bo. S lng v thnh phn protease c tit ra t vi sinh vt ph thuc vo tng loi, tng chng vi sinh vt, thm ch cc protese khc nhau cng c th c sinh ra t cng mt chng di cc iu kin nui cy khc nhau [17]. 1.2.1. Protease t n m men v n m m c Nm l nhm c kh nng to ra nhiu loi protease hn vi khun. Nhiu loi nm mc c kh nng tng hp mt lng ln protease c ng dng trong cng nghip thc phm, v d

nh: Aspergillus oryzae, Aspergillus tericola, Aspergillus fumigatus,

Penicillium chryfogenum,c bit Aspergillus oryzae l loi nm

mc c kh nng tng hp c 3 loi protease: protease axit, protease kim v protease trung tnh. T l cu t enzyme c th thay i ty thnh phn mi trng, v d: Aspergillus oryzae khi nui trong iu kin bnh thng th sinh tng hp ch yu protease kim, cn khi nui trong mi trng gim bt gluxit th ch yu to protease axit [3], [43]. Cc loi nm mc thuc chi Rhizopus c kh nng tng hp ch yu cc protease axit c kh nng thy phn protein pH 2,5-3,0 [3]. Mt s nm khc nh Aspergillus

cadidates, Penicillium camberti, Penicillium roqueforti,... cng c kh

nng tng hp protease lm ng t sa, c s dng trong lm pho mt. Bn cnh , ngun enzyme trong nm men cng rt phong ph v a dng, l i tng c con ngi bit n t rt sm. Mt s loi nm men sinh enzyme nh: Candida albicans v Candida

tropicalis l nhng vi sinh vt khi nhim vo c th vt ch gy

nhiu bnh c hi khi h thng min dch ca c th vt ch b tn thng, enzyme ngoi bo ca chng (aspartic protease) c xem ging nh yu t virus [91]. 1.2.2. Protease t x khu n S tng hp protease ca x khun t c nghin cu hn vi khun v nm mc. Tuy nhin ngi ta tm c 1 s chng x

khun c kh nng tng hp protease cao nh Streptomyces griceus,

S. fradiae, S. rimosus, [4], [13].

Cc ch phm protease t x khun c bit nhiu l Pronase (Nht) c tch chit t Streptomyces grieus. Enzyme ny c c tnh c hiu rng, c kh nng thy phn ti 90% lin kt peptide ca nhiu protein thnh amino acid. Lin X (c), ngi ta cng tch c ch phm tng t tStreptomyces

grieus c tn l Protelin. M, ch phm c sn xut c tn l

M-Zim dng trong sn xut da. Protease t Streptomyces

fradiae cng c hoat tnh elastase cao, do chng c dng

trong cng nghip ch bin tht. 1.2.3. Protease t vi khu n Serine protease l nhm enzyme c dng nhiu nht trong thng mi, ch yu l trung tnh v kim, c sinh ra t cc vi khun thuc chiBacillus, i din ca nhm ny l Bacillus

subtilis [43]. Ngoi ra cc serine protease cng c sinh ra t cc

nhm vi khun khc nhau nh: Thermus caldophilus, Aeromonas,

Escherichia v 1 s ging thuc Chlostridium. Cc protease trung

tnh hot ng trong gii pH hp (t 5 n 8) t bn nhit, sn phm phn gii ca cc protease ny t b ng hn so vi cc protease ng vt do tc ct trung bnh, v th rt c gi tr trong cng ngh thc phm [4]. Cc protease trung tnh t vi khun c tnh c hiu cao vi mi lin kt gia hai axit amin k nc. Nhit

 ti u thp rt c li cho vic phn gii thc n. Mt s metallo protease cn c cc ion kim loi cho qu trnh hot ng. Cc protease kim ca vi khun hot ng trong mi trng c pH cao, tnh c hiu c cht rng, nhit hot ng ti u 60oC, v th rt thch hp cho cng nghip cht ty ra. 1.2.4. Protease t virus Protease t virus c vai tr quan trng trong qu trnh lp rp cc thnh phn ca virus. Chng c th l serine protease, aspartic protease, cysteine protease nhng khng c metallo protease. Tt c cc protease ca virus u l endoprotease. Aspartyl protease ca Retrovirus tham gia vo qu trnh lp rp ht virus, to ra cc n v ng nht th hin nh tin thn ca cc protein v. Ngy nay ngi ta nghin cu rt nhiu v protease ca virus, cng nh cc cht c ch hot ng ca chng nhm cha cc bnh nguy him do virus gy ra nh ung th v AIDS