Mmepụta na njirimara biochemical nke oyster ero laccase NRC 620 na nyocha nke arụmọrụ ya na nkọwapụta ihe ọṅụṅụ apụl.

Mgbe ụbọchị iri abụọ na ise nke incubation static na 28°C gasịrị, laccase sitere na *Pleurotus ostreatus* NRC620 gosiri ọrụ kachasị elu na usoro ọdịbendị fungal. pH kacha mma na ụkpụrụ okpomọkụ maka enzyme a bụ 3.0 na 70°C, n'otu n'otu. Mgbe awa abụọ nke incubation na 40°C na 50°C gasịrị, ọrụ enzyme ahụ nọgidere na 68.33% na 59.61%, n'otu n'otu. Mgbe awa abụọ nke incubation na citrate-phosphate buffer (pH 7.0) gasịrị, ọrụ enzyme ahụ nọgidere na 100%. Mgbakwunye nke 10 mM MgSO₄ na CuSO₄ mụbara ọrụ enzyme ahụ site na ihe dị ka 21% na 35%, n'otu n'otu, ebe NaCl, MnCl₂, KCl, na CaCl₂ gbochiri ọrụ enzyme ahụ. Site n'iji ABTS mee ihe dị ka ihe e ji eme ihe, paramita kinetic (Km na Vmax) nke *Pleurotus ostreatus* NRC 620 laccase bụ 1.99 mM na 16,217 μmol min−1 L−1, n'otu n'otu. Ọgwụgwọ enzymatic nke ihe nlele ihe ọṅụṅụ apụl belatara pH na viscosity nke ukwuu, mbelata a mekwara ka oge nchekwa dịkwuo elu. Ọgwụgwọ Laccase butere obere mbelata na mkpokọta ọdịnaya phenolic nke ihe ọṅụṅụ apụl, mana ahụghị mbelata na ọrụ antioxidant.
N'afọ ndị na-adịbeghị anya, ndị nchọpụta elekwasịla anya n'itinye teknụzụ bayotekooliki akwụkwọ ndụ akwụkwọ ndụ n'ọrụ n'ụlọ ọrụ nri. Laccase bụ otu n'ime enzymes kachasị baa uru n'ụlọ ọrụ nri, na-achọta ojiji na mpaghara dịka nhazi ihe ọṅụṅụ, ime achịcha, ime ka mmanya kwụsie ike, na imeziwanye àgwà organoleptic nke ngwaahịa nri.¹Ọtụtụ osisi na obere nje na-emepụta laccoci,²na ero dịka deuteromycetes, ascomycetes, na basidiomycetes nwekwara ike imepụta laccase.³Laccase (EC 1.10.3.2) bụ oxidase acha anụnụ anụnụ nke na-ebelata oxygen molekul na mmiri site na iji usoro nke atọm ọla kọpa dị iche iche, si otú a na-eme ka ọtụtụ ihe mejupụtara phenolic na amines aromatic dị iche iche sie ike. N'oge mmepụta nke ihe ọṅụṅụ mkpụrụ osisi na akwụkwọ nri, mmetọ enzymatic na nke na-abụghị enzymatic browning bụ nsogbu dị oke mkpa.⁴Ebe ọ bụ na ihe ndị a na-emetụta agba, ụtọ, na isi nke ihe ọṅụṅụ ahụ n'ụzọ na-adịghị mma, a ghaghị iwepụ ha.⁵
N'ime mkpụrụ osisi niile, apụl bụ nke a na-erikarị n'ụwa niile nakwa na European Union. Na 2019, mmepụta apụl nọ n'ọkwa nke atọ n'ụwa niile, gafere nde tọn 87.⁶Apụl nwere ọtụtụ ihe mejupụtara phenolic, gụnyere flavonoids na phenolic acid dịka caffeic acid na chlorogenic acid.⁷Ebe ọ bụ na a na-aṅụ ihe ọṅụṅụ apụl n'ụdị doro anya ya, ihe dị ka pasentị 50 ruo 90 nke ihe ndị dị na phenolic na-efunahụ n'oge nhazi nzacha.⁸Taa, ndị ahịa na-ahọrọkarị ngwaahịa ndị a na-anaghị emecha nke ọma, dịka mmiri apụl dị n'ígwé ojii nke nwere ọtụtụ polyphenols. Agbanyeghị, n'ihi oke phenolic ya, ụdị mmiri apụl a na-adịkarị mfe ịgbanwe agba ma mee ka ọ gbaa ọchịchịrị.⁹A na-eji ọtụtụ teknụzụ dị iche iche, gụnyere ụzọ ọgwụgwọ okpomọkụ dịka pasteurization na 60–90°C, iji belata ma ọ bụ gbochie ọchịchịrị nke ihe ọṅụṅụ apụl.¹⁰Agbanyeghị, dịka nyocha nke Sauceda-Gálvez si kwuo¹¹, nhazi okpomọkụ nwere ike ibibi kemịkalụ ndị na-agbanwe agbanwe ma metụta àgwà organoleptic nke ihe ọṅụṅụ apụl. Ihe ndị ọzọ maka usoro nhazi okpomọkụ gụnyere oke carbon dioxide, radieshon ultraviolet, ultrasound, nrụgide hydrostatic dị elu, ma ọ bụ nhazi nrụgide dị elu.¹²Arụmọrụ nke teknụzụ ndị a na mmepụta nke ihe ọṅụṅụ mkpụrụ osisi kwesịrị ekwesị dabere na paramita ejiri na njirimara ngwaahịa. Ojiji ha n'ọtụtụ ebe na-ejedebe n'ihi oke ọnụ ahịa, mmetụta ọjọọ na ịdị mma nke ụfọdụ ngwaahịa nri, ma ọ bụ enweghị enzyme zuru oke.^13,14
Enwere ike iji Laccase mee ka ihe ọṅụṅụ mkpụrụ osisi sie ike ma mee ka ọ doo anya.¹⁵Gökmen na ndị ọzọ.¹⁶A na-atụ aro ka e jiri laccase mee ka ihe ọṅụṅụ mkpụrụ osisi doo anya n'ihi na ọ na-ewepụ ihe ndị dị na phenolic nke ọma site n'ịgbanwe ha ka ha bụrụ polymers ma ọ bụ oligomers nke akpụkpọ ahụ ọ bụla na-ewepụ ngwa ngwa, na-enye ihe ọṅụṅụ apụl ohere ịnọgide na-enwe agba na nghọta doro anya ruo izu isii na okpomọkụ 50°C. A na-eji laccase *Trichoderma* dị ọcha mee ihe na beads alumina ma jiri ya wepụ ihe ndị na-adịghị mma nke sitere na mmetọ nje nke ihe ọṅụṅụ apụl kpatara.¹⁷
Ihe dị ka pasentị 80-90 nke ihe ndị na-agbanwe agbanwe dị na ihe ọṅụṅụ apụl bụ esters na aldehydes, bụ́ ndị na-enye ihe ọṅụṅụ ahụ isi pụrụ iche.¹⁸E jiri eriri eke sitere na mkpụrụ aki oyibo ndị na-eto eto mee ka ihe ọṅụṅụ apụl dị nro, nke e ji *Trametes versicolor* mee ka ọ ghara ịgbagharị agbagharị.¹⁹Nnyocha ndị gara aga enyochaala etu mmiri apụl si kwụsie ike (agba na ntụ ntụ) site na iji usoro enweghị enzyme ma ọ bụ usoro mgbochi, ma ọ bụ na njikọta ya na ultrafiltration.^5,19Agbanyeghị, mmetụta nke laccos fungal nwere na njirimara physicochemical nke ihe ọṅụṅụ apụl n'oge nchekwa ka edoghị anya. Ya mere, ebumnuche nke ọmụmụ ihe a bụ inyocha mgbanwe ndị dị na njirimara physicochemical, ọdịnaya phenolic compound, na ọrụ antioxidant nke ihe ọṅụṅụ apụl mgbe ejiri laccos fungal na nchekwa izu abụọ dị na friji gwọọ. Laccos nwere ike ime ka ihe mejupụtara phenolic dị oxidize, nke na-eme ka ha nwee nkwa maka ojiji na usoro ụlọ ọrụ dị iche iche, gụnyere nkọwa ihe ọṅụṅụ. Ọmụmụ ihe a lere laccos sitere na *Pleurotus ostreatus* NRC 620, na-elekwasị anya na ọnọdụ kachasị mma maka ọrụ ha na ịdị irè ha na nkọwa ihe ọṅụṅụ. Ọ bụ ezie na nyocha na ero oyster (P. ostreatus NRC 620) ka dị oke, ọmụmụ ihe ndị gara aga enyochala enzymes sitere na isi mmalite fungal dị iche iche, dị ka Trametes versicolor na Ganoderma lucidum. Ebumnuche nke ọmụmụ ihe a bụ inyocha ojiji enzyme a nwere ike itinye n'ọrụ na ụlọ ọrụ nri ma gosipụta ihe pụrụ iche ya, karịsịa pH na okpomọkụ ya zuru oke.
E zụtara 2,2′-Azooxybis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) n'aka Sigma-Aldrich (Kanada). Ihe ndị ọzọ niile e ji mee ihe bụ nke nyocha.
Ebe Nchịkọta Omenala Microbial nke National Research Center nwetara ụdị ero oyster a ma ama bụ NRC620. Mgbe a gbasịrị obere ubi, a na-echekwa ụdị ero a na slants potato dextrose agar na 4°C. Usoro nkwadebe inoculum dị ka ndị a: a gbara mycelium dị ụbọchị iri, nke mepụtara nke ọma n'ime efere potato dextrose agar ma tinye ya na 28°C. Mgbe ụbọchị iri gachara, e wepụrụ blọk mycelium atọ dị 12mm-diamita site na mgbasa ozi agar site na iji ihe e ji ígwè mee ma tinye ya na karama Erlenmeyer 250-mL nwere ihe mkpuchi owu nwere 50 mL nke ihe ọkụkụ a gbanyere mkpọrọgwụ (pH 5.0, dịka Othman et al kọwara na mbụ.²⁰) E tinyere ihe ọkụkụ ndị ahụ n'ime akpa ọkụ na 28°C ruo ụbọchị iri na asatọ. E wee jiri akwụkwọ nzacha Whatman Nke 1 sachaa ọdịbendị ndị ahụ, ihe na-esi na ya pụta wee bụrụ isi iyi enzyme.
E jiri ABTS mee ihe dị ka ihe e ji eme ihe iji chọpụta ọrụ Laccase. Ngwakọta mmeghachi omume ahụ (2 mL) nwere 500 μL nke 0.3 mM ABTS (agbazere na 0.1 M sodium citrate buffer, pH 4.5) na ọnụọgụ enzyme achọrọ nke a gwakọtara na mmiri a gwakọtara agwakọta.^21,22Ebe ọ bụ na laccase nwere ike ime ka ABTS ghara ịdị ọcha na okpomọkụ ụlọ (28 °C ± 2), a chọpụtara na ABTS na-eme ka ọ ghara ịdị ọcha site n'ịtụ mmụba nke absorbance na 420 nm (ε)₄₂₀= 36,000 cm^-1 M ^-1) site na iji Agilent Carry-100 UV spectrophotometer. A chọrọ otu nkeji nke ọrụ laccase iji mee ka oxidize 1 μmol ABTS kwa nkeji. E jiri usoro Bradford chọpụta ntinye protein site na iji bovine serum albumin dị ka njikwa dị n'ime.^23,24
Mgbe e nwetasịrị enzyme ahụ site na ụdị ero oyster NRC 620, a tụrụ ọrụ ya n'oge dị iche iche nke ịkọ ihe ruo ụbọchị iri abụọ na ise n'okpuru ọnọdụ na-adịghị agbanwe agbanwe na 28 Celsius.
Iji mụọ mmetụta okpomọkụ na ọrụ laccase, e mere nnwale n'ogo okpomọkụ site na 20 ruo 90 Celsius. Tupu etinye enzyme ahụ ma malite mmeghachi omume ahụ, a gwakọtara ihe nchekwa ahụ (0.1 M sodium citrate, pH 4.5) na substrate (ABTS) ma tinye ya na mkpuchi maka nkeji ise na okpomọkụ dị iche iche. A tụlere nkwụsi ike okpomọkụ nke enzyme site na incubation na 0.05 M sodium phosphate buffer (pH 7.0) na 40, 50, 60, na 70 Celsius maka awa abụọ, n'otu n'otu. E mesịa, ejiri ihe ABTS substrate nyochaa ọrụ fọdụrụ.
E jiri ABTS nyochaa mmetụta pH na ọrụ laccase site na iji ABTS dị ka ihe na-eme ka mmiri citrate-phosphate dị na 0.1 M nke nwere pH dị n'etiti 2.5 na 7.0. E tinyere mmiri enzyme ahụ na 40°C ruo awa abụọ na citrate 0.1 M na Tris buffers (pH 3, 4, 6, na 7) iji chọpụta nkwụsi ike pH. A gbakọọ ọrụ fọdụrụ na ABTS dị ka ihe na-eme ka mmiri dị na ya mgbe a kpụchara ya.
A kpọchiri laccase ahụ ruo nkeji iri na sodium phosphate buffer (0.05 M, pH 7.0) nke nwere ọtụtụ ion ígwè (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, na Mn2+) na njupụta nke 2.5 mM na 10 mM, n'otu n'otu. E tinyekwara substrate (ABTS) iji malite mmeghachi omume ahụ, a tụlekwara ọrụ metụtara ya.
A tụrụ oxidation ABTS site na laccase na njupụta dị iche iche (0.025–3 mM) na pH 4.5 iji chọpụta paramita kinetic (Vmax na Km).^{ihe ndị na-agbanweghi agbanwe}E ji usoro Lineweaver-Burk gbakọọ nha nha Michaelis-Menten, nke na-egosi ọnụego mmeghachi omume dịka ọrụ nke mkpokọta substrate. E jiri ngwanrọ GraphPad Prism nke ụdị 6.01 gbakọọ ihe ndị na-agbanwe agbanwe site na usoro Lineweaver-Burk.
Mgbe e jiri mmiri pọmpụ sachaa apụl ndị ahụ nke ọma, a bepụrụ ha abụọ ma jiri ihe na-esi ísì ụtọ apụl Braun MP80 (nke e mere na Germany) tee ha mmiri. A na-ete mmiri ahụ site na akwa cheesecloth anọ. E tinyeghị enzymes ọ bụla na otu njikwa ahụ, ebe a na-etinye laccase 2.0% (nke a nwalere nke ọma) na mmiri apụl e mere ọhụrụ, nke e wee debe na okpomọkụ 4°C ruo izu abụọ.
A chọpụtara acidity nke a na-akpọ titratable acidity (TA) na pH dịka usoro Boulton et si dị.^al.27E jiri mita pH dijitalụ (JENWAY 3510 pH mita) tụọ pH nke ihe nlele ọ bụla. E jiri usoro a gbakọọ acidity titratable (TA) dabere na malic acid.
Ebe V na C bụ olu (mL) na ntinye (0.1 mol/L) nke ngwọta sodium hydroxide ejiri mee ihe na titration, n'otu n'otu. K bụ ọnụọgụ mgbanwe malic acid, nke hà nhata 0.067, na W bụ oke (g) nke ihe ọṅụṅụ apụl.
Ihe siri ike niile na-agbaze (^TDS) e ji ihe nlele PAL-1 pocket refractometer (ATAGO, Tokyo, Japan) chọpụta ọdịnaya nke ihe nlele mmiri niile. Mgbe a tụchara ya, e jiri mmiri deionized sachaa lenzi anya ahụ, a nwalekwara ihe nlele mmiri apụl ọ bụla ugboro atọ. A gbakọọ uru maka ihe nlele ọ bụla site na nkezi nha atọ ahụ. A gbakọọkwara nkezi ± ọkọlọtọ nke ihe nlele mmiri apụl ọ bụla site na nkezi nsonaazụ ndị a.
E jiri ihe nlele viscometer na-agbagharị agbagharị (RV, Rheotest 2, Germany) chọpụta etu ihe nlele mmiri apụl si dị. E tinyere ihe nlele ahụ n'ime silinda "S2″ nke viscometer ahụ. E gosipụtara viscosity doro anya site na mgbada nke nrụgide shear megide usoro shear rate, nke a gbakọrọ site na nrụgide shear na usoro ndị kwekọrọ na ya na ọnụego shear dị iche iche (site na 1.00 ruo 437.4 s⁻¹). Usoro maka ịgbakọ viscosity doro anya bụ dị ka ndị a:
Ebe η bụ viscosity doro anya (cP), τ bụ nrụgide shear (dyn/cm²), γ bụ ọnụego shear (sec⁻¹), a na-agbakọkwa (τ) site na iji ụkpụrụ torque (α) na cylinder (Z) site na iji usoro ndị a: τ = Z. α.
A chọpụtara ntụaka aja aja dịka usoro Meidav ​​​​et si dị^al.29A tụbara ihe nlele ihe ọṅụṅụ 10-ml na 2750 xg maka nkeji iri. A gwakọtara ihe ọṅụṅụ ahụ na 5 ml nke ethanol 95% nke 5 ml. A tụrụ mmiri nke ngwakọta ahụ na 420 nm site na iji Shimadzu UV spectrophotometer (UV-1601 PC).
E jiri Folin-Ciocalteu reagent chọpụta ihe dị n'ime phenolic (TPC) n'agba dị iche iche dịka Boulton et al si kọwaa.^[27]E wuru usoro ọkọlọtọ nke gallic acid maka mkpokọta site na 0 ruo 500 mg/L (^r²= 0.997). A na-egosipụta nsonaazụ ya dị ka ihe nhata gallic acid (mg GAE/mL).
Tinye 125 μL nke mmiri a gwakọtara agwakọta na 2850 μL nke mmiri FRAP na 25 μL nke ihe ọṅụṅụ apụl ma hapụ ngwakọta ahụ ka ọ nọrọ n'ọchịchịrị maka ya.³⁰Nkeji. Mgbe ahụ, tụọ mmịpụta ahụ na 593 nm site na iji Shimadzu UV spectrophotometer (UV-1601 PC). E kwadebere ihe na-eme FRAP site na ịgwakọta 300 mM acetate buffer (pH 3.6), 20 mM iron (III) chloride, na 10 mM 2,4,6-tris(2-pyridyl)triazine (TPTZ) (agbazere na 40 mM HCl) na oke nke 10:1:1. E mepụtara usoro ọkọlọtọ site na iji Trolox dị ka ọkọlọtọ (^R²= 0.999), a na-egosipụtakwa nsonaazụ ya dị ka μM Trolox/mL.
A chọpụtara na ihe ọṅụṅụ ndị a gwọrọ na ndị a na-anaghị agwọ ọrịa na-arụ ọrụ antioxidant site na iji usoro DPPH iji chọpụta ikike ha nwere ịchụpụ radicals efu nke DPPH.³¹E tinyere 1 ml nke ihe ngwọta DPPH (100 μM) na methanol gwakọta obere lita iri nke ihe ọṅụṅụ. Mgbe mmeghachi omume gasịrị n'ọchịchịrị ruo nkeji iri atọ, a tụrụ mmịpụta nke ngwakọta ahụ na 517 nm site na iji Shimadzu UV spectrophotometer (UV-1601 PC). E gosipụtara nsonaazụ ya dị ka ihe nhata trolox (μM trolox/ml) dabere na usoro nhazi (_^R2= 0.990).
Data e nwetara gosiri na a hụrụ mmepụta laccase kachasị na ero oyster NRC 620 ka ọ na-erule njedebe nke ụbọchị nke iri na asatọ nke ịgbara mmiri, na-eru ọrụ nke 1302 U/L. Nke a jere ozi dị ka ntọala maka ikpebi oge ịkụ ihe kacha mma maka mmepụta laccase (Foto 1). Ọ bụ ezie na mmepụta enzyme mụbara ka oge ịkụ ihe na-abawanye, ọnụego mmụba ahụ adabereghị kpọmkwem na oge ịkụ ihe; mgbe ụbọchị 21 gasịrị, ọrụ enzyme amụbaala naanị 90 U/L (ruo 1390 U/L). Ya mere, a họọrọ ụbọchị 18 dị ka oge ịkụ ihe kacha mma iji dozie mmepụta ngwaahịa na uru akụ na ụba nke oge ịkụ ihe.
Mmetụta nke oge ịkọ ihe na mkpụrụ laccase na Pleurotus ostreatus NRC 620. A gbara blọk mycelium fungal atọ (12 mm) n'ime 50 ml nke ihe na-adịghị ọcha wee kụọ ya na 28 Celsius maka oge dị iche iche.
Dịka ọmụmụ ndị ọzọ si dị, ihe anyị chọpụtara na-egosi na oge kachasị mma iji nweta oke mmiri ọgwụ nje site na ero nwere ike ịdị n'etiti ụbọchị 7 na 36.³²Dịka Ezike na ndị otu ya si kwuo.³³, *Trametes polyzona* WRF03 mepụtara oke laccase kachasị elu na njedebe nke ụbọchị nke itoolu nke ịgbara mmiri, yana ọrụ pụrụ iche nke 1637 U/mg protein. Ọzọkwa, Othman et al.³⁴chọpụtara na *Trichoderma harzianum* S7113 wepụtara nnukwu laccase n'ụbọchị nke ise nke ọdịbendị. Ọnụọgụ mmepụta laccase ruru ọkwa kachasị elu n'ụbọchị nke iri na anọ wee belata nwayọ nwayọ.³⁴Ọ bụ ezie na mmepụta enzyme nwekwara ike ime n'oge uto bụ isi, ọ na-abụkarị n'oge etiti ma na-akpalite site na oriri nke isi iyi carbon ma ọ bụ nitrogen.^34,35
Ọ bụ ezie na laccase sitere na Pleurotus ostreatus NRC 620 gosipụtara nnukwu ọrụ n'ofe oke okpomọkụ site na 50°C ruo 80°C, na-eru nso na oke ọrụ (69–98%), a hụrụ oke ọrụ ya na 70°C (Foto 2a). N'èzí oke okpomọkụ a, ọrụ enzyme belatara na ihe dị ka 70°C. Nsonaazụ ndị a na-egosi na enzyme ahụ na-arụ ọrụ n'oge oke okpomọkụ, ikekwe n'ihi na oke okpomọkụ na-eme ka ike kinetic nke mmeghachi omume dịkwuo elu.
Mmetụta nke okpomọkụ mmeghachi omume (a) na pH (b) na ọrụ laccase na *Pleurotus ostreatus* NRC 620. E nwetara okpomọkụ sitere na 20 ruo 90 °C site na itinye ngwakọta ahụ tupu oge eruo na okpomọkụ dị iche iche maka nkeji ise tupu etinye enzyme ahụ ma malite mmeghachi omume ahụ. E jiri ABTS nyochaa mmetụta nke pH na ọrụ laccase na ngwọta nwere 0.1 M citrate-phosphate buffer n'elu oke pH nke 2.5 ruo 7.0.
Dịka Ezike et si kwuo^al.33, okpomọkụ kacha mma maka *Trametes polyzona* WRF03 laccase bụ 55 °C, nke bụ otu ihe ahụ maka *Ganoderma lucidum*^laccase36ma yie okpomọkụ kacha mma (50 °C) maka *Trametes polyzona* KU-RNW02737^lakasị . ^Baldrian38na-ekwu na, dịka sistemụ enzyme ndị ọzọ na-emebi lignin, oke okpomọkụ kachasị mma maka laccase dị n'etiti 50 na 70 Celsius.
Nsonaazụ gosiri na enzyme ahụ gosipụtara ọrụ kachasị elu na pH 3.0, na-eru ọrụ 94% na pH 3.5. Agbanyeghị, ọ nọgidere na-arụ ọrụ n'ofe oke pH site na 2.5 ruo 7.0 (Foto 2b). Ọzọkwa, o gosipụtara ọrụ dị elu na ọnọdụ acidic ma e jiri ya tụnyere ọnọdụ na-anọpụ iche ma ọ bụ alkaline. Ọrụ ya nọgidere na-adị ma ọ dịkarịa ala 77% karịa oke pH site na 2.5 ruo 4.5, mana ọ ruru naanị ihe dị ka 38% na pH 7.0. pH kachasị mma maka laccase sitere na *Trametes polyzona* WRF03 bụ 4.533, nke bụ otu ihe ahụ dị ka pH maka laccase sitere na *Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, na *Trametes hirsuta* 41. Agbanyeghị, dịka nnyocha nke Chairin et al si kwuo.⁴², pH kacha mma maka laccase site na *Polymorpha f. sp.* WR710-1 bụ 2.2, ebe pH kacha mma maka laccase sitere na *Polymorpha f. sp.* IBL-04 bụ 5.043. Njikọ nke anions hydroxide (laccase inhibitor) na atọm ọla kọpa nke laccase T2/T3 nwere ike ịbụ ihe kpatara mbelata ọrụ laccase n'okpuru ọnọdụ pH na-anọpụ iche ma ọ bụ alkaline. Nke a nwere ike imebi nnyefe elektrọn dị n'ime site na etiti T1 gaa na etiti T2/T3, si otú a^mmachiọrụ enzyme 23,44
Site n'itinye enzyme ahụ n'ọnọdụ okpomọkụ dị iche iche, achọpụtara na ma oge incubation na okpomọkụ metụtara nkwụsi ike enzyme ahụ. Ihe pụrụ iche bụ na laccase sitere na *Trametes polyzona* NRC 620 gosipụtara nkwụsi ike dị elu na 40℃ na 50℃, na-ejigide 68.33% na 59.61% nke ọrụ mbụ ya, n'otu n'otu, mgbe nkeji 120 gasịrị (Foto 3a). N'ụzọ dị iche, n'okpuru otu ọnọdụ ahụ (40℃ na 50℃, nkeji 120), laccase sitere na *Trametes polyzona* WRF03 nọgidere na-ejigide 64.38% na 42.92% nke ọrụ ya, n'otu n'otu.³³N'ụzọ megidere nke ahụ, mmụba nke oge incubation na okpomọkụ belatara nkwụsi ike nke *Trametes polyzona* NRC 620 laccase; Mgbe incubation gasịrị na 60℃ na 70℃ ruo nkeji 60, ọrụ ya belatara ruo 39.24% na 1.72%, n'otu n'otu (Foto 3a). N'ikwekọ na nsonaazụ nnwale ahụ, laccase sitere na *Trametes polyzona* WRF03 gosipụtara nkwụsi ike dị elu na 40 na 50℃ n'oge usoro ọgwụgwọ okpomọkụ.³³N'otu aka ahụ, Lueangjaroenkit et^al.37na Chairin et^al.42kọrọ ọnọdụ nke laccase sitere na Trametes polyzona KURNW027 na Trametes polyzona WR710-1 na 50 °C ruo otu awa, n'otu n'otu. Dịka ihe na-eme ka ihe na-eme ka ihe dị ndụ dị mma nke dabara na ngalaba biotechnology dị iche iche, laccase kwesịrị inwe ezigbo nkwụsi ike na arụmọrụ n'ofe oke okpomọkụ dị iche iche.
Nkwụsi ike nke thermostatic (a) na pH (b) nke laccase sitere na *Pleurotus ostreatus* NRC 620. A tụlere nkwụsi ike nke thermostatic site na itinye ihe ngwọta enzyme na 0.05 M sodium phosphate buffer (pH 7.0) na 40, 50, 60, na 70 °C ruo awa 2, n'otu n'otu. A tụlere nkwụsi ike nke pH site na itinye ihe ngwọta enzyme na 0.1 M citrate buffer na Tris buffer (pH 3, 4, 6, na 7) na 40 °C maka awa 2. A gbakọọ ọrụ fọdụrụ site na iji ABTS dị ka ihe ndabere mgbe a kpọchara ya.
Iji chọpụta ọnọdụ kacha mma maka ojiji na nchekwa enzyme, anyị nyochara mmetụta nke pH na nkwụsi ike laccase. Ikpughe na ụkpụrụ pH dị iche iche metụtara nkwụsi ike nke nhazi protein ahụ nke ukwuu, si otú a na-emetụta nkwụsi ike na ọrụ nke molekul enzyme. Nsonaazụ gosiri na enzyme ahụ adịghị akwụsi ike n'okpuru ọnọdụ acidic, ebe o gosipụtara nkwụsi ike ka mma na ụkpụrụ pH dị elu (mpaghara na-anọpụ iche na alkaline). Na ụkpụrụ pH nke 7.0, 6.0, 4.0, na 3.0, ọnụego njigide enzyme mgbe nkeji 120 gasịrị bụ ihe dị ka 100%, 62.54%, 52.39%, na 11.14%, n'otu n'otu (Fig. 3b). *Strombus multisus* WRF03 laccase gosipụtara nkwụsi ike dị elu na ụkpụrụ pH na-anọpụ iche (5.5–6.5) na nkwụsi ike dị ala na ụkpụrụ pH acidic (n'okpuru 4.0). Mgbe nkeji 120 gachara na pH nke 5.5, 6.0, na 6.5, ọnụego njide enzyme dị ihe dị ka 82%, 100%, na 93%, n'otu n'otu.³³Khairin na ndị ọzọ.⁴²kwuru na laccase sitere na Trametes polyzona WR710-1 kwụsiri ike na oke pH nke 6.0 ruo 7.0, ebe Sayed et al.⁴⁵gosiri na laccase kwụsiri ike karịa n'okpuru ọnọdụ pH na-anọpụ iche. Agbanyeghị, laccase sitere na Cerrena unicolor gosikwara nkwụsi ike n'okpuru ọnọdụ alkaline (pH 9.0)⁴⁶Lakas ndị a mụrụ gosiri na ha kwụsiri ike nke ukwuu n'ọtụtụ pH dị iche iche. Nke a nwere ike ịbụ ihe dị mkpa maka ojiji ụlọ ọrụ mmepụta ihe.
Ebe ọ bụ na ụfọdụ ion ígwè nwere ma mmetụta mkpali na mgbochi na ọrụ enzyme, a ga-atụle mmetụta ha na ọrụ enzyme n'ọrụ ụlọ ọrụ mmepụta ihe. Nke a dị oke mkpa n'ihi na ion ígwè bụ ihe ndị na-emerụ gburugburu ebe obibi nke nwere ike imetụta nkwụsi ike na mmepụta nke enzymes ndị na-apụtaghị ìhè.⁴⁷Iji nyochaa mmetụta nke ọtụtụ ion ígwè na laccase sitere na *Pleurotus ostreatus* NRC 620, anyị mere nnwale ndị kwekọrọ na ya. Dịka egosiri na Foto 4, dabere na ụdị ígwè ejiri mee ihe, ịbawanye njupụta ion ígwè site na 2.5 mM ruo 10 mM metụtara ọrụ enzyme ahụ n'ụzọ na-adịghị mma. Dịka ọmụmaatụ,^Mg²⁺ , ^Co²⁺ , ^Zn²⁺, na^Cu²⁺nwere ike ịkpalite ma kwalite ọrụ nke enzyme ahụ,^Na⁺ , ^Mn²⁺ , ^Ca²⁺, na^K⁺nwere ike igbochi ọrụ enzyme ahụ. N'ime mkpokọta 10 mM, ion Cu²⁺ na Mg²⁺ bụ ndị kacha rụọ ọrụ laccase site na *Pleurotus ostreatus* NRC 620, na-enye ọkwa mkpali nke ihe dị ka 34% na 20%, n'otu n'otu. Agbanyeghị, na mkpokọta 10 mM, ion Ca²⁺ bụ ndị kacha egbochi laccase, na-ebelata ọrụ enzyme site na ihe dị ka 60%.
Mmetụta nke ion ígwè na ọrụ nke Pleurotus ostreatus NRC 620 laccase. E tinyere laccase ahụ n'ime sodium phosphate buffer (0.05 M, pH 7.0) nke nwere ion ígwè dị iche iche na njupụta nke 2.5 mM na 10 mM. E mesịa, a malitere mmeghachi omume ahụ site na mgbakwunye nke substrate (ABTS), mgbe nke ahụ gasịrị, a tụrụ ọrụ metụtara ya.
Nsonaazụ anyị kwekọrọ na nke ndị edemede ndị ọzọ chọpụtara na Mg²⁺ na Cu²⁺ na-eme ka ọrụ *Trametes polyzona* WRF03³ ka mma. Castaño et al.⁴⁸ chọpụtara na laccase sitere na *Xylaria* sp. na-akpali ruo n'ókè ụfọdụ site na ion ọla kọpa (Cu²⁺). Ọzọkwa, Foroutanfar et al.⁴⁹ na Si et al.⁵⁰ mere ọmụmụ ihe yiri nke ahụ na laccase sitere na *Paraconiothyrium variabile* na *Trametes pubescens*, n'otu n'otu. Ebe njikọ ọla kọpa nke ụdị II (T2) nke enzyme a nwere ike ijupụta na Cu²⁺ na ntinye enyere, nke nwere ike ịkọwa mkpali nke ọrụ laccase na ntinye Cu²⁺³⁹ dị elu. Ebe ọ bụ na ihe ndị dị ka ero ọcha na-ere ere bụ oxidases nwere ọtụtụ atọm ọla kọpa, mmetụta nke ion ọla kọpa na ọrụ laccase dị iche iche ma dịgasị iche site na mkpali na mgbochi ruo na-anọpụ iche.⁵¹ N'ụzọ dị iche, Zhou et al.^[52]kọrọ na^Cu²⁺egbochila ọrụ laccase nke termite dị n'okpuru ala Taiwan (Odontotermes formosanus). Agbanyeghị, laccase nke Cerena sp. HYB07^[53]na Clitocybe maxima^[54]ion ọla kọpa adịghị emetụta ya.
E gosipụtara nkọwapụta nke substrate site na paramita kinetic ya (Km na Vmax); ka njikọ nke substrate na enzyme sikwuo ike, otú ahụ ka uru Km na-ebelata ma nkọwapụta substrate na-abawanye.^{3, 21, 55}E jiri ngwanrọ GraphPad Prism 6.0 chọpụta paramita kinetic (Km na Vmax) nke laccase sitere na *Pleurotus ostreatus* NRC 620 site na iji usoro nhazi Lineweaver-Burk (Foto nke 5). Mgbe ejiri ABTS mee ihe dị ka ihe ndabere, nsonaazụ ya bụ 1.99 mM na 16217 μmol.^{nkeji⁻¹ L⁻¹,}n'otu n'otu. Elsayed na ndị otu ya.²¹kọrọ na uru Km maka oxidation ABTS bụ 0.1 mM na 0.064 mM, n'otu n'otu, na-egosi njikọ dị elu nke isoenzymes Lac A na Lac B maka ABTS. Ọzọkwa, uru Vmax bụ 0.182 μmol^nkeji⁻¹na 0.603 μmol^nkeji⁻¹, n'otu n'otu. Uru Km enwetara dị ala karịa nke Trametes polyzona WRF03 (8.66 mM); Ọzọkwa, uru Vmax ha (1429 mmol min⁻¹) bụkwa^alamgbe ị na-eji ABTS dị ka ihe na-eme ka ihe dị n'ime ala.33 N'otu aka ahụ, uru Km nke Lentinus squarrosulus MR13 na Trametes sp. AH28-2 laccase bụ 0.0714 mM na 0.025 mM, n'otu n'otu, uru Vmax bụkwa 0.0091 mM min−1 na 0.67 mM min−1 mg−1 (ma e jiri ya tụnyere ABTS)^{, n'otu n'otu.56,57}
A nyochachara mmetụta nke ntinye ABTS na ọrụ nke laccase site na *Pleurotus ostreatus* NRC 620, e wee see atụmatụ Lineweaver-Burk nke mgbanwe nke ọsọ mmeghachi omume mbụ megide ntinye ABTS. A tụrụ mmeghachi omume oxidation nke ABTS nwere njupụta dị iche iche (0.025–3.0 mM) nke laccase na pH 4.5 iji chọpụta paramita kinetic (Vmax na Km). A gbakọọ constants kinetic Michaelis-Menten site na iji Lineweaver-Burk plot nke reciprocal nke reciprocal reaction velocity megide substrate concentration. A gbakọọ constants kinetic site na plot Lineweaver-Burk site na iji ngwanrọ GraphPad Prism 6.01.
Enzymes ndị na-eme ka ihe dị n'ime ya doo anya, dịka pectinases, hydrolyze ihe pectic, na-ebelata viscosity na turbidity. Ha na-emebi polysaccharides nke ọma ma a na-ejikarị ha eme ihe na enzymes ndị ọzọ, dị ka cellulases na hemicellulases, iji melite mmepụta na idoanya. Agbanyeghị, pectinases anaghị elekwasị anya kpọmkwem na ngwakọta phenolic, bụ ndị bụ isi ihe na-eme ka turbidity na oxidative browning, karịsịa na ihe ọṅụṅụ dị ka apụl na mkpụrụ vaịn.⁵⁸N'ụzọ dị iche, laccases na-eme ka oxidation nke phenolic compounds ka mma, na-eme ka ha ghọọ molekul buru ibu, ndị na-anaghị agbaze nke enwere ike iwepụ site na sedimentation ma ọ bụ nzacha. Usoro a abụghị naanị na ọ na-eme ka idoanya dịkwuo mma kamakwa ọ na-agbatị ndụ ihe ọṅụṅụ site na ibelata ohere nke oxidative browning nke phenolic compounds kpatara. Ọzọkwa, enwere ike ime usoro nkọwa dabere na laccase n'okpuru ọnọdụ nhazi dị nro (pH 3.5–5.5, okpomọkụ 25–40 °C), na-eme ka ha dị mma maka ihe ọṅụṅụ dị nro na-emebighị ihe oriri ma ọ bụ organoleptic ha.⁵⁹Nnyocha egosila na ọgwụgwọ pectinase nwere ike ime ka ihe ọṅụṅụ dị ọcha n'ime awa 1-2, ebe ọgwụgwọ laccase na-achọkarị oge mmeghachi omume dị ogologo (awa 3-6) iji belata ihe ndị mejupụtara phenolic kpamkpam. Agbanyeghị, enwere ike ime ka usoro a ka mma site na ime ka enzyme ahụ ghara ịrụ ọrụ ma ọ bụ site na ijikọta laccase na usoro nhazi igwe.⁶⁰N'ọmụmụ ihe a, nchọpụta enzyme nke ihe e si na ya wepụta gosiri ọrụ laccase na α-amylase dị oke mkpa, ebe ọrụ pectinase na xylanase dị obere nke ukwuu, a chọpụtaghịkwa ọrụ cellulase. Ya mere, mbelata nke turbidity na ọdịnaya phenolic bụ isi n'ihi ọrụ laccase, ebe mgbanwe na viscosity nwere ike ịbụ n'ihi ọrụ amylase.
Tebụl nke 1 na-egosi paramita physicochemical nke ihe ọṅụṅụ apụl a mịkọrọ ọhụrụ na ihe nlele e ji laccase gwọọ. Nsonaazụ gosiri na mkpụrụ nke ihe ọṅụṅụ apụl a mịkọrọ ọhụrụ (71.59%) dị ala karịa nke ihe nlele e ji laccase gwọọ (87.34%). Nsonaazụ ndị a kwekọrọ na nchọpụta Pilnik na Orange.⁶¹, onye gosiri na ojiji nke enzymes n'ime nhazi mkpụrụ osisi nwere ike ime ka mkpụrụ osisi na-amịpụta nke ọma, melite nzacha, ma nweta ihe ọṅụṅụ dị mma, doro anya maka njupụta. Mmụba nke mkpụrụ osisi na-amịpụta bụ isi n'ihi mmụba nke ọdịnaya nke shuga na-agbaze na ihe ọṅụṅụ ahụ. N'oge enzymes hydrolysis nke mkpụrụ osisi, a na-ebibi mesoglea na pectin na mgbidi sel nke ngwaahịa ahụ ma gbanwee ha ka ha bụrụ ihe na-agbaze dị ka shuga na asịd na-anọpụ iche.^62.Uru pH nke mmiri apụl e tinyere na enzyme dị ala nke ukwuu karịa nke otu njikwa (P < 0.05), uru pH nke otu abụọ ahụ mụbara nke ukwuu n'oge nchekwa (Tebụl 1). Nsonaazụ ndị a kwekọrọ na nke Mark et al.⁶³, onye kwuru na pH nke ihe ọṅụṅụ mkpụrụ osisi cashew belatara mgbe echekwara ya mgbe ọgwụgwọ okpomọkụ gasịrị. Mmebi pectin na mmepụta nke galacturonic acid mgbe ọgwụgwọ enzyme gasịrị nwere ike ịbụ ihe kpatara mmụba nke pH n'oge nchekwa. pH nke ihe nlele ejiri enzyme gwọọ nọgidere n'etiti 4.05 na 4.31 n'oge nchekwa ahụ, ebe pH nke ihe ọṅụṅụ apụl a na-anaghị agwọ ọrịa dị n'etiti 4.12 na 4.33.
Asịd zuru oke (TA) nke ihe nlele ndị a na-agwọghị na nke a na-agwọ laccase gosiri na ọ na-ebelata ka oge nchekwa na-abawanye (Tebụl 1). E gosipụtara mbelata nke acidity n'ihi mgbanwe nke organic acid gaa na carbohydrates ma ọ bụ mmeghachi omume enzymatic, yana oxidation n'oge nchekwa ihe ọṅụṅụ.⁶⁴Asịd zuru oke nke ihe ọṅụṅụ apụl a na-achịkwa na ihe nlele e ji enzyme gwọọ dị ala karịa nke ihe ọṅụṅụ ndị ọzọ (ihe ọṅụṅụ strawberry 0.9%, ihe ọṅụṅụ plum 2.2%, ihe ọṅụṅụ kumquat 1.0%, ihe ọṅụṅụ aprịkọt 2.4%, ihe ọṅụṅụ oroma 0.8%), mana ọ dị ka nke ihe ọṅụṅụ ndị ọzọ (dịka ọmụmaatụ, ihe ọṅụṅụ ube 0.3%).⁶²Ihe dị iche iche a na-eme na mmiri apụl a mịpụtara ọhụrụ a na-anaghị agwọ ọrịa nwere ike ịbụ n'ihi ihe dị iche iche dịka ọnọdụ uto, ihe ndị metụtara mkpụrụ ndụ ihe nketa, ọkwa ntozu okè, na ụzọ esi edozi ya.⁶⁵Mbelata na mkpokọta acidity nke njikwa na ihe ọṅụṅụ apụl ejiri laccase mee kwekọrọ na nsonaazụ nke Singh et al.⁶⁶gbasara mbelata na mkpokọta acidity nke ihe ọṅụṅụ apụl Jin Nuo mgbe ụbọchị 74 nke nchekwa gasịrị. N'aka nke ọzọ, Oshmiansky na Wojdylo⁶⁷ahụghị mgbanwe ọ bụla dị mkpa na acidity nke ihe ọṅụṅụ apụl mgbe ọ na-amụ mmetụta nke ụzọ nkọwa ọdịnala.
Nsonaazụ e gosiri na Tebụl 1 na-egosi na uru zuru oke nke ihe ọṅụṅụ apụl a gwọrọ na laccase dị elu karịa nke ihe nlele a na-agwọghị. Nsonaazụ ndị a kwekọrọ na ọmụmụ ebipụtara.^{. 68}Ọzọkwa, Tebụl 1 na-egosi na uru TSS nke otu ihe ọṅụṅụ apụl njikwa bụ 9.58 na mmalite oge ma ruo 11.05 na njedebe nke oge nchekwa. Uru ndị a dị ala karịa uru TSS nke ihe ọṅụṅụ apụl ọhụrụ nke Hamid et al.^{. 69}(11.2 na 11.80, n'otu n'otu). Uru TSS nke ihe nlele ihe ọṅụṅụ apụl e ji laccase gwọọ mụbara nke ukwuu, malite na 11.23 wee ruo 12.93 mgbe izu abụọ nke nchekwa na 4°C gasịrị (Tebụl 1). A hụkwara mmụba yiri nke ahụ na TSS n'oge nchekwa na mkpụrụ osisi citrus, lemon, na oroma dị ụtọ. Mmụba na ihe siri ike niile na-agbaze (TSS) n'oge nchekwa nwere ike ịbụ n'ihi hydrolysis nke polysaccharides (starch) na monosaccharides (shuga), mmụba na njupụta n'ihi mmiri mmiri, na mmebi nke pectin na ihe ọṅụṅụ gaa na ihe siri ike na-agbaze. Mmụba na ihe siri ike niile na-agbaze (TSS) nwere ike ịbụ n'ihi mmụba na shuga na-agbaze, nke enwere ike ịmepụta site na mgbanwe nke pectin ma ọ bụ cellulose ka ọ bụrụ shuga na-agbaze site na pectin ma ọ bụ cellulose, n'otu n'otu, ma ọ bụ site na hydrolysis nke starch ka ọ bụrụ shuga, dịka Hamed et al. si kọọ.^69.A pụrụ ịhụ mmetụta nke laccase nwere n'ihe ndị dị na mmiri apụl n'anya, ebe ọ bụ na mmiri apụl e tinyere laccase na-egosi na ọ na-agbagharị nke ọma ma na-ebelata viscosity karịa mmiri a na-enyeghị ọgwụgwọ. A na-edekọ ihe a na Tebụl 1; viscosity nke ihe nlele e tinyere enzyme bụ 1.87 cP, ebe viscosity nke ihe nlele njikwa bụ 2.95 cP. Mbelata dị ukwuu a na viscosity nwere ike ịbụ n'ihi ikike dị elu nke ihe ndị dị ka pectin na nhazi nke usoro netwọk jikọtara ọnụ.
N'ọmụmụ ihe a, a nyochachara mmetụta nke laccase nwere na ntụ ntụ browning (BI) nke ihe ọṅụṅụ apụl site na iji spectrophotometer tụọ mmịpụta na 420 nm. A na-egosi nsonaazụ ya na Tebụl 1. N'oge nchekwa, BI nke ihe nlele ihe ọṅụṅụ apụl n'ime otu ndị a gwọrọ na ndị a na-agwọghị gosiri mmụba nwayọọ nwayọọ. BI na-egosipụta ogo nke ịgba aja aja ma nwee ike ije ozi dị ka^{ihe dị mkpa}ihe ngosi nke mmeghachi omume agba aja aja nke enzyme na nke na-abụghị enzyme. Ntinye ahụ mụbara nke ukwuu n'oge nchekwa (P < 0.05). Na njedebe nke nchekwa,_A420Uru nke ihe nlele ihe ọṅụṅụ apụl n'ime otu ndị a na-achịkwa na ndị a na-agwọ enzyme mụbara site na ihe dị ka 217% na 121%, n'otu n'otu (Tebụl 1). Nsonaazụ ya na-egosi na ọgwụgwọ enzyme nwere ike ibelata ogo agba aja aja nke ọma site na ihe dị ka 56%. Nsonaazụ nke Bezerra et al.^[19]] kwekọrọ na nsonaazụ anyị; Ha jiri eriri laccase-glutaraldehyde-coconut mee ka ihe ọṅụṅụ apụl dị ọcha, na-ebelata agba mbụ ya site na 61%.
Ọ bụ ezie na polyphenols dị na ihe ọṅụṅụ mkpụrụ osisi nwere mmetụta dị mma n'ahụ mmadụ na nri na ọgwụgwọ, ha nwekwara ike imeghachi omume na protein, na-eme ka ihe ọṅụṅụ ahụ jupụta n'ígwé ojii, sedimentation, ma ọ bụ turbidity, si otú a na-agbanwe ụtọ na isi nke ngwaahịa ahụ ma belata oge ọ na-adịru.⁷¹Ebumnobi nke ọmụmụ a bụ iji laccase sitere na Pleurotus ostreatus NRC 620 belata ọdịnaya phenolic compound nke ihe ọṅụṅụ apụl n'enweghị nsogbu. Nsonaazụ egosiri na Tebụl 1 na-egosi na mkpokọta ọdịnaya phenolic compound nke ihe ọṅụṅụ apụl ejiri laccase gwọọ belatara nke ukwuu tupu echekwa ya na 4 °C. Ọzọkwa, mkpokọta ọdịnaya phenolic compound belatara n'oge nchekwa na ihe nlele abụọ a mụrụ (Tebụl 1). Nnyocha nke Sandri et al.⁷²gosiri na ihe ọṅụṅụ apụl e tinyere na enzyme nwere ike ịnọgide na-arụ ọrụ antioxidant ya na ọdịnaya phenolic compound. Agbanyeghị, nsonaazụ nke nnyocha nke Lettera et al.⁷³gosi na ọgwụgwọ nke ihe ọṅụṅụ oroma na laccase fungal nwere ike ibelata ọdịnaya nke ihe mejupụtara phenolic dị na ya ruo 45%.
A gosila na ihe ndị dị na phenolic nwere ihe ndị dị ka free radicals scavenging, singlet oxygen reduction and quenching, hydrogen atom transfer, na electron enhancement to free radicals, na-eme ka ha bụrụ antioxidants siri ike.⁷⁴Ya mere, n'ọmụmụ ihe a, e jiri ụzọ DPPH na FRAP mee ihe iji nyochaa mmetụta nke laccase na ọrụ antioxidant nke ihe ọṅụṅụ apụl echekwara na friji ruo ụbọchị 14 (Tebụl 2). Ụzọ abụọ ahụ gosiri mmụba na ọrụ antioxidant n'oge nchekwa, nke nwere ike ịbụ n'ihi mmụba na ngwakọta phenolic efu ma ọ bụ mmepụta nke ngwaahịa mmeghachi omume Maillard (MRPs), ebe ngwaahịa mmeghachi omume Maillard nwere ike ịbụ ihe kpatara mmụba na ọrụ antioxidant.⁷⁵Mmeghachi omume na-adịghị mma nke na-abụghị nke enzymatic (gụnyere mmebi ascorbic acid, mmeghachi omume Maillard, na mmebi nke shuga nke acid-catalyzed) na-emepụta agba aja aja (melanoidins). Ngwaahịa mmebi ascorbic acid dị n'etiti na ngwaahịa mmebi shuga (dịka ngwakọta carbonyl) nwere ike imeghachi omume na amino acid site na mmeghachi omume Maillard.⁷⁶Ọ bụ ezie na a mụọla nke ọma otú e si agba mkpụrụ osisi na akwụkwọ nri n'oge a na-echekwa ha, nghọta anyị banyere mmeghachi omume ndị a ka dị oke.⁷⁷Ma e jiri ya tụnyere usoro FRAP, ihe ọṅụṅụ apụl e ji laccase gwọọ gosiri obere ọrụ antioxidant site na usoro DPPH (Tebụl 2), ọrụ antioxidant nke ihe nlele niile mụbara nke ukwuu ka oge nchekwa na-abawanye. E jiri ụzọ abụọ dị iche iche maka ịchọpụta ọrụ antioxidant mee ihe na ọmụmụ ihe a n'ihi na ụkpụrụ ha dị iche. Usoro DPPH na-atụ ikike iwepụ radicals efu, ebe usoro FRAP na-atụ ikike ibelata ion iron. Ya mere, a na-atụ aro ka e jiri ọtụtụ ụzọ maka ịchọpụta ọrụ antioxidant iji ghọta nke ọma ọrụ antioxidant nke ihe nlele a mụrụ.⁷⁸
Otu n'ime ihe ndị dị mkpa a chọpụtara n'ọmụmụ ihe a bụ na *Pleurotus ostreatus* laccase NRC 620 na-egosipụta ọrụ kacha mma na 70°C na pH 3.0. Ma e jiri ya tụnyere laccase fungal ndị ọzọ a na-ejikarị eme ihe maka ịkọwapụta ihe ọṅụṅụ, dịka *Trametes versicolor* na *Ganoderma lucidum* laccase, *P. ostreatus* NRC 620 na-egosipụta nkwụsi ike okpomọkụ dị elu na pH acidic karịa. Laccase sitere na *Trametes versicolor* na *Ganoderma lucidum* na-egosipụtakarị ọrụ kacha mma n'etiti 50-60°C na na pH dị n'etiti 3.5 na 5.0. Ọdịiche a nwere ike inye aka na mmezi arụmọrụ nke ihe ọṅụṅụ, karịsịa maka ihe ọṅụṅụ acidic ebe nkwụsi ike na obere pH dị oke mkpa. Njirimara pụrụ iche nke *P. Ma e jiri ya tụnyere laccase fungal ndị ọzọ a mụrụ, *Pleurotus ostreatus* NRC 620 na-egosipụta ikike ịrụ ọrụ nke ọma n'okpuru ọnọdụ siri ike karị. Okpomọkụ ọrụ ya dị elu na-egosi uru dị na ngwa ụlọ ọrụ mmepụta ihe, dịka ọnụego mmeghachi omume ngwa ngwa na mbelata mmetọ nje bacteria. Ala pH ya, nke dabara adaba na ọdịdị acidic nke ọtụtụ ihe ọṅụṅụ, nwere ike ịba uru na usoro nkọwa ihe ọṅụṅụ. Nsonaazụ ndị a na-egosi nyocha ọzọ maka ojiji buru ibu, na-eme ka *Pleurotus ostreatus* NRC 620 bụrụ ihe ọzọ bara uru maka isi mmalite laccase fungal ọdịnala. Ma e jiri ya tụnyere ọmụmụ ihe gara aga, anyị chọpụtara na okpomọkụ kachasị mma bụ 60°C na pH kacha mma bụ 3.0. Mgbe mmeghachi omume gasịrị na 60°C maka nkeji 80, *Ganoderma lucidum* laccase na-ejide⁴⁶Pasentị nke ọrụ ya.79 Dịka Kurniawati na Nicelle si kwuo⁸⁰, *Ganoderma lucidum* enzymes na-egosipụta nkwụsi ike dị mma ruo na nke dị mma na 25°C na ụkpụrụ pH sitere na 5.0 ruo 8.0, yana nkwụsi ike na pH 6.0 na okpomọkụ sitere na 10 ruo 30°C. N'ọmụmụ ihe a, anyị chọpụtara na pH na okpomọkụ kachasị mma maka ọrụ enzyme maka *Pleurotus ostreatus* bụ 3.0 na 70°C, n'otu n'otu. Mgbe a kpọchara ya na 40°C na 50°C ruo awa abụọ, enzyme ahụ nọgidere na-enwe 68.33% na 59.61% nke ọrụ ya, n'otu n'otu. Ọzọkwa, Pleurotus ostreatus NRC 620 laccase gosipụtara nnukwu ọrụ n'ofe oke okpomọkụ site na 50°C ruo 80°C, fọrọ nke nta ka ọ ruo oke ọrụ (69%–98%), ebe a hụrụ oke ọrụ na 70°C.
Na mmechi, oyster ero laccase NRC620, nke e nwetara n'okpuru ọnọdụ kwụsiri ike, gosipụtara ọrụ na nkwụsi ike kachasị mma n'ofe ọtụtụ pH na ọnọdụ okpomọkụ, na-egosi nkwụsi ike dị elu ma e jiri ya tụnyere isi mmalite enzyme ndị ọzọ. Mgbakwunye nke 10 mM MgSO₄ na CuSO₄ mụbara ọrụ enzyme site na ihe dị ka 21% na 35%, n'otu n'otu. Mgbe a na-etinye ya na mmiri apụl, enzyme ahụ belatara pH na viscosity, ebe ọdịnaya phenolic belatara naanị ntakịrị n'oge nchekwa.
Nsonaazụ ya na-egosi ike nke laccase nwere n'ụlọ ọrụ nri, ọkachasị n'ịkọwapụta ihe ọṅụṅụ. Site n'ịkwatu ihe ndị mejupụtara phenolic kpọmkwem, laccase anaghị ebelata naanị ntụ ntụ ma na-eme ka o doo anya kamakwa ọ na-ejigide ịdị mma nke ihe ọṅụṅụ mkpụrụ osisi n'okpuru ọnọdụ ọrụ dị nro. N'adịghị ka ihe ndị na-eme ka o doo anya ọdịnala dịka gelatin, bentonite, na silica gel, laccase anaghị emepụta ihe mkpofu ma ọ bụ wepụ isi ụtọ dị ụtọ na ihe ọṅụṅụ, na-eme ka ọ bụrụ nhọrọ kacha mma maka gburugburu ebe obibi na nke na-adịgide adịgide. Ọzọkwa, ma e jiri ya tụnyere enzymes na ụzọ nzacha ndị ọzọ, laccase na-enye ngwọta ezubere iche na nke dị ọnụ ala na-enweghị imebi àgwà ngwaahịa.
Kyomuhimbo, HD na Brink, HG. Ngwa na atụmatụ igbochi ihe eji eme ihe nke lakase nwere ọla kọpa; nyocha. Heliyon 9, e13156 (2023).