Laccase-Mediator System Siv Ib Tus Neeg Nruab Nrab Ntuj Ua Ib Tus Neeg Saib Xyuas Dawb Rau Lub Decolorization Ntawm Melanin Part 1

Abstract: Nyob rau hauv txoj kev tshawb no, lub laccase-mediator system (LMS) siv ib tug natural mediator yog tsim los ua ib tug whitening agent rau melanin decolorization. Xya lub ntuj nruab nrab tau siv los hloov cov khoom siv kho kom haum xeeb thiab ua tiav cov peev txheej qis redox ntawm laccase thiab txwv tsis pub nkag ntawm melanin mus rau qhov chaw nquag ntawm laccase. Kev ua haujlwm ntawm melanin decolorization ntawm laccases los ntawm Trametes versicolor (lacT) thiab Myceliophthora thermophila (lacM) tau txhim kho tau zoo siv cov neeg nruab nrab xws li acetosyringone, syringaldehyde, thiab acetovanillone, uas pom muaj cytotoxicity tsawg. Cov pab pawg methoxy thiab ketone ntawm cov neeg nruab nrab nruab nrab ua lub luag haujlwm tseem ceeb hauv melanin decolorization. Qhov tshwj xeeb tsis tu ncua ntawm lacT thiab lacM rau melanin decolorization tau txhim kho los ntawm 247 thiab 334, raws li, thaum acetosyringone tau siv los ua tus neeg nruab nrab. LMS siv lace thiab acetosyringone kuj tuaj yeem decolorize melanin tam sim no hauv cellulose hydrogel zaj duab xis, uas ua rau cov tawv nqaij mob. Tsis tas li ntawd, LMS tuaj yeem decolorize tsis tau tsuas yog hluavtaws eumelanin analogs npaj los ntawm oxidation ntawm tyrosine tab sis kuj ntuj melanin tsim los ntawm melanoma hlwb.

Raws li kev tshawb fawb cuam tshuam,cistancheyog ib hom tshuaj ntsuab uas hu ua "cov tshuaj ntsuab miracle uas ua rau lub neej ntev". Nws lub ntsiab tivthaiv yogcistanoside, uas muaj ntau yam teebmeem xws liantioxidant, anti-inflammatory, thiabkev tiv thaiv kev ua haujlwm txhawb nqa. Lub mechanism ntawm cistanche thiabtawv nqaij dawbnyob rau hauv cov nyhuv antioxidant ntawm cistancheglycosides. MelaninNyob rau hauv tib neeg daim tawv nqaij yog tsim los ntawm oxidation ntawm tyrosine catalyzed los ntawmtyrosinase, thiab cov tshuaj tiv thaiv oxidation yuav tsum muaj kev koom tes ntawm cov pa, yog li cov pa oxygen dawb radicals hauv lub cev dhau los ua ib qho tseem ceeb cuam tshuam rau kev tsim melanin. Cistanche muajcistanoside, uas yog ib qho antioxidant thiab tuaj yeem txo qhov tsim ntawm cov dawb radicals hauv lub cev, yog liinhibiting melanin ntau lawm.

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1. Taw qhia

Laccases (EC 1.10.3.2, benzenediol: dioxygen oxidoreductases) yog multicopper proteins uas catalyze oxidation ntawm ntau yam phenolic thiab non-phenolic tebchaw los ntawm ib tug radical-catalyzed cov tshuaj tiv thaiv los ntawm kev txo cov molecular oxygen [1,2]. Laccases tau siv los ua biocatalysts rau cov txheej txheem biodegradation, xws li bioremediation of dyes [3,4], pharmaceuticals [5,6], herbicides [7], thiab delignification [8-10]. Laccases kuj tau siv los catalyze lub polymerization ntawm dye precursors thiab organic compounds [11]. Tshwj xeeb, lawv cov khoom txaus nyiam, xws li qhov tsis tshua muaj substrate, kev siv cov pa oxygen raws li cov khoom siv hluav taws xob zaum kawg, kev tsim cov dej raws li cov khoom lag luam, thiab tsis muaj kev thov (lossis tsis muaj kev tsim tawm) ntawm peroxides, ua rau lawv nthuav dav hauv biotechnological thiab ib puag ncig teb [1,11,12].

Plaub tooj liab ions ntawm qhov chaw ua haujlwm tau koom nrog hauv kev ua haujlwm catalytic ntawm laccase. "Blue" tooj liab (T1 site) oxidizes substrate, thiab trinuclear tooj liab pawg (T2/T3) tau txais cov electrons los ntawm T1 qhov chaw los txo cov molecular oxygen [1,12,13]. Tshwj xeeb, lub peev xwm redox ntawm T1 site Cu yog suav tias yog qhov tseem ceeb hauv kev txiav txim siab lub peev xwm catalytic ntawm laccases [14]. Laccases muaj peev xwm redox tsawg (0.4–{11}}.8 V) piv rau ligninolytic peroxidases (tshaj 1 V) xws li manganese peroxidase thiab lignin peroxidase. Laccases tsis tuaj yeem ncaj qha oxidize non-phenolic substrates nrog lub peev xwm redox saum 1.3 V [13,14]. Yog li ntawd, kom kov yeej cov kev txwv ntawm laccase, laccase-mediator systems (LMS) siv cov khoom me me, xws li 2, 20 -azinobis (3- ethylbenzthiazoline-6-sulphonate) (ABTS), { {24}}hydroxy benzotriazole (HOBt), violuric acid (VLA), N-hydroxy phthalimide (HPI), N-hydroxy acetanilide (NHA), thiab TEMPO, uas ua raws li redox mediators, tau raug qhia [15–17].

Cov neeg nruab nrab no tso cai rau oxidation ntawm cov tebchaw loj ntawm kev sib txawv oxidation. Lub laccase-ABTS system oxidizes substrates los ntawm kev tsim cov cationic ABTS radical los ntawm kev hloov hluav taws xob (ET) mechanism. LMSs nrog HOBt, VLA, HPI, los yog NHA tsim nitroxyl radicals ntawm hydrogen atom hloov (HAT) mechanism [1,12,17]. Tsis tas li ntawd, cov neeg kho kom haum xeeb xws li TEMPO thiab nws cov analogs hnov ​​​​qab ntawm txoj hauv kev ionic los tsim oxoammonium ions [1,12,18]. Kev siv cov neeg nruab nrab tuaj yeem oxidize ntau qhov sib txuas hauv ntau daim ntawv thov, xws li zas xim degradation [3,4], tshuaj degradation [5,6], thiab lignin degradation [8-10]. Txawm li cas los xij, kev siv cov khoom siv hluavtaws hauv kev lag luam tau raug txwv vim lawv muaj peev xwm toxicity, tus nqi siab, thiab enzyme inactivation effect. Tsis ntev los no, lignin-derived phenolic molecules li natural mediators (piv txwv li, syringaldehyde, acetosyringone, vanillin, acetovanillone, methyl vanillaate, ferulic acid, sinapic acid, p-coumaric acid, thiab lwm yam) tau kawm los hloov cov hluavtaws mediators [1,12]. . Qhov zoo ntawm cov neeg kho kom haum xeeb yog tus nqi qis thiab tsis muaj tshuaj lom vim tias lawv tau txais los ntawm cov khoom siv hluav taws xob thiab rov ua dua tshiab [19].

Melanin yog ib pawg ntawm cov xim ntuj tsim los ntawm melanogenesis los ntawm oxidative polymerization ntawm tyrosine los ntawm melanocytes. Ntuj melanin tuaj yeem muab faib ua tsib pawg ntawm eumelanin, pheomelanin, tag nrho cov melanin, pheomelanin, thiab neuromelanin [20]. Tsis ntev los no, ntau yam kev kho mob thiab tshuaj electrochemical siv melanin lossis melanin precursors tau kawm [20,21]. Tib neeg cov tawv nqaij xim feem ntau txiav txim siab los ntawm qhov muaj melanin. Hauv kev lag luam tshuaj pleev ib ce, qhov ncaj qha depigmentation ntawm melanin siv cov enzymes tau raug npaj rau kev txhim kho cov tawv nqaij dawb. Ob peb peroxidases tau kawm los decolorize melanin. Woo et al. qhia tau hais tias hluavtaws melanin tuaj yeem ncaj qha decolorized los ntawm lignin peroxidase los ntawm P. chrysosporium [22]. Cov pab pawg Keneko thiab Mohorˇciˇc kuj tau tshaj tawm cov enzymatic decolorization ntawm melanin los ntawm manganese peroxidase cais los ntawm fungi (Sporotrichum pruinose thiab Phlebia radiata) [23,24]. Kim et al. qhia tias crude enzyme sib tov uas muaj manganese peroxidase, lignin peroxidase, thiab laccase pom melanin depigmentation kev ua [25]. Thaum peroxidases decolorize melanin, lawv xav tau hydrogen peroxide (H2O2) ua cov cofactor uas tuaj yeem ua rau tawv nqaij. Yog li, txhawm rau txo kev siv H2O2, cov piam thaj oxidase lossis laccase tau nkag mus rau hauv cov enzyme ua ke [26,27]. Laccases tuaj yeem decolorize melanin yam tsis tas siv hydrogen peroxide. Khammuang thiab Sarnthima tau tshaj tawm tias laccase los ntawm Lentinus polycarpous Lév tau pom melanin decolorization kev ua si siv ABTS, vanillin, thiab vanillic acid ua tus neeg nruab nrab [28].

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Khoom siv

2.2. Melanin Decolorization los ntawm LMS

Cov tshuaj saturated melanin (1.4 mg / mL) tau npaj los ntawm kev sib cais ntawm 3 mg hluavtaws melanin hauv 1.3 mL ntawm 10 mM NaOH. Cov tshuaj tau centrifuged ntawm 8500 rpm rau 5 min kom tshem tawm cov melanin undissolved, thiab cov supernatant yog diluted nrog 0.1 M citric acid phosphate buffer (pH 3, 4, 5, 5.5, 6, lossis 7) thiab siv los ua cov tshuaj substrate rau LMS. Qhov concentration ntawm melanin nyob rau hauv cov tshuaj substrate yog 63 µg / mL thiab spectrophotometrically paub tseeb ntawm 475 nm. Cov 0.8 mL ntawm melanin substrate tov yog tov nrog 0.1 mL mediator tov (0–1 mM) nyob rau hauv 1.5 mL Eppendorf raj. Cov tshuaj tiv thaiv melanin decolorization tau pib los ntawm kev ntxiv 0.1 mL ntawm cov tshuaj laccase (15.8 µg (0.6 U) lacT lossis 19.2 µg (1.8 U) lacM) rau kev sib xyaw ntawm melanin thiab tus neeg nruab nrab ntawm 25 ◦C hauv da dej ntawm 120 rpm. . Tom qab cov tshuaj tiv thaiv, cov tshuaj tiv thaiv sib tov yog centrifuged, thiab qhov absorbance ntawm supernatant yog ntsuas ntawm 475 nm. Qhov decolorization tawm los (feem pua ​​​​) tau suav nrog kev sib npaug hauv qab no:

Decolorization (%) {{0}} (A0 − At)/A0 × 100, (1)

2.3. Kinetic Study of Melanin Decolorization los ntawm LMS

2.4. Cytotoxicity ntawm Natural Mediators

B16F10 melanoma cell kab (Korea Cell Line Bank, Seoul, Kauslim Teb) tau siv los txiav txim siab txog cytotoxicity ntawm natural mediators rau LMS. Kev ntsuam xyuas nruab nrab liab (NR) tau ua los ntsuas qhov cytotoxicity ntawm cov neeg nruab nrab [29]. NR ntsuas qhov muaj peev xwm ntawm cov cell nyob lysosomes. Melanoma hlwb uas muaj qhov concentration ntawm 3 × 104 hlwb tau muab tso rau hauv txhua qhov dej ntawm 96-zoo phaj. Tom qab 24 teev ntawm kev cog qoob loo, cov hlwb raug kho nrog cov neeg nruab nrab (1, 2, 5, 10, 22, thiab 46 mM). Tom qab kev cog qoob loo ntxiv rau 2 hnub, cov hlwb tau kho nrog 50 µg / mL NR cov tshuaj yaj hauv DMEM thiab incubated rau 3 teev. Tom qab tshem tawm cov supernatant los ntawm kev nqus, NR desorb tov (1 feem pua ​​​​glacial acetic acid, 49 feem pua ​​​​ethanol, thiab 50 feem pua ​​​​ntawm cov dej distilled) tau siv rau xim extraction. Tom qab cov txheej txheem rho tawm, qhov hloov pauv ntawm qhov nqus tau ntsuas ntawm 540 nm.

2.5. Kev Npaj thiab Decolorization ntawm Melanin / Cellulose Hydrogel Film

Txhawm rau ntsuas qhov decolorization ntawm LMS rau cov yeeb yaj kiab melanin / cellulose, cov yeeb yaj kiab hydrogel npaj tau txiav rau hauv daim ntawv 1 × 2 cm. Cov yeeb yaj kiab hydrogel tau muab tso rau hauv 4 mL ntawm 0.1 M citric acid phosphate buffer (pH 5.5); Tom qab ntawd, 0.5 mL ntawm 1 mM acetosyringone thiab 0.5 mL ntawm lace solution (2.5 U) tau ntxiv rau qhov tsis. Cov tshuaj tiv thaiv de-colorization tau nqa tawm hauv da dej nrog co thaum 120 teev tsaus ntuj thiab 25 ◦C rau 3 teev. Tom qab cov tshuaj tiv thaiv, cov yeeb yaj kiab tau ntxuav nrog dej distilled thiab txuas mus rau sab hauv ntawm lub cuvette los ntsuas qhov kev hloov pauv hauv qhov ntsuas ntawm 400-800 nm siv lub UV / Vis spectrophotometer. Kev tswj cov tshuaj tiv thaiv tsis muaj lacM lossis cov neeg kho kom haum xeeb kuj tau ua raws li cov xwm txheej qub. Kev tso tawm ntawm melanin los ntawm zaj duab xis los yog xim hloov ntawm melanin / cellulose zaj duab xis tsis raug kuaj pom nyob rau hauv cov tshuaj tiv thaiv. Tsis tas li ntawd, qhov kev hloov ntawm cov xim tsis muaj (L *, a *, thiab b * qhov tseem ceeb) ntawm melanin / cellulose zaj duab xis tom qab decolorization cov tshuaj tiv thaiv los ntawm LMS kuj tau sau tseg siv lub colorimeter (KONICA MI NOLTA, Tokyo, Nyiv). Qhov ∆L (metric lightness sib txawv), ∆E (tag nrho cov xim sib txawv), YI (yellowness index), thiab WI (whiteness index) qhov tseem ceeb tau txais los ntawm kev sib npaug hauv qab no [30–32]:

2.6. Kev Npaj Ntuj Melanin

Ntuj melanin tau txais los ntawm B16F10 melanoma hlwb. Cov hlwb tau kho nrog alpha-melanocyte-stimulating hormone los tsim melanin. Tom qab 4 hnub ntawm incubation, lub hlwb raug ntes siv trypsin-EDTA thiab sonicated rau 10 min. Lub supernatant tau txais los ntawm centrifugation ntawm 8000 rpm rau 10 min thiab tom qab ntawd hloov mus rau pH 1.5 siv 6 M HCl. Cov tshuaj tau boiled ntawm 100 ◦C rau 4 h kom hydrolyze cov seem protein seem. Cov tshuaj uas muaj cov tshuaj melanin yog ntxuav nrog acetone, ua raws li chloroform thiab ethanol, thiab tom qab ntawd ntxuav nrog dej deionized kom tshem tawm cov seem, xws li cov hlwb, cov khoom siv xov xwm, thiab cov protein ntau [33,34]. Tag nrho cov txheej txheem ntxuav tau ua ntau tshaj ob zaug. Thaum kawg, ntuj melanin tau txais los ntawm khov-qhuav thiab siv los ua lub substrate rau LMS.

3. Cov txiaj ntsig thiab kev sib tham

3.1. Cov nyhuv ntawm Mediators ntawm Melanin Decolorization los ntawm LMS

Cov nyhuv ntawm ntau tus neeg nruab nrab ntawm cov tshuaj tiv thaiv melanin decolorization los ntawm LMS tau tshawb xyuas siv ob lub laccases los ntawm T. versicolor (lacT) thiab M. thermophila (lacM) (Daim duab 1).Thaum lacT tau siv yam tsis muaj tus neeg nruab nrab rau melanin decolorization, qhov decolorization tawm los tsuas yog 1 feem pua ​​​​tom qab 5 teev ntawm cov tshuaj tiv thaiv. Thaum HOBt tau siv los ua tus neeg nruab nrab rau lacT, cov txiaj ntsig decolorization tau txhim kho me ntsis rau 2 feem pua ​​​​tom qab 5 teev ntawm cov tshuaj tiv thaiv. Kev siv ntau yam khoom siv hluavtaws, xws li HOBt, ABTS, VLA, thiab TEMPO, nyob rau hauv laccase-catalyzed oxidation ntawm phenolic lossis non-phenolic tebchaw ua rau muaj txiaj ntsig zoo rau cov tshuaj tiv thaiv [10,15]. Thaum kev nkag mus ntawm lub hom phiaj sib txuas rau hauv qhov chaw nquag ntawm laccase raug txwv los ntawm lawv qhov kev cuam tshuam tsis zoo, cov neeg nruab nrab radicals tsim los ntawm laccase tuaj yeem ua kom zoo oxidize lub hom phiaj sib txuas los ntawm kev hloov hluav taws xob lossis hydrogen atom hloov tshuab [12]. HOBt yog ib qho ntawm feem ntau siv hluavtaws mediators hauv LMS vim nws lub peev xwm redox siab (1.1 V) [6]. Txawm li cas los xij, HOBt tsis yog cov khoom zoo nkauj zoo nkauj vim tias nws muaj peev xwm ua rau lub cev muaj zog thiab muaj peev xwm ua kom tsis muaj zog laccase. Yog li, peb xaiv xya tus neeg nruab nrab, acetosyringone, syringaldehyde, p-coumaric acid, vanillin, vanillic acid, vanillyl cawv, thiab acetovanillone, rau cov tshuaj melanin decolorization los ntawm LMS. Interestingly, tag nrho cov natural mediators ua raws li cov neeg kho kom zoo dua li HOBt rau melanin decolorization los ntawm tsis muaj. Thaum acetosyringone, syringaldehyde, thiab p-coumaric acid tau siv, cov decolorization yields yog 28 feem pua, 22 feem pua, thiab 18 feem pua, feem, tom qab 5 h ntawm cov tshuaj tiv thaiv. Cov txiaj ntsig no qhia tau hais tias muaj txiaj ntsig zoo ntawm cov neeg nruab nrab rau cov tshuaj melanin decolorization los ntawm LMS. Cov neeg nruab nrab hauv LMS yog oxidized rau mediator radicals los ntawm laccase, thiab cov mediator radicals induce oxidation thiab decolorization ntawm melanin. Thaum tsis muaj kev siv tsis muaj tus neeg nruab nrab rau melanin decolorization thaum lub sij hawm txaus cov tshuaj tiv thaiv, uas tuaj yeem ncav cuag lub xeev sib npaug, qhov decolorization tawm los yog 7 feem pua ​​​​tom qab 24 teev ntawm cov tshuaj tiv thaiv. Lub ntuj nruab nrab, tshwj tsis yog vanillic acid, ua raws li cov neeg kho kom zoo dua li HOBt rau melanin decolorization los ntawm lacT tom qab 24 teev ntawm cov tshuaj tiv thaiv. Lub decolorization tawm los tom qab 24 teev cov tshuaj tiv thaiv siv vanillic acid ua tus neeg nruab nrab qis dua li tom qab 5 teev ntawm cov tshuaj tiv thaiv. Qhov no yuav tshwm sim los ntawm qhov tsis tshua muaj kev ruaj ntseg ntawm oxidized radical daim ntawv ntawm vanillic acid. Thaum siv acetosyringone, syringaldehyde, thiab acetovanillone, cov decolorization yields yog 34 feem pua, 30 feem pua, thiab 31 feem pua, feem, tom qab 24 teev cov tshuaj tiv thaiv. p-Coumaric acid muaj txiaj ntsig zoo hauv kev txhim kho qhov pib cov tshuaj tiv thaiv ntau dua li acetovanillone, thaum acetovanillone ua rau muaj kev cuam tshuam ntau dua decolorization ntawm lub xeev equilibrium dua li p-coumaric acid.

Qhov cuam tshuam ntawm tus neeg nruab nrab ntawm cov tshuaj tiv thaiv decolorization los ntawm LMS siv lacM kuj zoo ib yam li qhov tau los ntawm LMS siv lacT. Thaum siv lace tsis muaj tus neeg nruab nrab rau melanin decolorization, qhov decolorization tawm los tsuas yog 2 feem pua ​​​​tom qab 5 teev ntawm cov tshuaj tiv thaiv. HOBt ua tus neeg nruab nrab rau lacM tsis txhim kho qhov decolorization tawm los thaum lub sij hawm 5 h cov tshuaj tiv thaiv. Tag nrho cov natural mediators, tshwj tsis yog p-coumaric acid thiab vanillin, ua raws li kev sib kho kom zoo ntawm melanin decolorization los ntawm lacM. Thaum siv acetosyringone thiab syringaldehyde, cov decolorization yields yog 25 feem pua ​​thiab 22 feem pua, feem, tom qab 5 h ntawm cov tshuaj tiv thaiv. p-Coumaric acid thiab vanillin tau siv los ua cov neeg kho kom zoo rau txhua tus, tab sis lawv tsis tuaj yeem txhim kho tus nqi decolorization hauv LMS siv lace. Qhov no tej zaum yuav tshwm sim los ntawm qhov qis substrate tshwj xeeb ntawm lacM rau p-coumaric acid thiab vanillin. Cov decolorization tawm los tom qab 24 teev ntawm cov tshuaj tiv thaiv ntawm lacM nrog p-coumaric acid thiab vanillin zoo ib yam li cov tau txais los ntawm lacT. Qhov no qhia tau hais tias cov ntaub ntawv oxidized ntawm p-coumaric acid thiab vanillin tuaj yeem decolorize melanin, txawm tias lawv cov oxidation tus nqi los ntawm lacM qis dua li ntawm lacT. Thaum lacM tsis muaj tus neeg nruab nrab siv rau melanin decolorization thaum lub sij hawm txaus cov tshuaj tiv thaiv, cov decolorization tawm los yog 5 feem pua ​​​​tom qab 24 teev ntawm cov tshuaj tiv thaiv. Lub ntuj nruab nrab, tshwj tsis yog vanillic acid, kuj ua raws li cov neeg kho kom zoo dua li HOBt rau melanin decolorization los ntawm lacM tom qab 24 teev ntawm cov tshuaj tiv thaiv. Thaum acetosyringone, syringaldehyde, thiab acetovanillone tau siv los ua tus neeg nruab nrab rau lacM, decolorization yields yog 34 feem pua, 28 feem pua, thiab 31 feem pua, raws li, tom qab 24 teev ntawm cov tshuaj tiv thaiv. Thaum vanillic acid tau siv los ua tus neeg nruab nrab rau ob qho tib si lacT thiab lacM, nws pom qhov qis tshaj decolorization tawm los. Qhov no yuav tshwm sim los ntawm qhov tsis tshua muaj kev ruaj ntseg ntawm oxidized radical daim ntawv ntawm vanillic acid. Khammuang thiab Sarnthima tau tshaj tawm tias vanillin thiab vanillic acid tuaj yeem siv los ua tus neeg nruab nrab rau melanin decolorization siv laccase los ntawm Lentinus polycarpous [28]. Txawm li cas los xij, lawv tau pom ntau qhov decolorization qis dua rau melanin dua li acetosyringone thaum lawv tau siv los ua tus neeg nruab nrab rau lacT thiab lace.

Cov txiaj ntsig no qhia tau tias cov neeg nruab nrab ntawm ntuj tsim muaj txiaj ntsig zoo rau melanin decolorization los ntawm LMS dua li HOBt. HOBt tau raug suav hais tias yog ib qho khoom siv hluav taws xob zoo rau laccase vim nws lub peev xwm redox siab thiab lub luag haujlwm catalytic ntawm N OH pawg ntawm HOBt [5]. Kev ua tau zoo ntawm cov neeg kho kom haum xeeb rau oxidize lub hom phiaj substrates yog nyob ntawm qhov muaj peev xwm tsim cov radicals ruaj khov nrog rau cov steric hindrance los ntawm cov khoom loj alkyl hloov dua li qhov redox peev xwm ntawm cov neeg kho kom haum xeeb [19,35]. Qhov tsis tshua muaj kev ruaj ntseg ntawm oxidized intermediate ntawm HOBt tau txiav txim los ntawm cyclic voltammetry [6]. Yog li ntawd, qhov qis decolorization tawm los ntawm LMS siv HOBt yuav tshwm sim los ntawm qhov tsis tshua muaj kev ruaj ntseg ntawm HOBt nyob rau hauv cov tshuaj tiv thaiv ntawm laccase. Txawm hais tias lub peev xwm redox ntawm syringaldehyde qis dua li ntawm HOBt, syringaldehyde pom tau tias muaj kev ruaj ntseg dua li HOBt [6].

Raws li pom nyob rau hauv daim duab 2, cov natural mediators siv nyob rau hauv cov hauj lwm no muaj ntau yam hloov pauv (xws li, hydroxyl, methoxy, carboxyl, ketone, los yog aldehyde) ntawm ntau txoj hauj lwm ntawm lub nplhaib benzene [12,19]. Cov neeg kho kom haum xeeb nrog ob pawg methoxy (acetosyringone thiab syringaldehyde) tau pom cov decolorization ntau dua li cov uas muaj ib pab pawg methoxy. Tus decolorization tus nqi tau los ntawm p-coumaric acid uas tsis muaj methoxy pawg yog nyob ntawm hom laccase. Cov p-coumaric acid nrog lacT pom qhov decolorization ntau dua li cov uas muaj ib pawg methoxy, thaum p-coumaric acid nrog lacM pom qhov decolorization qis tshaj plaws hauv 5 h cov tshuaj tiv thaiv. Fillat et al. kuj tau pom cov txiaj ntsig zoo sib xws rau kev decolorization ntawm flexographic inks los ntawm fungal laccases nrog natural mediators [36]. Cov phenolic natural mediators (acetosyringone, methyl syringe, thiab syringaldehyde) nrog ob methoxy hloov hauv lub nplhaib tau oxidized los ntawm laccase sai dua p-coumaric acid uas tsis muaj methoxy pawg. Qhov no qhia tau hais tias pawg methoxy ua lub luag haujlwm tseem ceeb dua li cov neeg pub khoom siv hluav taws xob ntau dua li ob daim ntawv cog lus ntawm cov saw hlau ntawm p-coumaric acid. Thaum cov neeg nruab nrab nrog ib pab pawg methoxy tau muab piv, cov txiaj ntsig decolorization tau nce hauv qhov kev txiav txim hauv qab no: acetovanillone> vanillin> vanillyl cawv> vanillic acid. Acetovanillone, uas muaj ib pawg ketone, tau pom ntau dua decolorization tus nqi thiab tawm los ntau dua li cov neeg nruab nrab nrog aldehyde, hydroxyl, thiab carboxyl pawg. Acetosyringone nrog ib pawg ketone kuj pom tias muaj qhov decolorization ntau dua thiab tawm los dua li syringaldehyde nrog ib pawg aldehyde.

Hauv cov kev sim hauv qab no, acetosyringone, syringaldehyde, thiab acetovanillone, uas pom tau tias muaj peev xwm ua kom melanin decolorization siab, tau raug xaiv los ua tus neeg nruab nrab rau LMS kom decolorize melanin. Cov nyhuv ntawm tus neeg nruab nrab ntawm cov tshuaj tiv thaiv decolorization los ntawm LMS tau tshawb xyuas lub sijhawm (Daim duab S1). Cov tshuaj tiv thaiv decolorization siv lacT nrog acetosyringone, syringaldehyde, thiab acetovanillone ua rau 21 feem pua, 18 feem pua, thiab 1 feem pua ​​decolorization yields tom qab 1 h cov tshuaj tiv thaiv, feem. Cov tshuaj tiv thaiv decolorization siv lacM nrog acetosyringone thiab syringaldehyde ua rau 19 feem pua ​​​​thiab 18 feem pua ​​​​ntawm decolorization yields tom qab 1 h ntawm cov tshuaj tiv thaiv, feem. Ob lub laccases pom zoo sib xws cov tshuaj tiv thaiv thaum siv tib tus neeg nruab nrab. Acetosyringone thiab syringaldehyde tau txhim kho qhov decolorization tus nqi thaum pib cov tshuaj tiv thaiv. Cov txiaj ntsig no qhia tau hais tias acetosyringone thiab syringaldehyde uas muaj cov pab pawg dimethoxy tau muaj txiaj ntsig zoo hauv kev txhim kho tus nqi pib ntawm decolorization los ntawm LMS dua li acetovanillone uas muaj ib pawg methoxy. Fillat et al. kuj tau tshaj tawm tias pawg methoxy nyob rau hauv lub nplhaib cov qauv ntawm cov neeg nruab nrab ua raws li cov accelerators rau oxidation ntawm substrates [36]. Ntawm qhov tod tes, qhov decolorization tawm los tom qab 24 teev ntawm cov tshuaj tiv thaiv los ntawm acetovanillone zoo ib yam li cov syringaldehyde, txawm hais tias acetovanillone me ntsis txhim kho cov tshuaj tiv thaiv.

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