Ngemva kwezinsuku ezingu-25 zokufukamela okungaguquki ku-28°C, i-laccase evela ku-*Pleurotus ostreatus* NRC620 ibonise umsebenzi ophezulu kakhulu endaweni yokukhulisa isikhunta. Amanani e-pH afanele kanye namazinga okushisa ale enzyme ayengu-3.0 no-70°C, ngokulandelana. Ngemva kwamahora ama-2 okufukamela ku-40°C kanye no-50°C, umsebenzi we-enzyme ugcine u-68.33% kanye no-59.61%, ngokulandelana. Ngemva kwamahora ama-2 okufukamela ku-citrate-phosphate buffer (pH 7.0), umsebenzi we-enzyme uhlale ku-100%. Ukwengezwa kwe-10 mM MgSO₄ kanye ne-CuSO₄ kwandise umsebenzi we-enzyme cishe ngo-21% kanye no-35%, ngokulandelana, kuyilapho i-NaCl, i-MnCl₂, i-KCl, kanye ne-CaCl₂ kuvimbele umsebenzi we-enzyme. Kusetshenziswa i-ABTS njenge-substrate, amapharamitha e-kinetic (Km kanye ne-Vmax) ye-*Pleurotus ostreatus* NRC 620 laccase ayengu-1.99 mM kanye no-16,217 μmol min−1 L−1, ngokulandelana. Ukwelashwa nge-enzyme kwamasampula ejusi le-apula kunciphisa kakhulu i-pH kanye ne-viscosity, futhi lokhu kuncipha kuhlobene nokwanda kwesikhathi sokugcina. Ukwelashwa nge-laccase kuholele ekwehleni okuncane kokuqukethwe kwe-phenolic okuphelele kwejusi le-apula, kodwa akukho kwehla komsebenzi we-antioxidant okubonwe.
Eminyakeni yamuva nje, abacwaningi bagxile ekusetshenzisweni kwe-biotechnology eluhlaza embonini yokudla. I-Laccase ingenye yama-enzyme awusizo kakhulu embonini yokudla, ithola ukusetshenziswa ezindaweni ezifana nokucubungula ujusi, ukubhaka, ukuzinzisa iwayini, kanye nokuthuthukisa izimfanelo ze-organoleptic zemikhiqizo yokudla.1Izitshalo eziningi eziphakeme kanye nama-microorganisms akhipha i-laccase,2futhi isikhunta esifana ne-deuteromycetes, i-ascomycetes, kanye ne-basidiomycetes singakhiqiza ne-laccase.3I-Laccase (EC 1.10.3.2) iyi-oxidase eluhlaza okwesibhakabhaka enciphisa i-oxygen yama-molecule ibe ngamanzi isebenzisa uhlelo oluqukethe ama-athomu amathathu ahlukene ethusi, ngaleyo ndlela i-oxidize ama-phenolic compounds ahlukahlukene kanye nama-aromatic amines. Ngesikhathi sokukhiqizwa kwamajusi ezithelo nemifino, ukuba nsundu kwe-enzymatic kanye nokungekona kwe-enzymatic kuyizinkinga ezibalulekile.4Njengoba lezi zinto zithinta kabi umbala, ukunambitheka, kanye nephunga lejusi, kumele zisuswe.5
Kuzo zonke izithelo, ama-apula yiwona adliwa kakhulu emhlabeni jikelele nase-European Union. Ngo-2019, ukukhiqizwa kwama-apula kwabekwa endaweni yesithathu emhlabeni jikelele, kudlula amathani ayizigidi ezingu-87.6Ama-apula aqukethe ama-phenolic compounds amaningi, okuhlanganisa ama-flavonoid nama-phenolic acid njenge-caffeic acid kanye ne-chlorogenic acid.7Ngenxa yokuthi ijusi lama-apula livame ukudliwa lisesimweni salo esicacile, cishe ama-50% kuya ku-90% ezingxenye ze-phenolic ziyalahleka ngesikhathi senqubo yokuhlunga.8Namuhla, abathengi bavame ukukhetha imikhiqizo engacutshungulwanga kahle, njengejusi lama-apula elinamafu elinokuqukethwe okuphezulu kwe-polyphenol. Kodwa-ke, ngenxa yokuqukethwe okuphezulu kwe-phenolic, lolu hlobo lwejusi lama-apula lusengozini enkulu yokushintsha umbala kanye nokuba mnyama.9Ubuchwepheshe obuhlukahlukene, okuhlanganisa nezindlela zokwelapha ukushisa ezifana nokufaka i-pasteurization ku-60–90°C, kusetshenziswa ukunciphisa noma ukuvimbela ukufiphala kwejusi lama-apula.10Nokho, ngokocwaningo lukaSauceda-Gálvez11, ukucubungula ukushisa kungabhubhisa amakhemikhali ashintshashintshayo futhi kuthinte izimfanelo ze-organoleptic zejusi le-apula. Ezinye izindlela zokucubungula ukushisa zifaka phakathi i-supercritical carbon dioxide, imisebe ye-ultraviolet, i-ultrasound, ingcindezi ephezulu ye-hydrostatic, noma i-homogenization ephezulu yengcindezi.12Ukusebenza kahle kwalezi zobuchwepheshe kanye nokukhiqiza amajusi ezithelo afanele kuncike kumingcele esetshenzisiwe kanye nezici zomkhiqizo. Ukusetshenziswa kwazo kabanzi kunqunyelwe yizindleko eziphakeme, imiphumela emibi ekhwalithini yemikhiqizo ethile yokudla, noma ukungasebenzi kahle kwama-enzyme.13,14
I-Laccase ingasetshenziswa ukuqinisa nokuhlanza ijusi yezithelo.15UGökmen nabanye.16batusa ukusetshenziswa kwe-laccase ukuze kucaciswe ijusi lezithelo ngoba isusa ngempumelelo ama-phenolic compounds ngokuwaguqula abe ama-polymers noma ama-oligomers asuswa kalula yinoma yiluphi ulwelwesi lwe-ultrafiltration, okuvumela ijusi le-apula ukuthi ligcine umbala ozinzile nokucaca kuze kube amasonto ayisithupha ku-50°C. I-laccase ehlanziwe *Trichoderma* yavinjwa kuma-alumina beads futhi yasetshenziswa ukususa ngokukhethekile ama-compounds anganambitheki abangelwa ukungcoliswa kwamagciwane ejusi le-apula.17
Cishe u-80-90% wezingxenye eziguquguqukayo zejusi lama-apula yi-esters kanye ne-aldehydes, okunikeza iphunga eliyingqayizivele kujusi.18I-Laccase evela ku-*Trametes versicolor* yavinjwa ukusekela okungabizi okwenziwe ngefayibha yemvelo evela kumagobolondo kakhukhunathi amancane ukuze kucaciswe ujusi wama-apula.19Izifundo zangaphambilini zihlole ukuzinza kwejusi lama-apula (umbala kanye nokugcwala) kusetshenziswa izindlela ezingenama-enzyme noma izindlela zokuvimbela ukuhamba, noma kuhlanganiswe ne-ultrafiltration.5,19Kodwa-ke, umphumela wama-laccase esikhunta ezimpahleni ze-physicochemical zejusi lama-apula ngesikhathi sokugcina awukacaci. Ngakho-ke, inhloso yalolu cwaningo kwakuwukuhlola ngokuhlola izinguquko ezimpahleni ze-physicochemical, okuqukethwe kwe-phenolic compound, kanye nomsebenzi we-antioxidant wejusi lama-apula ngemva kokwelashwa ngama-laccase esikhunta kanye nokugcinwa esiqandisini kwamasonto amabili. Ama-laccase anekhono lokuxubha amakhemikhali e-phenolic, okwenza athembise ukusetshenziswa ezinqubweni ezahlukene zezimboni, okuhlanganisa nokucaciswa kwejusi. Lolu cwaningo luhlole ama-laccase avela ku-*Pleurotus ostreatus* NRC 620, egxile ezimweni ezifanele zomsebenzi wawo kanye nokusebenza kahle ekucacisweni kwejusi. Ngenkathi ucwaningo ngamakhowe e-oyster (P. ostreatus NRC 620) lusalinganiselwe, izifundo zangaphambilini zihlole ama-enzyme avela emithonjeni eyahlukene yesikhunta, njenge-Trametes versicolor kanye ne-Ganoderma lucidum. Inhloso yalolu cwaningo kwakuwukuhlola ukusetshenziswa okungenzeka kwale enzyme embonini yokudla nokugqamisa izakhiwo zayo ezihlukile, ikakhulukazi i-pH yayo kanye nokushisa kwayo okuhle.
I-2,2′-Azooxybis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) ithengwe kwaSigma-Aldrich (Canada). Zonke ezinye izinto zokuhlaziya zazisezingeni lokuhlaziya.
Isikhungo Sokuqoqwa Kwama-Microbial Culture seNational Research Center sithole uhlobo olwaziwayo lwamakhowe e-oyster i-NRC620. Ngemva kokutshala i-subculture, lolu hlobo lwagcinwa ezitshalweni ze-potato dextrose agar ku-4°C. Indlela yokulungiselela i-inoculum yayilandelayo: i-mycelium eneminyaka eyi-10 ubudala, ekhule ngokugcwele yafakwa emapuletini e-potato dextrose agar futhi yafakwa kuma-28°C. Ngemva kwezinsuku eziyi-10, amabhlogo amathathu e-mycelial angu-12 mm ububanzi asuswa ku-agar media kusetshenziswa i-sterile metal punch futhi afakwa kuma-flasks e-Erlenmeyer angu-250-mL anama-plugs kakotini aqukethe i-50 mL ye-sterilized culture medium (pH 5.0, njengoba kuchaziwe ngaphambilini ngu-Othman et al.20). Ama-culture afakwa e-incubator ku-28°C izinsuku ezingu-18. Ama-culture ahlungwa ngephepha lokuhlunga le-Whatman No. 1, futhi i-supernatant ephumayo yasebenza njengomthombo we-enzyme.
Umsebenzi we-laccase unqunywe kusetshenziswa i-ABTS njenge-substrate. Ingxube yokusabela (2 mL) iqukethe ama-500 μL e-0.3 mM ABTS (ancibilikiswe ku-0.1 M sodium citrate buffer, i-pH 4.5) kanye nenani elidingekayo lesampula ye-enzyme exutshwe ngamanzi acwengekile.21,22Uma sibheka ukuthi i-laccase ingakhipha i-ABTS ekushiseni kwegumbi (28 °C ± 2), ukuchitheka kwe-ABTS kunqunywe ngokulinganisa ukwanda kokumuncwa ku-420 nm (ε420= 36,000 cm-1 M -1) kusetshenziswa i-Agilent Carry-100 UV spectrophotometer. Kwakudingeka iyunithi eyodwa yomsebenzi we-laccase ukuze kufakwe i-oxidize i-1 μmol ABTS ngomzuzu. Ukuhlushwa kwamaprotheni kunqunywe ngendlela yaseBradford kusetshenziswa i-bovine serum albumin njengokulawula kwangaphakathi.23,24
Ngemva kokuthola i-enzyme kusuka kuhlobo lwe-oyster mushroom NRC 620, umsebenzi wayo walinganiswa ngezikhathi ezahlukene zokulima izinsuku ezingu-25 ngaphansi kwezimo ezimile ku-28 °C.
Ukuze kufundwe umphumela wokushisa emsebenzini we-laccase, kwenziwa izivivinyo ezingeni lokushisa elisukela ku-20 kuya ku-90 °C. Ngaphambi kokufaka i-enzyme nokuqala ukusabela, i-buffer (0.1 M sodium citrate, pH 4.5) kanye ne-substrate (ABTS) kwaxutshwa futhi kwafakwa e-incubator imizuzu emi-5 emazingeni okushisa ahlukahlukene. Ukuqina kokushisa kwe-enzyme kwahlolwa ngokufaka e-incubator ku-0.05 M sodium phosphate buffer (pH 7.0) ku-40, 50, 60, kanye no-70 °C amahora ama-2, ngokulandelana. Umsebenzi osele wabe usuhlolwa kusetshenziswa i-substrate ye-ABTS.
Umphumela we-pH emsebenzini we-laccase uhlolwe kusetshenziswa i-ABTS njenge-substrate kuma-buffer e-citrate-phosphate angu-0.1 M ane-pH engu-2.5 kuya ku-7.0. Isixazululo se-enzyme safakwa ku-40°C amahora amabili kuma-buffer e-citrate angu-0.1 M kanye ne-Tris (pH 3, 4, 6, kanye no-7) ukuze kuhlolwe ukuqina kwe-pH. Umsebenzi osele ne-ABTS njenge-substrate ubalwa ngemva kokufakwa ku-incubation.
I-laccase yafakwa e-incubator imizuzu eyi-10 ku-sodium phosphate buffer (0.05 M, pH 7.0) equkethe ama-ion ensimbi ahlukahlukene (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, kanye ne-Mn2+) ngamanani angu-2.5 mM kanye no-10 mM, ngokulandelana. I-substrate (ABTS) yabe isingezwa ukuze kuqalwe ukusabela, futhi umsebenzi ohlobene wahlolwa.
Ukushiswa kwe-ABTS yi-laccase ngamanani ahlukahlukene (0.025–3 mM) kulinganiswe ku-pH 4.5 ukuze kutholakale amapharamitha e-kinetic (Vmax kanye ne-Km).ama-constantkwe-Michaelis-Menten equation kubalwe kusetshenziswa i-Lineweaver-Burk plot, edweba isilinganiso sokusabela njengomsebenzi wokuhlushwa kwe-substrate. Ama-kinetic constant abalwe kusukela ku-Lineweaver-Burk plot kusetshenziswa isofthiwe ye-GraphPad Prism version 6.01.
Ngemva kokuwageza kahle ama-apula ngamanzi ompompi, anqunywa phakathi futhi afakwa ujusi kusetshenziswa i-jusi ye-apula ye-Braun MP80 ezenzakalelayo ngokuphelele (eyenziwe eJalimane). Ijusi lahlungwa ngezingqimba ezine ze-cheesecloth. Awekho ama-enzyme angeziwe eqenjini lokulawula, kuyilapho i-2.0% laccase (i-concentration ephumelela kakhulu evivinyiwe) yafakwa kujusi we-apula osanda kulungiswa, owabe usugcinwa ku-4°C amasonto amabili.
I-Titratable acidity (TA) kanye ne-pH kunqunywe ngokwendlela kaBoulton et.al.27. I-pH yesampula ngayinye ilinganiswe kusetshenziswa imitha ye-pH yedijithali (imitha ye-pH ye-JENWAY 3510). I-Titratable acidity (TA) ibalwa ngokusekelwe ku-malic acid kusetshenziswa ifomula elandelayo.
Lapho i-V ne-C kuyivolumu (mL) kanye nokuhlushwa (0.1 mol/L) kwesisombululo se-sodium hydroxide esisetshenziswa ku-titration, ngokulandelana. I-K iyi-coefficient yokuguqulwa kwe-malic acid, elingana no-0.067, kanti i-W iyisisindo (g) sejusi le-apula.
Ingqikithi yezinto eziqinile ezincibilikayo (I-TDS) okuqukethwe kwawo wonke amasampula ejusi kunqunywe kusetshenziswa i-PAL-1 pocket refractometer (ATAGO, Tokyo, Japan). Ngemva kokulinganisa ngakunye, ilensi yokukhanya yahlanzwa ngamanzi acwengekile, futhi isampula ngayinye yejusi le-apula yahlolwa kathathu. Inani lesampula ngayinye labalwa ngokulinganisa izilinganiso ezintathu. Ukuphambuka okujwayelekile okungu-± kwesampula ngayinye yejusi le-apula nakho kwabalwa ngokulinganisa le miphumela.
Ukuqina kwe-viscoelasticity yamasampula ejusi le-apula kuhlolwe kusetshenziswa i-viscometer ejikelezayo (RV, Rheotest 2, Germany). Isampula yafakwa ngaphakathi kwesilinda "S2" se-viscometer. I-viscosity ebonakalayo imelelwe ukuthambeka kwe-shear stress vs shear rate curve, okwabalwa kusukela ku-shear stress kanye nama-curve ahambisanayo kumazinga ahlukahlukene e-shear (kusukela ku-1.00 kuya ku-437.4 s⁻¹). Ifomula yokubala i-viscosity ebonakalayo yile elandelayo:
Lapho u-η kuyi-viscosity ebonakalayo (cP), u-τ kuyi-shear stress (dyn/cm²), u-γ yi-shear rate (sec⁻¹), kanti u-(τ) ubalwa kusetshenziswa amanani e-torque (α) kanye nesilinda (Z) kusetshenziswa ifomula elandelayo: τ = Z . α.
Inkomba ye-browning inqunywe ngokwendlela kaMeidav etal.29. Isampula yejusi engu-10 ml yafakwa ku-centrifuge ku-2750 xg imizuzu eyi-10. I-5 ml ye-supernatant yejusi yaxutshwa ne-5 ml ye-ethanol engu-95%. Ukumuncwa kwengxube kwalinganiswa ku-420 nm kusetshenziswa i-Shimadzu UV spectrophotometer (UV-1601 PC).
Okuqukethwe kwe-phenolic okuphelele (i-TPC) kunqunywe ngombala kusetshenziswa i-reagent ye-Folin-Ciocalteu njengoba kuchazwe nguBoulton et al.[27]]. Ijika elijwayelekile le-gallic acid lakhiwe ukuze kube nokuhlushwa okusuka ku-0 kuya ku-500 mg/L (r²= 0.997). Imiphumela ivezwa njengezilinganiso ze-gallic acid (mg GAE/mL).
Engeza u-125 μL wamanzi acwengekile kanye no-2850 μL wesisombululo se-FRAP ku-25 μL wejusi le-apula bese ushiya ingxube ebumnyameni ukuze30min. Bese ulinganisa ukumuncwa ku-593 nm usebenzisa i-Shimadzu UV spectrophotometer (UV-1601 PC). I-reagent ye-FRAP yalungiswa ngokuxuba i-300 mM acetate buffer (pH 3.6), i-20 mM iron(III) chloride, kanye ne-10 mM 2,4,6-tris(2-pyridyl)triazine (TPTZ) (encibilikiswe ku-40 mM HCl) ngesilinganiso esingu-10:1:1. Ijika elijwayelekile lakhiqizwa kusetshenziswa i-Trolox njengejwayelekile (I-R²= 0.999), kanti imiphumela ivezwa njenge-μM Trolox/mL.
Umsebenzi we-antioxidant wamajusi aphathwe kanye nalawo angelashiwe wanqunywa kusetshenziswa indlela ye-DPPH ukuhlola ikhono lawo lokususa ama-free radical e-DPPH.31Ama-microliter ayishumi ejusi axutshwe ne-1 ml yesisombululo se-DPPH (100 μM) ku-methanol. Ngemva kokusabela ebumnyameni imizuzu engama-30, ukumuncwa kwengxube kwalinganiswa ku-517 nm kusetshenziswa i-Shimadzu UV spectrophotometer (UV-1601 PC). Imiphumela ivezwe njengezilinganiso ze-trolox (μM trolox/ml) ngokusekelwe ku-calibration curve (R2= 0.990).
Idatha etholakele ikhombisile ukuthi ukukhiqizwa okuphezulu kwe-laccase kwabonwa kumakhowe e-oyster e-NRC 620 ekupheleni kosuku lwe-18 lokubilisa, kwafinyelela umsebenzi ongu-1302 U/L. Lokhu kwasebenza njengesisekelo sokunquma isikhathi esifanele sokulima sokukhiqizwa kwe-laccase (Isithombe 1). Nakuba ukukhiqizwa kwama-enzyme kwanda ngesikhathi sokulima esandayo, izinga lokukhula lalingafani ngqo nesikhathi sokulima; ngemva kwezinsuku ezingu-21, umsebenzi wama-enzyme wawukhuphuke ngo-90 U/L kuphela (kuya ku-1390 U/L). Ngakho-ke, izinsuku ezingu-18 ekugcineni zakhethwa njengesikhathi esifanele sokulima ukuze kulinganiswe isivuno somkhiqizo nezinzuzo zomnotho zesikhathi sokulima esengeziwe.
Umphumela wesikhathi sokulima ekuvuneni kwe-laccase ku-Pleurotus ostreatus NRC 620. Amabhulokhi amathathu (12 mm) e-fungus mycelial afakwa ku-50 ml we-sterile medium abese ekhuliswa ku-28 °C ngezikhathi ezahlukene.
Ngokuhambisana nezinye izifundo, imiphumela yethu ikhombisa ukuthi isikhathi esifanele sokukhula ukuze kutholakale ukukhishwa okuphezulu kwe-laccase yi-fungus cishe singaba phakathi kwezinsuku eziyi-7 nezingama-36.32Ngokusho kuka-Ezike et al.33, *I-Trametes polyzona* I-WRF03 ikhiqize inani eliphakeme kakhulu le-laccase ekupheleni kosuku lwesishiyagalolunye lokubilisa, ngomsebenzi othize we-1637 U/mg protein. Ngaphezu kwalokho, u-Othman et al.34bathole ukuthi *i-Trichoderma harzianum* S7113 ikhipha inani elikhulu le-laccase ngosuku lwesihlanu lokukhuliswa. Izinga lokukhiqizwa kwe-laccase lafinyelela umsebenzi ophakeme ngosuku lweshumi nane labe selincipha kancane kancane.34Nakuba ukukhishwa kwama-enzyme kungenzeka futhi ngesikhathi sesigaba sokukhula esiyinhloko, kuvame ukuvuthwa ngesikhathi sesigaba esiphakathi futhi kubangelwa ukusetshenziswa komthombo wekhabhoni noma we-nitrogen.34,35
Nakuba i-laccase evela ku-Pleurotus ostreatus NRC 620 ibonise umsebenzi ophezulu ebangeni elibanzi lokushisa kusukela ku-50°C kuya ku-80°C, isondela emsebenzini ophezulu (69–98%), umsebenzi wayo omkhulu wabonwa ku-70°C (Isithombe 2a). Ngaphandle kwalolu hlu lokushisa, umsebenzi we-enzyme wehle cishe ku-70°C. Le miphumela iphakamisa ukuthi i-enzyme iyasebenza emazingeni okushisa aphezulu, mhlawumbe ngoba izinga lokushisa eliphezulu landisa amandla e-kinetic okusabela.
Umphumela wokushisa kokusabela (a) kanye ne-pH (b) emsebenzini we-laccase ku-*Pleurotus ostreatus* NRC 620. Amazinga okushisa asukela ku-20 kuya ku-90 °C afinyelelwe ngokufaka ingxube emazingeni okushisa ahlukene imizuzu emi-5 ngaphambi kokufaka i-enzyme nokuqala ukusabela. Umphumela we-pH emsebenzini we-laccase uhlolwe kusetshenziswa i-ABTS njenge-substrate ezixazululweni eziqukethe i-0.1 M citrate-phosphate buffer phezu kobubanzi be-pH obungu-2.5 kuya ku-7.0.
Ngokusho kuka-Ezike etal.33, izinga lokushisa elifanele kakhulu le-*Trametes polyzona* WRF03 laccase lingu-55 °C, okufana nalelo le-*Ganoderma lucidum*i-laccase36futhi kufana nokushisa okuhle kakhulu (50 °C) kwe-*Trametes polyzona* KU-RNW02737i-laccase . I-Baldrian38uphawula ukuthi, ngokuqondene nezinye izinhlelo ze-enzyme eziwohloza i-lignin, izinga lokushisa elifanele le-laccase liphakathi kuka-50 no-70 °C.
Imiphumela ikhombisile ukuthi i-enzyme ibonise umsebenzi ophezulu kakhulu ku-pH 3.0, yafinyelela umsebenzi ongu-94% ku-pH 3.5. Kodwa-ke, yahlala isebenza ebangeni elibanzi le-pH kusukela ku-2.5 kuya ku-7.0 (Isithombe 2b). Ngaphezu kwalokho, ibonise umsebenzi ophezulu ezimweni ze-acidic uma kuqhathaniswa nezimo ezingathathi hlangothi noma ze-alkaline. Umsebenzi wayo uhlale okungenani u-77% ngaphezu kobubanzi be-pH kusukela ku-2.5 kuya ku-4.5, kodwa wafinyelela cishe ku-38% kuphela ku-pH 7.0. I-pH engcono kakhulu ye-laccase evela ku-*Trametes polyzona* WRF03 yayingu-4.533, okufana ne-pH yama-laccase avela ku-*Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, kanye *Trametes hirsuta* 41. Kodwa-ke, ngokusho kocwaningo lukaChairin et al.42, i-pH efanele kakhulu ye-laccase evela ku-*Polymorpha f. sp.* I-WR710-1 ingu-2.2, kanti i-pH efanele kakhulu ye-laccase evela ku-*Polymorpha f. sp.* IBL-04 ingu-5.043. Ukubopha ama-anion e-hydroxide (i-laccase inhibitor) kuma-athomu ethusi e-T2/T3 laccase kungaba yimbangela yokwehla komsebenzi we-laccase ngaphansi kwezimo ze-pH ezingathathi hlangothi noma ze-alkaline. Lokhu kungaphazamisa ukudluliselwa kwe-electron yangaphakathi kusuka esikhungweni se-T1 kuya esikhungweni se-T2/T3, ngaleyo ndlelaukukhawulelaumsebenzi we-enzyme 23,44
Ngokufaka i-enzyme emazingeni okushisa ahlukene, kwatholakala ukuthi kokubili isikhathi sokufukamela kanye nokushisa kuthinte ukuqina kwe-enzyme. Okuphawulekayo ukuthi, i-laccase evela ku-*Trametes polyzona* NRC 620 ibonise ukuqina okuphezulu ku-40℃ kanye no-50℃, igcina u-68.33% kanye no-59.61% womsebenzi wayo wokuqala, ngokulandelana, ngemva kwemizuzu eyi-120 (Isithombe 3a). Ngokuphambene nalokho, ngaphansi kwezimo ezifanayo (40℃ kanye no-50℃, imizuzu eyi-120), i-laccase evela ku-*Trametes polyzona* WRF03 igcine u-64.38% kanye no-42.92% womsebenzi wayo, ngokulandelana.33Ngokuphambene nalokho, ukwanda kwesikhathi sokufukamela kanye nokushisa kunciphisa ukuzinza kwe-*Trametes polyzona* NRC 620 laccase; Ngemva kokufukamela ku-60℃ kanye no-70℃ imizuzu engama-60, umsebenzi wayo wehla wafika ku-39.24% kanye no-1.72%, ngokulandelana (Isithombe 3a). Ngokuhambisana nemiphumela yokuhlola, i-laccase evela ku-*Trametes polyzona* WRF03 ibonise ukuzinza okuphezulu ku-40 kanye no-50℃ kuyo yonke inqubo yokwelashwa kokushisa.33Ngokufanayo, uLueangjaroenkit etal.37kanye noSihlalo etal.42babike ukuzinza kwama-laccase avela ku-Trametes polyzona KURNW027 kanye ne-Trametes polyzona WR710-1 ku-50 °C ihora eli-1, ngokulandelana. Njenge-biocatalyst ewusizo esebenza emikhakheni eyahlukene ye-biotechnological, i-laccase kufanele ibe nokuzinza nokusebenza okuhle ebangeni elibanzi lokushisa.
Ukuqina kwe-thermostatic (a) kanye nokuqina kwe-pH (b) kwe-laccase evela ku-*Pleurotus ostreatus* NRC 620. Ukuqina kwe-thermostatic kuhlolwe ngokufaka isisombululo se-enzyme ku-0.05 M sodium phosphate buffer (pH 7.0) ku-40, 50, 60, kanye no-70 °C amahora ama-2, ngokulandelana. Ukuqina kwe-pH kuhlolwe ngokufaka isisombululo se-enzyme ku-0.1 M citrate buffer kanye ne-Tris buffer (pH 3, 4, 6, kanye no-7) ku-40 °C amahora ama-2. Umsebenzi osele ubalwa kusetshenziswa i-ABTS njenge-substrate ngemva kokufushanisa.
Ukuze sithole izimo ezifanele zokusetshenziswa nokugcinwa kwama-enzyme, sihlole umphumela we-pH ekuzinzeni kwe-laccase. Ukuchayeka kumanani ahlukene e-pH kuthinte kakhulu ukuzinza kwesakhiwo samaprotheni, ngaleyo ndlela kuthonye ukuzinza nomsebenzi we-molecule ye-enzyme. Imiphumela ibonise ukuthi i-enzyme yayingazinzi kangako ngaphansi kwezimo ze-asidi, kuyilapho ibonise ukuzinza okungcono kumanani aphezulu e-pH (izifunda ezingathathi hlangothi nezine-alkaline). Kumanani e-pH angu-7.0, 6.0, 4.0, kanye no-3.0, amazinga okugcinwa kwama-enzyme ngemva kwemizuzu engu-120 ayecishe abe ngu-100%, 62.54%, 52.39%, kanye no-11.14%, ngokulandelana (Isithombe 3b). *I-Strombus multisus* WRF03 laccase ibonise ukuzinza okuphezulu kumanani angathathi hlangothi e-pH (5.5–6.5) kanye nokuzinza okuphansi kumanani e-pH ane-asidi (ngaphansi kuka-4.0). Ngemva kwemizuzu eyi-120 kumanani e-pH angu-5.5, 6.0, kanye no-6.5, amazinga okugcinwa kwama-enzyme ayecishe abe ngu-82%, 100%, kanye no-93% ngokulandelana.33UKhairin nabanye.42waphawula ukuthi i-laccase evela ku-Trametes polyzona WR710-1 yayizinzile ebangeni le-pH elingu-6.0 kuya ku-7.0, kuyilapho uSayed et al.45kubonise ukuthi i-laccase yayizinzile kakhulu ngaphansi kwezimo ze-pH ezingathathi hlangothi. Kodwa-ke, i-laccase evela ku-Cerrena unicolor nayo ibonise ukuzinza ngaphansi kwezimo ze-alkaline (pH 9.0)46Ama-laccase afundwe abonise ukuzinza okuphezulu ebangeni elibanzi le-pH. Lokhu kungaba yisici esibalulekile ekusetshenzisweni kwezimboni.
Njengoba amanye ama-ion ensimbi enemiphumela yokuvuselela kanye neyokuvimbela emsebenzini wama-enzyme, imiphumela yawo emsebenzini wama-enzyme kumele icatshangelwe ekusetshenzisweni kwezimboni. Lokhu kubalulekile ngoba ama-ion ensimbi ayizinto ezivame ukungcola kwemvelo ezingathinta ukuzinza kanye nokwakheka kwama-enzyme angaphandle.47Ukuze sihlole imiphumela yama-ion amaningi ensimbi ku-laccase evela ku-*Pleurotus ostreatus* NRC 620, senze izivivinyo ezifanayo. Njengoba kuboniswe kuMfanekiso 4, kuye ngohlobo lwensimbi esetshenzisiwe, ukwandisa ukuhlushwa kwama-ion ensimbi kusuka ku-2.5 mM kuya ku-10 mM kuthinte kabi umsebenzi we-enzyme. Isibonelo,I-Mg²⁺ , I-Co²⁺ , I-Zn²⁺, futhiI-Cu²⁺kungavuselela futhi kusebenze umsebenzi we-enzyme, kuyilaphoNa⁺ , Mn²⁺ , I-Ca²⁺, futhiK⁺kungavimbela umsebenzi we-enzyme. Ekugxilweni okungu-10 mM, ama-ion e-Cu²⁺ kanye ne-Mg²⁺ ayeyi-activator enamandla kakhulu yomsebenzi we-laccase avela ku-*Pleurotus ostreatus* NRC 620, enikeza izinga lokusebenza elingaba ngu-34% kanye no-20%, ngokulandelana. Kodwa-ke, ekugxilweni okungu-10 mM, ama-ion e-Ca²⁺ ayeyi-inhibitor enamandla kakhulu ye-laccase, enciphisa umsebenzi we-enzyme cishe ngo-60%.
Umphumela wama-ion ensimbi emsebenzini we-Pleurotus ostreatus NRC 620 laccase. I-laccase yafakwa imizuzu eyi-10 ku-sodium phosphate buffer (0.05 M, pH 7.0) equkethe ama-ion ensimbi ahlukahlukene ekugxilweni okungu-2.5 mM kanye no-10 mM. Ukusabela kwabe sekuqala ngokufakwa kwe-substrate (ABTS), ngemva kwalokho umsebenzi ohlobene walinganiswa.
Imiphumela yethu ihambisana neyabanye ababhali abathole ukuthi i-Mg²⁺ kanye ne-Cu²⁺ kuthuthukisa umsebenzi we-*Trametes polyzona* WRF03³. UCastaño et al.⁴⁸ bathole ukuthi i-laccase evela ku-*Xylaria* sp. ivuselelwa ngezinga elithile ngama-ion ethusi (Cu²⁺). Ngaphezu kwalokho, uForoutanfar et al.⁴⁹ kanye noSi et al.⁵⁰ benze izifundo ezifanayo kuma-laccase avela ku-*Paraconiothyrium variabile* kanye *ne-Trametes pubescens*, ngokulandelana. Indawo yokubopha ithusi yohlobo lwe-II (T2) yale enzyme ingagcwala i-Cu²⁺ ekugxilweni okunikeziwe, okungachaza ukukhuthazwa komsebenzi we-laccase ekugxilweni okuphezulu kwe-Cu²⁺³⁹. Njengoba ama-laccase e-white rot fungi angama-oxidases aqukethe ama-athomu amaningi ethusi, imiphumela yama-ion ethusi emsebenzini we-laccase iyahlukahluka futhi isukela ekuvuseleleni nasekuvimbeleni kuya ekungathathi hlangothi.⁵¹ Ngokuphambene, uZhou nabanye.. [52]kubike ukuthiI-Cu²⁺kuvimbele umsebenzi we-laccase we-Taiwan subterranean termite (Odontotermes formosanus). Kodwa-ke, ama-laccase e-Cerena sp. HYB07[53]kanye ne-Clitocybe maxima[54]azizange zithinteke ngama-ion ethusi.
Ukucaciswa kwe-substrate kwamelelwa yimingcele yayo ye-kinetic (Km kanye ne-Vmax); lapho ukuqina kokubopha kwe-substrate ku-enzyme, kulapho inani le-Km liphansi futhi kulapho ukucaciswa kwe-substrate kuphakeme khona.3,21,55Amapharamitha e-kinetic (Km kanye ne-Vmax) e-laccase avela ku-*Pleurotus ostreatus* NRC 620 anqunywa kusetshenziswa isofthiwe ye-GraphPad Prism 6.0 ngokuhlela i-Lineweaver-Burk plot (Isithombe 5). Lapho kusetshenziswa i-ABTS njenge-substrate, imiphumela yayiyi-1.99 mM kanye ne-16217 μmol.min⁻¹ L⁻¹,ngokulandelana. U-Elsayed nabanye.21kubike ukuthi amanani e-Km e-oxidation ye-ABTS ayengu-0.1 mM kanye no-0.064 mM, ngokulandelana, okubonisa ukuhambisana okuphezulu kwama-isoenzyme e-Lac A kanye ne-Lac B e-ABTS. Ngaphezu kwalokho, amanani e-Vmax ayengu-0.182 μmolmin⁻¹kanye no-0.603 μmolmin⁻¹, ngokulandelana. Inani le-Km elitholiwe laliphansi kunele-Trametes polyzona WRF03 (8.66 mM); ngaphezu kwalokho, inani labo le-Vmax (1429 mmol min⁻¹) nalo laliphansi.phansilapho kusetshenziswa i-ABTS njenge-substrate.33 Ngokufanayo, amanani e-Km e-Lentinus squarrosulus MR13 kanye ne-Trametes sp. Amazinga e-AH28-2 laccase ayengu-0.0714 mM kanye no-0.025 mM, ngokulandelana, kanti amanani e-Vmax ayengu-0.0091 mM min−1 kanye no-0.67 mM min−1 mg−1 (uma kuqhathaniswa ne-ABTS), ngokulandelana.56,57
Umphumela wokuhlushwa kwe-ABTS emsebenzini we-laccase ovela ku-*Pleurotus ostreatus* NRC 620 uhlolwe, kwathi kwadwetshwa i-Lineweaver-Burk plot yokuphindaphindana kwejubane lokuqala lokusabela ngokumelene nokuhlushwa kwe-ABTS. Ukusabela kwe-oxidation kwe-ABTS okunokuhlushwa okuhlukile (0.025–3.0 mM) kwe-laccase kulinganiswe ku-pH 4.5 ukuze kutholakale amapharamitha e-kinetic (i-Vmax kanye ne-Km). Ama-Michaelis-Menten kinetic constants abalwe kusetshenziswa i-Lineweaver-Burk plot yokuphindaphindana kwejubane lokusabela ngokumelene nokuhlushwa kwe-substrate. Ama-kinetic constants abalwe kusukela ku-Lineweaver-Burk plot kusetshenziswa isofthiwe ye-GraphPad Prism 6.01.
Ama-enzyme acacisa ngokwesiko, njenge-pectinase, akhipha i-hydrolyze pectic substances, anciphisa i-viscosity kanye ne-turbidity. Aphula ngempumelelo ama-polysaccharide esakhiwo futhi avame ukusetshenziswa ngokuhlanganiswa namanye ama-enzyme, njenge-cellulases kanye ne-hemicellulases, ukuthuthukisa isivuno kanye nokucaca. Kodwa-ke, ama-pectinase awaqondisi ngqo ama-phenolic compounds, okuyiwona abangela kakhulu i-turbidity kanye ne-oxidative browning, ikakhulukazi kuma-juice afana ne-apula kanye nejusi lamagilebhisi.58Ngokuphambene nalokho, ama-laccase akhuthaza ukushiswa kwamakhemikhali e-phenolic, awenze abe ama-molecule amakhulu, angancibiliki angasuswa ngokuthungwa noma ngokuhlunga. Le ndlela ayigcini nje ngokuthuthukisa ukucaca kodwa futhi yandisa impilo yeshelufu yejusi ngokunciphisa amathuba okuba nombala we-oxidative obangelwa amakhemikhali e-phenolic. Ngaphezu kwalokho, izinqubo zokucacisa ezisekelwe ku-laccase zingenziwa ngaphansi kwezimo zokucubungula ezithambile (i-pH 3.5–5.5, izinga lokushisa elingu-25–40 °C), okwenza afanelekele amajusi athambile ngaphandle kokubeka engcupheni izakhiwo zawo zokudla okunempilo noma ze-organoleptic.59Izifundo zibonise ukuthi ukwelashwa nge-pectinase kungacacisa ijusi emahoreni angu-1-2, kuyilapho ukwelashwa nge-laccase ngokuvamile kudinga isikhathi eside sokuphendula (amahora angu-3-6) ukuze kuncishiswe ngokuphelele ama-phenolic compounds. Kodwa-ke, le nqubo ingathuthukiswa ngokuvimbela i-enzyme noma ngokuhlanganisa i-laccase nezindlela zokucacisa ngomshini.60Kulolu cwaningo, ukuprofayiliwa kwe-enzyme kwe-crude extract kwembule imisebenzi ebalulekile ye-laccase kanye ne-α-amylase, kuyilapho imisebenzi ye-pectinase kanye ne-xylanase yayiphansi kakhulu, futhi umsebenzi we-cellulase awutholakalanga. Ngakho-ke, ukwehla kokuqukethwe kwe-turbidity kanye ne-phenolic kwakubangelwa kakhulu isenzo se-laccase, kuyilapho ushintsho ku-viscosity lungaba ngenxa yesenzo se-amylase.
Ithebula 1 likhombisa amapharamitha e-physicochemical ejusi le-apula elisanda kukhanywa kanye namasampula aphathwe nge-laccase. Imiphumela ikhombisile ukuthi isivuno sejusi le-apula elisanda kukhanywa (71.59%) besiphansi kunesasampula esiphathwe nge-laccase (87.34%). Le miphumela ihambisana nokutholakele kwePilnik kanye ne-Orange.61, obonise ukuthi ukusetshenziswa kwama-enzyme ekucubungulweni kwezithelo kungakhuphula isivuno sejusi, kuthuthukise ukuhlunga, futhi kutholakale ijusi elisezingeni eliphezulu nelicacile lokuhlushwa. Ukwanda kwesivuno sejusi kungenxa yokwanda kokuqukethwe ushukela oncibilikayo kujusi. Ngesikhathi se-enzyme hydrolysis yezithelo, i-mesoglea ne-pectin ezindongeni zeseli zomkhiqizo kuyabhujiswa futhi kuguqulwe kube izinto ezincibilikayo njengoshukela ongathathi hlangothi nama-asidi.62.Inani le-pH lejusi le-apula eliphathwe nge-enzyme laliphansi kakhulu kuneleqembu lokulawula (P < 0.05), futhi inani le-pH lamaqembu womabili landa kakhulu ngesikhathi sokugcina (Ithebula 1). Le miphumela ihambisana naleyo kaMark et al.63, ophawule ukuthi i-pH yejusi yezithelo ze-cashew yehlile ngemva kokugcinwa ngemva kokwelashwa ngokushisa. Ukuwohloka kwe-pectin kanye nokwakheka kwe-galacturonic acid ngemva kokwelashwa nge-enzyme kungaba yimbangela yokwanda kwe-pH ngesikhathi sokugcinwa. I-pH yamasampula aphathwe nge-enzyme yahlala phakathi kuka-4.05 no-4.31 phakathi nokugcinwa, kuyilapho i-pH yejusi ye-apula engaphathwanga yayiphakathi kuka-4.12 no-4.33.
I-asidi iyonke (TA) yamasampula angelashwanga kanye nalawo aphathwe nge-laccase ibonise ukuthambekela okwehlayo ngokuhamba kwesikhathi sokugcina (Ithebula 1). Ukwehla kwe-asidi kubangelwa ukuguqulwa kwama-asidi e-organic abe ama-carbohydrate noma ukusabela kwe-enzymatic, kanye ne-oxidation ngesikhathi sokugcina ijusi.64I-acidity iyonke yejusi lama-apula elilawulwayo kanye namasampula aphathwe ngama-enzyme yayiphansi kuneyeminye ijusi (ijusi lama-strawberry 0.9%, ijusi lama-plum 2.2%, ijusi le-kumquat 1.0%, ijusi lama-apricot 2.4%, ijusi le-orange 0.8%), kodwa ifana neyeminye ijusi (isb., ijusi lama-pear 0.3%).62Lo mehluko kujusi we-apula osanda kuhlungwa ungabangelwa yizici ezahlukahlukene njengezimo zokukhula, izici zofuzo, izinga lokuvuthwa, kanye nezindlela zokucubungula.65Ukwehla kwe-acidity iyonke yokulawula kanye nejusi lama-apula aphathwe nge-laccase kuhambisana nemiphumela eyethulwe nguSingh et al.66mayelana nokwehla kwe-asidi iyonke yejusi le-apula i-Jin Nuo ngemva kwezinsuku ezingu-74 zokugcina. Ngakolunye uhlangothi, u-Oshmiansky no-Wojdylo67Akutholanga noma yiziphi izinguquko ezibalulekile ku-asidi yejusi le-apula lapho kufundwa umphumela wezindlela zokucacisa zendabuko.
Imiphumela eyethulwe kuThebula 1 ikhombisa ukuthi inani lama-solid ancibilikayo (i-TSS) eliphelele lejusi le-apula eliphathwe nge-laccase laliphezulu kunelesampula elingelashiwe. Le miphumela iyahambisana nezifundo ezishicilelwe.. 68Ngaphezu kwalokho, Ithebula 1 libonisa ukuthi inani le-TSS leqembu lejusi lama-apula elilawulayo lalingu-9.58 ngesikhathi sokuqala futhi lafinyelela ku-11.05 ekupheleni kwesikhathi sokugcina. Lawa manani aphansi kunamanani e-TSS ejusi lama-apula elisha elibikwe nguHamid et al.. 69(11.2 kanye no-11.80, ngokulandelana). Inani le-TSS lamasampula ejusi le-apula aphathwe nge-laccase landa kakhulu, kusukela ku-11.23 lafinyelela ku-12.93 ngemuva kwamasonto amabili okugcinwa ku-4°C (Ithebula 1). Ukwanda okufanayo ku-TSS ngesikhathi sokugcina kwabonwa nasezithelweni ze-citrus, ulamula, kanye nama-orange amnandi. Ukwanda kwezinto eziqinile ezincibilikayo (TSS) ngesikhathi sokugcina kungase kube ngenxa ye-hydrolysis yama-polysaccharides (isitashi) abe ama-monosaccharides (ushukela), ukwanda kokuhlushwa ngenxa yokuphelelwa amanzi yijusi, kanye nokuwohloka kwe-pectin kujusi abe izinto eziqinile ezincibilikayo. Ukwanda kwezinto eziqinile ezincibilikayo (TSS) kungenzeka kungenxa yokwanda koshukela oncibilikayo, okungase kwakhiwe ngokuguqulwa kwe-pectin noma i-cellulose kube ushukela oncibilikayo yi-pectin noma i-cellulase, ngokulandelana, noma nge-hydrolysis yesitashi sibe ushukela, njengoba kubikwe nguHamed et al.69.Umphumela we-laccase ezimpahleni zejusi le-apula ungabonakala ngokubonakalayo, njengoba ijusi le-apula eliphathwe nge-laccase libonisa ukugeleza okungcono kanye nokuthamba okuphansi kunejusi elingaphathwanga. Lokhu kuqashelwa kuqoshwe kuThebula 1; Ukuthamba kwesampula ephathwe nge-enzyme kwakungu-1.87 cP, kanti ukuthamba kwesampula yokulawula kwakungu-2.95 cP. Lokhu kwehla okukhulu kokuthamba kungenzeka ngenxa yamandla aphezulu okubamba amanzi ezinto ezifana ne-pectin kanye nokwakheka kwesakhiwo senethiwekhi esihlangene.
Kulolu cwaningo, umphumela we-laccase ku-browning index (BI) yejusi le-apula uhlolwe ngokulinganisa ukumuncwa ku-420 nm kusetshenziswa i-spectrophotometer. Imiphumela iboniswe kuThebula 1. Ngesikhathi sokugcina, i-BI yamasampula ejusi le-apula kokubili emaqenjini aphathwe kanye nalawo angelashwanga ibonise ukuthambekela okukhula kancane kancane. I-BI ibonisa izinga lokuba brown futhi ingasebenza njengeokubalulekileinkomba yokusabela kokunsundu kwe-enzymatic kanye nokungabi yi-enzymatic. Ukumuncwa kwanda kakhulu ngesikhathi sokugcina (P < 0.05). Ekupheleni kokugcina,I-A420Inani lamasampula ejusi le-apula emaqenjini okulawula kanye nalawa aphathwe ngama-enzyme likhuphuke cishe ngo-217% kanye no-121%, ngokulandelana (Ithebula 1). Imiphumela ikhombisa ukuthi ukwelashwa ngama-enzyme kunganciphisa ngempumelelo izinga lokuba mdaka cishe ngo-56%. Imiphumela kaBezerra et al.[19]] ziyahambisana nemiphumela yethu; Basebenzise i-laccase-glutaraldehyde-coconut fiber ukuze bacacise ijusi le-apula, benciphisa umbala walo wokuqala ngo-61%.
Nakuba ama-polyphenol kumajusi ezithelo anemiphumela emihle yokudla okunempilo nokwelapha emzimbeni womuntu, angasabela namaprotheni, okubangela ukufiphala kwejusi, ukubola kwenhlabathi, noma ukubola, ngaleyo ndlela kushintshe ukunambitheka nephunga lomkhiqizo futhi kunciphise isikhathi sawo sokuphelelwa yisikhathi.71Inhloso yalolu cwaningo kwakuwukunciphisa ngokuphephile okuqukethwe kwe-phenolic compound yejusi le-apula kusetshenziswa i-laccase evela ku-Pleurotus ostreatus NRC 620. Imiphumela eyethulwe kuThebula 1 ikhombisa ukuthi okuqukethwe kwe-phenolic compound yejusi le-apula eliphathwe nge-laccase kwehliswe kakhulu ngaphambi kokugcinwa ku-4 °C. Ngaphezu kwalokho, okuqukethwe kwe-phenolic compound nakho kwehle ngesikhathi sokugcinwa kuzo zombili izibonelo ezifundwe (Ithebula 1). Ucwaningo lukaSandri et al.72kubonise ukuthi ijusi le-apula eliphathwe ngama-enzyme lingagcina umsebenzi walo wokulwa ne-antioxidant kanye nokuqukethwe kwe-phenolic compound. Kodwa-ke, imiphumela yocwaningo lukaLettera et al.73khombisa ukuthi ukwelashwa kwejusi lewolintshi nge-fungus laccase kunganciphisa okuqukethwe kwamakhemikhali e-phenolic kuwo kufike ku-45%.
Kuye kwabonakala ukuthi ama-phenolic compounds anezakhiwo ezifana nokuqoqwa kwama-free radical, ukunciphisa nokucima i-singlet oxygen, ukudluliselwa kwama-athomu e-hydrogen, kanye nokunikelwa kwama-electron kuma-free radical, okwenza abe ama-antioxidants anamandla.74Ngakho-ke, kulolu cwaningo, izindlela ezisekelwe ku-DPPH kanye ne-FRAP zisetshenziswe ukuhlola umphumela we-laccase emsebenzini we-antioxidant wejusi le-apula eligcinwe esiqandisini izinsuku ezingu-14 (Ithebula 2). Zombili lezi zindlela zibonise ukwanda komsebenzi we-antioxidant ngesikhathi sokugcina, okungase kube ngenxa yokwanda kwamakhemikhali e-phenolic yamahhala noma ukwakheka kwemikhiqizo yokusabela kwe-Maillard (MRPs), kanye nemikhiqizo yokusabela kwe-Maillard okungenzeka ukuthi iyimbangela yokwanda komsebenzi we-antioxidant.75Ukusabela kokunsundu okungekhona okwe-enzyme (kufaka phakathi ukuchithwa kwe-ascorbic acid, ukusabela kwe-Maillard, kanye nokuchithwa koshukela okubangelwa yi-acid) kukhiqiza imibala ensundu (ama-melanoidin). Imikhiqizo yokunsundu ephakathi kwe-ascorbic acid kanye nemikhiqizo yokunsundu (njengezakhi ze-carbonyl) ingasabela nama-amino acid ngokusebenzisa ukusabela kwe-Maillard.76Nakuba ukunsunduza izithelo nemifino ngesikhathi sokugcina kuye kwafundwa kabanzi, ukuqonda kwethu ngalezi zindlela zokusabela kusalokhu kulinganiselwe.77Uma kuqhathaniswa nendlela ye-FRAP, ijusi le-apula eliphathwe nge-laccase libonise umsebenzi we-antioxidant ophansi kakhulu ngendlela ye-DPPH (Ithebula 2), kanti umsebenzi we-antioxidant wawo wonke amasampula wanda kakhulu ngesikhathi sokugcinwa esandisiwe. Kusetshenziswe izindlela ezimbili ezahlukene zokunquma umsebenzi we-antioxidant kulolu cwaningo ngoba izimiso zazo ziyahluka. Indlela ye-DPPH ilinganisa ikhono lokuqeda ama-free radicals, kuyilapho indlela ye-FRAP ilinganisa ikhono lokunciphisa ama-iron ion. Ngakho-ke, kunconywa ukusebenzisa izindlela eziningi zokunquma umsebenzi we-antioxidant ukuze uqonde kangcono umsebenzi we-antioxidant wamasampula afundwe.78
Enye yezinto ezibalulekile ezitholwe kulolu cwaningo ukuthi i-*Pleurotus ostreatus* laccase NRC 620 ibonisa umsebenzi omuhle kakhulu ku-70°C kanye ne-pH 3.0. Uma kuqhathaniswa nezinye i-laccases zesikhunta ezivame ukusetshenziswa ekucaciseni ijusi, njenge-*Trametes versicolor* kanye ne-*Ganoderma lucidum* laccases, i-*P. ostreatus* NRC 620 ibonisa ukuzinza okuphezulu kokushisa kanye ne-pH ene-asidi eningi. I-Laccases evela ku-*Trametes versicolor* kanye ne-*Ganoderma lucidum* ngokuvamile ibonisa umsebenzi omuhle kakhulu ebangeni eliphakathi kuka-50-60°C kanye namanani e-pH aphakathi kuka-3.5 no-5.0. Lo mehluko ungaba negalelo ekuthuthukisweni kokusebenza kahle kokucacisa ijusi, ikakhulukazi kumajusi ane-asidi lapho ukuzinza kwamanani e-pH aphansi kubalulekile. Isici esiyingqayizivele se-*P. Uma kuqhathaniswa nezinye i-laccases zesikhunta ezifundwe, i-*Pleurotus ostreatus* NRC 620 ibonisa ikhono lokusebenza kahle ngaphansi kwezimo ezinzima kakhulu. Izinga lokushisa eliphezulu lomsebenzi walo lisikisela izinzuzo ezingaba khona ekusetshenzisweni kwezimboni, njengezinga lokusabela okusheshayo kanye nokunciphisa ukungcoliswa kwamagciwane. I-pH yalo ephansi, efanelana kahle nesimo se-asidi samajusi amaningi, ingaba usizo ezinqubweni zokucacisa ujusi. Le miphumela iqinisekisa ukuhlolwa okwengeziwe kokusetshenziswa okukhulu, okwenza i-*Pleurotus ostreatus* NRC 620 ibe yindlela engcono kunemithombo yendabuko ye-laccase yesikhunta. Uma kuqhathaniswa nezifundo zangaphambilini, sithole ukuthi izinga lokushisa elihle lingu-60°C kanti i-pH enhle ingu-3.0. Ngemva kokusabela ku-60°C imizuzu engu-80, i-*Ganoderma lucidum* laccase igcinwe.46% womsebenzi wayo.79 Ngokusho kukaKurniawati noNicelle80, Ama-enzyme e-*Ganoderma lucidum* abonisa ukuzinza okuhle kakhulu kuya kokumaphakathi ku-25°C kanye namanani e-pH asukela ku-5.0 kuya ku-8.0, kanye nokuzinza ku-pH 6.0 kanye namazinga okushisa asukela ku-10 kuya ku-30°C. Kulolu cwaningo, sithole ukuthi i-pH kanye nezinga lokushisa elihle kakhulu lomsebenzi we-enzyme we-*Pleurotus ostreatus* kwakungu-3.0 no-70°C, ngokulandelana. Ngemva kokufukamela ku-40°C kanye no-50°C amahora amabili, i-enzyme igcine u-68.33% kanye no-59.61% womsebenzi wayo, ngokulandelana. Ngaphezu kwalokho, i-Pleurotus ostreatus NRC 620 laccase ibonise umsebenzi ophezulu ebangeni elibanzi lokushisa kusuka ku-50°C kuya ku-80°C, cishe yafinyelela umsebenzi omkhulu (69%–98%), kanti umsebenzi omkhulu ubonwe ku-70°C.
Ekuphetheni, i-oyster mushroom laccase NRC620, etholakale ngaphansi kwezimo ezimile, ibonise umsebenzi ofanele kanye nokuzinza kuzo zonke izinhlobo ze-pH kanye nezimo zokushisa, okubonisa ukuzinza okuphezulu uma kuqhathaniswa neminye imithombo ye-enzyme. Ukwengezwa kwe-10 mM MgSO₄ kanye ne-CuSO₄ kwandise umsebenzi we-enzyme cishe ngo-21% kanye no-35%, ngokulandelana. Lapho icutshungulwa ibe ujusi we-apula, i-enzyme yehlisa i-pH kanye ne-viscosity, kuyilapho okuqukethwe kwe-phenolic kwehla kancane ngesikhathi sokugcina.
Imiphumela iqinisekisa amandla e-laccase embonini yokudla, ikakhulukazi ekucaciseni iziphuzo. Ngokuhlukanisa ngqo ama-phenolic compounds, i-laccase ayigcini nje ngokunciphisa ukungcoliswa futhi ithuthukise ukucaca kodwa futhi igcina ikhwalithi yamajusi ezithelo ngaphansi kwezimo zokusebenza ezithambile. Ngokungafani nama-ejenti okucacisa endabuko njenge-gelatin, i-bentonite, ne-silica gel, i-laccase ayikhiqizi imfucuza noma isuse amaphunga amnandi eziphuzweni, okwenza kube inketho enobungani nemvelo futhi eqhubekayo. Ngaphezu kwalokho, uma kuqhathaniswa namanye ama-enzyme nezindlela zokuhlunga, i-laccase inikeza ikhambi eliqondiwe nelingabizi ngaphandle kokubeka engcupheni ikhwalithi yomkhiqizo.
UKyomuhimbo, i-HD kanye neBrink, i-HG. Ukusetshenziswa kanye namasu okuvimba ukuhamba kwe-laccases equkethe ithusi; isibuyekezo. Heliyon 9, e13156 (2023).
Isikhathi sokuthunyelwe: Disemba 15-2025