Kupanga ndi kulongosola kwa biochemical kwa bowa wa oyster laccase NRC 620 ndikuwunika momwe amagwirira ntchito powunikira bwino madzi a apulo.

Pambuyo pa masiku 25 a kuikidwa kwa static incubation pa 28°C, laccase yochokera ku *Pleurotus ostreatus* NRC620 inawonetsa ntchito yayikulu kwambiri mu fungi culture medium. pH yabwino kwambiri ndi kutentha kwa enzyme iyi inali 3.0 ndi 70°C, motsatana. Pambuyo pa maola awiri a kuikidwa kwa enzyme pa 40°C ndi 50°C, ntchito ya enzyme inasunga 68.33% ndi 59.61%, motsatana. Pambuyo pa maola awiri a kuikidwa kwa citrate-phosphate buffer (pH 7.0), ntchito ya enzyme inakhalabe pa 100%. Kuwonjezera kwa 10 mM MgSO₄ ndi CuSO₄ kunawonjezera ntchito ya enzyme ndi pafupifupi 21% ndi 35%, motsatana, pomwe NaCl, MnCl₂, KCl, ndi CaCl₂ zinaletsa ntchito ya enzyme. Pogwiritsa ntchito ABTS ngati substrate, ma kinetic parameter (Km ndi Vmax) a *Pleurotus ostreatus* NRC 620 laccase anali 1.99 mM ndi 16,217 μmol min−1 L−1, motsatana. Chithandizo cha enzymatic cha zitsanzo za madzi a apulo chinachepetsa kwambiri pH ndi kukhuthala, ndipo kuchepa kumeneku kunagwirizana ndi kuwonjezeka kwa nthawi yosungira. Chithandizo cha laccase chinapangitsa kuti kuchepa pang'ono kwa kuchuluka kwa phenolic mu madzi a apulo kuchepe, koma palibe kuchepa kwa ntchito ya antioxidant komwe kunawonedwa.
M'zaka zaposachedwa, ofufuza ayang'ana kwambiri pakugwiritsa ntchito ukadaulo wobiriwira wa zamoyo m'makampani azakudya. Laccase ndi imodzi mwa ma enzyme othandiza kwambiri m'makampani azakudya, yomwe imagwiritsa ntchito m'malo monga kukonza madzi, kuphika, kukhazikika kwa vinyo, komanso kukonza mawonekedwe a organoleptic azinthu zazakudya.¹Zomera zambiri zapamwamba ndi tizilombo toyambitsa matenda zimatulutsa laccase,²ndipo bowa monga deuteromycetes, ascomycetes, ndi basidiomycetes amathanso kupanga laccase.³Laccase (EC 1.10.3.2) ndi mtundu wa buluu wotchedwa oxidase womwe umachepetsa mpweya wa mamolekyu kukhala madzi pogwiritsa ntchito njira yokhala ndi maatomu atatu osiyanasiyana amkuwa, motero umapangitsa kuti mankhwala osiyanasiyana a phenolic ndi aromatic amines apangidwe. Pakupanga madzi a zipatso ndi ndiwo zamasamba, browning ya enzymatic ndi nonenzyme ndi nkhani zofunika kwambiri.⁴Popeza zinthuzi zimakhudza mtundu, kukoma, ndi fungo la madziwo molakwika, ziyenera kuchotsedwa.⁵
Pa zipatso zonse, maapulo ndi omwe amadyedwa kwambiri padziko lonse lapansi komanso ku European Union. Mu 2019, kupanga maapulo kunali pa nambala 3 padziko lonse lapansi, kupitirira matani 87 miliyoni.⁶Maapulo ali ndi mankhwala ambiri a phenolic, kuphatikizapo flavonoids ndi phenolic acids monga caffeic acid ndi chlorogenic acid.⁷Popeza madzi a apulo nthawi zambiri amamwedwa bwino, pafupifupi 50% mpaka 90% ya zinthu za phenolic zimatayika panthawi yosefera.⁸Masiku ano, ogula amakonda kusankha zinthu zosakonzedwa bwino, monga madzi a apulo okhala ndi mitambo okhala ndi polyphenol yambiri. Komabe, chifukwa cha kuchuluka kwa phenolic, mtundu uwu wa madzi a apulo umakhala wosavuta kusintha mtundu ndi mdima.⁹Maukadaulo osiyanasiyana, kuphatikizapo njira zotenthetsera monga kupha tizilombo toyambitsa matenda pa 60–90°C, amagwiritsidwa ntchito kuchepetsa kapena kupewa mdima wa madzi a apulo.¹⁰Komabe, malinga ndi kafukufuku wa Sauceda-Gálvez¹¹, kukonza kutentha kumatha kuwononga mankhwala osinthasintha ndikukhudza mawonekedwe a organoleptic a madzi a apulo. Njira zina zogwiritsira ntchito kutentha zimaphatikizapo supercritical carbon dioxide, ultraviolet radiation, ultrasound, high hydrostatic pressure, kapena high-pressure homogenization.¹²Kugwira ntchito bwino kwa ukadaulo uwu komanso kukolola kwa madzi abwino a zipatso kumadalira magawo omwe agwiritsidwa ntchito komanso mawonekedwe a mankhwalawo. Kugwiritsa ntchito kwawo kwambiri kumachepetsedwa ndi mtengo wokwera, zotsatira zoyipa pa mtundu wa zakudya zina, kapena kulephera kugwira ntchito bwino kwa ma enzyme.^13,14
Laccase ingagwiritsidwe ntchito kukhazikika ndi kuyeretsa madzi a zipatso.¹⁵Gökmen ndi ena.¹⁶Amalimbikitsa kugwiritsa ntchito laccase kuti madzi a zipatso awoneke bwino chifukwa amachotsa bwino mankhwala a phenolic powasandutsa ma polima kapena ma oligomer omwe amachotsedwa mosavuta ndi nembanemba iliyonse yofinyira, zomwe zimathandiza kuti madzi a apulo akhalebe ndi mtundu wokhazikika komanso womveka bwino kwa milungu isanu ndi umodzi pa 50°C. Laccase yoyeretsedwa ya *Trichoderma* idatsekedwa pa mikanda ya alumina ndipo idagwiritsidwa ntchito kuchotsa mosamala mankhwala osakoma omwe amayamba chifukwa cha kuipitsidwa kwa tizilombo toyambitsa matenda a madzi a apulo.¹⁷
Pafupifupi 80-90% ya zigawo zosasunthika za madzi a apulo ndi ma ester ndi aldehydes, zomwe zimapangitsa kuti madziwo akhale ndi fungo lapadera.¹⁸Laccase yochokera ku *Trametes versicolor* inalepheretsedwa kuyenda ndi chithandizo chotsika mtengo chopangidwa kuchokera ku ulusi wachilengedwe wochokera ku zipolopolo zazing'ono za kokonati kuti madzi a apulo amveke bwino.¹⁹Kafukufuku wakale adafufuza momwe madzi a apulo amakhalira olimba (mtundu ndi matope) pogwiritsa ntchito njira zopanda ma enzyme kapena njira zoletsa kuyenda, kapena kuphatikiza ndi ultrafiltration.^5,19Komabe, zotsatira za bowa laccases pa physicochemical ya madzi a apulo panthawi yosungira sizikudziwika bwino. Chifukwa chake, cholinga cha kafukufukuyu chinali kufufuza mwachidwi kusintha kwa physicochemical, kuchuluka kwa phenolic compound, ndi antioxidant activity ya madzi a apulo pambuyo pochiza ndi bowa laccases ndi kusunga mufiriji kwa milungu iwiri. Ma laccases ali ndi mphamvu yotulutsa phenolic compounds, zomwe zimapangitsa kuti azigwiritsidwa ntchito m'mafakitale osiyanasiyana, kuphatikizapo clearance ya madzi. Kafukufukuyu adafufuza ma laccases ochokera ku *Pleurotus ostreatus* NRC 620, kuyang'ana kwambiri pamikhalidwe yabwino yogwirira ntchito yawo komanso kugwira ntchito bwino poyeretsa madzi. Ngakhale kafukufuku wa bowa wa oyster (P. ostreatus NRC 620) akadali ochepa, maphunziro am'mbuyomu adafufuza ma enzymes ochokera kuzinthu zosiyanasiyana za bowa, monga Trametes versicolor ndi Ganoderma lucidum. Cholinga cha kafukufukuyu chinali kuwunika momwe enzyme iyi ingagwiritsire ntchito mumakampani azakudya ndikuwunikira mawonekedwe ake apadera, makamaka pH yake yoyenera komanso kutentha.
2,2′-Azooxybis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) idagulidwa ku Sigma-Aldrich (Canada). Ma reagents ena onse anali a kalasi yowunikira.
Malo Osonkhanitsira Ma Microbial Culture a National Research Center adapeza mtundu wodziwika bwino wa bowa wa oyster NRC620. Pambuyo pa ulimi wa subculture, mtundu uwu unasungidwa pa slants ya potato dextrose agar pa 4°C. Njira yokonzekera inoculum inali motere: mycelium ya masiku 10, yokhwima mokwanira, inayikidwa pa mbale za potato dextrose agar ndikuyikidwa pa 28°C. Patatha masiku 10, mabuloko atatu a mycelial a 12 mm-diameter adachotsedwa pa agar media pogwiritsa ntchito chopukutira chachitsulo chosabala ndipo adayikidwa mu 250-mL Erlenmeyer flasks ndi ma plugs a thonje okhala ndi 50 mL ya sterilized culture medium (pH 5.0, monga momwe Othman et al adafotokozera kale.²⁰). Zomerazo zinaikidwa mu chidebe chozizira pa 28°C kwa masiku 18. Zomerazo zinasefedwa kudzera mu pepala losefera la Whatman No. 1, ndipo supernatant yomwe inatuluka inakhala gwero la enzyme.
Kuchuluka kwa laccase kunapezeka pogwiritsa ntchito ABTS ngati substrate. Chisakanizo cha reaction (2 mL) chinali ndi 500 μL ya 0.3 mM ABTS (yosungunuka mu 0.1 M sodium citrate buffer, pH 4.5) ndi kuchuluka kwa enzyme komwe kumafunikira komwe kumachepetsedwa ndi madzi osungunuka.^21,22Poganizira kuti laccase imatha kupangitsa kuti ABTS iwonongeke kutentha kwa chipinda (28 °C ± 2), kupangika kwa ABTS kunatsimikiziridwa poyesa kuwonjezeka kwa kuyamwa kwa 420 nm (ε₄₂₀= 36,000 cm^-1 M ^-1) pogwiritsa ntchito Agilent Carry-100 UV spectrophotometer. Gawo limodzi la ntchito ya laccase linkafunika kuti lipangitse oxidize 1 μmol ABTS pamphindi. Kuchuluka kwa mapuloteni kunadziwika ndi njira ya Bradford pogwiritsa ntchito ng'ombe serum albumin ngati njira yowongolera mkati.^23,24
Pambuyo potenga enzyme kuchokera ku mtundu wa bowa wa oyster NRC 620, ntchito yake inayesedwa pa nthawi zosiyanasiyana zolima kwa masiku 25 pansi pa kutentha kwa 28 °C.
Kuti aphunzire momwe kutentha kumakhudzira ntchito ya laccase, mayeso anachitika pa kutentha kuyambira 20 mpaka 90 °C. Asanawonjezere enzyme ndikuyamba kuchitapo kanthu, buffer (0.1 M sodium citrate, pH 4.5) ndi substrate (ABTS) zinasakanizidwa ndikuyikidwa mu incubator kwa mphindi 5 pa kutentha kosiyanasiyana. Kukhazikika kwa kutentha kwa enzyme kunayesedwa mwa kuyika mu incubator mu 0.05 M sodium phosphate buffer (pH 7.0) pa 40, 50, 60, ndi 70 °C kwa maola awiri, motsatana. Kenako ntchito yotsalira inayesedwa pogwiritsa ntchito substrate ya ABTS.
Zotsatira za pH pa ntchito ya laccase zinayesedwa pogwiritsa ntchito ABTS ngati substrate mu 0.1 M citrate-phosphate buffers yokhala ndi pH range ya 2.5 mpaka 7.0. Yankho la enzyme linayikidwa pa 40°C kwa maola awiri mu 0.1 M citrate ndi Tris buffers (pH 3, 4, 6, ndi 7) kuti liwone kukhazikika kwa pH. Ntchito yotsalira ndi ABTS ngati substrate inawerengedwa pambuyo pa kukhazikika.
Laccase inayikidwa mu sodium phosphate buffer kwa mphindi 10 (0.05 M, pH 7.0) yokhala ndi ma ayoni osiyanasiyana achitsulo (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, ndi Mn2+) pamlingo wa 2.5 mM ndi 10 mM, motsatana. Kenako gawo lapansi (ABTS) linawonjezedwa kuti liyambe kuchitapo kanthu, ndipo ntchito yokhudzana nayo inayesedwa.
Kuchuluka kwa okosijeni wa ABTS ndi laccase pamlingo wosiyanasiyana (0.025–3 mM) kunayesedwa pa pH 4.5 kuti adziwe magawo a kinetic (Vmax ndi Km).^zosasinthikaMa equation a Michaelis-Menten adawerengedwa pogwiritsa ntchito Lineweaver-Burk plot, yomwe imafotokoza momwe reaction rate imayenderana ndi kuchuluka kwa substrate. Ma kinetic constants adawerengedwa kuchokera ku Lineweaver-Burk plot pogwiritsa ntchito pulogalamu ya GraphPad Prism version 6.01.
Pambuyo potsuka maapulo bwino ndi madzi apampopi, adadulidwa pakati ndikusakaniza ndi madzi pogwiritsa ntchito makina oyeretsera apulo a Braun MP80 okha (opangidwa ku Germany). Madziwo adasefedwa kudzera mu cheesecloth zinayi. Palibe ma enzyme omwe adawonjezedwa ku gulu lowongolera, pomwe 2.0% laccase (kuchuluka kogwira mtima kwambiri komwe kudayesedwa) kudawonjezedwa ku madzi a apulo okonzedwa kumene, omwe adasungidwa pa 4°C kwa milungu iwiri.
Titratable acidity (TA) ndi pH zinatsimikiziridwa motsatira njira ya Boulton et.^al.27. PH ya chitsanzo chilichonse inayesedwa pogwiritsa ntchito digito ya pH meter (JENWAY 3510 pH meter). Titratable acidity (TA) inawerengedwa kutengera malic acid pogwiritsa ntchito njira yotsatirayi.
Pamene V ndi C ndi voliyumu (mL) ndi kuchuluka (0.1 mol/L) kwa sodium hydroxide solution yomwe imagwiritsidwa ntchito mu titration, motsatana. K ndi malic acid conversion coefficient, yofanana ndi 0.067, ndipo W ndi kulemera (g) kwa madzi a apulo.
Zinthu zonse zosungunuka (^TDS) kuchuluka kwa zitsanzo zonse za madzi kunapezeka pogwiritsa ntchito PAL-1 pocket refractometer (ATAGO, Tokyo, Japan). Pambuyo poyesa kulikonse, lenzi yowunikira inkatsukidwa ndi madzi oyeretsedwa, ndipo chitsanzo chilichonse cha madzi a apulo chinayesedwa katatu. Mtengo wa chitsanzo chilichonse unawerengedwa poyesa miyeso itatu. Avereji ya ± standard deviation ya chitsanzo chilichonse cha madzi a apulo inawerengedwanso poyesa zotsatira izi.
Kukhuthala kwa zitsanzo za madzi a apulo kunayesedwa pogwiritsa ntchito rotational viscometer (RV, Rheotest 2, Germany). Chitsanzocho chinayikidwa mkati mwa silinda ya "S2" ya viscometer. Kukhuthala kooneka kunayimiridwa ndi kutsetsereka kwa shear stress vs shear rate curve, komwe kunawerengedwa kuchokera ku shear stress ndi ma curve ofanana pamitundu yosiyanasiyana ya shear rates (kuyambira 1.00 mpaka 437.4 s⁻¹). Njira yowerengera kukhuthala kooneka ndi iyi:
Kumene η ndi kukhuthala koonekera (cP), τ ndi kupsinjika kwa shear (dyn/cm²), γ ndi kuchuluka kwa shear (sec⁻¹), ndipo (τ) imawerengedwa pogwiritsa ntchito ma torque (α) ndi silinda (Z) pogwiritsa ntchito njira iyi: τ = Z . α.
Chizindikiro cha browning chinadziwika malinga ndi njira ya Meidav ​​​​et^al.29Chitsanzo cha madzi a 10 ml chinayikidwa mu centrifuge pa 2750 xg kwa mphindi 10. 5 ml ya madzi owonjezera anasakanizidwa ndi 5 ml ya 95% ethanol. Kuyamwa kwa chisakanizocho kunayesedwa pa 420 nm pogwiritsa ntchito Shimadzu UV spectrophotometer (UV-1601 PC).
Chiwerengero chonse cha phenolic (TPC) chinapezeka pogwiritsa ntchito utoto pogwiritsa ntchito Folin-Ciocalteu reagent monga momwe Boulton et al adafotokozera.^[27]]. Kapangidwe ka gallic acid kokhazikika kadapangidwa kuti kakhale ndi kuchuluka kwa 0 mpaka 500 mg/L (^r²= 0.997). Zotsatira zake zimafotokozedwa ngati gallic acid equivalents (mg GAE/mL).
Onjezani 125 μL ya madzi osungunuka ndi 2850 μL ya yankho la FRAP ku 25 μL ya madzi a apulo ndipo musiye chisakanizocho mumdima kwa nthawi yayitali.³⁰mphindi. Kenako yesani kuyamwa kwa madzi pa 593 nm pogwiritsa ntchito Shimadzu UV spectrophotometer (UV-1601 PC). Reagent ya FRAP inakonzedwa mwa kusakaniza 300 mM acetate buffer (pH 3.6), 20 mM iron(III) chloride, ndi 10 mM 2,4,6-tris(2-pyridyl)triazine (TPTZ) (yosungunuka mu 40 mM HCl) mu chiŵerengero cha 10:1:1. Mzere wokhazikika unapangidwa pogwiritsa ntchito Trolox ngati muyezo (^R²= 0.999), ndipo zotsatira zake zafotokozedwa ngati μM Trolox/mL.
Mphamvu ya antioxidant ya madzi ochiritsidwa ndi osachiritsidwa inapezeka pogwiritsa ntchito njira ya DPPH kuti iwone momwe amapezera ma free radicals a DPPH.³¹Ma microliters khumi a madzi adasakanizidwa ndi 1 ml ya yankho la DPPH (100 μM) mu methanol. Pambuyo pochitapo kanthu mumdima kwa mphindi 30, kuyamwa kwa chisakanizocho kunayesedwa pa 517 nm pogwiritsa ntchito Shimadzu UV spectrophotometer (UV-1601 PC). Zotsatira zake zidafotokozedwa ngati ma trolox equivalents (μM trolox/ml) kutengera calibration curve (_^R2= 0.990).
Deta yomwe idapezeka idawonetsa kuti kupanga laccase yayikulu kunawonedwa mu bowa wa oyster wa NRC 620 pofika kumapeto kwa tsiku la 18 la kuwira, kufika pa 1302 U/L. Izi zidagwira ntchito ngati maziko odziwira nthawi yoyenera yolima yopangira laccase (Chithunzi 1). Ngakhale kupanga ma enzyme kunawonjezeka ndi nthawi yowonjezera yolima, kuchuluka kwa kuwonjezeka sikunali kofanana ndi nthawi yolima; patatha masiku 21, ntchito ya ma enzyme idakwera ndi 90 U/L yokha (mpaka 1390 U/L). Chifukwa chake, masiku 18 adasankhidwa kukhala nthawi yoyenera yolima kuti agwirizane ndi phindu la zinthu ndi phindu lachuma la nthawi yowonjezera yolima.
Zotsatira za nthawi yolima pa zipatso za laccase mu Pleurotus ostreatus NRC 620. Mabuloko atatu (12 mm) a bowa a mycelial adabayidwa mu 50 ml ya malo osungiramo zinthu osabala kenako nkulimidwa pa 28 °C kwa nthawi zosiyanasiyana.
Mogwirizana ndi kafukufuku wina, zotsatira zathu zikusonyeza kuti nthawi yoyenera yolerera kuti bowa atulutse laccase kwambiri ndi bowa ikhoza kukhala pakati pa masiku 7 ndi 36.³²Malinga ndi Ezike et al.³³, *Trametes polyzona* WRF03 inapanga kuchuluka kwakukulu kwa laccase kumapeto kwa tsiku lachisanu ndi chinayi la kupesa, ndi ntchito yapadera ya 1637 U/mg protein. Komanso, Othman et al.³⁴adapeza kuti *Trichoderma harzianum* S7113 imatulutsa laccase yambiri patsiku lachisanu la ulimi. Kuchuluka kwa kupanga laccase kunafika pachimake pa tsiku la khumi ndi chinayi kenako kunachepa pang'onopang'ono.³⁴Ngakhale kuti kutulutsa ma enzyme kungachitikenso panthawi ya kukula kwakukulu, nthawi zambiri kumafika pachimake pa nthawi yapakati ndipo kumayambitsidwa ndi kugwiritsa ntchito gwero la kaboni kapena nayitrogeni.^34,35
Ngakhale kuti laccase yochokera ku Pleurotus ostreatus NRC 620 inachita zinthu zambiri pa kutentha kwakukulu kuyambira 50°C mpaka 80°C, ikuyandikira ntchito yaikulu (69–98%), ntchito yake yayikulu inawonedwa pa 70°C (Chithunzi 2a). Kunja kwa kutentha kumeneku, ntchito ya enzyme inachepa pa pafupifupi 70°C. Zotsatirazi zikusonyeza kuti enzyme imagwira ntchito pa kutentha kwakukulu, mwina chifukwa kutentha kwakukulu kumawonjezera mphamvu ya kinetic ya reaction.
Zotsatira za kutentha kwa reaction (a) ndi pH (b) pa ntchito ya laccase mu *Pleurotus ostreatus* NRC 620. Kutentha kuyambira 20 mpaka 90 °C kunapezeka mwa kuyika chisakanizocho pa kutentha kosiyanasiyana kwa mphindi 5 musanawonjezere enzyme ndikuyamba reaction. Zotsatira za pH pa ntchito ya laccase zinayesedwa pogwiritsa ntchito ABTS ngati substrate mu mayankho okhala ndi 0.1 M citrate-phosphate buffer pa pH range ya 2.5 mpaka 7.0.
Malinga ndi Ezike et^al.33, kutentha koyenera kwa *Trametes polyzona* WRF03 laccase ndi 55 °C, komwe ndi kofanana ndi kwa *Ganoderma lucidum*^laccase36ndipo mofanana ndi kutentha koyenera (50 °C) kwa *Trametes polyzona* KU-RNW02737^laccase . ^Baldrian38akunena kuti, ponena za machitidwe ena a enzyme omwe amawononga lignin, kutentha koyenera kwa laccase ndi pakati pa 50 ndi 70 °C.
Zotsatira zake zinasonyeza kuti enzymeyi inachita bwino kwambiri pa pH 3.0, kufika pa 94% ya ntchito pa pH 3.5. Komabe, inagwira ntchito pa pH yochuluka kuyambira 2.5 mpaka 7.0 (Chithunzi 2b). Kuphatikiza apo, inachita bwino kwambiri pa acidity poyerekeza ndi momwe zinthu zilili kapena za alkaline. Ntchito yake inakhalabe osachepera 77% kuposa pH yoyambira 2.5 mpaka 4.5, koma inangofika pafupifupi 38% pa pH 7.0. PH yabwino kwambiri ya laccase kuchokera ku *Trametes polyzona* WRF03 inali 4.533, yomwe ndi yofanana ndi pH ya laccases kuchokera ku *Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, ndi *Trametes hirsuta* 41. Komabe, malinga ndi kafukufuku wa Chairin et al.⁴², pH yabwino kwambiri ya laccase kuchokera ku *Polymorpha f. sp.* WR710-1 ndi 2.2, pomwe pH yabwino kwambiri ya laccase kuchokera ku *Polymorpha f. sp.* IBL-04 ndi 5.043. Kumangirira kwa ma hydroxide anions (laccase inhibitor) ku maatomu amkuwa a T2/T3 laccase kungakhale chifukwa cha kuchepa kwa ntchito ya laccase pansi pa mikhalidwe ya pH yopanda mbali kapena ya alkaline. Izi zitha kusokoneza kusamutsa kwa ma electron amkati kuchokera pakati pa T1 kupita pakati pa T2/T3, motero^kuchepetsantchito ya enzyme 23,44
Mwa kuyika enzymeyi pa kutentha kosiyanasiyana, zinapezeka kuti nthawi yoti ilowetsedwe komanso kutentha zonse zinakhudza kukhazikika kwa enzyme. Chodziwika bwino n'chakuti, laccase yochokera ku *Trametes polyzona* NRC 620 inasonyeza kukhazikika kwakukulu pa 40℃ ndi 50℃, kusunga 68.33% ndi 59.61% ya ntchito yake yoyambirira, motsatana, pambuyo pa mphindi 120 (Chithunzi 3a). Mosiyana ndi zimenezi, pansi pa mikhalidwe yomweyi (40℃ ndi 50℃, mphindi 120), laccase yochokera ku *Trametes polyzona* WRF03 inasunga 64.38% ndi 42.92% ya ntchito yake, motsatana.³³M'malo mwake, kuwonjezeka kwa nthawi yoyamwitsa ndi kutentha kunachepetsa kukhazikika kwa *Trametes polyzona* NRC 620 laccase; Pambuyo poyamwitsa pa 60℃ ndi 70℃ kwa mphindi 60, ntchito yake inachepa kufika pa 39.24% ndi 1.72%, motsatana (Chithunzi 3a). Mogwirizana ndi zotsatira za kuyesera, laccase yochokera ku *Trametes polyzona* WRF03 inasonyeza kukhazikika kwakukulu pa 40 ndi 50℃ panthawi yonse yochizira kutentha.³³Mofananamo, Lueangjaroenkit et^al.37ndi Chairin et^al.42adanenanso za kukhazikika kwa laccases kuchokera ku Trametes polyzona KURNW027 ndi Trametes polyzona WR710-1 pa 50 °C kwa ola limodzi motsatana. Monga biocatalyst yothandiza yogwiritsidwa ntchito m'magawo osiyanasiyana a biotechnology, laccase iyenera kukhala yokhazikika komanso yogwira ntchito bwino pa kutentha kwakukulu.
Kukhazikika kwa thermostatic (a) ndi kukhazikika kwa pH (b) kwa laccase kuchokera ku *Pleurotus ostreatus* NRC 620. Kukhazikika kwa thermostatic kunayesedwa mwa kuyika yankho la enzyme mu 0.05 M sodium phosphate buffer (pH 7.0) pa 40, 50, 60, ndi 70 °C kwa maola awiri, motsatana. Kukhazikika kwa pH kunayesedwa mwa kuyika yankho la enzyme mu 0.1 M citrate buffer ndi Tris buffer (pH 3, 4, 6, ndi 7) pa 40 °C kwa maola awiri. Ntchito yotsalira inawerengedwa pogwiritsa ntchito ABTS ngati substrate pambuyo pa kuyika.
Kuti tidziwe momwe ma enzyme amagwiritsidwira ntchito komanso kusungidwa bwino, tinafufuza momwe pH imakhudzira kukhazikika kwa laccase. Kukumana ndi ma pH osiyanasiyana kunakhudza kwambiri kukhazikika kwa kapangidwe ka mapuloteni, motero kunakhudza kukhazikika ndi ntchito ya molekyulu ya enzyme. Zotsatira zake zinasonyeza kuti enzymeyo sinali yokhazikika kwambiri pansi pa acidity, pomwe inasonyeza kukhazikika bwino pa ma pH apamwamba (malo osalowerera komanso a alkaline). Pa ma pH a 7.0, 6.0, 4.0, ndi 3.0, kuchuluka kwa ma enzyme osungidwa pambuyo pa mphindi 120 kunali pafupifupi 100%, 62.54%, 52.39%, ndi 11.14%, motsatana (Chithunzi 3b). *Strombus multisus* WRF03 laccase inasonyeza kukhazikika kwakukulu pa ma pH osalowerera (5.5–6.5) ndi kukhazikika kochepa pa ma pH osalowerera (pansi pa 4.0). Pambuyo pa mphindi 120 pa pH ya 5.5, 6.0, ndi 6.5, kuchuluka kwa ma enzyme osungidwa kunali pafupifupi 82%, 100%, ndi 93% motsatana.³³Khairin ndi ena.⁴²adazindikira kuti laccase yochokera ku Trametes polyzona WR710-1 inali yokhazikika pa pH ya 6.0 mpaka 7.0, pomwe Sayed et al.⁴⁵zinasonyeza kuti laccase inali yokhazikika bwino pansi pa pH yopanda mbali. Komabe, laccase yochokera ku Cerrena unicolor inasonyezanso kukhazikika pansi pa mikhalidwe ya alkaline (pH 9.0)⁴⁶Ma laccases omwe adaphunziridwa adawonetsa kukhazikika kwakukulu pa pH yochuluka. Ichi chikhoza kukhala chizindikiro chofunikira pa ntchito zamafakitale.
Popeza ma ayoni ena achitsulo ali ndi mphamvu zolimbikitsa komanso zoletsa ntchito ya ma enzyme, mphamvu zawo pa ntchito ya ma enzyme ziyenera kuganiziridwa m'mafakitale. Izi ndizofunikira chifukwa ma ayoni achitsulo ndi zinthu zodetsa zachilengedwe zomwe zingakhudze kukhazikika ndi kapangidwe ka ma enzymes akunja kwa thupi.⁴⁷Kuti tifufuze zotsatira za ma ayoni ambiri achitsulo pa laccase kuchokera ku *Pleurotus ostreatus* NRC 620, tinachita zoyeserera zofanana. Monga momwe zasonyezedwera pa Chithunzi 4, kutengera mtundu wa chitsulo chomwe chagwiritsidwa ntchito, kuwonjezera kuchuluka kwa ma ayoni achitsulo kuchokera pa 2.5 mM kufika pa 10 mM kunakhudza kwambiri ntchito ya enzyme. Mwachitsanzo,^Mg²⁺ , ^Co²⁺ , ^Zn²⁺ndi^Cu²⁺imatha kuyambitsa ndikuyambitsa ntchito ya ma enzyme,^Na⁺ , ^Mn²⁺ , ^Ca²⁺ndi^K⁺zimatha kuletsa ntchito ya enzyme. Pa kuchuluka kwa 10 mM, ma ioni a Cu²⁺ ndi Mg²⁺ anali oyambitsa mphamvu kwambiri ya ntchito ya laccase kuchokera ku *Pleurotus ostreatus* NRC 620, zomwe zimapangitsa kuti pakhale kuchuluka kwa ntchito ya laccase pafupifupi 34% ndi 20%, motsatana. Komabe, pa kuchuluka kwa 10 mM, ma ioni a Ca²⁺ anali oletsa mphamvu kwambiri ya laccase, zomwe zimachepetsa ntchito ya enzyme ndi pafupifupi 60%.
Zotsatira za ma ayoni achitsulo pa ntchito ya Pleurotus ostreatus NRC 620 laccase. Laccase inayikidwa mu incubator kwa mphindi 10 mu sodium phosphate buffer (0.05 M, pH 7.0) yokhala ndi ma ayoni achitsulo osiyanasiyana pamlingo wa 2.5 mM ndi 10 mM. Kenako yankho linayambitsidwa ndi kuwonjezera substrate (ABTS), pambuyo pake ntchito yofanana inayesedwa.
Zotsatira zathu zikugwirizana ndi za olemba ena omwe adapeza kuti Mg²⁺ ndi Cu²⁺ zimawonjezera ntchito ya *Trametes polyzona* WRF03³. Castaño et al.⁴⁸ adapeza kuti laccase yochokera ku *Xylaria* sp. imalimbikitsidwa pang'ono ndi ma ayoni amkuwa (Cu²⁺). Kuphatikiza apo, Foroutanfar et al.⁴⁹ ndi Si et al.⁵⁰ adachita maphunziro ofanana pa laccases ochokera ku *Paraconiothyrium variabile* ndi *Trametes pubescens*, motsatana. Mtundu wachiwiri wa copper-binding site (T2) wa enzyme iyi ukhoza kudzazidwa ndi Cu²⁺ pamlingo winawake, zomwe zitha kufotokoza kukondoweza kwa ntchito ya laccase pamlingo wapamwamba wa Cu²⁺³⁹. Popeza ma laccases a bowa woyera ndi ma oxidases okhala ndi ma atomu angapo amkuwa, zotsatira za ma ayoni amkuwa pa ntchito ya laccase ndizosiyanasiyana ndipo zimayambira pa zolimbikitsa komanso zoletsa mpaka zosalowerera.⁵¹ Mosiyana ndi zimenezi, Zhou et al.^{. [52]}adanenanso kuti^Cu²⁺inaletsa ntchito ya laccase ya chiswe cha pansi pa nthaka cha ku Taiwan (Odontotermes formosanus). Komabe, laccases ya Cerena sp. HYB07^[53]ndi Clitocybe maxima^[54]sizinakhudzidwe ndi ma ayoni amkuwa.
Kudziwika bwino kwa substrate kunayimiridwa ndi magawo ake a kinetic (Km ndi Vmax); kulimba kwa mgwirizano wa substrate ndi enzyme, kutsika kwa Km komanso kukwera kwa specificity ya substrate.^3,21,55Ma parameter a kinetic (Km ndi Vmax) a laccase ochokera ku *Pleurotus ostreatus* NRC 620 adapezeka pogwiritsa ntchito pulogalamu ya GraphPad Prism 6.0 pojambula chithunzi cha Lineweaver-Burk (Chithunzi 5). Pogwiritsa ntchito ABTS ngati substrate, zotsatira zake zinali 1.99 mM ndi 16217 μmol.^{mphindi⁻¹ L⁻¹,}Elsayed ndi ena.²¹adanenanso kuti ma Km ​​a okosijeni a ABTS anali 0.1 mM ndi 0.064 mM, motsatana, zomwe zikusonyeza kugwirizana kwakukulu kwa Lac A ndi Lac B isoenzymes a ABTS. Kuphatikiza apo, ma Vmax ​​adali 0.182 μmol.^mphindi⁻¹ndi 0.603 μmol^mphindi⁻¹, motsatana. Mtengo wa Km womwe unapezeka unali wotsika kuposa wa Trametes polyzona WRF03 (8.66 mM); komanso, mtengo wawo wa Vmax (1429 mmol min⁻¹) unalinso^pansipogwiritsa ntchito ABTS ngati substrate.33 Mofananamo, ma Km values ​​​​a Lentinus squarrosulus MR13 ndi Trametes sp. AH28-2 laccase concentrations anali 0.0714 mM ndi 0.025 mM, motsatana, ndipo ma Vmax values ​​​​anali 0.0091 mM min−1 ndi 0.67 mM min−1 mg−1 (poyerekeza ndi ABTS)^{, motsatana.56,57}
Zotsatira za kuchuluka kwa ABTS pa ntchito ya laccase kuchokera ku *Pleurotus ostreatus* NRC 620 zinafufuzidwa, ndipo chithunzi cha Lineweaver-Burk cha reciprocal ya liwiro loyamba la reaction motsutsana ndi kuchuluka kwa ABTS chinajambulidwa. Kuyankha kwa okosijeni kwa ABTS ndi kuchuluka kosiyana (0.025–3.0 mM) kwa laccase kunayesedwa pa pH 4.5 kuti adziwe magawo a kinetic (Vmax ndi Km). Zokhazikika za Michaelis-Menten kinetic zinawerengedwa pogwiritsa ntchito chithunzi cha Lineweaver-Burk cha reciprocal ya liwiro la reaction motsutsana ndi kuchuluka kwa substrate. Zokhazikika za kinetic zinawerengedwa kuchokera ku chithunzi cha Lineweaver-Burk pogwiritsa ntchito pulogalamu ya GraphPad Prism 6.01.
Ma enzyme oyeretsa mwachikhalidwe, monga ma pectinase, amachotsa zinthu za pectic, amachepetsa kukhuthala ndi kukhuthala. Amaphwanya bwino ma polysaccharide opangidwa ndipo nthawi zambiri amagwiritsidwa ntchito limodzi ndi ma enzyme ena, monga ma cellulases ndi ma hemicellulases, kuti akonze zokolola ndi kumveka bwino. Komabe, ma pectinase samayang'ana kwambiri mankhwala a phenolic, omwe ndi omwe amachititsa kuti turbidity ndi oxidative browning ziwonongeke, makamaka mu madzi monga apulo ndi madzi a mphesa.⁵⁸Mosiyana ndi zimenezi, ma laccase amalimbikitsa okosijeni wa mankhwala a phenolic, kuwapanga kukhala mamolekyu akuluakulu osasungunuka omwe angachotsedwe ndi sedimentation kapena kusefa. Njira imeneyi sikuti imangowonjezera kumveka bwino komanso imawonjezera moyo wa madzi mwa kuchepetsa mwayi wa okosijeni kufiira chifukwa cha mankhwala a phenolic. Kuphatikiza apo, njira zowunikira zochokera ku laccase zitha kuchitika pansi pa mikhalidwe yofatsa yokonza (pH 3.5–5.5, kutentha 25–40 °C), zomwe zimapangitsa kuti akhale oyenera madzi ofewa popanda kuwononga thanzi lawo kapena zinthu zina.⁵⁹Kafukufuku wasonyeza kuti chithandizo cha pectinase chimatha kuyeretsa madzi mkati mwa maola 1-2, pomwe chithandizo cha laccase nthawi zambiri chimafuna nthawi yayitali yochitirapo kanthu (maola 3-6) kuti achepetse kwathunthu mankhwala a phenolic. Komabe, njirayi ikhoza kukonzedwa bwino mwa kuletsa enzyme kapena kuphatikiza laccase ndi njira zowunikira zamakina.⁶⁰Mu kafukufukuyu, kufufuza kwa ma enzyme a chotsitsa chosaphika kunawonetsa ntchito zazikulu za laccase ndi α-amylase, pomwe ntchito za pectinase ndi xylanase zinali zochepa kwambiri, ndipo ntchito ya cellulase sinapezeke. Chifukwa chake, kuchepa kwa turbidity ndi kuchuluka kwa phenolic kunali makamaka chifukwa cha ntchito ya laccase, pomwe kusintha kwa viscosity kungakhale chifukwa cha ntchito ya amylase.
Gome 1 likuwonetsa magawo a physicochemical a madzi a apulo ofinyidwa kumene ndi zitsanzo zokonzedwa ndi laccase. Zotsatira zake zikuwonetsa kuti zokolola za madzi a apulo ofinyidwa kumene (71.59%) zinali zochepa kuposa za zitsanzo zokonzedwa ndi laccase (87.34%). Zotsatirazi zikugwirizana ndi zomwe zapezeka ndi Pilnik ndi Orange.⁶¹, omwe adanenanso kuti kugwiritsa ntchito ma enzyme pokonza zipatso kumatha kuwonjezera kuchuluka kwa madzi, kukonza kusefa, ndikupeza madzi abwino komanso omveka bwino kuti azigwira bwino ntchito. Kuwonjezeka kwa kuchuluka kwa madzi kumachitika makamaka chifukwa cha kuchuluka kwa shuga wosungunuka mu madzi. Pa nthawi ya enzyme hydrolysis ya zipatso, mesoglea ndi pectin m'makoma a maselo a chinthucho zimawonongeka ndikusandulika zinthu zosungunuka monga shuga wosalowerera komanso ma acid.^62.pH ya madzi a apulo omwe adachiritsidwa ndi enzyme inali yotsika kwambiri kuposa ya gulu lolamulira (P < 0.05), ndipo pH ya magulu onse awiri idakwera kwambiri panthawi yosungira (Table 1). Zotsatirazi zikugwirizana ndi za Mark et al.⁶³, amene adazindikira kuti pH ya madzi a zipatso za cashew inachepa pambuyo posungidwa pambuyo potenthedwa. Kuwonongeka kwa pectin ndi kupangika kwa galacturonic acid pambuyo pothandizidwa ndi ma enzyme kungakhale chifukwa cha kuwonjezeka kwa pH panthawi yosungidwa. pH ya zitsanzo zomwe zinatengedwa ndi ma enzyme inakhalabe pakati pa 4.05 ndi 4.31 nthawi yonse yosungidwa, pomwe pH ya madzi a apulo omwe sanatengedwe inali pakati pa 4.12 ndi 4.33.
Kuchuluka kwa asidi (TA) kwa zitsanzo zomwe sizinachiritsidwe komanso zomwe sizinalandire laccase kunawonetsa kuchepa kwa nthawi yosungira (Table 1). Kuchepa kwa asidi kunayambitsidwa ndi kusintha kwa ma organic acid kukhala chakudya kapena ma enzyme reaction, komanso okosijeni panthawi yosungira madzi.⁶⁴Asidi yonse ya madzi a apulo ndi zitsanzo zomwe zinaperekedwa ndi ma enzyme inali yotsika kuposa ya madzi ena (madzi a sitiroberi 0.9%, madzi a plum 2.2%, madzi a kumquat 1.0%, madzi a apricot 2.4%, madzi a lalanje 0.8%), koma yofanana ndi ya madzi ena (monga madzi a peyala 0.3%).⁶²Kusiyana kumeneku kwa madzi a apulo omwe angomalizidwa kumene osachiritsidwa kungakhale chifukwa cha zinthu zosiyanasiyana monga momwe amakulira, majini, kukula kwake, ndi njira zowakonzera.⁶⁵Kuchepa kwa asidi onse mu chakudya chowongolera ndi madzi a apulo okonzedwa ndi laccase kukugwirizana ndi zotsatira zomwe Singh et al.⁶⁶za kuchepa kwa asidi onse a madzi a apulo a Jin Nuo pambuyo pa masiku 74 osungidwa. Kumbali inayi, Oshmiansky ndi Wojdylo⁶⁷sanapeze kusintha kulikonse kwakukulu mu acidity ya madzi a apulo pophunzira momwe njira zachikhalidwe zowunikira zimakhudzira.
Zotsatira zomwe zaperekedwa mu Table 1 zikusonyeza kuti phindu lonse la madzi a apulo osungunuka (TSS) linali lalikulu kuposa la chitsanzo chomwe sichinachiritsidwe. Zotsatirazi zikugwirizana ndi maphunziro ofalitsidwa.^{. 68}Kuphatikiza apo, Gome 1 likuwonetsa kuti mtengo wa TSS wa gulu lolamulira la madzi a apulo unali 9.58 panthawi yoyamba ndipo unafika pa 11.05 kumapeto kwa nthawi yosungira. Mitengo iyi ndi yotsika kuposa mtengo wa TSS wa madzi atsopano a apulo omwe adanenedwa ndi Hamid et al.^{. 69}(11.2 ndi 11.80, motsatana). Mtengo wa TSS wa zitsanzo za madzi a apulo okonzedwa ndi laccase unakwera kwambiri, kuyambira pa 11.23 mpaka kufika pa 12.93 patatha milungu iwiri yosungidwa pa 4°C (Table 1). Kuwonjezeka kofanana kwa TSS panthawi yosungira kunawonedwanso mu zipatso za citrus, mandimu, ndi malalanje otsekemera. Kuwonjezeka kwa zinthu zonse zosungunuka (TSS) panthawi yosungira kungakhale chifukwa cha hydrolysis ya polysaccharides (wowuma) kukhala monosaccharides (shuga), kuwonjezeka kwa kuchuluka chifukwa cha kusowa madzi m'madzi, komanso kuwonongeka kwa pectin mu madzi kukhala zinthu zosungunuka. Kuwonjezeka kwa zinthu zonse zosungunuka (TSS) mwina chifukwa cha kuwonjezeka kwa shuga wosungunuka, komwe kungapangidwe ndi kusintha kwa pectin kapena cellulose kukhala shuga wosungunuka ndi pectin kapena cellulase, motsatana, kapena ndi hydrolysis ya wowuma kukhala shuga, monga momwe Hamed et al adanenera.^69.Zotsatira za laccase pa mphamvu ya madzi a apulo zitha kuwonedwa m'maso, chifukwa madzi a apulo okonzedwa ndi laccase amawonetsa kuyenda bwino komanso kukhuthala kochepa kuposa madzi osakonzedwa. Izi zalembedwa mu Gome 1; Kukhuthala kwa chitsanzo chokonzedwa ndi enzyme chinali 1.87 cP, pomwe kukhuthala kwa chitsanzo chowongolera chinali 2.95 cP. Kuchepa kwakukulu kwa kukhuthala kumeneku mwina chifukwa cha mphamvu yayikulu yosunga madzi ya zinthu zofanana ndi pectin komanso kupanga kapangidwe ka netiweki yogwirizana.
Mu kafukufukuyu, zotsatira za laccase pa browning index (BI) ya madzi a apulo zinafufuzidwa poyesa kuyamwa kwa madzi pa 420 nm pogwiritsa ntchito spectrophotometer. Zotsatira zake zawonetsedwa mu Table 1. Panthawi yosungira, BI ya zitsanzo za madzi a apulo m'magulu omwe adachiritsidwa ndi omwe sanalandire chithandizo inawonetsa kuwonjezeka pang'onopang'ono. BI imasonyeza kuchuluka kwa browning ndipo ikhoza kugwira ntchito ngati^chofunikachizindikiro cha kusintha kwa browning kwa enzymatic ndi non-enzyme. Kuyamwa kwa madzi kunawonjezeka kwambiri panthawi yosungira (P < 0.05). Pamapeto pa kusungira,_A420Kuchuluka kwa zitsanzo za madzi a apulo m'magulu owongolera ndi omwe amathandizidwa ndi ma enzyme kunakwera ndi pafupifupi 217% ndi 121%, motsatana (Table 1). Zotsatira zake zikusonyeza kuti chithandizo cha ma enzyme chingachepetse bwino kuchuluka kwa browning ndi pafupifupi 56%. Zotsatira za Bezerra et al.^[19][] zikugwirizana ndi zotsatira zathu; Anagwiritsa ntchito ulusi wa laccase-glutaraldehyde-kokonati kuti amveketse bwino madzi a apulo, zomwe zinachepetsa mtundu wake woyambirira ndi 61%.
Ngakhale kuti ma polyphenols omwe ali mu madzi a zipatso ali ndi zotsatira zabwino pa thanzi komanso pochiritsa thupi la munthu, amathanso kuchitapo kanthu ndi mapuloteni, zomwe zimapangitsa kuti madzi akhale ndi mitambo, kusungunuka kwa madzi, kapena kutayikira, motero kusintha kukoma ndi fungo la mankhwalawo ndikuchepetsa nthawi yake yosungira.⁷¹Cholinga cha kafukufukuyu chinali kuchepetsa mosamala kuchuluka kwa phenolic compound mu madzi a apulo pogwiritsa ntchito laccase kuchokera ku Pleurotus ostreatus NRC 620. Zotsatira zomwe zaperekedwa mu Table 1 zikuwonetsa kuti kuchuluka kwa phenolic compound mu madzi a apulo okonzedwa ndi laccase kunachepetsedwa kwambiri musanasungidwe pa 4 °C. Kuphatikiza apo, kuchuluka kwa phenolic compound nako kunachepa panthawi yosungidwa m'masampulu onse awiri omwe adaphunziridwa (Table 1). Kafukufuku wochitidwa ndi Sandri et al.⁷²zasonyeza kuti madzi a apulo omwe amathandizidwa ndi ma enzyme amatha kusunga ntchito yake yoteteza ku ma antioxidants komanso kuchuluka kwa phenolic compounds. Komabe, zotsatira za kafukufuku wopangidwa ndi Lettera et al.⁷³onetsani kuti kuchiza madzi a lalanje ndi bowa laccase kungachepetse kuchuluka kwa mankhwala a phenolic omwe ali mmenemo ndi 45%.
Mankhwala a phenolic awonetsedwa kuti ali ndi zinthu monga kuchotsa ma free radicals, kuchepetsa ndi kuzima kwa mpweya wa singlet, kusamutsa maatomu a hydrogen, komanso kupereka ma electron ku ma free radicals, zomwe zimapangitsa kuti akhale ma antioxidants amphamvu.⁷⁴Chifukwa chake, mu kafukufukuyu, njira zochokera ku DPPH ndi FRAP zidagwiritsidwa ntchito poyesa momwe laccase imakhudzira ntchito ya antioxidant ya madzi a apulo omwe amasungidwa mufiriji kwa masiku 14 (Table 2). Njira zonsezi zidawonetsa kuwonjezeka kwa ntchito ya antioxidant panthawi yosungidwa, zomwe zitha kukhala chifukwa cha kuwonjezeka kwa mankhwala a phenolic aulere kapena kupangika kwa mankhwala a Maillard reaction (MRPs), ndipo mankhwala a Maillard reaction mwina ndi omwe amachititsa kuwonjezeka kwa ntchito ya antioxidant.⁷⁵Machitidwe osagwiritsa ntchito ma enzyme browning (kuphatikizapo ascorbic acid degradation, Maillard reactions, ndi acid-catalyzed degradation of sugar) amapanga utoto wa bulauni (melanoidins). Zinthu zapakati zowononga ascorbic acid ndi zinthu zowononga shuga (monga carbonyl compounds) zimatha kuchitapo kanthu ndi amino acid kudzera mu Maillard reactions.⁷⁶Ngakhale kuti kufiira kwa zipatso ndi ndiwo zamasamba panthawi yosungira kwaphunziridwa kwambiri, kumvetsetsa kwathu za izi kumakhala kochepa.⁷⁷Poyerekeza ndi njira ya FRAP, madzi a apulo omwe adathiridwa ndi laccase adawonetsa mphamvu yotsika kwambiri ya antioxidant pogwiritsa ntchito njira ya DPPH (Table 2), ndipo mphamvu ya antioxidant ya zitsanzo zonse idawonjezeka kwambiri ndi nthawi yowonjezera yosungira. Njira ziwiri zosiyana zodziwira mphamvu ya antioxidant zidagwiritsidwa ntchito mu kafukufukuyu chifukwa mfundo zawo zimasiyana. Njira ya DPPH imayesa mphamvu yothetsa ma free radicals, pomwe njira ya FRAP imayesa mphamvu yochepetsera ayoni achitsulo. Chifukwa chake, tikulimbikitsidwa kugwiritsa ntchito njira zingapo zodziwira mphamvu ya antioxidant kuti timvetse bwino mphamvu ya antioxidant ya zitsanzo zomwe zaphunziridwa.⁷⁸
Chimodzi mwa zinthu zofunika kwambiri zomwe zapezeka mu kafukufukuyu ndichakuti *Pleurotus ostreatus* laccase NRC 620 imagwira ntchito bwino kwambiri pa 70°C ndi pH 3.0. Poyerekeza ndi ma laccase ena a bowa omwe amagwiritsidwa ntchito kwambiri poyeretsa madzi, monga *Trametes versicolor* ndi *Ganoderma lucidum* laccases, *P. ostreatus* NRC 620 imawonetsa kukhazikika kwa kutentha kwambiri komanso pH yokhala ndi asidi wambiri. Ma laccase ochokera ku *Trametes versicolor* ndi *Ganoderma lucidum* nthawi zambiri amakhala ndi ntchito yabwino kwambiri pakati pa 50-60°C komanso pa pH pakati pa 3.5 ndi 5.0. Kusiyana kumeneku kungathandize kuti madzi azikhala bwino, makamaka pamadzi a acidic pomwe kukhazikika pa pH yotsika ndikofunikira. Khalidwe lapadera la *P. Poyerekeza ndi ma laccase ena a bowa omwe aphunziridwa, *Pleurotus ostreatus* NRC 620 imasonyeza kuthekera kogwira ntchito bwino pamikhalidwe yovuta kwambiri. Kutentha kwake kwakukulu kwa ntchito kumasonyeza ubwino womwe ungakhalepo pa ntchito zamafakitale, monga kuchuluka kwa zochita mwachangu komanso kuchepa kwa kuipitsidwa kwa tizilombo toyambitsa matenda. pH yake yotsika, yomwe imagwirizana bwino ndi acidity ya madzi ambiri, ingakhale yothandiza pakuyeretsa madzi. Zotsatirazi zimatsimikizira kufufuza kwina kwa kugwiritsa ntchito kwakukulu, zomwe zimapangitsa kuti *Pleurotus ostreatus* NRC 620 ikhale njira ina yabwino m'malo mwa magwero achikhalidwe a bowa laccase. Poyerekeza ndi maphunziro am'mbuyomu, tapeza kuti kutentha kwabwino kwambiri ndi 60°C ndipo pH yabwino kwambiri ndi 3.0. Pambuyo pa kuchitapo kanthu pa 60°C kwa mphindi 80, *Ganoderma lucidum* laccase idasungidwa.⁴⁶% ya ntchito zake.79 Malinga ndi Kurniawati ndi Nicelle⁸⁰, Ma enzyme a *Ganoderma lucidum* amasonyeza kukhazikika bwino mpaka pang'ono pa 25°C ndi pH kuyambira 5.0 mpaka 8.0, ndi kukhazikika pa pH 6.0 ndi kutentha kuyambira 10 mpaka 30°C. Mu kafukufukuyu, tapeza kuti pH yabwino kwambiri ndi kutentha kwa ntchito ya enzyme ya *Pleurotus ostreatus* inali 3.0 ndi 70°C, motsatana. Pambuyo poikamo kutentha pa 40°C ndi 50°C kwa maola awiri, enzymeyo inasunga 68.33% ndi 59.61% ya ntchito yake, motsatana. Kuphatikiza apo, Pleurotus ostreatus NRC 620 laccase inachita ntchito yayikulu pa kutentha kwakukulu kuyambira 50°C mpaka 80°C, pafupifupi kufika pa ntchito yayikulu (69%–98%), ndipo ntchito yayikulu kwambiri inawonedwa pa 70°C.
Pomaliza, bowa wa oyster laccase NRC620, womwe unapezeka pansi pa mikhalidwe yosasunthika, unawonetsa ntchito yabwino kwambiri komanso kukhazikika pamitundu yosiyanasiyana ya pH ndi kutentha, kusonyeza kukhazikika kwakukulu poyerekeza ndi magwero ena a enzyme. Kuwonjezeredwa kwa 10 mM MgSO₄ ndi CuSO₄ kunawonjezera ntchito ya enzyme ndi pafupifupi 21% ndi 35%, motsatana. Ikakonzedwa kukhala madzi a apulo, enzymeyo inachepetsa pH ndi kukhuthala, pomwe kuchuluka kwa phenolic kunachepa pang'ono panthawi yosungira.
Zotsatirazi zikutsimikizira kuthekera kwa laccase mumakampani azakudya, makamaka pakuyeretsa zakumwa. Mwa kuswa makamaka mankhwala a phenolic, laccase sikuti imangochepetsa kukhuthala ndikuwongolera kumveka bwino komanso imasunga ubwino wa madzi a zipatso pansi pa mikhalidwe yofatsa yogwira ntchito. Mosiyana ndi zinthu zachikhalidwe zoyeretsa monga gelatin, bentonite, ndi silica gel, laccase sipanga zinyalala kapena kuchotsa fungo labwino kuchokera ku zakumwa, zomwe zimapangitsa kuti ikhale yosamala zachilengedwe komanso yokhazikika. Kuphatikiza apo, poyerekeza ndi ma enzyme ena ndi njira zosefera, laccase imapereka yankho lolunjika komanso lotsika mtengo popanda kuwononga ubwino wa malonda.
Kyomuhimbo, HD ndi Brink, HG. Kugwiritsa ntchito ndi njira zoletsa kuyenda kwa laccases zokhala ndi mkuwa; ndemanga. Heliyon 9, e13156 (2023).