Umusaruro n'imiterere ya biochemical y'ibihumyo bya oyster laccase NRC 620 n'isuzuma ry'ubushobozi bwabyo mu gusobanura neza umutobe w'amapera.

Nyuma y'iminsi 25 yo kwibasira mu buryo butaziguye kuri 28°C, laccase yo muri *Pleurotus ostreatus* NRC620 yagaragaje imikorere myiza cyane mu buryo bwo gukwirakwiza ibihumyo. pH nziza n'ubushyuhe kuri iyi enzyme byari 3.0 na 70°C, uko bikurikirana. Nyuma y'amasaha 2 yo kwibasira mu buryo butaziguye kuri 40°C na 50°C, imikorere ya enzyme yagumye kuri 68.33% na 59.61%, uko bikurikirana. Nyuma y'amasaha 2 yo kwibasira mu buryo butaziguye muri citrate-phosphate buffer (pH 7.0), imikorere ya enzyme yagumye kuri 100%. Kongeramo 10 mM MgSO₄ na CuSO₄ byongereye imikorere ya enzyme ku kigero cya 21% na 35%, uko bikurikirana, mu gihe NaCl, MnCl₂, KCl, na CaCl₂ byabujije imikorere ya enzyme. Hakoreshejwe ABTS nk'umusemburo, ibipimo bya kinetic (Km na Vmax) bya *Pleurotus ostreatus* NRC 620 laccase byari 1.99 mM na 16.217 μmol min−1 L−1, uko bikurikirana. Uburyo bwo kuvura ingero z'umutobe w'amapera bwagabanyije cyane pH na viscosity, kandi uku kugabanuka kwajyanye no kwiyongera kw'igihe cyo kubika. Uburyo bwo kuvura laccase bwagabanyije gato ingano ya phenolic mu mutobe w'amapera, ariko nta kugabanuka k'imikorere ya antioxidant kwagaragaye.
Mu myaka ya vuba aha, abashakashatsi bibanze ku ikoreshwa rya biotechnology ihumanya ikirere mu nganda z'ibiribwa. Laccase ni imwe muri enzymes z'ingirakamaro cyane mu nganda z'ibiribwa, ikaba ikoreshwa mu nzego nko gutunganya imitobe, guteka, kubungabunga divayi, no kunoza imiterere y'ibikomoka ku biribwa.¹Ibimera byinshi byo hejuru n'udukoko duto cyane bisohora laccase,²kandi ibihumyo nka deuteromycetes, ascomycetes, na basidiomycetes nabyo bishobora gutera laccase.³Laccase (EC 1.10.3.2) ni oxidase y'ubururu igabanya ogisijeni ya molekile ikayishyira mu mazi ikoresheje uburyo bugizwe n'atome eshatu zitandukanye z'umuringa, bityo ikagabanya ibintu bitandukanye bya phenolic na amines bihumura. Mu gihe cyo gukora imitobe y'imbuto n'imboga, gukaraba kw'ibinyabutabire n'ibitari ibya enzymatic ni ibibazo bikomeye.⁴Kubera ko ibi bintu bigira ingaruka mbi ku ibara, uburyohe, n'impumuro y'umutobe, bigomba gukurwaho.⁵
Mu mbuto zose, pome ni zo zikoreshwa cyane ku isi no mu Muryango w’Ubumwe bw’u Burayi. Mu 2019, umusaruro w’amapome wabaye uwa gatatu ku isi, urenga toni miliyoni 87.⁶Amapera arimo ibintu byinshi bya phenolic, birimo flavonoids na aside phenolic nka aside caffeic na aside chlorogenic.⁷Kubera ko umutobe w'amapera ukunze kunyobwa mu buryo bwawo busobanutse neza, hafi 50% kugeza 90% by'ibice bya phenolic biratakara mu gihe cyo kuyungurura.⁸Muri iki gihe, abaguzi bakunze guhitamo ibintu bidatunganyijwe cyane, nk'umutobe w'amapera ufite ibicu byinshi hamwe n'ingano nini ya polyphenol. Ariko, bitewe n'ingano yawo nini ya phenolic, ubu bwoko bw'umutobe w'amapera bukunze kwibasirwa cyane no guhinduka ibara ry'umukara.⁹Ikoranabuhanga ritandukanye, harimo n'uburyo bwo kuvura ubushyuhe nko gukaraba kuri dogere selisiyusi 60–90, rikoreshwa mu kugabanya cyangwa gukumira kwijima k'umutobe w'amapera.¹⁰Icyakora, ukurikije ubushakashatsi bwakozwe na Sauceda-Gálvez¹¹, gutunganya ubushyuhe bishobora gusenya imiti ihindagurika kandi bikagira ingaruka ku miterere y’umutobe w’amapera. Ubundi buryo bwo gutunganya ubushyuhe burimo supercritical carbon dioxide, imirasire ya ultraviolet, ultrasound, umuvuduko mwinshi wa hydrostatic, cyangwa homogenization y’umuvuduko mwinshi.¹²Ingufu z'ubu buryo bw'ikoranabuhanga n'umusaruro w'imitobe y'imbuto ikwiye biterwa n'ibipimo byakoreshejwe n'imiterere y'ibicuruzwa. Ikoreshwa ryabyo rikunze kugabanywa n'ibiciro biri hejuru, ingaruka mbi ku bwiza bw'ibiribwa bimwe na bimwe, cyangwa kudakora neza kwa enzymes.^13,14
Laccase ishobora gukoreshwa mu gutobesha no gutunganya umutobe w'imbuto.¹⁵Gökmen n'abandi.¹⁶Inama yo gukoresha laccase mu gutunganya umutobe w'imbuto kuko ikuraho neza ibintu bya phenolic ikabihinduramo polymer cyangwa oligomers bikurwaho byoroshye na ultrafiltration membrane iyo ari yo yose, bigatuma umutobe w'amapera ugumana ibara rihamye kandi risobanutse neza kugeza ku byumweru bitandatu kuri 50°C. Laccase ya *Trichoderma* yasukuwe yafunzwe ku dusaro twa alumina kandi ikoreshwa mu gukuraho ibintu bitari byiza byatewe n'udukoko twanduje umutobe w'amapera.¹⁷
Hafi 80-90% by'ibice by'umutobe w'amapera bihinduka ester na aldehydes, bitanga impumuro idasanzwe ku mutobe.¹⁸Laccase yo muri *Trametes versicolor* yafunzwe hakoreshejwe inkunga ihendutse ikozwe muri fibre karemano ikomoka ku bishishwa bito bya kokonati kugira ngo irusheho kunoza umutobe w'amapera.¹⁹Ubushakashatsi bwakozwe mbere bwakoze ubushakashatsi ku kuntu umutobe w’amapera uhoraho (ibara n’umuvuduko w’amazi) hakoreshejwe uburyo butagira enzyme cyangwa uburyo bwo guhagarika kugenda, cyangwa bugakoreshwa hamwe na ultrafiltration.^5,19Ariko, ingaruka za laccase z’ibihumyo ku miterere ya fizikikemike y’umutobe w’ibihumyo mu gihe cyo kubika ntizirasobanuka neza. Kubwibyo, intego y’ubu bushakashatsi kwari ugukora ubushakashatsi ku mpinduka mu miterere ya fizikikemike, ingano ya phenolic, n’imikorere ya antioxydant y’umutobe w’ibihumyo nyuma yo kuvurwa na laccase z’ibihumyo no kubika mu byumweru bibiri muri firigo. Laccase zifite ubushobozi bwo gukaranga ibihumyo bya phenolic, bituma zitanga icyizere cyo gukoreshwa mu nganda zitandukanye, harimo no gutunganya umutobe. Ubu bushakashatsi bwasuzumye laccase zo muri *Pleurotus ostreatus* NRC 620, zibanda ku miterere myiza y’imikorere yazo n’ubushobozi bwazo mu gutunganya umutobe. Nubwo ubushakashatsi ku bihumyo bya oyster (P. ostreatus NRC 620) bukiri buke, ubushakashatsi bwabanje bwasuzumye enzymes ziva ahantu hatandukanye ho gukamura, nka Trametes versicolor na Ganoderma lucidum. Intego y’ubu bushakashatsi kwari ugusuzuma ikoreshwa ry’iyi enzyme mu nganda z’ibiribwa no kugaragaza imiterere yayo yihariye, cyane cyane pH n’ubushyuhe byayo.
2,2′-Azooxybis (3-ethylbenzothiazoline-6-sulfonic aside) (ABTS) yaguzwe muri Sigma-Aldrich (Kanada). Izindi reagents zose zari zifite urwego rwo gusesengura.
Ikigo cy’Igihugu gishinzwe Ubushakashatsi cyabonye ubwoko bw’ibihumyo bya oyster buzwi cyane NRC620. Nyuma yo korora, ubu bwoko bwabitswe ku bimera bya dextrose agar kuri dogere selisiyusi 4. Uburyo bwo gutegura ibimera bwari ubu bukurikira: mycelium y’iminsi 10, yuzuye neza yatewe ku masahani ya dextrose agar y’ibimera hanyuma ishyirwa kuri dogere selisiyusi 28. Nyuma y’iminsi 10, uduce dutatu twa mm 12 tw’umurambararo twakuwe mu gitereko cya agar hakoreshejwe icyuma gikozwe mu cyuma gikozwe mu buryo bwa fertilizer maze dushyirwa mu macupa ya Erlenmeyer ya mL 250 hamwe n’udupfundikizo tw’ipamba turimo mL 50 z’uburyo bwa sterilized culture medium (pH 5.0, nk’uko byasobanuwe na Othman et al.²⁰). Amatungo yashyizwe mu bushyuhe bwa 28°C mu gihe cy'iminsi 18. Amatungo yasuzumwe binyuze mu mpapuro za Whatman No. 1, maze supernatant yavuyemo iba isoko y'imisemburo.
Igikorwa cya Laccase cyapimwe hakoreshejwe ABTS nk'umusemburo. Uruvange rw'ibisubizo (2 mL) rwarimo 500 μL ya 0.3 mM ABTS (yashongeshejwe muri 0.1 M sodium citrate buffer, pH 4.5) n'ingano ikenewe ya enzymes yakuwemo amazi yaciwe.^21,22Bitewe nuko laccase ishobora kugabanya ABTS mu bushyuhe bw'icyumba (28 °C ± 2), kugabanya ABTS byagenwe hapimwa ukwiyongera k'ubushyuhe kuri 420 nm (ε₄₂₀= cm 36,000^-1 M ^-1) hakoreshejwe Agilent Carry-100 UV spectrophotometer. Hakenewe igice kimwe cy'ibikorwa bya laccase kugira ngo oxidize 1 μmol ABTS ku munota. Igipimo cya poroteyine cyagenwe hakoreshejwe uburyo bwa Bradford hakoreshejwe albumin yo mu maraso y'inka nk'uburyo bwo kugenzura imbere.^23,24
Nyuma yo kubona enzyme ivuye mu bwoko bwa oyster mushrooms NRC 620, imikorere yayo yapimwe mu bihe bitandukanye byo guhinga mu gihe cy'iminsi 25 mu gihe cy'ubushyuhe buri kuri 28 °C.
Kugira ngo higwe ingaruka z'ubushyuhe ku bikorwa bya laccase, hakozwe igerageza mu bushyuhe buri hagati ya 20 na 90 °C. Mbere yo kongeramo enzyme no gutangira reaction, buffer (0.1 M sodium citrate, pH 4.5) na substrate (ABTS) byavanzwe hanyuma bishyirwa mu bushyuhe butandukanye mu minota 5. Ubushyuhe buhamye bwa enzyme bwapimwe hakoreshejwe incubation muri 0.05 M sodium phosphate buffer (pH 7.0) kuri 40, 50, 60, na 70 °C mu gihe cy'amasaha 2, uko bikurikirana. Nyuma hasuzumwe ibikorwa bisigaye hakoreshejwe substrate ya ABTS.
Ingaruka za pH ku bikorwa bya laccase zapimwe hakoreshejwe ABTS nk'umuyoboro mu buffer za 0.1 M citrate-phosphate zifite pH iri hagati ya 2.5 na 7.0. Umuti wa enzyme washyizwe muri 40°C mu gihe cy'amasaha abiri muri 0.1 M citrate na Tris buffers (pH 3, 4, 6, na 7) kugira ngo hamenyekane uburyo pH ihagaze. Ibikorwa bisigaye bya ABTS nk'umuyoboro byapimwe nyuma yo gukubitwa.
Laccase yashyizwe mu cyuma mu gihe cy'iminota 10 muri sodium phosphate buffer (0.05 M, pH 7.0) irimo ions zitandukanye z'icyuma (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, na Mn2+) ku gipimo cya 2.5 mM na 10 mM, uko bikurikirana. Hanyuma substrate (ABTS) yongewemo kugira ngo itangire reaction, maze igikorwa kigereranijwe kirasuzumwa.
Gupima ABTS oxidation hakoreshejwe laccase ku bipimo bitandukanye (0.025–3 mM) byapimwe kuri pH 4.5 kugira ngo hamenyekane ibipimo bya kinetic (Vmax na Km).^ibihorahobya equation ya Michaelis-Menten byabazwe hakoreshejwe igishushanyo cya Lineweaver-Burk, kigaragaza igipimo cy’ihinduka ry’ibisubizo nk’uko bigaragazwa n’icyerekezo cy’ifatizo. Ibipimo bya kinetic bibazwe bivuye kuri igishushanyo cya Lineweaver-Burk hakoreshejwe porogaramu ya GraphPad Prism verisiyo 6.01.
Nyuma yo koza neza pome n'amazi yo mu ijosi, zaciwemo kabiri hanyuma zishyirwamo umutobe hakoreshejwe imashini ikora neza ya Braun MP80 (yakorewe mu Budage). Umutobe wayunguruwe mu byiciro bine bya cheesecloth. Nta enzymes zongewe mu itsinda ry’abagenzuzi, mu gihe 2.0% laccase (icyiciro cyiza cyapimwe) cyongewe mu mutobe w’amapome wateguwe vuba, hanyuma ubikwa kuri dogere selisiyusi 4 mu gihe cy’ibyumweru bibiri.
Ubushyuhe bwa titratable (TA) na pH byagenwe hakurikijwe uburyo bwa Boulton et^al.27. pH ya buri gipimo yapimwe hakoreshejwe igipimo cya pH cya digitale (JENWAY 3510 pH meter). Ubusharire bushobora gupimwa (TA) bwabazwe hashingiwe kuri aside malic hakoreshejwe formula ikurikira.
Aho V na C ari ingano (mL) n'ingano (0.1 mol/L) by'umuti wa sodium hydroxide ukoreshwa muri titration, uko bikurikirana. K ni coefficient yo guhindura aside malic, ingana na 0.067, naho W ikaba uburemere (g) bw'umutobe w'amapera.
Ibintu byose bishobora gushonga (^TDS) ingano y'ibipimo byose by'umutobe byapimwe hakoreshejwe PAL-1 pocket refractometer (ATAGO, Tokyo, Ubuyapani). Nyuma ya buri gipimo, lenzi y'urumuri yogejwe n'amazi yakuwemo iayoni, kandi buri gipimo cy'umutobe w'amapera cyapimwe inshuro eshatu. Agaciro ka buri gipimo kabazwe hakoreshejwe impuzandengo y'ibipimo bitatu. Impuzandengo ± ihindagurika risanzwe kuri buri gipimo cy'umutobe w'amapera naryo ryabazwe hakoreshejwe impuzandengo y'ibi bisubizo.
Uburemere bw'ibipimo by'umutobe w'amapera bwapimwe hakoreshejwe icyuma gipima ubushyuhe (RV, Rheotest 2, Ubudage). Icyitegererezo cyashyizwe mu cyuma cya "S2" cya viscometer. Uburemere bugaragara bwagaragajwe n'umusemburo w'uburemere bw'uburemere bw'uburemere ugereranyije n'uburemere bw'uburemere bw'uburemere, bwabazwe uhereye ku buremere bw'uburemere n'uburemere bujyanye nabyo ku gipimo gitandukanye cyo gupima uburemere (kuva kuri 1.00 kugeza 437.4 s⁻¹). Uburyo bwo kubara uburemere bugaragara ni ubu bukurikira:
Aho η ari viscosity igaragara (cP), τ ni stress ya shear (dyn/cm²), γ ni igipimo cya shear (sec⁻¹), naho (τ) ikabarwa hakoreshejwe agaciro ka torque (α) na silinda (Z) hakoreshejwe formula ikurikira: τ = Z . α.
Igipimo cy'umukara cyagenwe hakurikijwe uburyo bwa Meidav ​​​​et^al.29. Icyitegererezo cy'umutobe wa mililitiro 10 cyashyizwe muri centrifuge kuri 2750 xg mu gihe cy'iminota 10. Mililitiro 5 z'umutobe wa supernatant zavanzwe na mililitiro 5 za ethanol ya 95%. Uburyo uruvange rw'umutobe rwapimwe kuri 420 nm hakoreshejwe icyuma gipima ubushyuhe cya Shimadzu (UV-1601 PC).
Igiteranyo cy'ibinyabutabire bya phenolic (TPC) cyagenwe hakoreshejwe ibara ry'umukara hakoreshejwe reagent ya Folin-Ciocalteu nk'uko byasobanuwe na Boulton et al.^[27]]. Hakozwe umurongo ngenderwaho wa aside gallic ku gipimo kiri hagati ya 0 na 500 mg/L (^r²= 0.997). Ibisubizo bigaragazwa nk'ibipimo bya aside gallic (mg GAE/mL).
Shyiramo 125 μL z'amazi yaciwe na 2850 μL by'umuti wa FRAP kuri 25 μL z'umutobe w'amapera hanyuma ureke imvange mu mwijima kugira ngo³⁰iminota. Hanyuma pima ubushobozi bwo kwinjiza amazi kuri 593 nm ukoresheje icyuma gipima UV cya Shimadzu (UV-1601 PC). Reagent ya FRAP yateguwe hifashishijwe kuvanga 300 mM acetate buffer (pH 3.6), 20 mM fer (III) chloride, na 10 mM 2,4,6-tris (2-pyridyl)triazine (TPTZ) (yashongeshejwe muri 40 mM HCl) mu gipimo cya 10: 1: 1. Umuvuduko usanzwe wakozwe hakoreshejwe Trolox nk'igipimo gisanzwe (^R²= 0.999), kandi ibisubizo byagaragajwe nka μM Trolox/mL.
Imikorere ya antioxydant y'imitobe yavuwe n'iyavuwe yagenwe hakoreshejwe uburyo bwa DPPH kugira ngo harebwe ubushobozi bwayo bwo gukuraho radicals zidafite DPPH.³¹Litiro icumi z'umutobe zavanzwe na ml 1 y'umuti wa DPPH (100 μM) muri methanol. Nyuma yo gukora mu mwijima mu gihe cy'iminota 30, ubushobozi bwo kwinjiza uruvange bwapimwe kuri 517 nm hakoreshejwe Shimadzu UV spectrophotometer (UV-1601 PC). Ibisubizo byagaragajwe nk'ibipimo bya trolox (μM trolox/ml) hashingiwe ku muvuduko wo gupima (_^R2= 0.990).
Amakuru yabonetse yagaragaje ko umusaruro ntarengwa wa laccase wagaragaye mu bihumyo bya oyster bya NRC 620 mu mpera z'umunsi wa 18 wo guhinga, bikagera ku ngano ya 1302 U/L. Ibi byashingiweho mu kugena igihe cyiza cyo guhinga cya laccase (Ishusho ya 1). Nubwo umusaruro wa enzymes wiyongereye uko igihe cyo guhinga cyiyongera, umuvuduko wo kwiyongera ntiwari ujyanye neza n'igihe cyo guhinga; nyuma y'iminsi 21, imikorere ya enzymes yari yiyongereyeho 90 U/L gusa (kugeza kuri 1390 U/L). Kubwibyo, iminsi 18 yatoranyijwe nk'igihe cyiza cyo guhinga kugira ngo umusaruro w'ibikomoka ku bihumyo uhuzwe n'inyungu z'ubukungu zo kongera igihe cyo guhinga.
Ingaruka z'igihe cyo guhinga ku musaruro wa laccase muri Pleurotus ostreatus NRC 620. Ibice bitatu (12 mm) by'ibihumyo byatewe muri 50 ml y'uburyo busanzwe butaribwa hanyuma bigahingwa kuri 28 °C mu bihe bitandukanye.
Nk’uko bigaragazwa n’ubundi bushakashatsi, ibyavuye mu bushakashatsi bwacu bigaragaza ko igihe cyiza cyo kwita ku bihumyo kugira ngo haboneke umusaruro mwinshi wa laccase gishobora kuba hagati y’iminsi 7 na 36.³²Nk’uko Ezike n’abandi babivuga.³³*Trametes polyzona* WRF03 yatanze ingano nyinshi ya laccase mu mpera z'umunsi wa cyenda wo guhisha, ifite imikorere yihariye ya poroteyine ya 1637 U/mg. Byongeye kandi, Othman et al.³⁴basanze *Trichoderma harzianum* S7113 yasohoraga ingano nini ya laccase ku munsi wa gatanu w’ubuhinzi. Umusaruro wa laccase wageze ku rwego rwo hejuru ku munsi wa cumi n’ine hanyuma ugabanuka buhoro buhoro.³⁴Nubwo gusohora enzymes bishobora no kubaho mu gihe cy’ikura ry’ibanze, akenshi birushaho kuzamuka mu gihe cy’itsinda ry’ibice bito kandi bigaterwa no gukoresha isoko ya karuboni cyangwa azote.^34,35
Nubwo laccase yo muri Pleurotus ostreatus NRC 620 yagaragaje imikorere myinshi mu bushyuhe bwinshi kuva kuri 50°C kugeza kuri 80°C, yegereje imikorere ikomeye (69–98%), imikorere yayo ntarengwa yagaragaye kuri 70°C (Ishusho ya 2a). Uretse ubushyuhe, imikorere ya enzyme yagabanutse kuri 70°C. Ibi bisubizo bigaragaza ko enzyme ikora ku bushyuhe bwinshi, bishoboka ko ari ukubera ko ubushyuhe bwinshi bwongera ingufu za kinetic z'ingaruka.
Ingaruka z'ubushyuhe bw'ibisubizo (a) na pH (b) ku bikorwa bya laccase muri *Pleurotus ostreatus* NRC 620. Ubushyuhe buri hagati ya 20 na 90 °C bwagezweho binyuze mu gushyira uruvange ku bushyuhe butandukanye mu minota 5 mbere yo kongeramo enzyme no gutangira gukora. Ingaruka za pH ku bikorwa bya laccase zapimwe hakoreshejwe ABTS nk'igice cy'ingenzi mu bisubizo birimo 0.1 M citrate-phosphate buffer ku rugero rwa pH ruri hagati ya 2.5 na 7.0.
Nk’uko Ezike et^al.33, ubushyuhe bwiza bwa *Trametes polyzona* WRF03 laccase ni 55 °C, ari na byo bimeze kuri *Ganoderma lucidum*.^laccase36kandi bisa n'ubushyuhe bwiza (50 °C) kuri *Trametes polyzona* KU-RNW02737^laccase . ^Baldrian38avuga ko, ku zindi sisitemu za enzymes zigabanya lignin, ubushyuhe bwiza bwa laccase buri hagati ya 50 na 70 °C.
Ibisubizo byagaragaje ko iyi enzyme yagaragaje imikorere myinshi kuri pH 3.0, igera kuri 94% by'ibikorwa kuri pH 3.5. Ariko, yakomeje gukora ku rugero rwa pH runini kuva kuri 2.5 kugeza kuri 7.0 (Ishusho ya 2b). Byongeye kandi, yagaragaje imikorere myinshi mu bihe bya aside ugereranije n'imimerere ya alkaline cyangwa iyo mu bihe bya aside. Umurimo wayo wakomeje kuba nibura 77% ugereranyije n'urugero rwa pH kuva kuri 2.5 kugeza kuri 4.5, ariko ugera kuri 38% gusa kuri pH 7.0. PH nziza kuri laccase kuva kuri *Trametes polyzona* WRF03 yari 4.533, ikaba ari imwe na pH kuri laccase kuva kuri *Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, na *Trametes hirsuta* 41. Ariko, nk'uko ubushakashatsi bwakozwe na Chairin et al.⁴², pH nziza kuri laccase iva kuri *Polymorpha f. sp.* WR710-1 ni 2.2, mu gihe pH nziza kuri laccase iva kuri *Polymorpha f. sp.* IBL-04 ari 5.043. Gufatana kwa anions za hydroxide (laccase inhibitor) kuri atome z'umuringa za laccase ya T2/T3 bishobora kuba impamvu yo kugabanuka k'imikorere ya laccase mu gihe pH idafite aho ibogamiye cyangwa ya alkaline. Ibi bishobora kubangamira ihererekanya rya electron imbere kuva hagati ya T1 kugera hagati ya T2/T3, bityo^kugabanyaimikorere ya enzyme23,44
Mu gushyira iyi enzyme mu bushyuhe butandukanye, byagaragaye ko igihe cyo kuyishyira mu bushyuhe n'ubushyuhe byagize ingaruka ku kudahinduka kwa enzyme. Ikigaragara ni uko laccase yo muri *Trametes polyzona* NRC 620 yagaragaje kudahinduka cyane kuri 40℃ na 50℃, igumana 68.33% na 59.61% by'ibikorwa byayo bya mbere, nyuma y'iminota 120 (Ishusho ya 3a). Mu buryo bunyuranye, mu bihe bimwe (40℃ na 50℃, iminota 120), laccase yo muri *Trametes polyzona* WRF03 yagumanye 64.38% na 42.92% by'ibikorwa byayo, uko bikurikirana.³³Ibinyuranye n'ibyo, kongera igihe cyo gushyushya no gushyushya byagabanyije ubushobozi bwo gushyushya bwa *Trametes polyzona* NRC 620 laccase; Nyuma yo gushyushya kuri 60℃ na 70℃ mu minota 60, imikorere yayo yagabanutse igera kuri 39.24% na 1.72%, uko bikurikirana (Ishusho ya 3a). Dukurikije ibyavuye mu bushakashatsi, laccase ya *Trametes polyzona* WRF03 yagaragaje ubushobozi bwo gushyushya kuri 40℃ na 50℃ mu gihe cyose cyo kuvura ubushyuhe.³³Mu buryo nk'ubwo, Lueangjaroenkit et^al.37na Chairin et^al.42batangaje ko laccase ihamye ya Trametes polyzona KURNW027 na Trametes polyzona WR710-1 kuri 50 °C mu gihe cy'isaha imwe, uko bikurikirana. Nk'umuti w'ingirakamaro wa biocatalyst ukoreshwa mu nzego zitandukanye z'ikoranabuhanga rya biotechnological, laccase igomba kugira umutekano n'imikorere myiza mu bushyuhe bwinshi.
Ubudahangarwa bwa Thermostatic (a) na pH (b) bya laccase bivuye kuri *Pleurotus ostreatus* NRC 620. Ubudahangarwa bwa Thermostatic bwapimwe hakoreshejwe uburyo bwa enzyme muri 0.05 M sodium phosphate buffer (pH 7.0) kuri 40, 50, 60, na 70 °C mu gihe cy'amasaha 2, uko bikurikirana. Ubudahangarwa bwa pH bwapimwe hakoreshejwe uburyo bwa enzyme muri 0.1 M citrate buffer na Tris buffer (pH 3, 4, 6, na 7) kuri 40 °C mu gihe cy'amasaha 2. Igikorwa gisigaye cyabazwe hakoreshejwe ABTS nk'igice nyuma yo gukubitwa.
Kugira ngo tumenye imiterere myiza yo gukoresha no kubika enzyme, twakoze ubushakashatsi ku ngaruka za pH ku kudahinduka kwa laccase. Guhura n'agaciro gatandukanye ka pH byagize ingaruka zikomeye ku kudahinduka kwa poroteyine, bityo bigira ingaruka ku kudahinduka no gukora kwa enzyme. Ibisubizo byagaragaje ko enzyme itari ihamye cyane mu bihe bya aside, mu gihe yagaragaje kudahinduka neza mu bihe bya pH iri hejuru (uturere tudafite aho duhuriye n'aside na alkaline). Ku gaciro ka pH ka 7.0, 6.0, 4.0, na 3.0, igipimo cyo kugumana enzyme nyuma y'iminota 120 cyari hafi 100%, 62.54%, 52.39%, na 11.14%, uko bikurikirana (Ishusho ya 3b). *Strombus multisus* WRF03 laccase yagaragaje kudahinduka cyane mu bihe bya pH iri hagati (5.5–6.5) n'uguhagarara kugabanuka mu bihe bya pH iri hagati (munsi ya 4.0). Nyuma y'iminota 120 ku gipimo cya pH cya 5.5, 6.0, na 6.5, igipimo cyo kubika imisemburo cyari hafi 82%, 100%, na 93% uko bikurikirana.³³Khairin n'abandi.⁴²yavuze ko laccase ya Trametes polyzona WR710-1 yari ihamye mu rugero rwa pH ruri hagati ya 6.0 na 7.0, mu gihe Sayed n'abandi.⁴⁵byagaragaje ko laccase yari ihamye kurushaho mu gihe cy’imiterere ya pH idafite aho ibogamiye. Ariko, laccase yo muri Cerrena unicolor nayo yagaragaje ko ihamye mu gihe cy’imiterere ya alkaline (pH 9.0)⁴⁶. Inyigo zakozweho zagaragaje ko zihamye cyane mu rugero rwa pH runini. Ibi bishobora kuba ingenzi mu bikorwa by'inganda.
Kubera ko ioni zimwe na zimwe z’icyuma zigira ingaruka zo gukangura no gukumira ibikorwa bya enzyme, ingaruka zazo ku bikorwa bya enzyme zigomba kwitabwaho mu nganda. Ibi ni ingenzi kuko ioni z’icyuma ari ibintu bisanzwe bihumanya ibidukikije bishobora kugira ingaruka ku ituze no gukora kwa enzymes zo hanze y’umubiri.⁴⁷Kugira ngo dukore ubushakashatsi ku ngaruka za iyoni nyinshi z'icyuma kuri laccase ikomoka kuri *Pleurotus ostreatus* NRC 620, twakoze igerageza rijyanye nayo. Nkuko bigaragara ku Ishusho ya 4, bitewe n'ubwoko bw'icyuma cyakoreshejwe, kongera ingano ya iyoni z'icyuma kuva kuri 2.5 mM kugeza kuri 10 mM byagize ingaruka mbi ku mikorere ya enzyme. Urugero,^Mg²⁺ , ^Co²⁺ , ^Zn²⁺, na^Cu²⁺bishobora gukangura no gukora imikorere ya enzyme, mu gihe^Na⁺ , ^Mn²⁺ , ^Ca²⁺, na^K⁺byashoboraga kubuza imikorere ya enzyme. Ku gipimo cya 10 mM, Cu²⁺ na Mg²⁺ ions ni byo byateraga imbaraga cyane mu mikorere ya laccase bivuye kuri *Pleurotus ostreatus* NRC 620, bitanga urugero rwo gukora rwa hafi 34% na 20%, uko bikurikirana. Ariko, ku gipimo cya 10 mM, ions za Ca²⁺ ni zo zateraga imbaraga cyane mu mikorere ya laccase, bigabanya imikorere ya enzyme ku kigero cya 60%.
Ingaruka za iyoni z'icyuma ku mikorere ya Pleurotus ostreatus NRC 620 laccase. Laccase yashyizwe mu cyuma mu gihe cy'iminota 10 muri sodium phosphate buffer (0.05 M, pH 7.0) irimo iyoni z'icyuma zitandukanye ku gipimo cya 2.5 mM na 10 mM. Nyuma y'ibyo, igisubizo cyatangijwe no kongeramo substrate (ABTS), hanyuma igikorwa cyapimwe.
Ibisubizo byacu birahuye n'iby'abandi banditsi basanze Mg²⁺ na Cu²⁺ byongera imikorere ya *Trametes polyzona* WRF03³. Castaño n'abandi.⁴⁸ basanze laccase iva kuri *Xylaria* sp. iterwa imbaraga na iyoni z'umuringa (Cu²⁺). Byongeye kandi, Foroutanfar n'abandi.⁴⁹ na Si n'abandi.⁵⁰ bakoze ubushakashatsi nk'ubwo kuri laccase iva kuri *Paraconiothyrium variabile* na *Trametes pubescens*, uko bikurikirana. Ubwoko bwa kabiri bw'umuringa-bufatanye (T2) bw'iyi enzyme bushobora kuzuzwa na Cu²⁺ ku rugero runaka, bishobora gusobanura uburyo laccase ikora ku rugero ruri hejuru rwa Cu²⁺³⁹. Kubera ko fungi zo mu bwoko bwa white rot laccases ari oxidases zirimo atome nyinshi z'umuringa, ingaruka za iyoni z'umuringa ku mikorere ya laccase ziratandukanye kandi ziratandukanye kuva ku zitera imbaraga no kuzibuza kugeza ku zitagira aho zibogamiye.⁵¹ Ibinyuranye n'ibyo, Zhou n'abandi^{. [52]}yavuze ko^Cu²⁺yabujije imikorere ya laccase y'inyenzi zo munsi y'ubutaka zo muri Taiwan (Odontotermes formosanus). Ariko, laccase ya Cerena sp. HYB07^[53]na Clitocybe maxima^[54]ntabwo byagizweho ingaruka na iyoni z'umuringa.
Uburyo bwo gupima ibintu mu buryo busanzwe bwagaragajwe n'ibipimo byayo bya kinetic (Km na Vmax); uko uburyo bwo gufatanya ibintu mu buryo bukomeye na enzyme burushaho gukomera, niko agaciro ka Km kagabanuka kandi uburyo bwo gupima ibintu mu buryo busanzwe burushaho kwiyongera.^3,21,55Ibipimo bya kinetic (Km na Vmax) bya laccase bivuye kuri *Pleurotus ostreatus* NRC 620 byagenwe hakoreshejwe porogaramu ya GraphPad Prism 6.0 hashingiwe ku ishusho ya Lineweaver-Burk (Ishusho ya 5). Mu gukoresha ABTS nk'ishingiro, ibisubizo byari 1.99 mM na 16217 μmol.^{min⁻¹ L⁻¹,}Elsayed n'abandi.²¹bavuze ko agaciro ka Km kuri oxidation ya ABTS kari 0.1 mM na 0.064 mM, uko bikurikirana, bigaragaza ko isoenzymes za Lac A na Lac B kuri ABTS zifatanye cyane. Byongeye kandi, agaciro ka Vmax kari 0.182 μmol^min⁻¹na 0.603 μmol^min⁻¹, uko bikurikirana. Agaciro ka Km kabonetse kari hasi ugereranyije n'aka Trametes polyzona WRF03 (8.66 mM); byongeye kandi, agaciro ka Vmax kabo (1429 mmol min⁻¹) nako kari^hasiiyo hakoreshejwe ABTS nk'umusemburo.33 Mu buryo nk'ubwo, agaciro ka Km ka Lentinus squarrosulus MR13 na Trametes sp. AH28-2 laccase kiyongereyeho kari 0.0714 mM na 0.025 mM, uko bikurikirana, naho agaciro ka Vmax kari 0.0091 mM min−1 na 0.67 mM min−1 mg−1 (ugereranyije na ABTS)^{, uko bikurikirana. 56,57}
Ingaruka z'ubwinshi bwa ABTS ku bikorwa bya laccase bivuye kuri *Pleurotus ostreatus* NRC 620 zarasuzumwe, maze hagaragazwa ishusho ya Lineweaver-Burk y'umuvuduko w'itangira ry'inyigo ugereranyije n'ubwinshi bwa ABTS. Uburyo bwo gupima okiside ya ABTS ifite ibipimo bitandukanye (0.025–3.0 mM) bya laccase bwapimwe kuri pH 4.5 kugira ngo hamenyekane ibipimo bya kinetic (Vmax na Km). Ibipimo bya kinetic bya Michaelis-Menten byabazwe hakoreshejwe ishusho ya Lineweaver-Burk y'umuvuduko w'inyigo ugereranyije n'ubwinshi bwa substrate. Ibipimo bya kinetic byabazwe bivuye kuri porogaramu ya Lineweaver-Burk hakoreshejwe porogaramu ya GraphPad Prism 6.01.
Enzyme zisanzwe zitunganya ibintu, nka pectinases, hydrolyze pectic substances, zigabanya ubukana n'umukungugu. Zisenya neza polysaccharides z'imiterere kandi zikunze gukoreshwa hamwe n'izindi enzymes, nka cellulases na hemicellulases, kugira ngo zirusheho gutanga umusaruro no kugaragara neza. Ariko, pectinases ntizibanda ku buryo bwihariye ku bintu bya phenolic, ari nabyo bigira uruhare runini mu gutuma umukungugu uhinduka umukara, cyane cyane mu mitobe nka pome n'umutobe w'imizabibu.⁵⁸Mu buryo bunyuranye, laccases zitera ogisijeni y’ibintu bya phenolic, zigahindura molekile nini, zidashonga zishobora gukurwaho hakoreshejwe sedimentation cyangwa filtration. Ubu buryo ntibutuma umutobe urushaho kuba mwiza gusa, ahubwo bunatuma igihe cyo kubaho cy’umutobe kigabanuka binyuze mu kugabanya amahirwe yo guhindagurika kw’ibintu bya phenolic. Byongeye kandi, inzira zo gusesengura zishingiye kuri laccases zishobora gukorwa mu buryo bworoshye bwo gutunganya (pH 3.5–5.5, ubushyuhe bwa 25–40 °C), bigatuma zikwiriye imitobe yoroshye idahungabanya imiterere yazo y’imirire cyangwa imiterere y’umubiri.⁵⁹Ubushakashatsi bwagaragaje ko uburyo bwo kuvura pectinase bushobora gusobanura neza umutobe mu masaha 1-2, mu gihe uburyo bwo kuvura laccase busanzwe busaba igihe kirekire cyo gusubiza (amasaha 3-6) kugira ngo bigabanye burundu ibintu bya phenolic. Ariko, ubu buryo bushobora kunozwa binyuze mu guhagarika enzyme cyangwa guhuza laccase n'uburyo bwo kuvura bwa mechanical.⁶⁰Muri ubu bushakashatsi, isesengura rya enzymes ku gicuruzwa cyakozwe mu buryo bwa "crooked extract" ryagaragaje ibikorwa bikomeye bya laccase na α-amylase, mu gihe ibikorwa bya pectinase na xylanase byari bike cyane, kandi ibikorwa bya cellulase ntibyagaragaye. Kubwibyo, kugabanuka k'umukungugu n'ingano ya phenolic byatewe ahanini n'imikorere ya laccase, mu gihe impinduka mu buryo bwa "viscosity" zishobora guterwa n'imikorere ya amylase.
Imbonerahamwe ya 1 igaragaza ibipimo bya fiziki y'umutobe w'amapera uherutse gukamurwa n'ibipimo bya laccase. Ibisubizo byagaragaje ko umusaruro w'umutobe w'amapera uherutse gukamurwa (71.59%) wari muto ugereranije n'uw'ibipimo bya laccase (87.34%). Ibi bisubizo bihuye n'ibyavuye muri Pilnik na Orange.⁶¹, wagaragaje ko ikoreshwa rya enzymes mu gutunganya imbuto rishobora kongera umusaruro w'umutobe, kunoza uburyo bwo kuyungurura, no kubona umutobe mwiza kandi usobanutse neza kugira ngo urusheho kuba mwiza. Ubwiyongere bw'umusaruro w'umutobe buterwa ahanini n'ubwiyongere bw'isukari ishongesha mu mutobe. Mu gihe cy'amazi y'imbuto, mesoglea na pectin mu rukuta rw'uturemangingo tw'umusaruro birasenywa bigahinduka ibintu bishongesha nk'isukari na aside.^62.Agaciro ka pH k'umutobe w'amapera wavuwe na enzyme kari hasi cyane ugereranyije n'itsinda ryagenzuwe (P < 0.05), kandi agaciro ka pH k'amatsinda yombi kiyongereye cyane mu gihe cyo kubika (Imbonerahamwe ya 1). Ibi bisubizo birahuye n'ibyavuzwe na Mark et al.⁶³, wavuze ko pH y'umutobe w'imbuto za cashew yagabanutse nyuma yo kubibika nyuma yo kubikoresha ubushyuhe. Kwangirika kwa pectine no kwirema kwa aside galacturonic nyuma yo kubikoresha bishobora kuba impamvu y'ubwiyongere bwa pH mu gihe cyo kubibika. pH y'ibipimo byavuwe na enzymes yagumye hagati ya 4.05 na 4.31 mu gihe cyose cyo kubibika, mu gihe pH y'umutobe w'amapera utavuwe yari hagati ya 4.12 na 4.33.
Uburemere bw'uburemere (TA) bw'ibipimo byavuwe n'ibitaravuwe byagaragaje ko bigenda bigabanuka uko igihe cyo kubika cyagendaga cyiyongera (Imbonerahamwe ya 1). Kugabanuka kw'uburemere byatewe n'ihinduka rya aside organic muri carbohydrate cyangwa enzyme reactions, ndetse no gushonga mu gihe cyo kubika umutobe.⁶⁴Aside yose y'umutobe w'amapera n'ibipimo byavuwe na enzymes yari hasi ugereranyije n'indi mitobe (umutobe wa sitroberi 0.9%, umutobe wa plum 2.2%, umutobe wa kumquat 1.0%, umutobe wa apricot 2.4%, umutobe w'icunga 0.8%), ariko isa n'iy'indi mitobe (urugero: umutobe w'amapera 0.3%).⁶²Iri tandukaniro mu mutobe w'amapera ukiri muto utavuwe rishobora guterwa n'ibintu bitandukanye nko mu mikurire, ibintu bigize imiterere y'uturemangingo, urwego rwo gukura, n'uburyo bwo kuwutunganya.⁶⁵Kugabanuka k'ubushyuhe bwose mu kugenzura no gukoresha umutobe w'amapera wavuwe na laccase bihuye n'ibyavuye mu bushakashatsi bwatanzwe na Singh et al.⁶⁶ku bijyanye no kugabanuka kwa aside yose y'umutobe w'amapera wa Jin Nuo nyuma y'iminsi 74 yo kubika. Ku rundi ruhande, Oshmiansky na Wojdylo⁶⁷Nta mpinduka zikomeye zabonetse mu bushyuhe bw'umutobe w'amapera ubwo bigaga ingaruka z'uburyo gakondo bwo gusobanura neza.
Ibisubizo byagaragajwe mu mbonerahamwe ya 1 bigaragaza ko agaciro k'ibinyabutabire bishongeshwa (TSS) by'umutobe w'amapera wavuwe na laccase kari hejuru ugereranyije n'icyitegererezo kitavuwe. Ibi bisubizo birahuye n'ubushakashatsi bwasohotse.^{. 68}Byongeye kandi, Imbonerahamwe ya 1 igaragaza ko agaciro ka TSS k'umutobe w'amapera wari 9.58 mu gihe cya mbere kandi kageze kuri 11.05 mu mpera z'igihe cyo kubika. Izi gaciro ziri hasi ugereranyije n'agaciro ka TSS k'umutobe w'amapera mushya wavuzwe na Hamid et al.^{. 69}(11.2 na 11.80, uko bikurikirana). Agaciro ka TSS k'ibipimo by'umutobe w'amapera byavuwe na laccase kiyongereye cyane, guhera kuri 11.23 kugera kuri 12.93 nyuma y'ibyumweru bibiri bibitswe kuri 4°C (Imbonerahamwe ya 1). Ukwiyongera nk'uku kwa TSS mu gihe cyo kubika kwagaragaye no mu mbuto z'indimu, indimu, n'amacunga meza. Ukwiyongera kw'ibintu byose bishongesha (TSS) mu gihe cyo kubika bishobora guterwa na hydrolysis ya polysaccharides (starch) ikaba monosaccharides (isukari), kwiyongera k'ubwinshi bitewe no kubura amazi mu mutobe, no kwangirika kwa pectini mu mutobe ikaba ibintu byose bishongesha. Ukwiyongera kw'ibintu byose bishongesha (TSS) birashoboka guterwa n'ubwiyongere bw'isukari ishongesha, ishobora guterwa no guhindura pectini cyangwa cellulose ikaba isukari ishongesha na pectini cyangwa cellulase, uko bikurikirana, cyangwa hidrolysis y'ibishyimbo ikaba isukari, nk'uko byatangajwe na Hamed et al.^69.Ingaruka za laccase ku miterere y'umutobe w'amapera zishobora kugaragara mu maso, kuko umutobe w'amapera wavuwe na laccase ugaragaza ubushobozi bwo gutemba neza no kuba muto kurusha umutobe utavuwe. Iki gitekerezo cyanditswe mu mbonerahamwe ya 1; Ubushobozi bw'icyitegererezo cyavuwe na enzyme bwari 1.87 cP, mu gihe ubushobozi bw'icyitegererezo cyari 2.95 cP. Uku kugabanuka gukomeye kw'ubushobozi bw'ubushobozi bushoboka guterwa n'ubushobozi bwinshi bw'ibintu bimeze nka pectine mu gufata amazi no gushyiraho imiterere y'urusobe rw'amahuriro.
Muri ubu bushakashatsi, ingaruka za laccase ku gipimo cy’umutobe w’amapera (BI) zakoreweho ubushakashatsi hapimwa uburyo amazi yakwinjiramo kuri 420 nm hakoreshejwe spectrophotometer. Ibisubizo bigaragara mu mbonerahamwe ya 1. Mu gihe cyo kubika, BI y’ibipimo by’umutobe w’amapera haba mu matsinda yavuwe n’ayavuwe yagaragaje ko agenda yiyongera buhoro buhoro. BI igaragaza urwego rw’umuhondo kandi ishobora gukora nk’aho^ingenziikimenyetso cy'uko ibintu bihinduka nk'ibinyabutabire n'ibitari ibinyabutabire. Uburyo bwo kubikuramo bwiyongereye cyane mu gihe cyo kubibika (P < 0.05). Mu mpera yo kubibika,_A420Agaciro k'ibipimo by'umutobe w'amapera mu matsinda yagenzuwe n'ayavuwe na enzymes kiyongereyeho hafi 217% na 121%, uko bikurikirana (Imbonerahamwe ya 1). Ibisubizo bigaragaza ko kuvura enzymes bishobora kugabanya neza urugero rw'umukara ku kigero cya 56%. Ibisubizo bya Bezerra et al.^[19]] bihuye n'ibyavuye mu bushakashatsi bwacu; Bakoresheje fibre ya laccase-glutaraldehyde-coconut kugira ngo basobanure neza umutobe w'amapera, bagabanya ibara ryawo ry'umwimerere ho 61%.
Nubwo polyphenols mu mitobe y'imbuto zigira ingaruka nziza ku mirire no kuvura umubiri w'umuntu, zishobora no kugira ingaruka kuri poroteyine, bigatuma umutobe uhinduka ibicu, uhinduka nk'umusenyi, cyangwa uhinduka nk'umukungugu, bityo bigatuma uburyohe n'impumuro y'umusaruro bigabanuka kandi bikagabanya igihe cyo kubikwa.⁷¹Intego y'ubu bushakashatsi kwari ukugabanya mu buryo bwizewe ingano y'ibinyabutabire bya phenolic mu mutobe w'amapera hakoreshejwe laccase yo muri Pleurotus ostreatus NRC 620. Ibisubizo byagaragajwe mu mbonerahamwe ya 1 bigaragaza ko ingano y'ibinyabutabire bya phenolic mu mutobe w'amapera wavuwe na laccase yagabanutse cyane mbere yo kubika kuri 4 °C. Byongeye kandi, ingano y'ibinyabutabire bya phenolic nayo yagabanutse mu gihe cyo kubika mu ngero zombi zakoreweho ubushakashatsi (Imbonerahamwe ya 1). Ubushakashatsi bwakozwe na Sandri et al.⁷²byagaragaje ko umutobe w'amapera uvuwe na enzymes ushobora kugumana imikorere yawo ya antioxydant hamwe n'ibikubiye mu mvange ya phenolic. Ariko, ibyavuye mu bushakashatsi bwakozwe na Lettera et al.⁷³bigaragaza ko kuvura umutobe w'icunga hakoreshejwe fungi ya laccase bishobora kugabanya ingano y'ibintu bya phenolic biyirimo kugeza kuri 45%.
Byagaragaye ko ibintu bya phenolic bifite imiterere nko gushakisha uturemangingo tw’ubuntu, kugabanya no kuzimya ogisijeni, kohereza atome ya hydrogen, no gutanga electron kuri uturemangingo tw’ubuntu, bigatuma tuba antioxydants zikomeye.⁷⁴Kubwibyo, muri ubu bushakashatsi, uburyo bwa DPPH na FRAP bwakoreshejwe mu gusuzuma ingaruka za laccase ku mikorere ya antioxydant y'umutobe w'amapera wabitswe muri firigo mu gihe cy'iminsi 14 (Imbonerahamwe ya 2). Ubwo buryo bwombi bwagaragaje ukwiyongera kw'imikorere ya antioxydant mu gihe cyo kubika, bishobora guterwa n'ubwiyongere bw'ibinyabutabire bya phenolic cyangwa ikorwa rya Maillard reaction products (MRPs), aho Maillard reaction products zishobora kuba ari zo zitera kwiyongera kw'imikorere ya antioxydant.⁷⁵Uburyo bwo kwisiga ibara ry’umukara butari ubwo mu buryo bwa enzymes (harimo no kwangirika kwa aside ascorbic, Maillard reactions, no kwangirika kw’isukari bikomoka kuri aside) bitanga ibara ry’umukara (melanoidins). Ibicuruzwa byo kwangirika kwa aside ascorbic hamwe n’ibicuruzwa byo kwangirika kw’isukari (nk’ibinyabutabire bya karuboni) bishobora kugira ingaruka ku biyobyabwenge bya aside amine binyuze mu buryo bwa Maillard reactions.⁷⁶Nubwo gukaraba imbuto n'imboga mu gihe cyo kubibika byigwa cyane, uburyo dusobanukirwa ibi bisubizo biracyari bike.⁷⁷Ugereranyije n'uburyo bwa FRAP, umutobe w'amapera wavuwe na laccase wagaragaje imikorere mike cyane ya antioxydant ukoresheje uburyo bwa DPPH (Imbonerahamwe ya 2), kandi imikorere ya antioxydant y'ibipimo byose yariyongereye cyane uko igihe cyo kubika cyagendaga cyiyongera. Muri ubu bushakashatsi hakoreshejwe uburyo bubiri butandukanye bwo kumenya imikorere ya antioxydant kuko amahame yabwo atandukanye. Uburyo bwa DPPH bupima ubushobozi bwo kugabanya radicals libre, mu gihe uburyo bwa FRAP bupima ubushobozi bwo kugabanya iyoni z'icyuma. Kubwibyo, ni byiza gukoresha uburyo bwinshi bwo kumenya imikorere ya antioxydant kugira ngo usobanukirwe neza imikorere ya antioxydant y'ibipimo byakoreweho ubushakashatsi.⁷⁸
Kimwe mu byavumbuwe n'ubu bushakashatsi ni uko *Pleurotus ostreatus* laccase NRC 620 igaragaza imikorere myiza kuri 70°C na pH 3.0. Ugereranyije n'izindi laccase z'ibihumyo zikunze gukoreshwa mu gutunganya umutobe, nka *Trametes versicolor* na *Ganoderma lucidum* laccases, *P. ostreatus* NRC 620 igaragaza ubushyuhe bwinshi kandi ifite pH ikungahaye kuri aside. Laccase zikomoka kuri *Trametes versicolor* na *Ganoderma lucidum* zikunze kugaragaza imikorere myiza hagati ya 50-60°C no ku gipimo cya pH kiri hagati ya 3.5 na 5.0. Iri tandukaniro rishobora kugira uruhare mu kunoza imikorere myiza y'umutobe, cyane cyane ku mitobe ishaje aho kudahindagurika ku gipimo cya pH kiri hasi ari ingenzi cyane. Ikiranga cyihariye cya *P. Ugereranyije n'izindi laccase z'ibihumyo zakoreweho ubushakashatsi, *Pleurotus ostreatus* NRC 620 igaragaza ubushobozi bwo gukora neza mu bihe bigoye kurushaho. Ubushyuhe bwinshi bw'imikorere yayo bugaragaza ibyiza bishobora kubaho mu nganda, nko kwihuta mu mikorere no kugabanya ubwandu bwa mikorobe. pH yayo iri hasi, ijyanye neza n'imiterere ya aside y'imitobe myinshi, ishobora kuba ingirakamaro mu mikorere yo gutunganya imitobe. Ibi bisubizo byemeza ko hagomba gukorwa ubushakashatsi bwinshi, bigatuma *Pleurotus ostreatus* NRC 620 iba ubundi buryo bwiza bwo gukoresha mu gusiga ifu gakondo ya laccase. Ugereranyije n'ubushakashatsi bwakozwe mbere, twasanze ubushyuhe bwiza ari 60°C naho pH nziza ikaba 3.0. Nyuma yo gukora kuri 60°C mu minota 80, *Ganoderma lucidum* laccase yagumanye.⁴⁶% by'ibikorwa byayo.79 Dukurikije Kurniawati na Nicelle⁸⁰, Enzymes za *Ganoderma lucidum* zigaragaza ubushyuhe bwiza cyane kugeza ku bunini kuri 25°C na pH kuva kuri 5.0 kugeza kuri 8.0, no guhagarara kuri pH 6.0 n'ubushyuhe kuva kuri 10 kugeza kuri 30°C. Muri ubu bushakashatsi, twasanze pH nziza n'ubushyuhe ku bikorwa bya enzyme kuri *Pleurotus ostreatus* byari 3.0 na 70°C, uko bikurikirana. Nyuma yo gushyuha kuri 40°C na 50°C mu gihe cy'amasaha abiri, enzyme yagumanye 68.33% na 59.61% by'ibikorwa byayo, uko bikurikirana. Byongeye kandi, Pleurotus ostreatus NRC 620 laccase yagaragaje ubushyuhe bwinshi kuva kuri 50°C kugeza kuri 80°C, hafi kugera ku bushyuhe bwinshi (69%–98%), aho ubushyuhe bwinshi bwagaragaye kuri 70°C.
Mu gusoza, ibihumyo bya oyster mushroom laccase NRC620, byabonetse mu bihe bidahinduka, byagaragaje imikorere myiza n'ubudahangarwa mu bihe bitandukanye bya pH n'ubushyuhe, bigaragaza ubudahangarwa bwiza ugereranije n'izindi enzyme. Kongeramo 10 mM MgSO₄ na CuSO₄ byongereye imikorere ya enzyme ku kigero cya 21% na 35%, uko bikurikirana. Iyo byatunganyijwe mu mutobe w'amapera, enzyme yagabanyije pH n'ubudahangarwa, mu gihe ingano ya phenolic yagabanutseho gato mu gihe cyo kubika.
Ibisubizo byemeza ubushobozi bwa laccase mu nganda z'ibiribwa, cyane cyane mu gutunganya ibinyobwa. Mu gusenya by'umwihariko ibintu bya phenolic, laccase ntigabanya gusa ubushyuhe no kunoza imiterere y'ibinyobwa, ahubwo inakomeza ubwiza bw'imitobe y'imbuto mu gihe cyoroheje. Bitandukanye n'ibintu bisanzwe bitunganya ibinyobwa nka gelatin, bentonite, na silica gel, laccase ntabwo ikora imyanda cyangwa ngo ikuremo impumuro nziza mu binyobwa, bigatuma iba amahitamo meza kandi arambye. Byongeye kandi, ugereranije n'izindi enzymes n'uburyo bwo kuyungurura, laccase itanga igisubizo cyihariye kandi gihendutse kitabangamiye ireme ry'ibicuruzwa.
Kyomuhimbo, HD na Brink, HG. Imikoreshereze n'ingamba zo guhagarika laccases irimo umuringa; isuzuma. Heliyon 9, e13156 (2023).