Kev tsim khoom thiab kev tshuaj xyuas biochemical ntawm oyster mushroom laccase NRC 620 thiab kev ntsuam xyuas nws cov txiaj ntsig zoo hauv kev ua kom cov kua txiv apple ntshiab.

Tom qab 25 hnub ntawm kev sib xyaw ua ke ntawm 28 ° C, laccase los ntawm *Pleurotus ostreatus* NRC620 tau qhia txog kev ua haujlwm siab tshaj plaws hauv cov kab lis kev cai fungal. Qhov zoo tshaj plaws pH thiab qhov kub thiab txias rau cov enzyme no yog 3.0 thiab 70 ° C, feem. Tom qab 2 teev ntawm kev sib xyaw ua ke ntawm 40 ° C thiab 50 ° C, qhov kev ua haujlwm enzyme khaws cia 68.33% thiab 59.61%, feem. Tom qab 2 teev ntawm kev sib xyaw ua ke hauv citrate-phosphate buffer (pH 7.0), qhov kev ua haujlwm enzyme tseem nyob ntawm 100%. Qhov ntxiv ntawm 10 mM MgSO₄ thiab CuSO₄ ua rau qhov kev ua haujlwm enzyme nce ntxiv kwv yees li 21% thiab 35%, feem, thaum NaCl, MnCl₂, KCl, thiab CaCl₂ inhibited qhov kev ua haujlwm enzyme. Siv ABTS ua ib qho substrate, cov kinetic parameters (Km thiab Vmax) ntawm *Pleurotus ostreatus* NRC 620 laccase yog 1.99 mM thiab 16,217 μmol min−1 L−1, raws li. Kev kho enzymatic ntawm cov kua txiv apple txo qis pH thiab viscosity, thiab qhov kev txo qis no cuam tshuam nrog kev nce ntxiv ntawm lub sijhawm khaws cia. Kev kho Laccase ua rau muaj kev txo qis me ntsis ntawm tag nrho cov phenolic ntsiab lus ntawm kua txiv apple, tab sis tsis muaj kev txo qis ntawm cov antioxidant activity tau pom.
Nyob rau xyoo tas los no, cov kws tshawb fawb tau tsom mus rau kev siv cov thev naus laus zis ntsuab hauv kev lag luam zaub mov. Laccase yog ib qho ntawm cov enzymes muaj txiaj ntsig zoo tshaj plaws hauv kev lag luam zaub mov, nrhiav kev siv hauv cov cheeb tsam xws li kev ua kua txiv hmab txiv ntoo, ci, kev ruaj khov cawv, thiab kev txhim kho cov khoom noj khoom haus zoo.¹Ntau cov nroj tsuag siab dua thiab cov kab mob me me tso tawm laccase,²thiab cov nceb xws li deuteromycetes, ascomycetes, thiab basidiomycetes kuj tseem tuaj yeem tsim cov laccase.³Laccase (EC 1.10.3.2) yog ib qho tshuaj oxidase xiav uas txo cov pa oxygen molecular rau hauv dej siv ib lub tshuab uas muaj peb lub atoms tooj liab sib txawv, yog li ntawd oxidizing ntau yam phenolic compounds thiab aromatic amines. Thaum lub sijhawm tsim cov kua txiv hmab txiv ntoo thiab zaub, enzymatic thiab nonenzymatic browning yog cov teeb meem tseem ceeb.⁴Vim tias cov tshuaj no cuam tshuam tsis zoo rau cov xim, saj, thiab aroma ntawm cov kua txiv, lawv yuav tsum tau muab tshem tawm.⁵
Ntawm txhua yam txiv hmab txiv ntoo, txiv apples yog cov txiv uas noj ntau tshaj plaws thoob ntiaj teb thiab hauv European Union. Xyoo 2019, kev tsim txiv apples tau qeb thib peb thoob ntiaj teb, tshaj 87 lab tonnes.⁶Cov txiv apples muaj ntau yam phenolic compounds, suav nrog flavonoids thiab phenolic acids xws li caffeic acid thiab chlorogenic acid.⁷Vim tias cov kua txiv apple feem ntau noj ua cov kua ntshiab, kwv yees li 50% txog 90% ntawm cov khoom phenolic ploj mus thaum lub sijhawm lim dej.⁸Niaj hnub nim no, cov neeg siv khoom feem ntau xaiv cov khoom uas tsis tau ua tiav ntau, xws li kua txiv apple uas muaj cov polyphenol ntau. Txawm li cas los xij, vim nws muaj cov phenolic ntau, hom kua txiv apple no yooj yim hloov xim thiab tsaus dua.⁹Muaj ntau yam thev naus laus zis, suav nrog cov txheej txheem kho cua sov xws li pasteurization ntawm 60–90°C, siv los txo lossis tiv thaiv kom cov kua txiv apple tsaus ntuj.¹⁰Txawm li cas los xij, raws li kev tshawb fawb los ntawm Sauceda-Gálvez¹¹, kev ua cov txheej txheem thermal tuaj yeem rhuav tshem cov tshuaj lom neeg uas tsis muaj zog thiab cuam tshuam rau cov yam ntxwv ntawm cov kua txiv apple. Lwm txoj hauv kev rau kev ua cov txheej txheem thermal suav nrog supercritical carbon dioxide, ultraviolet radiation, ultrasound, siab hydrostatic siab, lossis siab siab homogenization.¹²Qhov ua tau zoo ntawm cov thev naus laus zis no thiab cov kua txiv hmab txiv ntoo tsim nyog nyob ntawm cov yam ntxwv siv thiab cov yam ntxwv ntawm cov khoom. Lawv siv dav dav yog txwv los ntawm cov nqi siab, cov teebmeem tsis zoo rau qhov zoo ntawm qee cov khoom noj, lossis tsis ua haujlwm ntawm cov enzyme txaus.^13,14
Laccase siv tau los ua kom cov kua txiv hmab txiv ntoo ruaj khov thiab ua kom huv.¹⁵Gökmen thiab lwm tus.¹⁶pom zoo kom siv cov laccase rau kev ua kom cov kua txiv hmab txiv ntoo ntshiab vim nws tshem tawm cov tshuaj phenolic los ntawm kev hloov lawv mus ua cov polymers lossis oligomers uas yooj yim tshem tawm los ntawm txhua lub ultrafiltration membrane, ua rau cov kua txiv apple tswj tau cov xim ruaj khov thiab meej meej txog li rau lub lis piam ntawm 50°C. Cov *Trichoderma* laccase ntshiab tau raug immobilized rau ntawm cov hlaws alumina thiab siv los xaiv cov tshuaj tsis qab uas tshwm sim los ntawm kev kis kab mob ntawm cov kua txiv apple.¹⁷
Kwv yees li 80-90% ntawm cov khoom sib xyaw ua ke ntawm cov kua txiv apple yog esters thiab aldehydes, uas muab cov ntxhiab tsw tshwj xeeb rau cov kua txiv.¹⁸Laccase los ntawm *Trametes versicolor* raug khi rau ntawm ib qho kev txhawb nqa pheej yig uas ua los ntawm cov fiber ntuj los ntawm cov plhaub txiv maj phaub hluas rau kev ua kom kua txiv apple ntshiab.¹⁹Cov kev tshawb fawb yav dhau los tau tshawb nrhiav qhov kev ruaj khov ntawm cov kua txiv apple (xim thiab qhov turbidity) siv cov txheej txheem tsis muaj enzyme lossis immobilization, lossis ua ke nrog ultrafiltration.^5,19Txawm li cas los xij, qhov cuam tshuam ntawm cov fungal laccases rau cov khoom siv physicochemical ntawm cov kua txiv apple thaum lub sijhawm khaws cia tseem tsis meej. Yog li ntawd, lub hom phiaj ntawm txoj kev tshawb fawb no yog los sim tshawb nrhiav cov kev hloov pauv hauv cov khoom siv physicochemical, cov ntsiab lus phenolic compound, thiab cov haujlwm antioxidant ntawm cov kua txiv apple tom qab kho nrog fungal laccases thiab ob lub lis piam cia hauv tub yees. Laccases muaj peev xwm oxidize cov phenolic compounds, uas ua rau lawv cog lus tias yuav siv tau rau ntau yam txheej txheem kev lag luam, suav nrog kev ua kom cov kua txiv hmab txiv ntoo meej. Txoj kev tshawb fawb no tau tshuaj xyuas cov laccases los ntawm *Pleurotus ostreatus* NRC 620, tsom mus rau qhov xwm txheej zoo tshaj plaws rau lawv cov haujlwm thiab kev ua haujlwm zoo hauv kev ua kom cov kua txiv hmab txiv ntoo meej. Thaum kev tshawb fawb txog cov nceb oyster (P. ostreatus NRC 620) tseem tsawg, cov kev tshawb fawb yav dhau los tau tshuaj xyuas cov enzymes los ntawm ntau qhov chaw fungal, xws li Trametes versicolor thiab Ganoderma lucidum. Lub hom phiaj ntawm txoj kev tshawb fawb no yog los ntsuas qhov kev siv tau ntawm cov enzyme no hauv kev lag luam zaub mov thiab qhia txog nws cov khoom tshwj xeeb, tshwj xeeb tshaj yog nws cov pH thiab kub zoo tagnrho.
2,2′-Azooxybis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) tau yuav los ntawm Sigma-Aldrich (Canada). Tag nrho lwm cov tshuaj reagents yog qib analytical.
Lub Chaw Tshawb Fawb Hauv Tebchaws Lub Chaw Sau Kab Mob Me Me tau txais cov kab mob oyster nceb NRC620 uas paub lawm. Tom qab kev cog qoob loo me me, cov kab mob no tau khaws cia rau ntawm cov qos yaj ywm dextrose agar slants ntawm 4 ° C. Txoj kev npaj cov tshuaj inoculum yog raws li nram no: 10-hnub-laus, mycelium uas loj hlob tag nrho tau txhaj tshuaj rau ntawm cov phaj qos yaj ywm dextrose agar thiab incubated ntawm 28 ° C. Tom qab 10 hnub, peb lub thaiv mycelial 12-mm-txoj kab uas hla tau raug tshem tawm ntawm cov xov xwm agar siv lub tshuab xuas hlau sterile thiab muab tso rau hauv 250-mL Erlenmeyer flasks nrog cov paj rwb plugs uas muaj 50 mL ntawm cov kab lis kev cai sterilized (pH 5.0, raws li tau piav qhia ua ntej los ntawm Othman et al.²⁰). Cov kab lis kev cai tau muab tso rau hauv qhov kub thiab txias li ntawm 28°C rau 18 hnub. Cov kab lis kev cai tom qab ntawd tau lim los ntawm daim ntawv lim dej Whatman No. 1, thiab cov khoom noj uas tshwm sim los ntawm cov kab mob no tau ua lub hauv paus enzyme.
Kev ua haujlwm ntawm laccase tau txiav txim siab siv ABTS ua lub substrate. Cov tshuaj sib tov (2 mL) muaj 500 μL ntawm 0.3 mM ABTS (yaj hauv 0.1 M sodium citrate buffer, pH 4.5) thiab qhov ntau ntawm cov qauv enzyme uas xav tau diluted nrog dej distilled.^21,22Xav txog tias laccase tuaj yeem oxidize ABTS ntawm chav tsev kub (28 ° C ± 2), ABTS oxidation tau txiav txim siab los ntawm kev ntsuas qhov nce ntawm absorbance ntawm 420 nm (ε₄₂₀= 36,000 cm^-1 M ^-1) siv Agilent Carry-100 UV spectrophotometer. Ib chav ntawm laccase kev ua ub no yuav tsum tau oxidize 1 μmol ABTS ib feeb. Protein concentration tau txiav txim siab los ntawm Bradford txoj kev siv bovine serum albumin ua kev tswj hwm sab hauv.^23,24
Tom qab tau txais cov enzyme los ntawm cov nceb oyster hom NRC 620, nws cov haujlwm tau ntsuas ntawm ntau lub sijhawm cog qoob loo rau 25 hnub nyob rau hauv cov xwm txheej zoo li qub ntawm 28 ° C.
Txhawm rau kawm txog qhov cuam tshuam ntawm qhov kub thiab txias rau kev ua haujlwm ntawm laccase, kev sim tau ua nyob rau hauv qhov kub thiab txias ntawm 20 txog 90 ° C. Ua ntej ntxiv cov enzyme thiab pib qhov kev tshuaj tiv thaiv, lub buffer (0.1 M sodium citrate, pH 4.5) thiab substrate (ABTS) tau sib xyaw thiab incubated rau 5 feeb ntawm ntau qhov kub thiab txias. Kev ruaj khov ntawm enzyme thermal tau soj ntsuam los ntawm kev incubation hauv 0.05 M sodium phosphate buffer (pH 7.0) ntawm 40, 50, 60, thiab 70 ° C rau 2 teev, raws li. Kev ua haujlwm seem tau soj ntsuam siv ABTS substrate.
Qhov cuam tshuam ntawm pH rau kev ua haujlwm ntawm laccase tau soj ntsuam siv ABTS ua lub substrate hauv 0.1 M citrate-phosphate buffers nrog pH ntau ntawm 2.5 txog 7.0. Cov kua enzyme tau incubated ntawm 40 ° C rau ob teev hauv 0.1 M citrate thiab Tris buffers (pH 3, 4, 6, thiab 7) los ntsuas qhov ruaj khov ntawm pH. Cov dej num seem nrog ABTS ua lub substrate tau suav tom qab incubation.
Cov laccase tau muab tso rau hauv sodium phosphate buffer (0.05 M, pH 7.0) rau 10 feeb uas muaj ntau yam hlau ions (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, thiab Mn2+) ntawm qhov concentration ntawm 2.5 mM thiab 10 mM, raws li. Tom qab ntawd, cov substrate (ABTS) tau ntxiv rau kom pib qhov kev ua haujlwm, thiab qhov kev ua haujlwm sib piv tau raug soj ntsuam.
ABTS oxidation los ntawm laccase ntawm ntau qhov concentration (0.025–3 mM) tau ntsuas ntawm pH 4.5 los txiav txim siab cov kev ntsuas kinetic (Vmax thiab Km). Lub kinetic^{cov tsis hloov pauv}ntawm Michaelis-Menten equation tau suav los ntawm kev siv Lineweaver-Burk plot, uas plot qhov sib piv ntawm qhov nrawm ntawm cov tshuaj tiv thaiv raws li kev ua haujlwm ntawm qhov concentration ntawm substrate. Cov kinetic constants tau suav los ntawm Lineweaver-Burk plot siv GraphPad Prism version 6.01 software.
Tom qab ntxuav cov txiv apples kom huv si nrog dej kais, lawv raug txiav ua ob nrab thiab kua txiv los ntawm kev siv lub tshuab kua txiv apple Braun MP80 uas ua haujlwm tsis siv neeg (ua nyob rau hauv lub teb chaws Yelemees). Cov kua txiv tau lim los ntawm plaub txheej ntawm cheesecloth. Tsis muaj cov enzymes ntxiv rau pawg tswj, thaum 2.0% laccase (qhov concentration zoo tshaj plaws uas tau sim) tau ntxiv rau cov kua txiv apple tshiab, uas tom qab ntawd khaws cia rau ntawm 4°C rau ob lub lis piam.
Titratable acidity (TA) thiab pH tau txiav txim siab raws li txoj kev ntawm Boulton et al.^{lwm yam.27}. Lub pH ntawm txhua tus qauv tau ntsuas siv lub ntsuas pH digital (JENWAY 3510 pH meter). Titratable acidity (TA) tau suav raws li malic acid siv cov mis hauv qab no.
Qhov twg V thiab C yog qhov ntim (mL) thiab qhov concentration (0.1 mol/L) ntawm cov kua sodium hydroxide siv hauv titration, raws li. K yog tus coefficient hloov pauv malic acid, sib npaug rau 0.067, thiab W yog qhov hnyav (g) ntawm kua txiv apple.
Tag nrho cov khoom soluble (^TDS) cov ntsiab lus ntawm tag nrho cov kua txiv hmab txiv ntoo tau txiav txim siab siv PAL-1 hnab tshos refractometer (ATAGO, Tokyo, Nyiv). Tom qab txhua qhov kev ntsuas, lub lens optical tau ntxuav nrog dej deionized, thiab txhua tus qauv kua txiv apple tau kuaj peb zaug. Tus nqi rau txhua tus qauv tau suav los ntawm kev nruab nrab ntawm peb qhov kev ntsuas. Qhov nruab nrab ± tus qauv sib txawv rau txhua tus qauv kua txiv apple kuj tau suav los ntawm kev nruab nrab cov txiaj ntsig no.
Qhov viscoelasticity ntawm cov kua txiv apple tau raug soj ntsuam siv lub rotational viscometer (RV, Rheotest 2, Lub Tebchaws Yelemees). Cov qauv tau muab tso rau hauv lub tog raj kheej "S2" ntawm lub viscometer. Qhov viscosity pom tseeb tau sawv cev los ntawm qhov nqes hav ntawm qhov shear stress piv rau shear rate curve, uas tau suav los ntawm qhov shear stress thiab cov curves sib xws ntawm ntau qhov shear rates (los ntawm 1.00 txog 437.4 s⁻¹). Cov mis rau kev xam viscosity pom tseeb yog raws li nram no:
Qhov twg η yog qhov pom tseeb viscosity (cP), τ yog qhov kev ntxhov siab shear (dyn / cm²), γ yog tus nqi shear (sec⁻¹), thiab (τ) yog xam siv cov nqi torque (α) thiab lub tog raj kheej (Z) siv cov mis hauv qab no: τ = Z. α.
Tus ntsuas xim av tau txiav txim siab raws li txoj kev ntawm Meidav ​​​​et^{lwm yam.29}. Ib qho kua txiv 10-ml raug centrifuged ntawm 2750 xg rau 10 feeb. 5 ml ntawm cov kua txiv supernatant tau sib xyaw nrog 5 ml ntawm 95% ethanol. Qhov absorbance ntawm qhov sib tov tau ntsuas ntawm 420 nm siv Shimadzu UV spectrophotometer (UV-1601 PC).
Tag nrho cov phenolic cov ntsiab lus (TPC) tau txiav txim siab siv cov xim siv cov Folin-Ciocalteu reagent raws li tau piav qhia los ntawm Boulton et al.^[27]]. Ib qho nkhaus txheem ntawm gallic acid tau tsim rau cov concentration ntawm 0 txog 500 mg / L (^r²= 0.997). Cov txiaj ntsig tau qhia ua gallic acid sib npaug (mg GAE/mL).
Ntxiv 125 μL ntawm dej distilled thiab 2850 μL ntawm cov kua FRAP rau 25 μL ntawm kua txiv apple thiab cia cov khoom sib tov nyob hauv qhov tsaus ntuj rau³⁰min. Tom qab ntawd ntsuas qhov absorbance ntawm 593 nm siv Shimadzu UV spectrophotometer (UV-1601 PC). Lub FRAP reagent tau npaj los ntawm kev sib tov 300 mM acetate buffer (pH 3.6), 20 mM iron(III) chloride, thiab 10 mM 2,4,6-tris(2-pyridyl)triazine (TPTZ) (yaj hauv 40 mM HCl) hauv qhov sib piv ntawm 10:1:1. Ib qho nkhaus txheem tau tsim los ntawm kev siv Trolox ua tus qauv (^R²= 0.999), thiab cov txiaj ntsig tau qhia ua μM Trolox/mL.
Cov kua txiv hmab txiv ntoo uas tau kho thiab tsis tau kho tau txiav txim siab siv txoj kev DPPH los ntsuas lawv lub peev xwm los tshem tawm DPPH cov free radicals.³¹Kaum microliters ntawm cov kua txiv hmab txiv ntoo tau sib xyaw nrog 1 ml ntawm DPPH kua (100 μM) hauv methanol. Tom qab ua tshuaj hauv qhov tsaus ntuj rau 30 feeb, qhov nqus ntawm qhov sib tov tau ntsuas ntawm 517 nm siv Shimadzu UV spectrophotometer (UV-1601 PC). Cov txiaj ntsig tau qhia ua trolox sib npaug (μM trolox/ml) raws li kab calibration (_^R2= 0.990).
Cov ntaub ntawv tau txais qhia tau tias qhov siab tshaj plaws ntawm cov nceb oyster NRC 620 thaum kawg ntawm hnub 18 ntawm kev fermentation, ncav cuag kev ua haujlwm ntawm 1302 U / L. Qhov no tau ua lub hauv paus rau kev txiav txim siab lub sijhawm cog qoob loo zoo tshaj plaws rau kev tsim laccase (Daim Duab 1). Txawm hais tias kev tsim cov enzyme nce ntxiv nrog kev nce sijhawm cog qoob loo, qhov nrawm ntawm kev nce tsis yog qhov sib piv ncaj qha rau lub sijhawm cog qoob loo; tom qab 21 hnub, kev ua haujlwm enzyme tau nce tsuas yog 90 U / L (mus rau 1390 U / L). Yog li ntawd, 18 hnub tau raug xaiv thaum kawg ua lub sijhawm cog qoob loo zoo tshaj plaws los sib npaug cov khoom tsim tawm nrog cov txiaj ntsig kev lag luam ntawm kev nce sijhawm cog qoob loo.
Cov nyhuv ntawm lub sijhawm cog qoob loo rau laccase cov qoob loo hauv Pleurotus ostreatus NRC 620. Peb (12 mm) fungal mycelial blocks tau inoculated rau hauv 50 ml ntawm sterile medium thiab tom qab ntawd cultivated ntawm 28 ° C rau ntau lub sijhawm sib txawv.
Sib xws nrog lwm cov kev tshawb fawb, peb cov txiaj ntsig qhia tau hais tias lub sijhawm zoo tshaj plaws rau kev cog qoob loo kom ua tiav qhov siab tshaj plaws ntawm cov laccase secretion los ntawm cov fungi yuav yog ntawm 7 thiab 36 hnub.³²Raws li Ezike et al.³³, *Trametes polyzona* WRF03 tsim cov laccase ntau tshaj plaws thaum kawg ntawm hnub cuaj ntawm kev fermentation, nrog rau kev ua haujlwm tshwj xeeb ntawm 1637 U / mg protein. Ntxiv mus, Othman et al.³⁴pom tias *Trichoderma harzianum* S7113 tso tawm ntau cov laccase rau hnub thib tsib ntawm kev cog qoob loo. Qhov kev tsim cov laccase tau mus txog qhov siab tshaj plaws rau hnub thib kaum plaub thiab tom qab ntawd maj mam txo qis.³⁴Txawm hais tias kev tso tawm ntawm cov enzyme kuj tseem tuaj yeem tshwm sim thaum lub sijhawm loj hlob tseem ceeb, nws feem ntau nce siab tshaj plaws thaum lub sijhawm nruab nrab thiab yog qhov tshwm sim los ntawm kev noj cov pa roj carbon lossis nitrogen.^34,35
Txawm hais tias laccase los ntawm Pleurotus ostreatus NRC 620 tau ua haujlwm siab dhau ntawm qhov kub thiab txias ntawm 50 ° C txog 80 ° C, ze rau qhov ua haujlwm siab tshaj plaws (69–98%), nws qhov ua haujlwm siab tshaj plaws tau pom ntawm 70 ° C (Daim Duab 2a). Sab nraum qhov kub thiab txias no, qhov ua haujlwm ntawm enzyme txo qis ntawm kwv yees li 70 ° C. Cov txiaj ntsig no qhia tias cov enzyme ua haujlwm ntawm qhov kub thiab txias siab, tej zaum vim tias qhov kub thiab txias siab ua rau lub zog kinetic ntawm cov tshuaj tiv thaiv nce ntxiv.
Cov nyhuv ntawm qhov kub thiab txias ntawm cov tshuaj tiv thaiv (a) thiab pH (b) ntawm kev ua haujlwm ntawm laccase hauv *Pleurotus ostreatus* NRC 620. Qhov kub thiab txias txij li 20 txog 90 °C tau ua tiav los ntawm kev ua kom cov khoom sib xyaw ua ntej ntawm qhov kub thiab txias sib txawv rau 5 feeb ua ntej ntxiv cov enzyme thiab pib ua cov tshuaj tiv thaiv. Cov nyhuv ntawm pH ntawm kev ua haujlwm ntawm laccase tau soj ntsuam siv ABTS ua cov khoom siv hauv cov tshuaj uas muaj 0.1 M citrate-phosphate buffer hla pH ntau ntawm 2.5 txog 7.0.
Raws li Ezike thiab^al.33, qhov kub zoo tshaj plaws rau *Trametes polyzona* WRF03 laccase yog 55 °C, uas yog tib yam li qhov rau *Ganoderma lucidum*^laccase36thiab zoo ib yam li qhov kub zoo tshaj plaws (50 °C) rau *Trametes polyzona* KU-RNW02737^laccase . ^Baldrian38sau tseg tias, zoo li lwm cov kab ke enzyme uas rhuav tshem lignin, qhov kub zoo tshaj plaws rau laccase yog ntawm 50 thiab 70 ° C.
Cov txiaj ntsig qhia tau hais tias cov enzyme ua haujlwm siab tshaj plaws ntawm pH 3.0, ncav cuag 94% kev ua haujlwm ntawm pH 3.5. Txawm li cas los xij, nws tseem ua haujlwm ntawm ntau qhov pH txij li 2.5 txog 7.0 (Daim Duab 2b). Ntxiv mus, nws ua haujlwm siab dua hauv cov xwm txheej acidic piv rau cov xwm txheej nruab nrab lossis alkaline. Nws ua haujlwm tseem tsawg kawg 77% ntawm pH txij li 2.5 txog 4.5, tab sis tsuas yog ncav cuag kwv yees li 38% ntawm pH 7.0. Qhov pH zoo tshaj plaws rau laccase los ntawm *Trametes polyzona* WRF03 yog 4.533, uas yog tib yam li pH rau laccases los ntawm *Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, thiab *Trametes hirsuta* 41. Txawm li cas los xij, raws li kev tshawb fawb los ntawm Chairin et al.⁴², qhov pH zoo tshaj plaws rau laccase los ntawm *Polymorpha f. sp.* WR710-1 yog 2.2, thaum qhov pH zoo tshaj plaws rau laccase los ntawm *Polymorpha f. sp.* IBL-04 yog 5.043. Kev khi ntawm hydroxide anions (laccase inhibitor) rau cov tooj liab atoms ntawm T2/T3 laccase tej zaum yuav yog qhov ua rau laccase ua haujlwm tsawg dua nyob rau hauv cov xwm txheej pH nruab nrab lossis alkaline. Qhov no yuav cuam tshuam kev hloov pauv electron sab hauv los ntawm T1 chaw mus rau T2/T3 chaw, yog li ntawd^txwvqhov kev ua haujlwm ntawm cov enzyme23,44
Los ntawm kev ua kom cov enzyme nyob rau ntawm qhov kub sib txawv, nws pom tias ob qho tib si lub sijhawm ua kom lub cev thiab qhov kub thiab txias cuam tshuam rau qhov ruaj khov ntawm enzyme. Qhov tseem ceeb, laccase los ntawm *Trametes polyzona* NRC 620 tau pom tias muaj kev ruaj khov siab dua ntawm 40 ℃ thiab 50 ℃, khaws cia 68.33% thiab 59.61% ntawm nws cov haujlwm pib, raws li, tom qab 120 feeb (Daim Duab 3a). Qhov sib piv, nyob rau hauv tib qho xwm txheej (40 ℃ thiab 50 ℃, 120 feeb), laccase los ntawm *Trametes polyzona* WRF03 khaws cia 64.38% thiab 42.92% ntawm nws cov haujlwm, raws li.³³Ntawm qhov tsis sib xws, kev nce lub sijhawm thiab qhov kub thiab txias ua rau txo qhov ruaj khov ntawm *Trametes polyzona* NRC 620 laccase; Tom qab kev sib xyaw ntawm 60 ℃ thiab 70 ℃ rau 60 feeb, nws cov haujlwm txo qis mus rau 39.24% thiab 1.72%, raws li (Daim Duab 3a). Sib xws nrog cov txiaj ntsig ntawm kev sim, laccase los ntawm *Trametes polyzona* WRF03 tau qhia txog kev ruaj khov siab dua ntawm 40 thiab 50 ℃ thoob plaws hauv cov txheej txheem kho cua sov.³³Ib yam li ntawd, Lueangjaroenkit et^al.37thiab Chairin et^al.42tau tshaj tawm txog kev ruaj khov ntawm laccases los ntawm Trametes polyzona KURNW027 thiab Trametes polyzona WR710-1 ntawm 50 ° C rau 1 teev, raws li. Raws li ib qho biocatalyst muaj txiaj ntsig siv tau rau ntau yam biotechnological teb, laccase yuav tsum muaj kev ruaj khov zoo thiab kev ua tau zoo dhau ntawm ntau qhov kub thiab txias.
Kev ruaj khov ntawm thermostatic (a) thiab pH stability (b) ntawm laccase los ntawm *Pleurotus ostreatus* NRC 620. Kev ruaj khov ntawm thermostatic tau soj ntsuam los ntawm kev incubating cov kua enzyme hauv 0.05 M sodium phosphate buffer (pH 7.0) ntawm 40, 50, 60, thiab 70 °C rau 2 teev, raws li. Kev ruaj khov ntawm pH tau soj ntsuam los ntawm kev incubating cov kua enzyme hauv 0.1 M citrate buffer thiab Tris buffer (pH 3, 4, 6, thiab 7) ntawm 40 °C rau 2 teev. Kev ua haujlwm seem tau suav siv ABTS ua lub substrate tom qab incubation.
Txhawm rau txiav txim siab qhov zoo tshaj plaws rau kev siv thiab khaws cia enzyme, peb tau tshawb xyuas qhov cuam tshuam ntawm pH rau kev ruaj khov ntawm laccase. Kev raug rau cov nqi pH sib txawv cuam tshuam rau kev ruaj khov ntawm cov qauv protein, yog li cuam tshuam rau kev ruaj khov thiab kev ua haujlwm ntawm cov enzyme molecule. Cov txiaj ntsig tau qhia tias cov enzyme tsis ruaj khov nyob rau hauv cov xwm txheej acidic, thaum nws qhia tau tias muaj kev ruaj khov zoo dua ntawm cov nqi pH siab dua (cheeb tsam nruab nrab thiab alkaline). Ntawm cov nqi pH ntawm 7.0, 6.0, 4.0, thiab 3.0, cov nqi khaws cia enzyme tom qab 120 feeb yog kwv yees li 100%, 62.54%, 52.39%, thiab 11.14%, feem (Daim Duab 3b). *Strombus multisus* WRF03 laccase tau qhia txog kev ruaj khov siab dua ntawm cov nqi pH nruab nrab (5.5–6.5) thiab kev ruaj khov qis dua ntawm cov nqi pH acidic (qis dua 4.0). Tom qab 120 feeb ntawm pH tus nqi ntawm 5.5, 6.0, thiab 6.5, cov nqi khaws cia enzyme yog kwv yees li 82%, 100%, thiab 93%, raws li.³³Khairin thiab lwm tus.⁴²tau sau tseg tias laccase los ntawm Trametes polyzona WR710-1 ruaj khov hauv pH ntau ntawm 6.0 txog 7.0, thaum Sayed et al.⁴⁵qhia tau tias laccase ruaj khov dua nyob rau hauv cov xwm txheej pH nruab nrab. Txawm li cas los xij, laccase los ntawm Cerrena unicolor kuj tau pom tias muaj kev ruaj khov nyob rau hauv cov xwm txheej alkaline (pH 9.0).⁴⁶Cov laccases uas tau kawm tau qhia tias muaj kev ruaj khov siab dhau ntawm ntau yam pH. Qhov no yuav yog ib qho tseem ceeb rau kev siv hauv kev lag luam.
Vim tias qee cov hlau ions muaj ob qho tib si stimulatory thiab inhibitory cuam tshuam rau kev ua haujlwm ntawm enzyme, lawv cov teebmeem rau kev ua haujlwm ntawm enzyme yuav tsum tau xav txog hauv kev siv hauv kev lag luam. Qhov no yog qhov tseem ceeb vim tias cov hlau ions yog cov pa phem ib puag ncig uas tuaj yeem cuam tshuam rau kev ruaj khov thiab kev tsim cov enzymes extracellular.⁴⁷Txhawm rau tshawb nrhiav cov teebmeem ntawm ntau cov hlau ions rau laccase los ntawm *Pleurotus ostreatus* NRC 620, peb tau ua cov kev sim sib xws. Raws li pom hauv Daim Duab 4, nyob ntawm hom hlau siv, kev nce qhov concentration ntawm cov hlau ion los ntawm 2.5 mM mus rau 10 mM cuam tshuam tsis zoo rau kev ua haujlwm ntawm enzyme. Piv txwv li,^Mg²⁺ , ^Co²⁺ , ^Zn²⁺, thiab^Cu²⁺tuaj yeem ua kom muaj zog thiab ua kom cov enzyme ua haujlwm zoo,^Na⁺ , ^Mn²⁺ , ^Ca²⁺, thiab^K⁺yuav tiv thaiv tau qhov kev ua haujlwm ntawm cov enzyme. Ntawm qhov concentration ntawm 10 mM, Cu²⁺ thiab Mg²⁺ ions yog cov activators muaj zog tshaj plaws ntawm laccase kev ua haujlwm los ntawm *Pleurotus ostreatus* NRC 620, muab qib kev ua haujlwm ntawm kwv yees li 34% thiab 20%, raws li. Txawm li cas los xij, ntawm qhov concentration ntawm 10 mM, Ca²⁺ ions yog cov inhibitor muaj zog tshaj plaws ntawm laccase, txo cov kev ua haujlwm ntawm enzyme los ntawm kwv yees li 60%.
Cov nyhuv ntawm cov hlau ions rau kev ua haujlwm ntawm Pleurotus ostreatus NRC 620 laccase. Cov laccase tau muab tso rau hauv sodium phosphate buffer (0.05 M, pH 7.0) rau 10 feeb uas muaj ntau yam hlau ions ntawm qhov concentration ntawm 2.5 mM thiab 10 mM. Tom qab ntawd qhov kev tshuaj tiv thaiv tau pib los ntawm kev ntxiv cov substrate (ABTS), tom qab ntawd qhov kev ua haujlwm sib piv tau ntsuas.
Peb cov txiaj ntsig zoo ib yam li lwm cov kws sau ntawv uas pom tias Mg²⁺ thiab Cu²⁺ ua rau cov haujlwm ntawm *Trametes polyzona* WRF03³ zoo dua. Castaño et al.⁴⁸ pom tias laccase los ntawm *Xylaria* sp. raug txhawb nqa qee qhov los ntawm cov ions tooj liab (Cu²⁺). Ntxiv mus, Foroutanfar et al.⁴⁹ thiab Si et al.⁵⁰ tau ua cov kev tshawb fawb zoo sib xws ntawm laccases los ntawm *Paraconiothyrium variabile* thiab *Trametes pubescens*, raws li. Hom II tooj liab-khi qhov chaw (T2) ntawm cov enzyme no tuaj yeem saturated nrog Cu²⁺ ntawm qhov concentration, uas tej zaum yuav piav qhia txog kev txhawb nqa ntawm laccase kev ua haujlwm ntawm Cu²⁺³⁹ ntau dua. Vim tias cov kab mob dawb rot laccases yog oxidases uas muaj ntau lub atoms tooj liab, cov teebmeem ntawm cov ions tooj liab rau kev ua haujlwm laccase yog ntau yam thiab ntau yam los ntawm kev txhawb nqa thiab kev txwv tsis pub mus rau qhov nruab nrab.⁵¹ Hauv kev sib piv, Zhou et al^{. [52]}tau tshaj tawm tias^Cu²⁺inhibited lub laccase ua haujlwm ntawm Taiwan subterranean termite (Odontotermes formosanus). Txawm li cas los xij, laccases ntawm Cerena sp. HYB07^[53]thiab Clitocybe maxima^[54]tsis raug cuam tshuam los ntawm cov ions tooj liab.
Qhov tshwj xeeb ntawm cov substrate tau sawv cev los ntawm nws cov kev teeb tsa kinetic (Km thiab Vmax); qhov muaj zog ntawm qhov kev sib txuas ntawm cov substrate rau cov enzyme, qhov qis dua tus nqi Km thiab qhov siab dua qhov tshwj xeeb ntawm cov substrate.^{3, 21, 55}Cov kev ntsuas kinetic (Km thiab Vmax) ntawm laccase los ntawm *Pleurotus ostreatus* NRC 620 tau txiav txim siab siv GraphPad Prism 6.0 software los ntawm kev kos duab Lineweaver-Burk plot (Daim Duab 5). Thaum siv ABTS ua lub substrate, cov txiaj ntsig yog 1.99 mM thiab 16217 μmol.^{feeb⁻¹ L⁻¹,}raws li. Elsayed et al.²¹tau tshaj tawm tias cov nqi Km rau ABTS oxidation yog 0.1 mM thiab 0.064 mM, feem, qhia txog qhov siab ntawm Lac A thiab Lac B isoenzymes rau ABTS. Ntxiv mus, cov nqi Vmax yog 0.182 μmol^feeb⁻¹thiab 0.603 μmol^feeb⁻¹, raws li. Tus nqi Km tau txais qis dua li ntawm Trametes polyzona WRF03 (8.66 mM); ntxiv rau, lawv tus nqi Vmax (1429 mmol min⁻¹) kuj yog^{qis dua}thaum siv ABTS ua ib qho substrate.33 Ib yam li ntawd, cov nqi Km ntawm Lentinus squarrosulus MR13 thiab Trametes sp. AH28-2 laccase concentrations yog 0.0714 mM thiab 0.025 mM, feem, thiab cov nqi Vmax yog 0.0091 mM min−1 thiab 0.67 mM min−1 mg−1 (piv rau ABTS)^{, feem.56,57}
Qhov cuam tshuam ntawm ABTS concentration rau kev ua haujlwm ntawm laccase los ntawm *Pleurotus ostreatus* NRC 620 tau tshawb nrhiav, thiab ib daim duab Lineweaver-Burk ntawm qhov sib piv ntawm qhov ceev ntawm qhov tshuaj tiv thaiv thawj zaug piv rau ABTS concentration tau plotted. Qhov tshuaj tiv thaiv oxidation ntawm ABTS nrog cov concentration sib txawv (0.025–3.0 mM) ntawm laccase tau ntsuas ntawm pH 4.5 los txiav txim siab cov kev teeb tsa kinetic (Vmax thiab Km). Cov Michaelis-Menten kinetic constants tau suav los ntawm kev siv Lineweaver-Burk plotting ntawm qhov sib piv ntawm qhov ceev ntawm qhov tshuaj tiv thaiv piv rau qhov concentration ntawm substrate. Cov kinetic constants tau suav los ntawm Lineweaver-Burk plotting siv GraphPad Prism 6.01 software.
Cov enzymes ua kom pom tseeb ib txwm muaj, xws li pectinases, hydrolyze cov tshuaj pectic, txo qhov viscosity thiab turbidity. Lawv ua tau zoo rhuav tshem cov polysaccharides thiab feem ntau siv ua ke nrog lwm cov enzymes, xws li cellulases thiab hemicellulases, los txhim kho cov txiaj ntsig thiab kev pom tseeb. Txawm li cas los xij, pectinases tsis tsom mus rau cov tshuaj phenolic, uas yog cov ua rau muaj turbidity thiab oxidative browning, tshwj xeeb tshaj yog nyob rau hauv cov kua txiv xws li kua txiv apple thiab kua txiv hmab.⁵⁸Qhov sib txawv, laccases catalyze qhov oxidation ntawm phenolic compounds, polymerizing lawv mus rau hauv cov loj dua, insoluble molecules uas tuaj yeem tshem tawm los ntawm sedimentation lossis filtration. Lub tshuab no tsis yog tsuas yog txhim kho qhov meej xwb tab sis kuj ntev lub txee lub neej ntawm cov kua txiv los ntawm kev txo qhov tshwm sim ntawm oxidative browning los ntawm phenolic compounds. Ntxiv mus, cov txheej txheem clarification raws li laccase tuaj yeem ua tiav nyob rau hauv cov xwm txheej ua tiav me me (pH 3.5–5.5, kub 25–40 ° C), ua rau lawv haum rau cov kua txiv hmab txiv ntoo tsis muaj kev cuam tshuam rau lawv cov khoom noj khoom haus lossis organoleptic.⁵⁹Cov kev tshawb fawb tau qhia tias kev kho pectinase tuaj yeem ua rau cov kua txiv ntshiab hauv 1-2 teev, thaum kev kho laccase feem ntau xav tau lub sijhawm teb ntev dua (3-6 teev) kom txo cov tshuaj phenolic tag nrho. Txawm li cas los xij, cov txheej txheem no tuaj yeem ua kom zoo tshaj plaws los ntawm kev ua kom cov enzyme tsis txav lossis los ntawm kev sib xyaw laccase nrog cov txheej txheem ua kom huv.⁶⁰Hauv txoj kev tshawb no, kev tshuaj xyuas cov enzyme ntawm cov kua txiv hmab txiv ntoo nyoos qhia tau tias muaj cov haujlwm tseem ceeb ntawm laccase thiab α-amylase, thaum cov haujlwm ntawm pectinase thiab xylanase qis heev, thiab cov haujlwm ntawm cellulase tsis pom. Yog li ntawd, qhov txo qis ntawm qhov turbidity thiab cov ntsiab lus phenolic feem ntau yog vim qhov kev ua ntawm laccase, thaum qhov kev hloov pauv ntawm viscosity tuaj yeem yog ib feem vim qhov kev ua ntawm amylase.
Rooj 1 qhia txog cov yam ntxwv ntawm cov kua txiv apple tshiab uas tau muab rho tawm thiab cov qauv uas tau kho nrog laccase. Cov txiaj ntsig tau qhia tias cov kua txiv apple tshiab uas tau muab rho tawm (71.59%) qis dua li cov qauv uas tau kho nrog laccase (87.34%). Cov txiaj ntsig no sib xws nrog cov kev tshawb pom ntawm Pilnik thiab Orange.⁶¹, uas tau qhia tias kev siv cov enzymes hauv kev ua cov txiv hmab txiv ntoo tuaj yeem ua rau cov kua txiv ntau ntxiv, txhim kho kev lim dej, thiab tau txais cov kua txiv zoo, ntshiab rau kev sib xyaw. Qhov nce ntawm cov kua txiv ntau ntxiv yog vim muaj kev nce ntxiv ntawm cov suab thaj uas yaj tau hauv cov kua txiv. Thaum lub sijhawm enzymatic hydrolysis ntawm cov txiv hmab txiv ntoo, mesoglea thiab pectin hauv cov phab ntsa cell ntawm cov khoom raug rhuav tshem thiab hloov mus ua cov tshuaj yaj tau xws li cov suab thaj tsis muaj zog thiab cov kua qaub.^62.Tus nqi pH ntawm cov kua txiv apple uas tau kho nrog enzyme qis dua li ntawm pawg tswj (P < 0.05), thiab tus nqi pH ntawm ob pawg tau nce ntxiv ntau thaum lub sijhawm khaws cia (Rooj 1). Cov txiaj ntsig no sib xws nrog cov txiaj ntsig ntawm Mark et al.⁶³, uas tau sau tseg tias pH ntawm cov kua txiv cashew txo qis tom qab khaws cia tom qab kho cua sov. Pectin degradation thiab galacturonic acid tsim tom qab kho enzyme tej zaum yuav yog lub luag haujlwm rau qhov nce hauv pH thaum khaws cia. Lub pH ntawm cov qauv kho enzyme tseem nyob nruab nrab ntawm 4.05 thiab 4.31 thoob plaws hauv kev khaws cia, thaum lub pH ntawm cov kua txiv apple tsis kho nyob nruab nrab ntawm 4.12 thiab 4.33.
Tag nrho cov kua qaub (TA) ntawm cov qauv tsis tau kho thiab cov qauv kho laccase tau pom tias muaj kev hloov pauv tsawg zuj zus nrog lub sijhawm khaws cia ntev dua (Rooj 1). Qhov kev txo qis ntawm cov kua qaub yog vim muaj kev hloov pauv ntawm cov organic acids mus rau cov carbohydrates lossis cov tshuaj enzymatic, nrog rau kev oxidation thaum lub sijhawm khaws cia kua txiv.⁶⁴Tag nrho cov kua qaub ntawm cov kua txiv apple tswj thiab cov qauv kho enzyme qis dua li lwm cov kua txiv (kua txiv pos nphuab 0.9%, kua txiv plum 2.2%, kua txiv kumquat 1.0%, kua txiv apricot 2.4%, kua txiv kab ntxwv 0.8%), tab sis zoo ib yam li lwm cov kua txiv (piv txwv li, kua txiv pear 0.3%).⁶²Cov qhov sib txawv ntawm cov kua txiv apple uas tsis tau kho tshiab no tej zaum yuav yog vim muaj ntau yam xws li tej yam kev loj hlob, cov yam ntxwv ntawm caj ces, qib kev loj hlob, thiab cov txheej txheem ua.⁶⁵Qhov txo qis ntawm tag nrho cov acidity ntawm kev tswj hwm thiab kua txiv apple kho laccase yog sib xws nrog cov txiaj ntsig los ntawm Singh et al.⁶⁶txog qhov txo qis ntawm tag nrho cov kua qaub ntawm Jin Nuo kua txiv tom qab 74 hnub ntawm kev khaws cia. Ntawm qhov tod tes, Oshmiansky thiab Wojdylo⁶⁷tsis pom muaj kev hloov pauv tseem ceeb hauv qhov acidity ntawm kua txiv apple thaum kawm txog cov txiaj ntsig ntawm cov txheej txheem ua kom pom tseeb ib txwm muaj.
Cov txiaj ntsig tau muab tso rau hauv Rooj 1 qhia tau tias cov khoom khov uas yaj tau tag nrho (TSS) ntawm cov kua txiv apple uas kho nrog laccase siab dua li cov qauv uas tsis kho. Cov txiaj ntsig no sib xws nrog cov kev tshawb fawb luam tawm^{. 68}Ntxiv mus, Rooj 1 qhia tau hais tias tus nqi TSS ntawm pawg kua txiv apple tswj yog 9.58 ntawm lub sijhawm pib thiab mus txog 11.05 thaum kawg ntawm lub sijhawm khaws cia. Cov nqi no qis dua tus nqi TSS ntawm cov kua txiv apple tshiab uas Hamid et al tau tshaj tawm.^{. 69}(11.2 thiab 11.80, raws li). Tus nqi TSS ntawm cov kua txiv apple uas kho nrog laccase tau nce ntxiv ntau heev, pib ntawm 11.23 thiab mus txog 12.93 tom qab ob lub lis piam khaws cia ntawm 4°C (Rooj 1). Kev nce zoo sib xws hauv TSS thaum lub sijhawm khaws cia kuj tau pom hauv cov txiv hmab txiv ntoo citrus, txiv qaub, thiab txiv kab ntxwv qab zib. Kev nce hauv cov khoom khov tag nrho (TSS) thaum lub sijhawm khaws cia tej zaum yuav yog vim hydrolysis ntawm polysaccharides (starch) rau monosaccharides (qab zib), kev nce hauv concentration vim kua txiv qhuav, thiab kev puas tsuaj ntawm pectin hauv cov kua txiv rau cov khoom khov. Kev nce hauv cov khoom khov tag nrho (TSS) tej zaum yuav yog vim muaj kev nce hauv cov suab thaj soluble, uas tej zaum yuav tsim los ntawm kev hloov pauv ntawm pectin lossis cellulose rau cov suab thaj soluble los ntawm pectin lossis cellulase, raws li, lossis los ntawm hydrolysis ntawm starch rau qab zib, raws li Hamed et al tau tshaj tawm.^69.Qhov cuam tshuam ntawm laccase rau cov yam ntxwv ntawm cov kua txiv apple tuaj yeem pom tau los ntawm kev pom, vim tias cov kua txiv apple uas kho laccase muaj qhov ntws zoo dua thiab viscosity qis dua li cov kua txiv uas tsis kho. Qhov kev soj ntsuam no tau sau tseg hauv Rooj 1; Qhov viscosity ntawm cov qauv kho enzyme yog 1.87 cP, thaum qhov viscosity ntawm cov qauv tswj yog 2.95 cP. Qhov kev txo qis tseem ceeb ntawm viscosity no yuav yog vim muaj peev xwm tuav dej ntau dua ntawm cov tshuaj pectin zoo li thiab kev tsim cov qauv sib koom ua ke.
Hauv kev tshawb fawb no, qhov cuam tshuam ntawm laccase rau qhov ntsuas xim av (BI) ntawm cov kua txiv apple tau tshawb xyuas los ntawm kev ntsuas qhov absorbance ntawm 420 nm siv lub spectrophotometer. Cov txiaj ntsig tau pom hauv Rooj 1. Thaum lub sijhawm khaws cia, BI ntawm cov qauv kua txiv apple hauv ob pawg kho thiab tsis kho tau qhia txog qhov sib txawv maj mam nce ntxiv. BI qhia txog qib ntawm xim av thiab tuaj yeem ua haujlwm ua^{ib qho tseem ceeb}qhia txog kev ua kom xim av enzymatic thiab tsis yog enzymatic. Qhov absorbance nce ntxiv ntau thaum lub sijhawm khaws cia (P < 0.05). Thaum kawg ntawm kev khaws cia, qhov_A420tus nqi ntawm cov kua txiv apple hauv cov pab pawg tswj thiab cov pab pawg kho enzyme tau nce ntxiv txog li 217% thiab 121%, raws li (Rooj 1). Cov txiaj ntsig qhia tau tias kev kho enzyme tuaj yeem txo qhov degree browning los ntawm kwv yees li 56%. Cov txiaj ntsig ntawm Bezerra et al.^[19]] yog sib xws nrog peb cov txiaj ntsig; Lawv siv cov laccase-glutaraldehyde-txiv maj phaub fiber los ua kom cov kua txiv apple meej, txo nws cov xim qub los ntawm 61%.
Txawm hais tias cov polyphenols hauv cov kua txiv hmab txiv ntoo muaj cov txiaj ntsig zoo rau kev noj qab haus huv thiab kev kho mob rau tib neeg lub cev, lawv kuj tuaj yeem ua rau cov protein ua rau cov kua txiv pos nphuab, ua rau cov av noo, lossis ua rau cov kua txiv tsis huv, ua rau cov saj thiab aroma ntawm cov khoom hloov pauv thiab txo nws lub sijhawm khaws cia.⁷¹Lub hom phiaj ntawm txoj kev tshawb no yog kom txo cov phenolic compound hauv cov kua txiv apple siv laccase los ntawm Pleurotus ostreatus NRC 620 kom muaj kev nyab xeeb. Cov txiaj ntsig tau nthuav tawm hauv Rooj 1 qhia tau tias tag nrho cov phenolic compound hauv cov kua txiv apple kho laccase tau txo qis ua ntej khaws cia ntawm 4 ° C. Ntxiv mus, tag nrho cov phenolic compound kuj tau txo qis thaum khaws cia hauv ob qho qauv uas tau kawm (Rooj 1). Kev tshawb fawb los ntawm Sandri et al.⁷²qhia tau hais tias cov kua txiv apple uas tau kho nrog enzyme tuaj yeem khaws nws cov kev ua haujlwm antioxidant thiab cov ntsiab lus phenolic compound. Txawm li cas los xij, cov txiaj ntsig ntawm kev tshawb fawb los ntawm Lettera et al.⁷³qhia tias kev kho cov kua txiv kab ntxwv nrog cov fungal laccase tuaj yeem txo cov ntsiab lus ntawm cov tshuaj phenolic hauv nws txog li 45%.
Cov tshuaj phenolic tau pom tias muaj cov khoom xws li kev tshem tawm cov dawb radicals, kev txo qis oxygen thiab quenching, kev hloov pauv hydrogen atom, thiab kev pub electron rau cov dawb radicals, ua rau lawv muaj zog antioxidants.⁷⁴Yog li ntawd, hauv txoj kev tshawb fawb no, DPPH thiab FRAP-raws li cov txheej txheem tau siv los ntsuas qhov cuam tshuam ntawm laccase rau kev ua haujlwm antioxidant ntawm cov kua txiv apple khaws cia rau hauv lub tub yees rau 14 hnub (Rooj 2). Ob txoj kev tau qhia txog kev nce ntxiv ntawm kev ua haujlwm antioxidant thaum lub sijhawm khaws cia, uas tej zaum yuav yog vim muaj kev nce ntxiv ntawm cov tshuaj phenolic dawb lossis kev tsim cov khoom lag luam Maillard reaction (MRPs), nrog rau cov khoom lag luam Maillard reaction yuav yog qhov ua rau muaj kev nce ntxiv ntawm kev ua haujlwm antioxidant.⁷⁵Cov tshuaj tiv thaiv xim av uas tsis yog enzymatic (suav nrog kev puas tsuaj ascorbic acid, Maillard reactions, thiab acid-catalyzed degradation ntawm cov suab thaj) tsim cov xim av (melanoidins). Cov khoom siv puas tsuaj ascorbic acid nruab nrab thiab cov khoom siv puas tsuaj qab zib (xws li carbonyl compounds) tuaj yeem cuam tshuam nrog amino acids los ntawm Maillard reactions.⁷⁶Txawm hais tias kev ua xim av ntawm cov txiv hmab txiv ntoo thiab zaub thaum lub sijhawm khaws cia tau raug kawm ntau yam, peb txoj kev nkag siab txog cov kev tshuaj tiv thaiv no tseem tsawg.⁷⁷Piv nrog rau txoj kev FRAP, kua txiv apple uas kho nrog laccase tau qhia tias muaj kev ua haujlwm antioxidant qis dua los ntawm txoj kev DPPH (Rooj 2), thiab kev ua haujlwm antioxidant ntawm txhua tus qauv tau nce ntxiv ntau nrog rau lub sijhawm khaws cia ntev dua. Ob txoj kev sib txawv rau kev txiav txim siab txog kev ua haujlwm antioxidant tau siv hauv kev tshawb fawb no vim tias lawv cov ntsiab cai sib txawv. Txoj kev DPPH ntsuas qhov muaj peev xwm los tshem tawm cov free radicals, thaum txoj kev FRAP ntsuas qhov muaj peev xwm los txo cov hlau ions. Yog li ntawd, nws raug pom zoo kom siv ntau txoj hauv kev los txiav txim siab txog kev ua haujlwm antioxidant kom nkag siab zoo dua txog kev ua haujlwm antioxidant ntawm cov qauv uas tau kawm.⁷⁸
Ib qho ntawm cov ntsiab lus tseem ceeb ntawm txoj kev tshawb no yog tias *Pleurotus ostreatus* laccase NRC 620 ua haujlwm zoo tshaj plaws ntawm 70 ° C thiab pH 3.0. Piv nrog rau lwm cov fungal laccases uas feem ntau siv rau kev ua kom cov kua txiv ntshiab, xws li *Trametes versicolor* thiab *Ganoderma lucidum* laccases, *P. ostreatus* NRC 620 ua haujlwm zoo dua thiab muaj pH acidic ntau dua. Laccases los ntawm *Trametes versicolor* thiab *Ganoderma lucidum* feem ntau ua haujlwm zoo tshaj plaws hauv qhov ntau ntawm 50-60 ° C thiab ntawm pH tus nqi ntawm 3.5 thiab 5.0. Qhov sib txawv no yuav pab txhawb rau kev ua haujlwm zoo dua ntawm kev ua kom cov kua txiv ntshiab, tshwj xeeb tshaj yog rau cov kua txiv acidic uas kev ruaj khov ntawm pH qis dua yog qhov tseem ceeb. Lub yam ntxwv tshwj xeeb ntawm *P. Piv nrog rau lwm cov fungal laccases uas tau kawm, *Pleurotus ostreatus* NRC 620 ua haujlwm tau zoo hauv qab cov xwm txheej nyuaj dua. Nws qhov kub siab dua ntawm kev ua haujlwm zoo qhia txog qhov zoo ntawm kev siv hauv kev lag luam, xws li kev ua haujlwm sai dua thiab txo cov kab mob me me. Nws cov pH qis, uas haum rau qhov acidic ntawm ntau cov kua txiv hmab txiv ntoo, tej zaum yuav pab tau rau cov txheej txheem ua kom cov kua txiv hmab txiv ntoo huv. Cov txiaj ntsig no ua pov thawj ntxiv rau kev tshawb nrhiav rau kev siv loj, ua rau *Pleurotus ostreatus* NRC 620 yog lwm txoj hauv kev zoo rau cov tshuaj fungal laccase ib txwm muaj. Piv rau cov kev tshawb fawb yav dhau los, peb pom tias qhov kub zoo tshaj plaws yog 60 ° C thiab pH zoo tshaj plaws yog 3.0. Tom qab ua haujlwm ntawm 60 ° C rau 80 feeb, *Ganoderma lucidum* laccase khaws cia.⁴⁶% ntawm nws cov haujlwm.79 Raws li Kurniawati thiab Nicelle⁸⁰, *Ganoderma lucidum* enzymes qhia tau zoo heev rau qhov ruaj khov nruab nrab ntawm 25 ° C thiab pH tus nqi xws li 5.0 txog 8.0, thiab ruaj khov ntawm pH 6.0 thiab kub xws li 10 txog 30 ° C. Hauv kev tshawb fawb no, peb pom tias pH thiab kub zoo tshaj plaws rau kev ua haujlwm enzyme rau *Pleurotus ostreatus* yog 3.0 thiab 70 ° C, raws li. Tom qab incubation ntawm 40 ° C thiab 50 ° C rau ob teev, cov enzyme khaws cia 68.33% thiab 59.61% ntawm nws cov haujlwm, raws li. Ntxiv mus, Pleurotus ostreatus NRC 620 laccase tau qhia txog kev ua haujlwm siab dhau ntawm qhov kub thiab txias dav dav ntawm 50 ° C txog 80 ° C, yuav luag ncav cuag qhov siab tshaj plaws (69% -98%), nrog rau qhov siab tshaj plaws pom ntawm 70 ° C.
Xaus lus, oyster mushroom laccase NRC620, tau txais nyob rau hauv cov xwm txheej zoo li qub, tau qhia txog kev ua haujlwm zoo tshaj plaws thiab kev ruaj khov thoob plaws ntau yam pH thiab qhov kub thiab txias, qhia txog kev ruaj khov zoo dua piv rau lwm cov enzyme qhov chaw. Kev ntxiv 10 mM MgSO₄ thiab CuSO₄ ua rau muaj kev ua haujlwm ntawm enzyme ntau ntxiv kwv yees li 21% thiab 35%, raws li. Thaum ua tiav rau hauv kua txiv apple, cov enzyme txo qis pH thiab viscosity, thaum cov ntsiab lus phenolic txo qis me ntsis thaum lub sijhawm khaws cia.
Cov txiaj ntsig tau lees paub qhov muaj peev xwm ntawm laccase hauv kev lag luam khoom noj, tshwj xeeb tshaj yog hauv kev ua kom dej haus huv. Los ntawm kev rhuav tshem cov tshuaj phenolic, laccase tsis tsuas yog txo qhov turbidity thiab txhim kho qhov meej xwb tab sis kuj tswj tau qhov zoo ntawm cov kua txiv hmab txiv ntoo hauv qab kev ua haujlwm tsis muaj zog. Tsis zoo li cov neeg sawv cev ua kom huv ib txwm muaj xws li gelatin, bentonite, thiab silica gel, laccase tsis tsim cov khib nyiab lossis tshem tawm cov ntxhiab tsw qab ntawm cov dej haus, ua rau nws yog ib qho kev xaiv zoo dua rau ib puag ncig thiab ruaj khov. Ntxiv mus, piv rau lwm cov enzymes thiab cov txheej txheem lim dej, laccase muab cov kev daws teeb meem uas tsom mus rau thiab pheej yig yam tsis muaj kev cuam tshuam rau qhov zoo ntawm cov khoom.
Kyomuhimbo, HD thiab Brink, HG. Cov ntawv thov thiab cov tswv yim immobilization ntawm tooj liab-muaj laccases; kev tshuaj xyuas. Heliyon 9, e13156 (2023).