Emva kweentsuku ezingama-25 zokufunxwa okungashukumiyo kwi-28°C, i-laccase evela kwi-*Pleurotus ostreatus* NRC620 ibonise umsebenzi ophezulu kwi-fungus culture medium. Amaxabiso aphezulu e-pH kunye nobushushu bale enzyme yayiyi-3.0 kunye ne-70°C, ngokwahlukeneyo. Emva kweeyure ezi-2 zokufunxwa kwi-40°C kunye ne-50°C, umsebenzi we-enzyme ugcine i-68.33% kunye ne-59.61%, ngokwahlukeneyo. Emva kweeyure ezi-2 zokufunxwa kwi-citrate-phosphate buffer (pH 7.0), umsebenzi we-enzyme wahlala kwi-100%. Ukongezwa kwe-10 mM MgSO₄ kunye ne-CuSO₄ kwandisa umsebenzi we-enzyme malunga ne-21% kunye ne-35%, ngokwahlukeneyo, ngelixa i-NaCl, i-MnCl₂, i-KCl, kunye ne-CaCl₂ zithintele umsebenzi we-enzyme. Ukusebenzisa i-ABTS njenge-substrate, iiparameter ze-kinetic (Km kunye ne-Vmax) ze-*Pleurotus ostreatus* NRC 620 laccase zaziyi-1.99 mM kunye ne-16,217 μmol min−1 L−1, ngokulandelelana. Unyango lwe-enzyme lweesampuli zejusi yeapile lunciphise kakhulu i-pH kunye ne-viscosity, kwaye oku kuncipha kuhambelana nokwanda kwexesha lokugcina. Unyango lwe-laccase lubangele ukwehla okuncinci kumxholo we-phenolic iyonke yejusi yeapile, kodwa akukho kuncipha komsebenzi we-antioxidant okubonwe.
Kwiminyaka yakutshanje, abaphandi baye bagxila ekusetyenzisweni kwe-biotechnology eluhlaza kwishishini lokutya. I-Laccase yenye yee-enzymes eziluncedo kakhulu kwishishini lokutya, ifumana ukusetyenziswa kwiindawo ezifana nokucubungula ijusi, ukubhaka, ukuzinzisa iwayini, kunye nokuphucula iimpawu ze-organoleptic zeemveliso zokutya.1Izityalo ezininzi eziphezulu kunye neentsholongwane zikhupha i-laccase,2kwaye iifungi ezifana ne-deuteromycetes, i-ascomycetes, kunye ne-basidiomycetes nazo zinokuvelisa i-laccase.3I-Laccase (EC 1.10.3.2) yi-oxidase eluhlaza okwesibhakabhaka enciphisa i-oksijini yemolekyuli ibe ngamanzi isebenzisa inkqubo equlathe ii-athomu ezintathu ezahlukeneyo zobhedu, ngaloo ndlela i-oxidation kwiikhompawundi ezahlukeneyo ze-phenolic kunye ne-aromatic amines. Ngexesha lokuveliswa kweejusi zeziqhamo nemifuno, i-enzymatic kunye ne-nonenzymatic browning ziingxaki ezibalulekileyo.4Ekubeni ezi zinto zichaphazela kakubi umbala, incasa, kunye nevumba lejusi, kufuneka zisuswe.5
Kuzo zonke iziqhamo, ii-apile zezona zityiwa kakhulu kwihlabathi liphela nakwi-European Union. Ngo-2019, imveliso yee-apile yabekwa kwindawo yesithathu kwihlabathi liphela, idlula iitoni ezingama-87 ezigidi.6Ama-apile aqulathe iikhompawundi ezininzi ze-phenolic, kuquka ii-flavonoids kunye nee-phenolic acids ezifana ne-caffeic acid kunye ne-chlorogenic acid.7Ngenxa yokuba ijusi yeapile idla ngokutyiwa ikwimo yayo ecacileyo, malunga ne-50% ukuya kwi-90% yeenxalenye ze-phenolic ziyalahleka ngexesha lenkqubo yokuhluza.8Namhlanje, abathengi bathanda ukukhetha iimveliso ezingacutshungulwanga kakhulu, ezifana nejusi yeapile enamafu enomxholo ophezulu we-polyphenol. Nangona kunjalo, ngenxa yomxholo wayo ophezulu we-phenolic, olu hlobo lwejusi yeapile luchaphazeleka kakhulu kukutshintsha kombala kunye nokuba mnyama.9Ubuchwepheshe obahlukeneyo, kuquka iindlela zokunyanga ubushushu ezifana nokuthambisa ii-apile kwiqondo lobushushu elingama-60–90°C, busetyenziswa ukunciphisa okanye ukuthintela ukuba mnyama kwejusi yeapile.10Nangona kunjalo, ngokophando olwenziwe nguSauceda-Gálvez11, ukucubungula ubushushu kunokutshabalalisa iikhemikhali eziguquguqukayo kwaye kuchaphazele iimpawu ze-organoleptic zejusi yeapile. Ezinye iindlela zokucubungula ubushushu ziquka i-supercritical carbon dioxide, i-ultraviolet radiation, i-ultrasound, uxinzelelo oluphezulu lwe-hydrostatic, okanye i-high-pressure homogenization.12Ukusebenza kakuhle kwezi teknoloji kunye nemveliso yeejusi zeziqhamo ezifanelekileyo kuxhomekeke kwiiparameter ezisetyenzisiweyo kunye neempawu zemveliso. Ukusetyenziswa kwazo ngokubanzi kunqunyelwe ziindleko eziphezulu, iziphumo ezimbi kumgangatho wezinye iimveliso zokutya, okanye ukungasebenzi kakuhle kwee-enzyme.13,14
I-Laccase ingasetyenziselwa ukuzinzisa nokucacisa ijusi yeziqhamo.15UGökmen nabanye.16Ndicebisa ukusetyenziswa kwe-laccase ukucacisa ijusi yeziqhamo kuba isusa ngempumelelo iikhompawundi ze-phenolic ngokuziguqula zibe zii-polymers okanye ii-oligomers ezisuswa lula yiyo nayiphi na i-membrane ye-ultrafiltration, ivumela ijusi yeapile ukuba igcine umbala ozinzileyo kunye nokucaca kangangeeveki ezintandathu kwi-50°C. I-laccase ecociweyo *Trichoderma* yayingashukumi kwiibhead ze-alumina kwaye yasetyenziselwa ukususa ngokukhethekileyo iikhompawundi ezingenancasa ezibangelwa kukungcola kweentsholongwane zejusi yeapile.17
Malunga ne-80-90% yezinto eziguquguqukayo zejusi yeapile zii-esters kunye nee-aldehydes, ezinika ivumba elikhethekileyo kwijusi.18I-Laccase evela kwi *Trametes versicolor* ayizange ikwazi ukuhambahamba ngenkxaso engabizi kakhulu eyenziwe ngefayibha yendalo evela kwiigobolondo zekhokhonathi eziselula ukuze kucaciswe ijusi yeapile.19Izifundo zangaphambili ziphande ukuzinza kwejusi yeapile (umbala kunye nokudumba) kusetyenziswa iindlela ezingenazo iienzymes okanye iindlela zokuthintela ukuhamba, okanye xa zidibene ne-ultrafiltration.5,19Nangona kunjalo, impembelelo ye-fungus laccases kwi-physicochemical properties yejusi yeapile ngexesha lokugcinwa kwayo ayicaci. Ke ngoko, injongo yolu phononongo yayikukuphanda ngovavanyo utshintsho kwi-physicochemical properties, umxholo we-phenolic compound, kunye nomsebenzi we-antioxidant wejusi yeapile emva konyango nge-fungus laccases kunye nokugcinwa efrijini kweeveki ezimbini. I-Laccases inamandla okuxuba ii-phenolic compounds, nto leyo eyenza ukuba zithembise ukusetyenziswa kwiinkqubo ezahlukeneyo zoshishino, kubandakanya ukucaciswa kwejusi. Olu phononongo luhlolisise ii-laccases ezivela kwi-*Pleurotus ostreatus* NRC 620, zigxile kwiimeko ezifanelekileyo zomsebenzi wazo kunye nokusebenza kakuhle ekucacisweni kwejusi. Ngelixa uphando malunga nee-oyster mushrooms (P. ostreatus NRC 620) lusancinci, izifundo zangaphambili zihlolisise ii-enzymes ezivela kwimithombo eyahlukeneyo ye-fungus, njengeTrametes versicolor kunye neGanoderma lucidum. Injongo yolu phononongo yayikukuvavanya ukusetyenziswa okunokwenzeka kwale enzyme kwishishini lokutya kunye nokugqamisa iimpawu zayo ezikhethekileyo, ngakumbi i-pH kunye nobushushu bayo obufanelekileyo.
I-2,2′-Azooxybis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) ithengwe kwiSigma-Aldrich (eKhanada). Zonke ezinye ii-reagents bezikumgangatho wohlalutyo.
IZiko leSizwe loPhando lweZiko leSizwe loQokelelo lweeMicrobial Culture lifumene uhlobo lwe-oyster mushroom oludumileyo i-NRC620. Emva kokulima okuncinci, olu hlobo lwagcinwa kwi-potato dextrose agar slants kwi-4°C. Indlela yokulungiselela i-inoculum yayilandelayo: i-mycelium eneentsuku ezili-10 ubudala, ephuhlileyo yafakwa kwi-potato dextrose agar plates yaza yafakwa kwi-28°C. Emva kweentsuku ezili-10, iibhloko ezintathu ze-mycelial ezinobubanzi obuyi-12 mm zasuswa kwi-agar media kusetyenziswa i-sterile metal punch zaza zafakwa kwi-250-mL Erlenmeyer flasks ezine-cotton plugs eziqulethe i-50 mL ye-sterilized culture medium (pH 5.0, njengoko kuchaziwe ngaphambili ngu-Othman et al.20Ezi zityalo zafakwa kwi-incubator kuma-28°C kangangeentsuku ezili-18. Ezi zityalo zahluzwa ngephepha lesihluzo leWhatman No. 1, kwaye i-supernatant ephumayo yasebenza njengomthombo we-enzyme.
Umsebenzi we-laccase ufunyenwe kusetyenziswa i-ABTS njengesiseko. Umxube we-reaction (2 mL) uqulethe i-500 μL ye-0.3 mM ABTS (enyibilikiswe kwi-0.1 M sodium citrate buffer, pH 4.5) kunye nomlinganiselo ofunekayo wesampulu ye-enzyme exutywe namanzi acocekileyo.21,22Xa kucingwa ukuba i-laccase inokuyinyibilikisa i-ABTS kubushushu begumbi (28 °C ± 2), ukunyibilikisa i-ABTS kumiselwe ngokulinganisa ukwanda kokufunxwa kwi-420 nm (ε420= 36,000 cm-1 M -1) kusetyenziswa i-Agilent Carry-100 UV spectrophotometer. Kwakufuneka iyunithi enye yomsebenzi we-laccase ukuze kufakwe i-oxidize i-1 μmol ABTS ngomzuzu. Uxinzelelo lweproteni lumiselwe ngendlela yaseBradford kusetyenziswa i-bovine serum albumin njengolawulo lwangaphakathi.23,24
Emva kokufumana i-enzyme kwi-oyster mushroom strain NRC 620, umsebenzi wayo ulinganiswe ngamaxesha ahlukeneyo okulima kangangeentsuku ezingama-25 phantsi kweemeko ezingashukumiyo kwi-28 °C.
Ukuze kufundwe impembelelo yobushushu kumsebenzi we-laccase, kwenziwe uvavanyo kumanqanaba obushushu ukusuka kwi-20 ukuya kwi-90 °C. Ngaphambi kokongeza i-enzyme kunye nokuqalisa i-reaction, i-buffer (0.1 M sodium citrate, pH 4.5) kunye ne-substrate (ABTS) zaxutywa zaza zafakwa kwi-incubator imizuzu emi-5 kubushushu obahlukeneyo. Uzinzo lobushushu be-enzyme luhlolwe ngokufaka kwi-incubator kwi-0.05 M sodium phosphate buffer (pH 7.0) kwi-40, 50, 60, kunye ne-70 °C iiyure ezi-2, ngokulandelelana. Umsebenzi oseleyo emva koko wavavanywa kusetyenziswa i-substrate ye-ABTS.
Impembelelo ye-pH kumsebenzi we-laccase ihlolwe kusetyenziswa i-ABTS njenge-substrate kwi-0.1 M citrate-phosphate buffers enoluhlu lwe-pH oluyi-2.5 ukuya kwi-7.0. Isisombululo se-enzyme sifakwe kwi-40°C kangangeeyure ezimbini kwi-0.1 M citrate kunye ne-Tris buffers (pH 3, 4, 6, kunye ne-7) ukuvavanya uzinzo lwe-pH. Umsebenzi oseleyo kunye ne-ABTS njenge-substrate ubalwe emva kokufakelwa.
I-laccase yafakwa kwi-incubator imizuzu eli-10 kwi-sodium phosphate buffer (0.05 M, pH 7.0) equlethe ii-ion zesinyithi ezahlukeneyo (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, kunye neMn2+) kumanqanaba e-2.5 mM kunye ne-10 mM, ngokulandelelana. Emva koko i-substrate (ABTS) yongezwa ukuqala ukusabela, kwaye umsebenzi ohambelanayo wavavanywa.
I-oxidation ye-ABTS yi-laccase kumanqanaba ahlukeneyo (0.025–3 mM) ilinganiswe kwi-pH 4.5 ukuze kuchongwe iiparameter ze-kinetic (Vmax kunye ne-Km).izinto ezingaguqukiyoye-Michaelis-Menten equation ibalwe kusetyenziswa i-Lineweaver-Burk plot, edweba ireciprocal ye-reaction rate njengomsebenzi woxinzelelo lwe-substrate. Ii-kinetic constants zibalwe kwi-Lineweaver-Burk plot kusetyenziswa isoftware yeGraphPad Prism version 6.01.
Emva kokuhlamba kakuhle ama-apile ngamanzi epompo, anqunyulwa phakathi aze afakwe ijusi kusetyenziswa i-comprehensive Braun MP80 apple juicer (eyenziwe eJamani). Ijusi yahluzwa ngeengqimba ezine ze-cheesecloth. Akukho enzymes zongezwa kwiqela lolawulo, ngelixa i-2.0% laccase (uxinzelelo olusebenzayo oluvavanyiweyo) yongezwa kwijusi ye-apile esandula ukwenziwa, eyathi emva koko yagcinwa kwi-4°C kangangeeveki ezimbini.
I-Titratable acidity (TA) kunye ne-pH zichongiwe ngokwendlela kaBoulton et.al.27. I-pH yesampulu nganye ilinganiswe kusetyenziswa imitha ye-pH yedijithali (i-JENWAY 3510 pH meter). I-Titratable acidity (TA) ibalwe ngokusekelwe kwi-malic acid kusetyenziswa le fomyula ilandelayo.
Apho i-V kunye ne-C ziyi-volume (mL) kunye noxinzelelo (0.1 mol/L) yesisombululo se-sodium hydroxide esisetyenziswa kwi-titration, ngokulandelelana. I-K yi-malic acid conversion coefficient, elingana ne-0.067, kwaye i-W yi-mass (g) yejusi yeapile.
Zonke izinto ezinyibilikayo (I-TDS) umxholo wazo zonke iisampuli zejusi umiselwe kusetyenziswa i-PAL-1 pocket refractometer (ATAGO, Tokyo, Japan). Emva kokulinganisa ngakunye, ilensi ye-optical ihlanjwe ngamanzi acocekileyo, kwaye isampuli nganye yejusi yeapile ivavanywe kathathu. Ixabiso lesampuli nganye libalwe ngokulinganisa imilinganiselo emithathu. Umlinganiselo ophakathi ± ukuphambuka okuqhelekileyo kwisampuli nganye yejusi yeapile ubalwa ngokulinganisa ezi ziphumo.
Ukuqina kwe-viscoelasticity yeesampuli zejusi yeapile kuhlolwe kusetyenziswa i-viscometer ejikelezayo (RV, Rheotest 2, eJamani). Isampuli ibekwe ngaphakathi kwi-"S2″ cylinder ye-viscometer. I-viscosity ebonakalayo imelwe yi-slope ye-shear stress vs shear rate curve, eyabalwa ukusuka kwi-shear stress kunye ne-curves ezihambelanayo kwi-shear rates ezahlukeneyo (ukusuka kwi-1.00 ukuya kwi-437.4 s⁻¹). Ifomula yokubala i-viscosity ebonakalayo yile ilandelayo:
Apho i-η yi-viscosity ebonakalayo (cP), i-τ yi-shear stress (dyn/cm²), i-γ yi-shear rate (sec⁻¹), kwaye i-(τ) ibalwa kusetyenziswa amaxabiso e-torque (α) kunye ne-cylinder (Z) kusetyenziswa le fomyula ilandelayo: τ = Z . α.
Isalathisi se-browning simiselwe ngokwendlela kaMeidav etal.29. Isampulu yejusi ye-10-ml ixutywe nge-centrifuge kwi-2750 xg kangangemizuzu eli-10. I-5 ml ye-juice supernatant ixutywe ne-5 ml ye-95% ye-ethanol. Ukufunxwa komxube kulinganiswe kwi-420 nm kusetyenziswa i-Shimadzu UV spectrophotometer (UV-1601 PC).
Umxholo we-phenolic opheleleyo (TPC) uchongiwe ngokwemibala kusetyenziswa i-reagent ye-Folin-Ciocalteu njengoko kuchaziwe nguBoulton et al.[27]]. Igophe eliqhelekileyo le-gallic acid lenzelwe amazinga ukusuka kwi-0 ukuya kwi-500 mg/L (r²= 0.997). Iziphumo zibonakaliswa njengee-gallic acid equivalents (mg GAE/mL).
Yongeza i-125 μL yamanzi acociweyo kunye ne-2850 μL yesisombululo se-FRAP kwi-25 μL yejusi yeapile uze uyishiye le mxube ebumnyameni ukuze30min. Emva koko ulinganise ukufunxwa kwi-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, kunye ne-10 mM 2,4,6-tris(2-pyridyl)triazine (TPTZ) (enyibilikiswe kwi-40 mM HCl) ngomlinganiselo we-10:1:1. I-curve esemgangathweni yenziwe kusetyenziswa i-Trolox njengomgangatho (I-R²= 0.999), kwaye iziphumo zichazwe njenge-μM Trolox/mL.
Umsebenzi we-antioxidant weejusi ezinyangiweyo nezinganyangwanga umiselwe kusetyenziswa indlela ye-DPPH ukuvavanya amandla azo okususa ii-free radicals ze-DPPH.31Ii-microliters ezilishumi zejusi zixutywe ne-1 ml yesisombululo se-DPPH (100 μM) kwi-methanol. Emva kokusabela ebumnyameni imizuzu engama-30, ukufunxwa komxube kulinganiswe kwi-517 nm kusetyenziswa i-Shimadzu UV spectrophotometer (UV-1601 PC). Iziphumo zichazwe njengee-trolox equivalents (μM trolox/ml) ngokusekelwe kwi-calibration curve (R2= 0.990).
Idatha efunyenweyo ibonise ukuba imveliso ephezulu ye-laccase ibonwe kwi-NRC 620 oyster mushrooms ekupheleni kosuku lwe-18 lokubila, ifikelela kumsebenzi we-1302 U/L. Oku kusebenze njengesiseko sokumisela ixesha elifanelekileyo lokulima lokuvelisa i-laccase (Umfanekiso 1). Nangona imveliso ye-enzyme inyuke ngokwanda kwexesha lokulima, izinga lokukhula lalingafani ngokuthe ngqo nexesha lokulima; emva kweentsuku ezingama-21, umsebenzi we-enzyme wanda nge-90 U/L kuphela (ukuya kwi-1390 U/L). Ke ngoko, iintsuku ezili-18 ekugqibeleni zakhethwa njengexesha elifanelekileyo lokulima ukulinganisela isivuno semveliso kunye neenzuzo zoqoqosho zokwanda kwexesha lokulima.
Isiphumo sexesha lokulima kwisivuno se-laccase kwi-Pleurotus ostreatus NRC 620. Iibhloko ezintathu (12 mm) ze-fungus mycelial zifakwe kwi-50 ml ye-sterile medium zaza zakhuliswa kwi-28 °C ngamaxesha ahlukeneyo.
Ngokuhambelana nezinye izifundo, iziphumo zethu zibonisa ukuba ixesha elifanelekileyo lokukhulisa i-laccase linokuba phakathi kweentsuku ezisi-7 nezingama-36.32Ngokutsho kuka-Ezike et al.33, *I-Trametes polyzona* I-WRF03 ivelise inani eliphezulu le-laccase ekupheleni kosuku lwesithoba lokubilisa, ngomsebenzi othile we-1637 U/mg protein. Ngaphezu koko, u-Othman et al.34bafumanise ukuba *iTrichoderma harzianum* S7113 ikhupha inani elikhulu le-laccase ngosuku lwesihlanu lokulima. Izinga lokuveliswa kwe-laccase lifikelele kwincopho yomsebenzi ngosuku lweshumi elinesine laza lancipha kancinci kancinci.34Nangona ukukhutshwa kwee-enzymes kunokwenzeka nakwisigaba sokukhula esiphambili, kudla ngokuba yincopho ngexesha lesigaba esiphakathi kwaye kubangelwa kukusetyenziswa komthombo wekhabhoni okanye we-nitrogen.34,35
Nangona i-laccase evela kwi-Pleurotus ostreatus NRC 620 ibonise umsebenzi ophezulu kumanqanaba obushushu ahlukeneyo ukusuka kwi-50°C ukuya kwi-80°C, isondela kumsebenzi ophezulu (69–98%), umsebenzi wayo ophezulu ubonwe kwi-70°C (Umzobo 2a). Ngaphandle kolu luhlu lobushushu, umsebenzi we-enzyme wehle malunga ne-70°C. Ezi ziphumo zibonisa ukuba i-enzyme iyasebenza kumazinga obushushu aphezulu, mhlawumbi ngenxa yokuba ubushushu obuphezulu bunyusa amandla e-kinetic e-reaction.
Isiphumo sobushushu bokusabela (a) kunye ne-pH (b) kumsebenzi we-laccase kwi-*Pleurotus ostreatus* NRC 620. Amaqondo obushushu aqala kwi-20 ukuya kwi-90 °C afezekiswe ngokufaka umxube kubushushu obahlukeneyo imizuzu emi-5 ngaphambi kokongeza i-enzyme kunye nokuqalisa i-reaction. Isiphumo se-pH kumsebenzi we-laccase sivavanywe kusetyenziswa i-ABTS njenge-substrate kwizisombululo eziqulethe i-0.1 M citrate-phosphate buffer kuluhlu lwe-pH oluyi-2.5 ukuya kwi-7.0.
Ngokutsho kuka-Ezike etal.33, ubushushu obufanelekileyo kwi *Trametes polyzona* WRF03 laccase yi-55 °C, nto leyo efanayo neyo kwi *Ganoderma lucidum*laccase36kwaye ifana nobushushu obufanelekileyo (50 °C) kwi *Trametes polyzona* KU-RNW02737i-laccase . I-Baldrian38uqaphela ukuba, ngokunxulumene nezinye iinkqubo ze-enzyme eziwohlokayo ze-lignin, ubushushu obufanelekileyo be-laccase buphakathi kwama-50 nama-70 °C.
Iziphumo zibonise ukuba i-enzyme ibonakalise umsebenzi ophezulu kwi-pH 3.0, ifikelela kwi-94% yomsebenzi kwi-pH 3.5. Nangona kunjalo, yahlala isebenza kuluhlu olubanzi lwe-pH ukusuka kwi-2.5 ukuya kwi-7.0 (Umfanekiso 2b). Ngaphezu koko, ibonakalise umsebenzi ophezulu kwiimeko ze-acidic xa kuthelekiswa neemeko ezingathathi cala okanye ze-alkaline. Umsebenzi wayo uhlale ubuncinci yi-77% kuluhlu lwe-pH ukusuka kwi-2.5 ukuya kwi-4.5, kodwa ifikelele kuphela malunga ne-38% kwi-pH 7.0. I-pH efanelekileyo ye-laccase evela kwi-*Trametes polyzona* WRF03 yayiyi-4.533, efana ne-pH ye-laccases evela kwi-*Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, kunye *Trametes hirsuta* 41. Nangona kunjalo, ngokutsho kophando lukaChairin et al.42, i-pH efanelekileyo ye-laccase evela kwi-*Polymorpha f. sp.* I-WR710-1 yi-2.2, ngelixa i-pH efanelekileyo ye-laccase evela kwi-*Polymorpha f. sp.* IBL-04 yi-5.043. Ukubopha ii-hydroxide anions (i-laccase inhibitor) kwiiathom zobhedu ze-T2/T3 laccase kusenokuba sisizathu sokuncipha komsebenzi we-laccase phantsi kweemeko ze-pH ezingathathi cala okanye ze-alkaline. Oku kunokuphazamisa ukudluliselwa kwe-electron yangaphakathi ukusuka kwiziko le-T1 ukuya kwiziko le-T2/T3, ngaloo ndlelaukunciphisaumsebenzi we-enzyme23,44
Ngokufaka i-enzyme kumaqondo obushushu ahlukeneyo, kwafunyaniswa ukuba ixesha lokufunxa kunye nobushushu zichaphazele uzinzo lwe-enzyme. Okuphawulekayo kukuba, i-laccase evela kwi-*Trametes polyzona* NRC 620 ibonakalise uzinzo oluphezulu kwi-40℃ kunye ne-50℃, igcina i-68.33% kunye ne-59.61% yomsebenzi wayo wokuqala, ngokulandelelana, emva kwemizuzu eyi-120 (Umfanekiso 3a). Ngokwahlukileyo koko, phantsi kweemeko ezifanayo (40℃ kunye ne-50℃, imizuzu eyi-120), i-laccase evela kwi-*Trametes polyzona* WRF03 igcine i-64.38% kunye ne-42.92% yomsebenzi wayo, ngokulandelelana.33Ngokwahlukileyo koko, ukwanda kwexesha lokufunxa kunye nobushushu kunciphisa ukuzinza kwe-*Trametes polyzona* NRC 620 laccase; Emva kokufunxa kwi-60℃ kunye ne-70℃ imizuzu engama-60, umsebenzi wayo wehla waya kwi-39.24% kunye ne-1.72%, ngokulandelanayo (Umfanekiso 3a). Ngokuhambelana neziphumo zovavanyo, i-laccase evela kwi-*Trametes polyzona* WRF03 ibonise ukuzinza okuphezulu kwi-40 kunye ne-50℃ kuyo yonke inkqubo yonyango lobushushu.33Ngokufanayo, uLueangjaroenkit etal.37kunye noSihlalo etal.42babike ukuzinza kwee-laccases ezivela kwi-Trametes polyzona KURNW027 kunye ne-Trametes polyzona WR710-1 kwi-50 °C kangangeyure e-1, ngokulandelelana. Njenge-biocatalyst eluncedo esebenza kwiindawo ezahlukeneyo ze-biotechnology, i-laccase kufuneka ibe nokuzinza okuhle kunye nokusebenza kakuhle kumanqanaba obushushu abanzi.
Uzinzo lwe-thermostatic (a) kunye nozinzo lwe-pH (b) lwe-laccase evela kwi-*Pleurotus ostreatus* NRC 620. Uzinzo lwe-thermostatic luvavanywe ngokufaka isisombululo se-enzyme kwi-0.05 M sodium phosphate buffer (pH 7.0) kwi-40, 50, 60, kunye ne-70 °C kangangeeyure ezi-2, ngokulandelelana. Uzinzo lwe-pH luvavanywe ngokufaka isisombululo se-enzyme kwi-0.1 M citrate buffer kunye ne-Tris buffer (pH 3, 4, 6, kunye ne-7) kwi-40 °C kangangeeyure ezi-2. Umsebenzi oseleyo ubalwe kusetyenziswa i-ABTS njenge-substrate emva kokufaka.
Ukuze sifumanise iimeko ezifanelekileyo zokusetyenziswa kunye nokugcinwa kwee-enzyme, siphande ngempembelelo ye-pH ekuzinzeni kwe-laccase. Ukuvezwa kwiixabiso ezahlukeneyo ze-pH kuchaphazele kakhulu uzinzo lwesakhiwo seprotheni, ngaloo ndlela kuchaphazele uzinzo kunye nomsebenzi wemolekyuli ye-enzyme. Iziphumo zibonise ukuba i-enzyme yayingazinzanga kangako phantsi kweemeko ze-asidi, ngelixa ibonise uzinzo olungcono kwiixabiso eziphezulu ze-pH (imimandla engathathi hlangothi neye-alkaline). Kwiixabiso ze-pH ze-7.0, 6.0, 4.0, kunye ne-3.0, amazinga okugcinwa kwee-enzyme emva kwemizuzu eyi-120 ayemalunga ne-100%, 62.54%, 52.39%, kunye ne-11.14%, ngokulandelanayo (Umzobo 3b). *I-Strombus multisus* WRF03 laccase ibonise uzinzo oluphezulu kwiixabiso ze-pH ezingathathi hlangothi (5.5–6.5) kunye nokuzinza okuphantsi kwiixabiso ze-pH ezingathathi hlangothi (ngaphantsi kwe-4.0). Emva kwemizuzu eli-120 kwixabiso le-pH eliyi-5.5, 6.0, kunye ne-6.5, amazinga okugcinwa kwee-enzyme ayemalunga ne-82%, 100%, kunye ne-93%, ngokwahlukeneyo.33UKhairin nabanye.42baqaphele ukuba i-laccase evela kwi-Trametes polyzona WR710-1 yayizinzile kuluhlu lwe-pH oluyi-6.0 ukuya kwi-7.0, ngelixa uSayed et al.45ibonise ukuba i-laccase yayizinzile ngakumbi phantsi kweemeko ze-pH ezingathathi hlangothi. Nangona kunjalo, i-laccase evela kwi-Cerrena unicolor ikwabonakalise ukuzinza phantsi kweemeko ze-alkaline (pH 9.0)46Ii-laccases ezifundweyo zibonise uzinzo oluphezulu kuluhlu olubanzi lwe-pH. Oku kungaba luphawu olubalulekileyo kwizicelo zoshishino.
Ekubeni ezinye ii-ion zesinyithi zineziphumo zokuvuselela kunye nezokuthintela kumsebenzi we-enzyme, iziphumo zazo kumsebenzi we-enzyme kufuneka ziqwalaselwe kwizicelo zoshishino. Oku kubalulekile kuba ii-ion zesinyithi zizinto ezingcolisayo eziqhelekileyo kwindalo ezinokuchaphazela uzinzo kunye nokuhlanganiswa kwee-enzymes ezingaphandle kweseli.47Ukuze siphande ngemiphumo yee-ion ezininzi zesinyithi kwi-laccase evela kwi-*Pleurotus ostreatus* NRC 620, senze iimvavanyo ezifanayo. Njengoko kubonisiwe kuMfanekiso 4, ngokuxhomekeke kuhlobo lwesinyithi esisetyenzisiweyo, ukwandisa uxinzelelo lwee-ion zesinyithi ukusuka kwi-2.5 mM ukuya kwi-10 mM kuyichaphazele kakubi umsebenzi we-enzyme. Umzekelo,I-Mg²⁺ , I-Co²⁺ , Zn²⁺kunyeI-Cu²⁺inokuvuselela kwaye ivuselele umsebenzi we-enzyme, ngelixaNa⁺ , Mn²⁺ , I-Ca²⁺kunyeK⁺inokuthintela umsebenzi we-enzyme. Kwi-concentration ye-10 mM, ii-ion ze-Cu²⁺ kunye ne-Mg²⁺ bezizezona zivuselelayo zinamandla zomsebenzi we-laccase ezivela kwi-*Pleurotus ostreatus* NRC 620, zibonelela nge-activation degree emalunga ne-34% kunye ne-20%, ngokulandelelana. Nangona kunjalo, kwi-concentration ye-10 mM, ii-ion ze-Ca²⁺ bezizezona zithintelayo i-laccase, zinciphisa umsebenzi we-enzyme malunga ne-60%.
Impembelelo yee-ion zesinyithi kumsebenzi we-Pleurotus ostreatus NRC 620 laccase. I-laccase yafakwa imizuzu eli-10 kwi-sodium phosphate buffer (0.05 M, pH 7.0) equlethe ii-ion zesinyithi ezahlukeneyo kumanqanaba e-2.5 mM kunye ne-10 mM. Emva koko impendulo yaqaliswa ngokongezwa kwe-substrate (ABTS), emva koko umsebenzi ohambelanayo walinganiswa.
Iziphumo zethu zihambelana nezo zabanye ababhali abafumanise ukuba iMg²⁺ kunye neCu²⁺ ziyawuphucula umsebenzi we *Trametes polyzona* WRF03³. UCastaño et al.⁴⁸ bafumanise ukuba i-laccase evela kwi *Xylaria* sp. ivuselelwa ngandlela thile zii-ion ze-copper (Cu²⁺). Ngaphezu koko, uForoutanfar et al.⁴⁹ kunye noSi et al.⁵⁰ benze izifundo ezifanayo kwi-laccases ezivela kwi *Paraconiothyrium variabile* kunye *Trametes pubescens*, ngokwahlukeneyo. Indawo yokubopha i-copper-binding (T2) yohlobo lwesibini (i-T2) yale enzyme inokugcwala yiCu²⁺ kuxinzelelo oluthile, olunokuchaza ukukhuthazwa komsebenzi we-laccase kuxinzelelo oluphezulu lweCu²⁺³⁹. Ekubeni ii-white rot fungi laccases ziyi-oxidases eziqulethe ii-athomu ezininzi ze-copper, iziphumo zee-copper ion kumsebenzi we-laccase zahlukile kwaye ziqala ekuvuseleleni nasekuthinteleni ukuya ekungathathi hlangothi.⁵¹ Ngokwahlukileyo koko, uZhou et al.. [52]ingxelo yokubaI-Cu²⁺ithintele umsebenzi we-laccase we-Taiwan subterranean termite (Odontotermes formosanus). Nangona kunjalo, i-laccases yeCerena sp. HYB07[53]kunye neClitocybe maxima[54]azichaphazelekanga zii-ion zekopolo.
Ukucaciswa kwe-substrate kubonakaliswe ziiparameter zayo ze-kinetic (Km kunye ne-Vmax); okukhona ukuqina kokubopha kwe-substrate kwi-enzyme, kokukhona ixabiso le-Km liphantsi kwaye kokukhona ukucaciswa kwe-substrate kuphezulu.3,21,55Iiparameter ze-kinetic (Km kunye ne-Vmax) ze-laccase ezivela kwi-*Pleurotus ostreatus* NRC 620 zichongiwe kusetyenziswa isoftware yeGraphPad Prism 6.0 ngokuzoba iploti yeLineweaver-Burk (Umfanekiso 5). Xa kusetyenziswa i-ABTS njenge-substrate, iziphumo zaba yi-1.99 mM kunye ne-16217 μmol.min⁻¹ L⁻¹,ngokulandelelana. UElsayed et al.21Ixele ukuba amaxabiso e-Km e-ABTS oxidation yayiyi-0.1 mM kunye ne-0.064 mM, ngokwahlukeneyo, nto leyo ebonisa ukuba i-Lac A kunye ne-Lac B isoenzymes zinamandla kakhulu kwi-ABTS. Ngaphezu koko, amaxabiso e-Vmax yayiyi-0.182 μmol.min⁻¹kunye ne-0.603 μmolmin⁻¹, ngokulandelelana. Ixabiso le-Km elifunyenweyo beliphantsi kunelo leTrametes polyzona WRF03 (8.66 mM); ngaphezulu koko, ixabiso labo le-Vmax (1429 mmol min⁻¹) nalo beliphantsi.ezantsixa kusetyenziswa i-ABTS njenge-substrate.33 Ngokufanayo, amaxabiso e-Km e-Lentinus squarrosulus MR13 kunye ne-Trametes sp. Amanqanaba e-AH28-2 laccase ayeyi-0.0714 mM kunye ne-0.025 mM, ngokwahlukeneyo, kwaye amaxabiso e-Vmax ayeyi-0.0091 mM min−1 kunye ne-0.67 mM min−1 mg−1 (ngokumalunga ne-ABTS), ngokulandelelana.56,57
Isiphumo soxinzelelo lwe-ABTS kumsebenzi we-laccase evela kwi-*Pleurotus ostreatus* NRC 620 siphandwe, kwaye kwadweliswa iploti ye-Lineweaver-Burk yesantya sokuqala sokusabela ngokuchasene noxinzelelo lwe-ABTS. Impendulo ye-oxidation ye-ABTS enezinga ezahlukeneyo (0.025–3.0 mM) ye-laccase ilinganiswe kwi-pH 4.5 ukumisela iiparameter ze-kinetic (Vmax kunye ne-Km). Ii-Michaelis-Menten kinetic constants zibalwe kusetyenziswa iploti ye-Lineweaver-Burk yesantya sokusabela ngokuchasene noxinzelelo lwe-substrate. Ii-kinetic constants zibalwe kwiploti ye-Lineweaver-Burk kusetyenziswa isoftware yeGraphPad Prism 6.01.
Iienzymes zemveli zokucacisa, ezifana neepectinase, i-hydrolyze pectic substances, zinciphisa i-viscosity kunye ne-turbidity. Ziyaphula ngempumelelo ii-polysaccharides zesakhiwo kwaye zihlala zisetyenziswa kunye nezinye ii-enzymes, ezifana nee-cellulases kunye nee-hemicellulases, ukuphucula isivuno kunye nokucaca. Nangona kunjalo, ii-pectinase azijolisi ngokukodwa kwiikhompawundi ze-phenolic, ezizezona zinto ziphambili ezibangela i-turbidity kunye ne-oxidative browning, ngakumbi kwiijusi ezifana nejusi ye-apile kunye nediliya.58Ngokwahlukileyo koko, ii-laccases zivuselela i-oxidation yee-phenolic compounds, zizenza i-polymer zibe zii-molecules ezinkulu, ezinganyibilikiyo ezinokususwa ngokutyibilika okanye ngokuhluzwa. Le ndlela ayiphuculi nje kuphela ukucaca kodwa yandisa ubomi beshelufu yejusi ngokunciphisa amathuba okuba i-oxidative browning ebangelwa zii-phenolic compounds. Ngaphezu koko, iinkqubo zokucacisa ezisekelwe kwi-laccase zinokwenziwa phantsi kweemeko zokucubungula ezithambileyo (pH 3.5–5.5, ubushushu obuyi-25–40 °C), nto leyo ezenza zifaneleke kwiijusi ezithambileyo ngaphandle kokuphazamisa iipropati zazo zesondlo okanye ze-organoleptic.59Izifundo zibonise ukuba unyango lwe-pectinase lunokucacisa ijusi kwiiyure ezi-1-2, ngelixa unyango lwe-laccase ludla ngokufuna ixesha elide lokuphendula (iiyure ezi-3-6) ukuze kuncitshiswe ngokupheleleyo iikhompawundi ze-phenolic. Nangona kunjalo, le nkqubo inokulungiswa ngokuthintela i-enzyme okanye ngokudibanisa i-laccase neendlela zokucacisa ngoomatshini.60Kolu phononongo, iprofayili ye-enzyme ye-crude extract ibonakalise imisebenzi ebalulekileyo ye-laccase kunye ne-α-amylase, ngelixa imisebenzi ye-pectinase kunye ne-xylanase yayiphantsi kakhulu, kwaye umsebenzi we-cellulase awuzange ufunyanwe. Ke ngoko, ukuncipha kwe-turbidity kunye nomxholo we-phenolic kwakubangelwa ikakhulu yintshukumo ye-laccase, ngelixa utshintsho kwi-viscosity lunokuba ngenxa yesenzo se-amylase.
Itheyibhile 1 ibonisa iiparameter ze-physicochemical zeesampuli zejusi yeapile ezisandula ukucocwa kunye neesampuli eziphathwe nge-laccase. Iziphumo zibonise ukuba isivuno sejusi yeapile esandula ukucocwa (71.59%) besingaphantsi kuneso seesampuli eziphathwe nge-laccase (87.34%). Ezi ziphumo ziyahambelana neziphumo zikaPilnik no-Orange.61, obonise ukuba ukusetyenziswa kwee-enzymes ekucutshungulweni kweziqhamo kunokunyusa isivuno sejusi, kuphucule ukuhluzwa, kwaye kufumaneke ijusi esemgangathweni ophezulu necacileyo yokugxila. Ukwanda kwesivuno sejusi kubangelwa ikakhulu kukwanda komxholo weeswekile ezinyibilikayo kwijusi. Ngexesha le-enzyme hydrolysis yeziqhamo, i-mesoglea kunye ne-pectin eludongeni lweseli yemveliso ziyatshatyalaliswa kwaye ziguqulwe zibe zizinto ezinyibilikayo ezifana neeswekile ezingathathi cala kunye nee-asidi.62.Ixabiso le-pH yejusi yeapile ephathwe nge-enzyme yayiphantsi kakhulu kunelo leqela lolawulo (P < 0.05), kwaye ixabiso le-pH lamaqela omabini landa kakhulu ngexesha lokugcinwa (Itheyibhile 1). Ezi ziphumo zihambelana nezo zikaMark et al.63, ophawule ukuba i-pH yejusi yeziqhamo ze-cashew yehlile emva kokugcinwa emva konyango lobushushu. Ukonakala kwe-pectin kunye nokwakheka kwe-galacturonic acid emva konyango lwe-enzyme kunokubangela ukunyuka kwe-pH ngexesha lokugcinwa. I-pH yeesampuli eziphathwe nge-enzyme zihlale ziphakathi kwe-4.05 kunye ne-4.31 ngexesha lokugcinwa, ngelixa i-pH yejusi ye-apile enganyangwanga yayiphakathi kwe-4.12 kunye ne-4.33.
I-acidity iyonke (TA) yeesampuli ezinganyangwanga kunye nezinganyangwanga nge-laccase ibonise umkhwa wokuncipha ngokuhamba kwexesha lokugcina (Itheyibhile 1). Ukwehla kwe-acidity kubangelwe kukuguqulwa kwee-organic acids zibe yi-carbohydrates okanye ii-enzyme reactions, kunye ne-oxidation ngexesha lokugcina ijusi.64I-asidi iyonke yejusi yeapile elawulwayo kunye neesampuli eziphathwe nge-enzyme yayingaphantsi kuneyezinye iijusi (ijusi yestrawberry 0.9%, ijusi yeplum 2.2%, ijusi yekumquat 1.0%, ijusi yeapricot 2.4%, ijusi yeorenji 0.8%), kodwa ifana neyezinye iijusi (umz., ijusi yepere 0.3%).62Olu mahluko kwijusi yeapile esandul’ ukucocwa enganyangwanga lunokubangelwa zizinto ezahlukeneyo ezifana neemeko zokukhula, izinto zemfuza, inqanaba lokuvuthwa, kunye neendlela zokuyilungisa.65Ukwehla kwe-asidi iyonke yokulawula kunye nejusi yeapile ephathwe nge-laccase kuhambelana neziphumo ezivezwe nguSingh et al.66malunga nokwehla kwe-asidi iyonke yejusi yeapile iJin Nuo emva kweentsuku ezingama-74 zokugcina. Kwelinye icala, uOshmiansky noWojdylo67Ayifumananga naluphi na utshintsho olubalulekileyo kwi-asidi yejusi yeapile xa befunda impembelelo yeendlela zemveli zokucacisa.
Iziphumo eziboniswe kwiTheyibhile 1 zibonisa ukuba ixabiso le-total solids (TSS) lejusi yeapile ephathwe nge-laccase laliphezulu kunelesampuli engakhange inyangwe. Ezi ziphumo ziyahambelana nezifundo ezipapashweyo.. 68Ngaphezu koko, iTheyibhile 1 ibonisa ukuba ixabiso le-TSS leqela lejusi yeapile elilawulayo laliyi-9.58 ngexesha lokuqala kwaye lafikelela kwi-11.05 ekupheleni kwexesha lokugcina. La maxabiso aphantsi kunexabiso le-TSS lejusi yeapile entsha echazwe nguHamid et al.. 69(11.2 kunye ne-11.80, ngokulandelelana). Ixabiso le-TSS leesampuli zejusi yeapile ephathwe nge-laccase landa kakhulu, liqala kwi-11.23 laza lafikelela kwi-12.93 emva kweeveki ezimbini zokugcina kwi-4°C (Itheyibhile 1). Ukwanda okufanayo kwi-TSS ngexesha lokugcina kwabonwa nakwiziqhamo ze-citrus, iilamuni, kunye neeorenji ezimnandi. Ukwanda kwe-total solids soluble (TSS) ngexesha lokugcina kusenokubangelwa yi-hydrolysis ye-polysaccharides (isitatshi) ibe yi-monosaccharides (iswekile), ukwanda koxinzelelo ngenxa yokuphelelwa ngamanzi kwijusi, kunye nokuwohloka kwe-pectin kwijusi ibe yi-solids soluble. Ukwanda kwe-total solids soluble (TSS) kusenokwenzeka ukuba kungenxa yokwanda kwe-soluble sugar, enokwenziwa kukuguqulwa kwe-pectin okanye i-cellulose ibe yi-soluble sugar yi-pectin okanye i-cellulase, ngokulandelelana, okanye yi-hydrolysis ye-starch ibe yiswekile, njengoko kubikwe nguHamed et al.69.Isiphumo se-laccase kwiipropati zejusi yeapile sinokubonwa ngokubonakalayo, njengoko ijusi yeapile ephathwe nge-laccase ibonisa ukuhamba kakuhle kunye nokukhanya okuphantsi kunejusi enganyangwanga. Olu qwalaselo lurekhodwe kwiTheyibhile 1; Ukukhanya kwesampulu ephathwe nge-enzyme yayiyi-1.87 cP, ngelixa ukukhanya kwesampulu yokulawula yayiyi-2.95 cP. Oku kuncipha okukhulu kokukhanya kusenokwenzeka ukuba kungenxa yomthamo ophezulu wokubamba amanzi wezinto ezifana ne-pectin kunye nokwenziwa kwesakhiwo senethiwekhi esidibeneyo.
Kolu phononongo, isiphumo se-laccase kwi-browning index (BI) yejusi yeapile sihlolwe ngokulinganisa ukufunxwa kwi-420 nm kusetyenziswa i-spectrophotometer. Iziphumo ziboniswe kwiTheyibhile 1. Ngexesha lokugcina, i-BI yeesampulu zejusi yeapile kumaqela anyangiweyo nalawo anganyangwanga ibonise umkhwa okhulayo kancinci kancinci. I-BI ibonisa inqanaba lokuba mdaka kwaye ingasebenza njengeinto ebalulekileyoisibonisi seempendulo ze-enzymatic kunye ne-non-enzyme browning. Ukufunxwa kwanda kakhulu ngexesha lokugcina (P < 0.05). Ekupheleni kokugcina,A420Ixabiso leesampuli zejusi yeapile kumaqela alawulwayo naphethwe ziienzyme linyuke malunga ne-217% kunye ne-121%, ngokulandelelana (Itheyibhile 1). Iziphumo zibonisa ukuba unyango lweeenzyme lunokunciphisa ngempumelelo inqanaba le-browning malunga ne-56%. Iziphumo zikaBezerra et al.[19]] ziyahambelana neziphumo zethu; Basebenzise i-laccase-glutaraldehyde-coconut fiber ukucacisa ijusi yeapile, benciphisa umbala wayo wokuqala nge-61%.
Nangona iipolyphenols ezikwijusi yeziqhamo zinefuthe elihle kwisondlo nakwimpilo yomzimba womntu, zinokusabela neeproteni, zibangele ukuba ijusi ibe namafu, ukubola, okanye ukudumba, ngaloo ndlela zitshintshe incasa nevumba lemveliso kwaye zinciphise ixesha layo lokuphelelwa.71Injongo yolu phononongo yayikukunciphisa ngokukhuselekileyo umxholo we-phenolic compound yejusi yeapile kusetyenziswa i-laccase evela kwi-Pleurotus ostreatus NRC 620. Iziphumo ezivezwe kwiTheyibhile 1 zibonisa ukuba umxholo we-phenolic compound iyonke yejusi yeapile ephathwe nge-laccase wehliswe kakhulu ngaphambi kokugcinwa kwi-4 °C. Ngaphezu koko, umxholo we-phenolic compound iyonke nawo wehla ngexesha lokugcinwa kuzo zombini iisampuli ezifundweyo (Itheyibhile 1). Uphando olwenziwe nguSandri et al.72ibonise ukuba ijusi yeapile ephathwe ngee-enzyme inokugcina umsebenzi wayo we-antioxidant kunye nomxholo we-phenolic compound. Nangona kunjalo, iziphumo zophando lukaLettera et al.73bonisa ukuba unyango lwejusi yeorenji nge-fungus laccase lunokunciphisa umxholo wee-phenolic compounds kuyo ukuya kuthi ga kwi-45%.
Iikhompawundi zephenolic zibonakalisiwe zineempawu ezifana nokususa ii-free radicals, ukunciphisa kunye nokucima i-singlet oxygen, ukudluliselwa kwee-atom ze-hydrogen, kunye nokunikezelwa kwee-electron kwi-free radicals, nto leyo eyenza zibe zii-antioxidants ezinamandla.74Ngoko ke, kolu phononongo, iindlela ezisekelwe kwi-DPPH kunye ne-FRAP zisetyenzisiwe ukuvavanya impembelelo ye-laccase kumsebenzi we-antioxidant wejusi yeapile egcinwe efrijini kangangeentsuku ezili-14 (Itheyibhile 2). Zombini ezi ndlela zibonise ukwanda komsebenzi we-antioxidant ngexesha lokugcina, okunokubangelwa kukwanda kwee-phenolic compounds zasimahla okanye ukwenziwa kweemveliso ze-Maillard reaction (MRPs), apho iimveliso ze-Maillard reaction zisenokuba yimbangela yokwanda komsebenzi we-antioxidant.75Iimpendulo ezingezizo ze-enzyme browning (kuquka ukubola kwe-ascorbic acid, ii-Maillard reactions, kunye nokubola kweswekile okubangelwa yi-acid) zivelisa imibala emdaka (ii-melanoidins). Iimveliso zokubola kwe-ascorbic acid eziphakathi kunye neemveliso zokubola kweswekile (ezifana nee-carbonyl compounds) zinokusabela kunye nee-amino acids ngokusebenzisa ii-Maillard reactions.76Nangona ukujika kweziqhamo nemifuno ibe mdaka ngexesha lokugcinwa kwayo kufundwe ngokubanzi, ukuqonda kwethu ezi mpendulo kusalinganiselwe.77Xa kuthelekiswa nendlela ye-FRAP, ijusi yeapile ephathwe nge-laccase ibonise umsebenzi ophantsi kakhulu we-antioxidant ngendlela ye-DPPH (Itheyibhile 2), kwaye umsebenzi we-antioxidant wazo zonke iisampuli wanda kakhulu ngokuhamba kwexesha lokugcina. Iindlela ezimbini ezahlukeneyo zokumisela umsebenzi we-antioxidant zisetyenzisiwe kolu phononongo kuba imigaqo yazo yahlukile. Indlela ye-DPPH ilinganisa amandla okuphelisa ii-free radicals, ngelixa indlela ye-FRAP ilinganisa amandla okunciphisa ii-ion zesinyithi. Ke ngoko, kuyacetyiswa ukusebenzisa iindlela ezininzi zokumisela umsebenzi we-antioxidant ukuze uqonde ngcono umsebenzi we-antioxidant weesampuli ezifundweyo.78
Esinye seziphumo eziphambili zolu phononongo kukuba *Pleurotus ostreatus* laccase NRC 620 ibonisa umsebenzi olungileyo kwi-70°C kunye ne-pH 3.0. Xa kuthelekiswa nezinye ii-laccases zefungus ezisetyenziswa rhoqo ekucaciseni ijusi, ezifana ne *Trametes versicolor* kunye ne *Ganoderma lucidum* laccases, *P. ostreatus* NRC 620 ibonisa uzinzo oluphezulu lobushushu kunye ne-pH ene-asidi engakumbi. Ii-Laccases ezivela kwi *Trametes versicolor* kunye ne *Ganoderma lucidum* zihlala zibonisa umsebenzi olungileyo kuluhlu lwe-50-60°C kunye namaxabiso e-pH aphakathi kwe-3.5 kunye ne-5.0. Lo mahluko unokuba negalelo ekuphuculeni ukusebenza kakuhle kwejusi, ngakumbi kwiijusi ze-asidi apho ukuzinza kwixabiso le-pH eliphantsi kubalulekile. Uphawu olukhethekileyo lwe *P. Xa kuthelekiswa nezinye ii-laccases zefungus ezifundwe, *Pleurotus ostreatus* NRC 620 ibonisa amandla okusebenza ngempumelelo phantsi kweemeko ezinzima ngakumbi. Ubushushu bayo obuphezulu bomsebenzi bubonisa iingenelo ezinokubakho kwizicelo zoshishino, ezinje ngamazinga okusabela ngokukhawuleza kunye nokunciphisa ungcoliseko lweentsholongwane. I-pH yayo ephantsi, ehambelana kakuhle nemvelo ye-asidi yeejusi ezininzi, inokuba luncedo kwiinkqubo zokucacisa ijusi. Ezi ziphumo ziqinisekisa uphando olongezelelekileyo lokusetyenziswa okukhulu, okwenza *Pleurotus ostreatus* NRC 620 ibe yindlela efanelekileyo endaweni yemithombo ye-laccase yesikhunta yendabuko. Xa kuthelekiswa nezifundo zangaphambili, sifumanise ukuba ubushushu obufanelekileyo yi-60°C kwaye i-pH efanelekileyo yi-3.0. Emva kokusabela kwi-60°C imizuzu engama-80, *Ganoderma lucidum* laccase igcinwe.46% yomsebenzi wayo.79 Ngokutsho kukaKurniawati noNicelle80, ii-enzymes ze-*Ganoderma lucidum* zibonakalisa uzinzo olugqwesileyo ukuya koluphakathi kwi-25°C kunye namaxabiso e-pH aqala kwi-5.0 ukuya kwi-8.0, kunye nozinzo kwi-pH 6.0 kunye namaqondo obushushu aqala kwi-10 ukuya kwi-30°C. Kolu phononongo, sifumanise ukuba i-pH kunye namaqondo obushushu afanelekileyo kumsebenzi we-enzyme we-*Pleurotus ostreatus* yayiyi-3.0 kunye ne-70°C, ngokwahlukeneyo. Emva kokufunxwa kwi-40°C kunye ne-50°C iiyure ezimbini, i-enzyme igcine i-68.33% kunye ne-59.61% yomsebenzi wayo, ngokwahlukeneyo. Ngaphezu koko, i-Pleurotus ostreatus NRC 620 laccase ibonakalise umsebenzi ophezulu kumanqanaba obushushu abanzi ukusuka kwi-50°C ukuya kwi-80°C, phantse ifikelele kumsebenzi ophezulu (69%–98%), kunye nomsebenzi ophezulu obonwe kwi-70°C.
Ukuqukumbela, i-oyster mushroom laccase NRC620, efunyenwe phantsi kweemeko ezizinzileyo, ibonise umsebenzi olungileyo kunye nozinzo kuluhlu lwe-pH kunye neemeko zobushushu, ibonisa uzinzo oluphezulu xa kuthelekiswa neminye imithombo ye-enzyme. Ukongezwa kwe-10 mM MgSO₄ kunye ne-CuSO₄ kwandisa umsebenzi we-enzyme malunga ne-21% kunye ne-35%, ngokulandelelana. Xa icutshungulwa ibe yijusi yeapile, i-enzyme yanciphisa i-pH kunye ne-viscosity, ngelixa umxholo we-phenolic wehla kancinci ngexesha lokugcina.
Iziphumo ziqinisekisa amandla e-laccase kwishishini lokutya, ngakumbi ekucaciseni iziselo. Ngokuqhekeza ngokukodwa iikhompawundi ze-phenolic, i-laccase ayinciphisi nje kuphela ukudumba kwaye iphucula ukucaca kodwa ikwagcina umgangatho weejusi zeziqhamo phantsi kweemeko zokusebenza ezithambileyo. Ngokungafaniyo neearhente zokucacisa zemveli ezifana ne-gelatin, i-bentonite, kunye ne-silica gel, i-laccase ayivelisi inkunkuma okanye isuse amavumba amnandi kwiziselo, nto leyo eyenza ibe lukhetho olunobuhlobo nokusingqongileyo noluzinzileyo. Ngaphezu koko, xa kuthelekiswa nezinye ii-enzymes kunye neendlela zokucoca, i-laccase inikezela ngesisombululo esijolise kuso nesingabizi kakhulu ngaphandle kokubeka emngciphekweni umgangatho wemveliso.
UKyomuhimbo, i-HD kunye neBrink, i-HG. Ukusetyenziswa kunye namaqhinga okuthintela ukuhamba kwe-laccases equlethe ubhedu; uphononongo. Heliyon 9, e13156 (2023).
Ixesha leposi: Disemba-15-2025