Ma hope o 25 mau lā o ka hoʻomoʻa static ma 28°C, ua hōʻike ʻo laccase mai *Pleurotus ostreatus* NRC620 i ka hana kiʻekiʻe loa ma ka medium moʻomeheu fungal. ʻO nā waiwai pH a me ka mahana kūpono no kēia enzyme he 3.0 a me 70°C, kēlā me kēia. Ma hope o 2 mau hola o ka hoʻomoʻa ʻana ma 40°C a me 50°C, ua mālama ka hana enzyme i 68.33% a me 59.61%, kēlā me kēia. Ma hope o 2 mau hola o ka hoʻomoʻa ʻana i loko o ka citrate-phosphate buffer (pH 7.0), ua noho ka hana enzyme ma 100%. ʻO ka hoʻohui ʻana o 10 mM MgSO₄ a me CuSO₄ i hoʻonui i ka hana enzyme ma kahi o 21% a me 35%, kēlā me kēia, ʻoiai ʻo NaCl, MnCl₂, KCl, a me CaCl₂ i kāohi i ka hana enzyme. Ma ka hoʻohana ʻana iā ABTS ma ke ʻano he substrate, ʻo nā ʻano kinetic (Km a me Vmax) o *Pleurotus ostreatus* NRC 620 laccase he 1.99 mM a me 16,217 μmol min−1 L−1, kēlā me kēia. Ua hōʻemi nui ka mālama ʻana i nā laʻana wai ʻāpala i ka pH a me ka viscosity, a ua pili kēia hōʻemi me ka hoʻonui ʻana i ka manawa mālama. Ua hopena ka mālama ʻana iā Laccase i ka emi iki ʻana o ka nui o ka phenolic o ka wai ʻāpala, akā ʻaʻohe hōʻemi i ka hana antioxidant i ʻike ʻia.
I nā makahiki i hala iho nei, ua kālele nā mea noiʻi i ka hoʻohana ʻana o ka biotechnology ʻōmaʻomaʻo i ka ʻoihana meaʻai. ʻO Laccase kekahi o nā enzymes pono loa i ka ʻoihana meaʻai, e loaʻa ana nā noi ma nā wahi e like me ka hana ʻana i ka wai, ka hoʻomoʻa ʻana, ka hoʻopaʻa ʻana i ka waina, a me ka hoʻomaikaʻi ʻana i nā ʻano organoleptic o nā huahana meaʻai.1He nui nā mea kanu kiʻekiʻe a me nā microorganisms e hoʻopuka i ka laccase,2a me nā fungi e like me deuteromycetes, ascomycetes, a me basidiomycetes hiki ke hana i ka laccase.3ʻO Laccase (EC 1.10.3.2) kahi oxidase polū e hoʻemi ana i ka oxygen molekala i ka wai me ka hoʻohana ʻana i kahi ʻōnaehana i haku ʻia me ʻekolu mau ʻātoma keleawe like ʻole, a laila e oxidizing i nā hui phenolic like ʻole a me nā amines ʻala. I ka wā o ka hana ʻana i nā wai huaʻai a me nā mea kanu, he mau pilikia koʻikoʻi ka browning enzymatic a me ka nonenzymatic.4ʻOiai he hopena maikaʻi ʻole kēia mau mea i ke kala, ka ʻono, a me ke ʻala o ka wai, pono e wehe ʻia.5
ʻO nā huaʻai a pau, ʻo nā ʻāpala ka mea i hoʻopau nui ʻia ma ka honua holoʻokoʻa a ma ka ʻAhahui ʻEulopa. I ka makahiki 2019, ua helu ʻia ka hana ʻāpala ma ke kolu o ka honua, ma mua o 87 miliona tone.6Loaʻa i nā ʻāpala nā pūhui phenolic he nui, me nā flavonoids a me nā waikawa phenolic e like me ka caffeic acid a me ka chlorogenic acid.7Ma muli o ka ʻai pinepine ʻia ʻana o ka wai ʻāpala i kona ʻano moakāka, ua nalowale ma kahi o 50% a 90% o nā ʻāpana phenolic i ka wā o ke kaʻina kānana.8I kēia mau lā, koho pinepine nā mea kūʻai aku i nā huahana i hana liʻiliʻi ʻia, e like me ka wai ʻāpala noe me ka nui o ka polyphenol. Eia nō naʻe, ma muli o kona nui o ka phenolic, he mea maʻalahi loa kēia ʻano wai ʻāpala i ka hoʻololi ʻana i ke kala a me ka ʻeleʻele.9Hoʻohana ʻia nā ʻenehana like ʻole, me nā ʻano hana wela e like me ka pasteurization ma 60–90°C, e hōʻemi a pale aku paha i ka ʻeleʻele ʻana o ka wai ʻāpala.10Eia naʻe, e like me ka noiʻi a Sauceda-Gálvez11, hiki i ka hana wela ke hoʻopau i nā kemika volatile a hoʻopilikia i nā ʻano organoleptic o ka wai ʻāpala. ʻO nā koho ʻē aʻe i nā hana hana wela e komo pū me ka supercritical carbon dioxide, ultraviolet radiation, ultrasound, high hydrostatic pressure, a i ʻole ka homogenization kiʻekiʻe.12ʻO ka pono o kēia mau ʻenehana a me ka loaʻa ʻana o nā wai hua kūpono e pili ana i nā palena i hoʻohana ʻia a me nā ʻano huahana. Ua kaupalena ʻia ko lākou hoʻohana nui ʻia e nā kumukūʻai kiʻekiʻe, nā hopena maikaʻi ʻole i ka maikaʻi o kekahi mau huahana meaʻai, a i ʻole ka hoʻopau ʻana o ka enzyme i lawa ʻole.13,14
Hiki ke hoʻohana ʻia ʻo Laccase e hoʻopaʻa a hoʻomālamalama i ka wai hua.15Gökmen et al.16Paipai i ka hoʻohana ʻana i ka laccase no ka hoʻomālamalama ʻana i ka wai huaʻai no ka mea hoʻoneʻe pono ia i nā pūhui phenolic ma ka hoʻololi ʻana iā lākou i polymers a i ʻole oligomers i maʻalahi ke wehe ʻia e kekahi membrane ultrafiltration, e ʻae ana i ka wai ʻāpala e mālama i ke kala paʻa a me ka maopopo no ʻeono pule ma 50°C. Ua hoʻopaʻa ʻia ka laccase *Trichoderma* i hoʻomaʻemaʻe ʻia ma luna o nā momi alumina a hoʻohana ʻia e wehe koho i nā pūhui ʻono ʻole i hoʻokumu ʻia e ka haumia microbial o ka wai ʻāpala.17
Ma kahi o 80-90% o nā ʻāpana lele o ka wai ʻāpala he mau esters a me nā aldehydes, nā mea e hāʻawi i kahi ʻala kū hoʻokahi i ka wai.18Ua hoʻopaʻa ʻia ka Laccase mai *Trametes versicolor* ma luna o kahi kākoʻo makepono i hana ʻia mai ka olonā maoli mai nā pūpū niu ʻōpio no ka hoʻomālamalama ʻana i ka wai ʻāpala.19Ua noiʻi nā haʻawina mua i ka hoʻopaʻa ʻana o ka wai ʻāpala (kala a me ka turbidity) me ka hoʻohana ʻana i nā ʻano enzyme-free a i ʻole immobilization, a i ʻole i ka hui pū ʻana me ka ultrafiltration.5,19Eia nō naʻe, ʻaʻole maopopo ka hopena o nā laccases fungal i nā waiwai physicochemical o ka wai ʻāpala i ka wā e mālama ai. No laila, ʻo ka pahuhopu o kēia haʻawina, ʻo ia ke noiʻi hoʻokolohua i nā loli i nā waiwai physicochemical, ka nui o ka hui phenolic, a me ka hana antioxidant o ka wai ʻāpala ma hope o ka mālama ʻana me nā laccases fungal a me ka mālama ʻana i ʻelua pule i loko o ka pahu hau. Loaʻa i nā laccases ka hiki ke hoʻopau i nā hui phenolic, kahi e hoʻohiki ai lākou no ka hoʻohana ʻana i nā kaʻina hana like ʻole, me ka hoʻomālamalama ʻana i ka wai. Ua nānā kēia haʻawina i nā laccases mai *Pleurotus ostreatus* NRC 620, e kālele ana i nā kūlana kūpono no kā lākou hana a me ka pono i ka hoʻomālamalama ʻana i ka wai. ʻOiai ua kaupalena ʻia ka noiʻi ʻana i nā ʻōhiʻo oyster (P. ostreatus NRC 620), ua nānā nā haʻawina mua i nā enzymes mai nā kumu fungal like ʻole, e like me Trametes versicolor a me Ganoderma lucidum. ʻO ka pahuhopu o kēia haʻawina, ʻo ia ke loiloi i ka hiki ke hoʻohana ʻia o kēia enzyme i ka ʻoihana meaʻai a hōʻike i kona mau waiwai kūikawā, ʻoiai kona pH kūpono a me ka mahana.
Ua kūʻai ʻia ʻo 2,2′-Azooxybis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) mai Sigma-Aldrich (Kanada). ʻO nā reagents ʻē aʻe a pau o ke ʻano analytical.
Ua loaʻa i ke Kikowaena Hōʻiliʻili Moʻomeheu Microbial o ke Kikowaena Noiʻi Lahui ke ʻano mushroom oyster i ʻike ʻia ʻo NRC620. Ma hope o ka subculture, ua mālama ʻia kēia ʻano ma luna o nā slants agar dextrose potato ma 4°C. Penei ke ʻano hoʻomākaukau inoculum: Ua hoʻokomo ʻia ka mycelium 10 lā, i hoʻomohala piha ʻia ma luna o nā papa agar dextrose potato a hoʻomoʻa ʻia ma 28°C. Ma hope o 10 mau lā, ua wehe ʻia ʻekolu mau poloka mycelial 12-mm-diameter mai ka media agar me ka hoʻohana ʻana i kahi punch metala sterile a waiho ʻia i loko o nā ʻōmole Erlenmeyer 250-mL me nā plug pulupulu e loaʻa ana he 50 mL o ka medium culture sterilized (pH 5.0, e like me ka mea i wehewehe mua ʻia e Othman et al.20). Ua hoʻomoʻa ʻia nā moʻomeheu ma 28°C no 18 mau lā. A laila ua kānana ʻia nā moʻomeheu ma o ka pepa kānana Whatman No. 1, a ʻo ka supernatant hopena i lawelawe ʻia ma ke ʻano he kumu enzyme.
Ua hoʻoholo ʻia ka hana Laccase me ka hoʻohana ʻana iā ABTS ma ke ʻano he substrate. ʻO ka hui hopena (2 mL) aia he 500 μL o 0.3 mM ABTS (i hoʻoheheʻe ʻia i loko o 0.1 M sodium citrate buffer, pH 4.5) a me ka nui o ka hāpana enzyme i hoʻoheheʻe ʻia me ka wai distilled.21,22Ke noʻonoʻo nei hiki i ka laccase ke hoʻoheheʻe i ka ABTS ma ka mahana o ka lumi (28 °C ± 2), ua hoʻoholo ʻia ka hoʻoheheʻe ʻana o ka ABTS ma ke ana ʻana i ka hoʻonui ʻana o ka absorbance ma 420 nm (ε420= 36,000 kenimika-1 M -1) me ka hoʻohana ʻana i kahi spectrophotometer UV Agilent Carry-100. Pono hoʻokahi anakahi o ka hana laccase e hoʻopau i ka 1 μmol ABTS i kēlā me kēia minuke. Ua hoʻoholo ʻia ka nui o ka protein e ke ʻano Bradford me ka hoʻohana ʻana i ka bovine serum albumin ma ke ʻano he kaohi kūloko.23,24
Ma hope o ka loaʻa ʻana o ka enzyme mai ke ʻano mushroom oyster NRC 620, ua ana ʻia kāna hana ma nā wā mahi like ʻole no 25 mau lā ma lalo o nā kūlana paʻa ma 28 °C.
No ke aʻo ʻana i ka hopena o ka mahana ma ka hana laccase, ua hana ʻia nā hoʻokolohua ma ka pae mahana mai 20 a 90 °C. Ma mua o ka hoʻohui ʻana i ka enzyme a me ka hoʻomaka ʻana i ka hopena, ua hui pū ʻia ka buffer (0.1 M sodium citrate, pH 4.5) a me ka substrate (ABTS) a ua hoʻomoʻa ʻia no 5 mau minuke ma nā mahana like ʻole. Ua loiloi ʻia ke kūpaʻa wela o ka enzyme ma o ka hoʻomoʻa ʻana i loko o 0.05 M sodium phosphate buffer (pH 7.0) ma 40, 50, 60, a me 70 °C no 2 mau hola, kēlā me kēia. A laila ua loiloi ʻia ka hana koena me ka hoʻohana ʻana i ka substrate ABTS.
Ua loiloi ʻia ka hopena o ka pH ma ka hana laccase me ka hoʻohana ʻana iā ABTS ma ke ʻano he substrate i loko o 0.1 M citrate-phosphate buffers me kahi pae pH o 2.5 a 7.0. Ua hoʻomoʻa ʻia ka hopena enzyme ma 40°C no ʻelua hola i loko o 0.1 M citrate a me Tris buffers (pH 3, 4, 6, a me 7) e loiloi i ke kūpaʻa pH. Ua helu ʻia ka hana koena me ABTS ma ke ʻano he substrate ma hope o ka hoʻomoʻa ʻana.
Ua hoʻomoʻa ʻia ka laccase no 10 min i loko o ka sodium phosphate buffer (0.05 M, pH 7.0) e loaʻa ana nā ʻano ion metala like ʻole (Mg2+, Cu2+, Co2+, Ca2+, Zn2+, K+, Na+, a me Mn2+) ma nā ʻano he 2.5 mM a me 10 mM, kēlā me kēia. A laila ua hoʻohui ʻia ka substrate (ABTS) e hoʻomaka i ka hopena, a ua loiloi ʻia ka hana pili.
Ua ana ʻia ka hoʻoheheʻe ʻana o ABTS e ka laccase ma nā ʻano like ʻole (0.025–3 mM) ma ka pH 4.5 e hoʻoholo ai i nā ʻano kinetic (Vmax a me Km). ʻO ka kineticnā mea mauo ka hoʻohālikelike Michaelis-Menten ua helu ʻia me ka hoʻohana ʻana i kahi kiʻi Lineweaver-Burk, kahi e kiʻi ai i ka reciprocal o ka helu hopena ma ke ʻano he hana o ka substrate concentration. Ua helu ʻia nā kinetic constants mai ka Lineweaver-Burk plot me ka hoʻohana ʻana i ka polokalamu GraphPad Prism mana 6.01.
Ma hope o ka holoi pono ʻana i nā ʻāpala me ka wai paipa, ua ʻoki ʻia i ka hapalua a ua hoʻoinu ʻia ka wai me ka hoʻohana ʻana i ka mea hoʻoinu ʻāpala Braun MP80 piha piha (i hana ʻia ma Kelemania). Ua kānana ʻia ka wai ma o ʻehā papa o ka cheesecloth. ʻAʻohe enzymes i hoʻohui ʻia i ka hui kaohi, ʻoiai ua hoʻohui ʻia ka 2.0% laccase (ka nui kūpono loa i hoʻāʻo ʻia) i ka wai ʻāpala i hoʻomākaukau hou ʻia, a laila mālama ʻia ma 4°C no ʻelua pule.
Ua hoʻoholo ʻia ka acidity Titratable (TA) a me ka pH e like me ke ʻano o Boulton et al.al.27Ua ana ʻia ka pH o kēlā me kēia hāpana me ka hoʻohana ʻana i ka mika pH kikohoʻe (mika pH JENWAY 3510). Ua helu ʻia ka waikawa Titratable (TA) ma muli o ka waikawa malic me ka hoʻohana ʻana i ke ʻano hana ma lalo nei.
Ma kahi o V a me C ka nui (mL) a me ka nui (0.1 mol/L) o ka hopena sodium hydroxide i hoʻohana ʻia i ka titration, kēlā me kēia. ʻO K ka coefficient hoʻololi waikawa malic, like me 0.067, a ʻo W ka nuipa (g) o ka wai ʻāpala.
ʻO ka huina o nā mea paʻa hiki ke hoʻoheheʻe ʻia (Nā TDS) ua hoʻoholo ʻia ka nui o nā hāpana wai āpau me ka hoʻohana ʻana i kahi refractometer pocket PAL-1 (ATAGO, Tokyo, Iapana). Ma hope o kēlā me kēia ana, ua holoi ʻia ka lens optical me ka wai deionized, a ua hoʻāʻo ʻia kēlā me kēia hāpana wai ʻāpala i ʻekolu manawa. Ua helu ʻia ka waiwai no kēlā me kēia hāpana ma ka awelika ʻana i nā ana ʻekolu. Ua helu ʻia hoʻi ka awelika ± standard deviation no kēlā me kēia hāpana wai ʻāpala ma ka awelika ʻana i kēia mau hopena.
Ua loiloi ʻia ka viscoelasticity o nā hāpana wai ʻāpala me ka hoʻohana ʻana i kahi viscometer rotational (RV, Rheotest 2, Kelemānia). Ua waiho ʻia ka hāpana i loko o ka cylinder "S2" o ka viscometer. Ua hōʻike ʻia ka viscosity ʻike ʻia e ka pali o ke kuʻekuʻe koʻikoʻi shear versus shear rate curve, i helu ʻia mai ke koʻikoʻi shear a me nā kuʻekuʻe e pili ana i nā ʻano shear rates like ʻole (mai 1.00 a 437.4 s⁻¹). ʻO ke ʻano hana no ka helu ʻana i ka viscosity ʻike ʻia penei:
Ma kahi o η ka viscosity apparent (cP), ʻo τ ka shear stress (dyn/cm²), ʻo γ ka shear rate (sec⁻¹), a ua helu ʻia ʻo (τ) me ka hoʻohana ʻana i nā waiwai torque (α) a me ka cylinder (Z) me ka hoʻohana ʻana i kēia formula: τ = Z. α.
Ua hoʻoholo ʻia ka helu browning e like me ke ʻano o Meidav etal.29Ua centrifuged ʻia kahi hāpana wai 10-ml ma 2750 xg no 10 min. Ua hui pū ʻia ʻo 5 ml o ka supernatant wai me 5 ml o 95% ethanol. Ua ana ʻia ka absorbance o ka hui ʻana ma 420 nm me ka hoʻohana ʻana i ka Shimadzu UV spectrophotometer (UV-1601 PC).
Ua hoʻoholo ʻia ka nui o ka phenolic content (TPC) ma ke ʻano colorimetrically me ka hoʻohana ʻana i ka reagent Folin-Ciocalteu e like me ka mea i wehewehe ʻia e Boulton et al.[27]Ua kūkulu ʻia kahi piʻo maʻamau o ka waikawa gallic no nā ʻano mai 0 a 500 mg/L (r²= 0.997). Hōʻike ʻia nā hopena ma ke ʻano he mau kūlike waikawa gallic (mg GAE/mL).
E hoʻohui i 125 μL o ka wai i hoʻoheheʻe ʻia a me 2850 μL o ka hopena FRAP i 25 μL o ka wai ʻāpala a waiho i ka hui ʻana i ka pouli no30min. A laila e ana i ka absorbance ma 593 nm me ka hoʻohana ʻana i ka Shimadzu UV spectrophotometer (UV-1601 PC). Ua hoʻomākaukau ʻia ka reagent FRAP ma ke kāwili ʻana i ka 300 mM acetate buffer (pH 3.6), 20 mM iron(III) chloride, a me 10 mM 2,4,6-tris(2-pyridyl)triazine (TPTZ) (i hoʻoheheʻe ʻia i loko o 40 mM HCl) ma ka lakio o 10:1:1. Ua hana ʻia kahi piʻo maʻamau me ka hoʻohana ʻana iā Trolox ma ke ʻano he maʻamau (R²= 0.999), a ua hōʻike ʻia nā hopena ma ke ʻano he μM Trolox/mL.
Ua hoʻoholo ʻia ka hana antioxidant o nā wai i mālama ʻia a me nā wai i mālama ʻole ʻia me ka hoʻohana ʻana i ke ʻano DPPH e loiloi i ko lākou hiki ke scavenge i nā radical manuahi DPPH.31Ua hui ʻia he ʻumi microliters o ka wai me 1 ml o kahi hopena DPPH (100 μM) i loko o ka methanol. Ma hope o ka hana ʻana i ka pouli no 30 min, ua ana ʻia ka absorbance o ka hui ʻana ma 517 nm me ka hoʻohana ʻana i kahi spectrophotometer Shimadzu UV (UV-1601 PC). Ua hōʻike ʻia nā hopena ma ke ʻano he trolox equivalents (μM trolox/ml) ma muli o kahi piʻo calibration (R2= 0.990).
Ua hōʻike ʻia ka ʻikepili i loaʻa ua ʻike ʻia ka nui o ka hana laccase ma nā halo ʻoyster NRC 620 ma ka hopena o ka lā 18 o ka fermentation, a hiki i ka hana o 1302 U/L. Ua lawelawe kēia ma ke ʻano he kumu no ka hoʻoholo ʻana i ka manawa mahi kūpono no ka hana laccase (Kiʻi 1). ʻOiai ua piʻi ka hana enzyme me ka hoʻonui ʻana i ka manawa mahi, ʻaʻole kūlike ka nui o ka hoʻonui ʻana i ka manawa mahi; ma hope o 21 mau lā, ua piʻi wale ka hana enzyme ma 90 U/L (a i 1390 U/L). No laila, ua koho hope ʻia he 18 mau lā ʻo ia ka manawa mahi kūpono e kaulike ai i ka hua huahana me nā pono waiwai o ka hoʻonui ʻana i ka manawa mahi.
Ka hopena o ka manawa mahiʻai ma ka hua laccase ma Pleurotus ostreatus NRC 620. ʻEkolu (12 mm) mau poloka mycelial fungal i hoʻokomo ʻia i loko o 50 ml o ka medium sterile a laila mahiʻai ʻia ma 28 °C no nā manawa like ʻole.
E like me nā haʻawina ʻē aʻe, hōʻike kā mākou mau hopena ʻo ka manawa kūpono e hoʻokō ai i ka hoʻokuʻu ʻana o ka laccase e nā fungi he 7 a 36 mau lā paha.32Wahi a Ezike et al.33, *Trametes polyzona* WRF03 i hana i ka nui loa o ka laccase ma ka hopena o ka lā ʻeiwa o ka fermentation, me kahi hana kikoʻī o 1637 U/mg protein. Eia kekahi, ʻo Othman et al.34ua ʻike ʻia ua hoʻokuʻu ʻo *Trichoderma harzianum* S7113 i ka nui o ka laccase i ka lā ʻelima o ka hoʻoulu ʻana. Ua hōʻea ka nui o ka hana laccase i kahi hana kiʻekiʻe loa i ka lā ʻumikumamāhā a laila ua emi mālie.34ʻOiai hiki ke hana ʻia ka hoʻokuʻu ʻana o ka enzyme i ka wā ulu nui, ʻoi aku ka nui i ka wā waena a hoʻoulu ʻia e ka hoʻohana ʻana i kahi kumu kalapona a naikokene paha.34,35
ʻOiai ua hōʻike ka laccase mai Pleurotus ostreatus NRC 620 i ka hana kiʻekiʻe ma luna o kahi ākea o ka mahana mai 50°C a 80°C, kokoke i ka hana kiʻekiʻe (69–98%), ua ʻike ʻia kāna hana kiʻekiʻe loa ma 70°C (Kiʻi 2a). Ma waho o kēia pae mahana, ua emi ka hana enzyme ma kahi o 70°C. Hōʻike kēia mau hopena ua hana ka enzyme ma nā mahana kiʻekiʻe, ma muli paha o ka hoʻonui ʻana o ka mahana kiʻekiʻe i ka ikehu kinetic o ka hopena.
Ka hopena o ka mahana o ka hopena (a) a me ka pH (b) ma ka hana laccase ma *Pleurotus ostreatus* NRC 620. Ua hoʻokō ʻia nā mahana mai 20 a 90 °C ma ka hoʻomoʻa mua ʻana i ka hui ʻana ma nā mahana like ʻole no 5 min ma mua o ka hoʻohui ʻana i ka enzyme a hoʻomaka i ka hopena. Ua loiloi ʻia ka hopena o ka pH ma ka hana laccase me ka hoʻohana ʻana iā ABTS ma ke ʻano he substrate i loko o nā hopena e loaʻa ana ka 0.1 M citrate-phosphate buffer ma luna o kahi pae pH o 2.5 a 7.0.
Wahi a Ezike etal.33, ʻo ka mahana kūpono no *Trametes polyzona* WRF03 laccase he 55 °C, ʻo ia ka mea like me ko *Ganoderma lucidum*laccase36a like me ka mahana kūpono (50 °C) no *Trametes polyzona* KU-RNW02737laccase . Baldrian38hoʻomaopopo ʻia, e like me nā ʻōnaehana enzyme lignin-degrading ʻē aʻe, ʻo ka pae mahana kūpono no ka laccase ma waena o 50 a me 70 °C.
Ua hōʻike nā hopena ua hōʻike ka enzyme i ka hana kiʻekiʻe loa ma ka pH 3.0, e hōʻea ana i ka 94% hana ma ka pH 3.5. Eia naʻe, ua noho ikaika ia ma luna o kahi ākea pH mai 2.5 a 7.0 (Kiʻi 2b). Eia kekahi, ua hōʻike ʻo ia i ka hana kiʻekiʻe ma nā kūlana acidic i hoʻohālikelike ʻia me nā kūlana neutral a alkaline paha. Ua noho kāna hana ma ka liʻiliʻi he 77% ma luna o ka pae pH mai 2.5 a 4.5, akā ua hiki wale i kahi 38% ma ka pH 7.0. ʻO ka pH kūpono no ka laccase mai *Trametes polyzona* WRF03 he 4.533, ʻo ia ka pH like me ka pH no nā laccases mai *Trametes polyzona* KU-RNW02737, *Trichoderma harzanium* 39, *Pleurotus* sp. 40, a me *Trametes hirsuta* 41. Eia naʻe, e like me ke aʻo ʻana a Chairin et al.42, ʻo ka pH kūpono no ka laccase mai *Polymorpha f. sp.* WR710-1 he 2.2, ʻoiai ʻo ka pH kūpono no ka laccase mai *Polymorpha f. sp.* IBL-04 he 5.043. ʻO ka hoʻopaʻa ʻana o nā anions hydroxide (laccase inhibitor) i nā ʻātoma keleawe o T2/T3 laccase paha ke kumu o ka emi ʻana o ka hana laccase ma lalo o nā kūlana pH kūlike ʻole a alkaline paha. Hiki i kēia ke hoʻopilikia i ka hoʻoili ʻana o nā electron kūloko mai ke kikowaena T1 a i ke kikowaena T2/T3, no laila.kaupalenaka hana enzyme23,44
Ma ka hoʻomoe ʻana i ka enzyme ma nā mahana like ʻole, ua ʻike ʻia ua hoʻopilikia ka manawa hoʻomoe a me ka mahana i ke kūpaʻa o ka enzyme. ʻO ka mea nui, ua hōʻike ka laccase mai *Trametes polyzona* NRC 620 i ke kūpaʻa kiʻekiʻe ma 40℃ a me 50℃, e mālama ana i ka 68.33% a me 59.61% o kāna hana mua, ma hope o 120 mau minuke (Kiʻi 3a). I ka hoʻohālikelike ʻana, ma lalo o nā kūlana like (40℃ a me 50℃, 120 mau minuke), ua mālama ka laccase mai *Trametes polyzona* WRF03 i ka 64.38% a me 42.92% o kāna hana, kēlā me kēia.33I ka ʻaoʻao ʻē aʻe, ʻo ka hoʻonui ʻana i ka manawa hoʻomoʻa a me ka mahana i hoʻemi i ke kūpaʻa o *Trametes polyzona* NRC 620 laccase; Ma hope o ka hoʻomoʻa ʻana ma 60℃ a me 70℃ no 60 mau minuke, ua emi kāna hana i 39.24% a me 1.72%, kēlā me kēia (Kiʻi 3a). E like me nā hopena hoʻokolohua, ua hōʻike ʻo laccase mai *Trametes polyzona* WRF03 i ke kūpaʻa kiʻekiʻe ma 40 a me 50℃ ma o ke kaʻina hana wela.33Pēlā nō, ʻo Lueangjaroenkit etal.37a me Chairin etal.42ua hōʻike ʻia ke kūpaʻa o nā laccases mai Trametes polyzona KURNW027 a me Trametes polyzona WR710-1 ma 50 °C no 1 hola, kēlā me kēia. Ma ke ʻano he biocatalyst pono e pili ana i nā ʻano biotechnological like ʻole, pono i ka laccase ke kūpaʻa maikaʻi a me ka hana ma luna o kahi ākea ākea.
ʻO ke kūpaʻa thermostatic (a) a me ke kūpaʻa pH (b) o ka laccase mai *Pleurotus ostreatus* NRC 620. Ua loiloi ʻia ke kūpaʻa thermostatic ma ka hoʻomoʻa ʻana i ka hopena enzyme i loko o ka 0.05 M sodium phosphate buffer (pH 7.0) ma 40, 50, 60, a me 70 °C no 2 h, kēlā me kēia. Ua loiloi ʻia ke kūpaʻa pH ma ka hoʻomoʻa ʻana i ka hopena enzyme i loko o ka 0.1 M citrate buffer a me ka Tris buffer (pH 3, 4, 6, a me 7) ma 40 °C no 2 h. Ua helu ʻia ka hana koena me ka hoʻohana ʻana iā ABTS ma ke ʻano he substrate ma hope o ka hoʻomoʻa ʻana.
No ka hoʻoholo ʻana i nā kūlana kūpono no ka hoʻohana ʻana a me ka mālama ʻana i ka enzyme, ua noiʻi mākou i ka hopena o ka pH ma ke kūpaʻa o ka laccase. ʻO ka ʻike ʻana i nā waiwai pH like ʻole ua hoʻopilikia nui i ke kūpaʻa o ka ʻano protein, a no laila e hoʻopilikia ai i ke kūpaʻa a me ka hana o ka mole enzyme. Ua hōʻike nā hopena ua emi ka paʻa o ka enzyme ma lalo o nā kūlana acidic, ʻoiai ua hōʻike ʻo ia i ke kūpaʻa maikaʻi ma nā waiwai pH kiʻekiʻe (nā ʻāpana neutral a me alkaline). Ma nā waiwai pH o 7.0, 6.0, 4.0, a me 3.0, ʻo nā helu paʻa enzyme ma hope o 120 mau minuke ma kahi o 100%, 62.54%, 52.39%, a me 11.14%, kēlā me kēia (Kiʻi 3b). *Strombus multisus* WRF03 laccase i hōʻike i ke kūpaʻa kiʻekiʻe ma nā waiwai pH neutral (5.5–6.5) a me ke kūpaʻa haʻahaʻa ma nā waiwai pH acidic (ma lalo o 4.0). Ma hope o 120 mau minuke ma nā waiwai pH o 5.5, 6.0, a me 6.5, ʻo ka nui o ka paʻa ʻana o ka enzyme ma kahi o 82%, 100%, a me 93%, kēlā me kēia.33Khairin et al.42ua ʻike ʻia ua paʻa ka laccase mai Trametes polyzona WR710-1 ma ka pae pH o 6.0 a 7.0, ʻoiai ʻo Sayed et al.45ua hōʻike ʻia ua ʻoi aku ka paʻa o ka laccase ma lalo o nā kūlana pH kūlike ʻole. Eia nō naʻe, ua hōʻike pū ka laccase mai Cerrena unicolor i ke kūpaʻa ma lalo o nā kūlana alkaline (pH 9.0)46Ua hōʻike nā laccases i aʻo ʻia i ke kūpaʻa kiʻekiʻe ma luna o kahi pae pH ākea. He ʻano koʻikoʻi paha kēia no nā noi ʻoihana.
ʻOiai he hopena hoʻoulu a me ka hopena pale o kekahi mau iona metala i ka hana enzyme, pono e noʻonoʻo ʻia ko lākou hopena ma ka hana enzyme i nā noi ʻoihana. He mea koʻikoʻi kēia no ka mea he mau mea haumia kaiapuni maʻamau nā iona metala e hiki ke hoʻopilikia i ke kūpaʻa a me ka synthesis o nā enzymes extracellular.47No ka noiʻi ʻana i nā hopena o nā ion metala he nui ma ka laccase mai *Pleurotus ostreatus* NRC 620, ua hana mākou i nā hoʻokolohua like. E like me ka mea i hōʻike ʻia ma ke Kiʻi 4, ma muli o ke ʻano o ka metala i hoʻohana ʻia, ʻo ka hoʻonui ʻana i ka nui o ka ion metala mai 2.5 mM a i 10 mM ua hoʻopilikia maikaʻi ʻole i ka hana enzyme. No ka laʻana,Mg²⁺ , Co²⁺ , Zn²⁺, a meCu²⁺hiki ke hoʻoulu a hoʻāla i ka hana enzyme, ʻoiaiʻAe⁺ , Mn²⁺ , Ca²⁺, a meK⁺hiki ke kāohi i ka hana enzyme. Ma kahi o 10 mM, ʻo nā ion Cu²⁺ a me Mg²⁺ nā mea hoʻoulu ikaika loa o ka hana laccase mai *Pleurotus ostreatus* NRC 620, e hāʻawi ana i kahi kekelē hoʻāla o kahi o 34% a me 20%, kēlā me kēia. Eia nō naʻe, ma kahi o 10 mM, ʻo nā ion Ca²⁺ ka mea hoʻōki ikaika loa o ka laccase, e hōʻemi ana i ka hana enzyme ma kahi o 60%.
Ka hopena o nā ion metala ma ka hana o Pleurotus ostreatus NRC 620 laccase. Ua hoʻomoʻa ʻia ka laccase no 10 mau minuke i loko o ka sodium phosphate buffer (0.05 M, pH 7.0) e loaʻa ana nā ion metala like ʻole ma nā ʻano o 2.5 mM a me 10 mM. A laila ua hoʻomaka ʻia ka hopena ma ka hoʻohui ʻana o ka substrate (ABTS), ma hope o ke ana ʻia ʻana o ka hana pili.
Ua kūlike kā mākou mau hopena me nā mea kākau ʻē aʻe i ʻike ua hoʻonui ʻo Mg²⁺ a me Cu²⁺ i ka hana o *Trametes polyzona* WRF03³. Ua ʻike ʻo Castaño et al.⁴⁸ ua hoʻoulu ʻia ka laccase mai *Xylaria* sp. i kekahi ʻano e nā ion keleawe (Cu²⁺). Eia kekahi, ua hana ʻo Foroutanfar et al.⁴⁹ lāua ʻo Si et al.⁵⁰ i nā haʻawina like ma nā laccases mai *Paraconiothyrium variabile* a me *Trametes pubescens*, kēlā me kēia. Hiki ke hoʻopiha ʻia ke kahua hoʻopaʻa keleawe ʻano II (T2) o kēia enzyme me Cu²⁺ ma kahi ʻano i hāʻawi ʻia, kahi e wehewehe ai i ka hoʻoulu ʻana o ka hana laccase ma nā ʻano Cu²⁺³⁹ kiʻekiʻe. ʻOiai ʻo nā laccases fungi pala keʻokeʻo he mau oxidases e loaʻa ana nā ʻātoma keleawe he nui, ʻokoʻa nā hopena o nā ion keleawe ma ka hana laccase a mai ka hoʻoulu ʻana a me ka inhibitory a i ka neutral.⁵¹ I ka hoʻohālikelike ʻana, ʻo Zhou et al.[52]ua hōʻike ʻiaCu²⁺ua kāohi i ka hana laccase o ka ʻukulele lalo o Taiwan (Odontotermes formosanus). Eia naʻe, nā laccases o Cerena sp. HYB07[53]a me Clitocybe maxima[54]ʻaʻole i hoʻopilikia ʻia e nā iona keleawe.
Ua hōʻike ʻia ke ʻano kikoʻī o ka substrate e kona mau palena kinetic (Km a me Vmax); ʻo ka ikaika o ka pilina paʻa o ka substrate i ka enzyme, ʻo ka haʻahaʻa o ka waiwai Km a ʻo ke kiʻekiʻe o ka ʻano kikoʻī o ka substrate.3,21,55Ua hoʻoholo ʻia nā ʻano kinetic (Km a me Vmax) o laccase mai *Pleurotus ostreatus* NRC 620 me ka hoʻohana ʻana i ka polokalamu GraphPad Prism 6.0 ma ke kaha kiʻi ʻana i ka Lineweaver-Burk plot (Kiʻi 5). I ka hoʻohana ʻana iā ABTS ma ke ʻano he substrate, ʻo nā hopena he 1.99 mM a me 16217 μmol.minuke⁻¹ L⁻¹,kēlā me kēia. Elsayed et al.21ua hōʻike ʻia ʻo nā waiwai Km no ka hoʻomake ʻana o ABTS he 0.1 mM a me 0.064 mM, kēlā me kēia, e hōʻike ana i kahi pilina kiʻekiʻe o nā isoenzymes Lac A a me Lac B no ABTS. Eia kekahi, ʻo nā waiwai Vmax he 0.182 μmolminuke⁻¹a me 0.603 μmolminuke⁻¹, kēlā me kēia. ʻOi aku ka haʻahaʻa o ka waiwai Km i loaʻa ma mua o Trametes polyzona WRF03 (8.66 mM); eia kekahi, ʻo kā lākou waiwai Vmax (1429 mmol min⁻¹) ua like pū kekahihaʻahaʻai ka wā e hoʻohana ai iā ABTS ma ke ʻano he substrate.33 Pēlā nō, ʻo nā waiwai Km o Lentinus squarrosulus MR13 a me Trametes sp. ʻO AH28-2 laccase concentrations he 0.0714 mM a me 0.025 mM, kēlā me kēia, a ʻo nā waiwai Vmax he 0.0091 mM min−1 a me 0.67 mM min−1 mg−1 (e pili ana iā ABTS), kēlā me kēia.56,57
Ua noiʻi ʻia ka hopena o ka nui o ABTS ma ka hana o ka laccase mai *Pleurotus ostreatus* NRC 620, a ua hoʻolālā ʻia kahi palapala Lineweaver-Burk o ka reciprocal o ka wikiwiki o ka hopena mua e kūʻē i ka nui o ABTS. Ua ana ʻia ka hopena oxidation o ABTS me nā ʻano like ʻole (0.025–3.0 mM) o ka laccase ma ka pH 4.5 e hoʻoholo i nā ʻano kinetic (Vmax a me Km). Ua helu ʻia nā Michaelis-Menten kinetic constants me ka hoʻohana ʻana i ka palapala Lineweaver-Burk o ka reciprocal o ka wikiwiki o ka hopena e kūʻē i ka nui o ka substrate. Ua helu ʻia nā kinetic constants mai ka palapala Lineweaver-Burk me ka hoʻohana ʻana i ka polokalamu GraphPad Prism 6.01.
ʻO nā enzymes hoʻomaʻamaʻa kuʻuna, e like me nā pectinases, hydrolyze i nā mea pectic, e hōʻemi ana i ka viscosity a me ka turbidity. Hoʻopau maikaʻi lākou i nā polysaccharides structural a hoʻohana pinepine ʻia me nā enzymes ʻē aʻe, e like me nā cellulases a me nā hemicellulases, e hoʻomaikaʻi i ka hua a me ka maopopo. Eia nō naʻe, ʻaʻole kuhikuhi pono nā pectinases i nā hui phenolic, ʻo ia nā mea nui e hāʻawi i ka turbidity a me ka oxidative browning, ʻoi aku hoʻi i nā wai e like me ka ʻāpala a me ka wai hua waina.58I ka hoʻohālikelike ʻana, hoʻoulu nā laccases i ka oxidation o nā pūhui phenolic, polymerizing iā lākou i loko o nā molekala nui a hiki ke hoʻoheheʻe ʻia e hiki ke wehe ʻia e ka sedimentation a i ʻole ka filtration. ʻAʻole wale kēia ʻano hana e hoʻomaikaʻi akā hoʻolōʻihi pū i ke ola o ka wai ma ka hōʻemi ʻana i ka likelike o ka oxidative browning i hoʻokumu ʻia e nā pūhui phenolic. Eia kekahi, hiki ke hoʻokō ʻia nā kaʻina hana hoʻomālamalama e pili ana i ka laccase ma lalo o nā kūlana hana akahai (pH 3.5–5.5, mahana 25–40 °C), e kūpono ai no nā wai palupalu me ka ʻole o ka hoʻopilikia ʻana i kā lākou mau waiwai nutritional a organoleptic paha.59Ua hōʻike ʻia nā haʻawina e hiki i ka mālama ʻana i ka pectinase ke hoʻomālamalama i ka wai i loko o 1-2 mau hola, ʻoiai ʻo ka mālama ʻana i ka laccase e koi pinepine i kahi manawa hopena lōʻihi (3-6 mau hola) e hōʻemi loa i nā hui phenolic. Eia nō naʻe, hiki ke hoʻomaikaʻi ʻia kēia kaʻina hana ma ka hoʻopaʻa ʻana i ka enzyme a i ʻole ma ka hoʻohui ʻana i ka laccase me nā ʻano hoʻomālamalama mechanical.60Ma kēia haʻawina, ua hōʻike ka profiling enzyme o ka extract maka i nā hana koʻikoʻi o ka laccase a me ka α-amylase, ʻoiai ua haʻahaʻa loa nā hana pectinase a me xylanase, a ʻaʻole i ʻike ʻia ka hana cellulase. No laila, ʻo ka emi ʻana o ka turbidity a me ka ʻike phenolic ma muli nui o ka hana a ka laccase, ʻoiai ʻo ka loli o ka viscosity hiki ke lilo i hapa ma muli o ka hana a ka amylase.
Hōʻike ka Papa 1 i nā ʻano physicochemical o ka wai ʻāpala hou i ʻomi ʻia a me nā laʻana i mālama ʻia me ka laccase. Ua hōʻike nā hopena ua haʻahaʻa ka hua o ka wai ʻāpala hou i ʻomi ʻia (71.59%) ma mua o nā laʻana i mālama ʻia me ka laccase (87.34%). Kūlike kēia mau hopena me nā ʻike o Pilnik lāua ʻo Orange.61, nāna i hōʻike e hiki i ka hoʻohana ʻana i nā enzymes i ka hana ʻana i nā hua ke hoʻonui i ka hua wai, hoʻomaikaʻi i ka kānana, a loaʻa ka wai maikaʻi a maopopo no ka noʻonoʻo ʻana. ʻO ka hoʻonui ʻana i ka hua wai ma muli nui o ka hoʻonui ʻana i ka nui o nā kō hiki ke hoʻoheheʻe ʻia i loko o ka wai. I ka wā o ka hydrolysis enzymatic o nā hua, ua luku ʻia ka mesoglea a me ka pectin i loko o nā paia cell o ka huahana a hoʻololi ʻia i nā mea hiki ke hoʻoheheʻe ʻia e like me nā kō kūlohelohe a me nā waikawa.62.ʻOi aku ka haʻahaʻa o ka waiwai pH o ka wai ʻāpala i mālama ʻia me ka enzyme ma mua o ka hui kaohi (P < 0.05), a ua piʻi nui ka waiwai pH o nā hui ʻelua i ka wā o ka mālama ʻana (Papa 1). Kūlike kēia mau hopena me nā hopena o Mark et al.63, nāna i ʻike ua emi ka pH o ka wai hua cashew ma hope o ka mālama ʻana ma hope o ka mālama ʻana i ka wela. ʻO ka hoʻohaʻahaʻa pectin a me ka hoʻokumu ʻana o ka waikawa galacturonic ma hope o ka mālama ʻana i ka enzyme ke kuleana no ka hoʻonui ʻana o ka pH i ka wā e mālama ai. Ua noho ka pH o nā laʻana i mālama ʻia me ka enzyme ma waena o 4.05 a me 4.31 i ka wā o ka mālama ʻana, ʻoiai ʻo ka pH o ka wai ʻāpala i mālama ʻole ʻia ma waena o 4.12 a me 4.33.
Ua hōʻike ka huina acidity (TA) o nā laʻana i mālama ʻole ʻia a me nā laʻana i mālama ʻole ʻia me ka laccase i kahi ʻano emi me ka hoʻonui ʻana i ka manawa mālama (Papa 1). Ua pili ka emi ʻana o ka acidity i ka hoʻololi ʻana o nā waikawa organik i nā kalapona a i ʻole nā hana enzymatic, a me ka oxidation i ka wā e mālama ai i ka wai.64ʻOi aku ka haʻahaʻa o ka huina waikawa o ka wai ʻāpala kaohi a me nā laʻana i mālama ʻia me ka enzyme ma mua o nā wai ʻē aʻe (wai ʻōhelopapa 0.9%, wai plum 2.2%, wai kumquat 1.0%, wai ʻapelika 2.4%, wai ʻalani 0.8%), akā ua like ia me nā wai ʻē aʻe (e laʻa, wai pea 0.3%).62ʻO kēia mau ʻokoʻa o ka wai ʻāpala hou i ʻomi ʻole ʻia ma muli paha o nā kumu like ʻole e like me nā kūlana ulu, nā kumu hoʻoilina, ka pae oʻo, a me nā ʻano hana.65ʻO ka emi ʻana o ka acidity holoʻokoʻa o ka wai ʻāpala kaohi a me ka laccase i mālama ʻia e kūlike me nā hopena i hōʻike ʻia e Singh et al.66e pili ana i ka emi ʻana o ka waikawa holoʻokoʻa o ka wai ʻāpala Jin Nuo ma hope o 74 mau lā o ka mālama ʻana. Ma ka ʻaoʻao ʻē aʻe, ʻo Oshmiansky lāua ʻo Wojdylo67ʻaʻole i loaʻa kekahi mau loli koʻikoʻi i ka waikawa o ka wai ʻāpala i ka wā e aʻo ai i ka hopena o nā ʻano hoʻomālamalama kuʻuna.
Hōʻike nā hopena i hōʻike ʻia ma ka Papa 1 ua ʻoi aku ka nui o ka waiwai o nā mea paʻa hiki ke hoʻoheheʻe ʻia (TSS) o ka wai ʻāpala i mālama ʻia me ka laccase ma mua o ka laʻana i mālama ʻole ʻia. Kūlike kēia mau hopena me nā haʻawina i paʻi ʻia..68Eia kekahi, hōʻike ka Papa 1 ʻo ka waiwai TSS o ka hui wai ʻāpala kaohi he 9.58 i ka manawa mua a hiki i ka 11.05 ma ka hopena o ka manawa mālama. ʻOi aku ka haʻahaʻa o kēia mau waiwai ma mua o nā waiwai TSS o ka wai ʻāpala hou i hōʻike ʻia e Hamid et al..69(11.2 a me 11.80, kēlā me kēia). Ua piʻi nui ka waiwai TSS o nā hāpana wai ʻāpala i mālama ʻia me ka laccase, e hoʻomaka ana mai 11.23 a hiki i ka 12.93 ma hope o ʻelua pule o ka mālama ʻana ma 4°C (Papa 1). Ua ʻike ʻia hoʻi ka piʻi like ʻana o ka TSS i ka wā o ka mālama ʻana i nā hua citrus, nā lemona, a me nā ʻalani ʻono. ʻO ka piʻi ʻana o nā mea paʻa hiki ke hoʻoheheʻe ʻia (TSS) i ka wā o ka mālama ʻana ma muli paha o ka hydrolysis o polysaccharides (starch) i monosaccharides (sugars), ka piʻi ʻana o ka nui ma muli o ka dehydration o ka wai, a me ka hōʻino ʻana o ka pectin i loko o ka wai i nā mea paʻa hiki ke hoʻoheheʻe ʻia. ʻO ka piʻi ʻana o nā mea paʻa hiki ke hoʻoheheʻe ʻia (TSS) ma muli paha o ka piʻi ʻana o nā kō hiki ke hoʻoheheʻe ʻia, hiki ke hoʻokumu ʻia e ka hoʻololi ʻana o ka pectin a i ʻole cellulose i nā kō hiki ke hoʻoheheʻe ʻia e ka pectin a i ʻole cellulase, kēlā me kēia, a i ʻole e ka hydrolysis o ka starch i nā kō, e like me ka hōʻike a Hamed et al.69.Hiki ke ʻike maka ʻia ka hopena o ka laccase ma nā waiwai o ka wai ʻāpala, ʻoiai ʻo ka wai ʻāpala i mālama ʻia me ka laccase e hōʻike ana i ka flowability maikaʻi a me ka viscosity haʻahaʻa ma mua o ka wai i mālama ʻole ʻia. Ua hoʻopaʻa ʻia kēia ʻike ma ka Papa 1; ʻO ka viscosity o ka hāpana i mālama ʻia me ka enzyme he 1.87 cP, ʻoiai ʻo ka viscosity o ka hāpana kaohi he 2.95 cP. ʻO kēia emi nui o ka viscosity ma muli paha o ka hiki ke paʻa wai o nā mea like me ka pectin a me ka hoʻokumu ʻana o kahi ʻano pūnaewele cohesive.
Ma kēia haʻawina, ua noiʻi ʻia ka hopena o ka laccase ma ka index browning (BI) o ka wai ʻāpala ma ke ana ʻana i ka absorbance ma 420 nm me ka hoʻohana ʻana i kahi spectrophotometer. Hōʻike ʻia nā hopena ma ka Papa 1. I ka wā e mālama ai, ua hōʻike ka BI o nā laʻana wai ʻāpala i nā hui i mālama ʻia a me nā hui i mālama ʻole ʻia i kahi ʻano piʻi mālie. Hōʻike ka BI i ke kiʻekiʻe o ka browning a hiki ke lawelawe ma ke ʻano hehe mea nuihōʻailona o nā hopena browning enzymatic a me nā non-enzymatic. Ua piʻi nui ka absorbance i ka wā mālama (P < 0.05). I ka pau ʻana o ka mālama ʻana, ʻo kaA420ua piʻi ka waiwai o nā hāpana wai ʻāpala i loko o nā hui kaohi a me nā hui i mālama ʻia me ka enzyme ma kahi o 217% a me 121%, kēlā me kēia (Papa 1). Hōʻike nā hopena e hiki i ka mālama ʻana i ka enzyme ke hōʻemi pono i ke kekelē browning ma kahi o 56%. ʻO nā hopena o Bezerra et al.[19]] kūlike me kā mākou mau hopena; Ua hoʻohana lākou i ka laccase-glutaraldehyde-coconut fiber e hoʻomālamalama i ka wai ʻāpala, e hōʻemi ana i kona kala mua ma 61%.
ʻOiai ʻo nā polyphenols i loko o nā wai huaʻai he hopena maikaʻi i ka meaʻai a me ka therapeutic i ke kino o ke kanaka, hiki iā lākou ke hana pū me nā protein, e hana ana i ka wai ʻeleʻele, ka sedimentation, a i ʻole ka turbidity, a laila e hoʻololi ai i ka ʻono a me ke ʻala o ka huahana a hoʻemi i kona ola papa.71ʻO ka pahuhopu o kēia noiʻi, ʻo ia ke hōʻemi palekana i ka nui o ka hui phenolic o ka wai ʻāpala me ka hoʻohana ʻana i ka laccase mai Pleurotus ostreatus NRC 620. Hōʻike nā hopena i hōʻike ʻia ma ka Papa 1 ua hōʻemi nui ʻia ka nui o ka hui phenolic o ka wai ʻāpala i mālama ʻia me ka laccase ma mua o ka mālama ʻana ma 4 °C. Eia kekahi, ua emi pū ka nui o ka hui phenolic i ka wā o ka mālama ʻana i nā laʻana ʻelua i aʻo ʻia (Papa 1). Noiʻi na Sandri et al.72ua hōʻike ʻia e hiki i ka wai ʻāpala i mālama ʻia me ka enzyme ke mālama i kāna hana antioxidant a me ka nui o ka hui phenolic. Eia nō naʻe, ʻo nā hopena o kahi noiʻi na Lettera et al.73hōʻike i ka mālama ʻana i ka wai ʻalani me ka fungal laccase hiki ke hōʻemi i ka nui o nā pūhui phenolic i loko a hiki i ka 45%.
Ua hōʻike ʻia nā pūhui Phenolic he mau waiwai e like me ka scavenging radical free, ka hoʻemi ʻana o ka oxygen singlet a me ka quenching, ka hoʻoili ʻana o ka ʻātoma hydrogen, a me ka hāʻawi ʻana i nā electron i nā radical free, e hoʻolilo iā lākou i mau antioxidants ikaika.74No laila, ma kēia haʻawina, ua hoʻohana ʻia nā ʻano hana DPPH a me FRAP e loiloi i ka hopena o ka laccase ma ka hana antioxidant o ka wai ʻāpala i mālama ʻia i loko o ka pahu hau no 14 mau lā (Papa 2). Ua hōʻike nā ʻano hana ʻelua i ka piʻi ʻana o ka hana antioxidant i ka wā e mālama ai, hiki ke kumu o ka piʻi ʻana o nā pūhui phenolic manuahi a i ʻole ka hoʻokumu ʻia ʻana o nā huahana hana Maillard (MRP), me nā huahana hana Maillard paha ke kumu o ka piʻi ʻana o ka hana antioxidant.75ʻO nā hopena browning non-enzymatic (me ka ascorbic acid degradation, Maillard reactions, a me ka acid-catalyzed degradation of sugars) e hana i nā pigments brown (melanoidins). Hiki i nā huahana ascorbic acid degradation waena a me nā huahana sugar degradation (e like me nā carbonyl compounds) ke hana me nā amino acids ma o nā Maillard reactions.76ʻOiai ua aʻo nui ʻia ka hoʻololi ʻana o nā huaʻai a me nā mea kanu i ka wā mālama ʻana, ua kaupalena ʻia ko mākou ʻike i kēia mau hopena.77I ka hoʻohālikelike ʻia me ke ʻano FRAP, ua hōʻike ka wai ʻāpala i mālama ʻia me ka laccase i ka hana antioxidant haʻahaʻa loa e ke ʻano DPPH (Papa 2), a ua piʻi nui ka hana antioxidant o nā laʻana āpau me ka hoʻonui ʻana i ka manawa mālama. Ua hoʻohana ʻia ʻelua mau ʻano like ʻole no ka hoʻoholo ʻana i ka hana antioxidant ma kēia haʻawina no ka mea ʻokoʻa ko lākou mau kumumanaʻo. Ana ke ʻano DPPH i ka hiki ke hoʻopau i nā radical manuahi, ʻoiai ke ʻano FRAP i ka hiki ke hōʻemi i nā ion hao. No laila, ua ʻōlelo ʻia e hoʻohana i nā ʻano he nui no ka hoʻoholo ʻana i ka hana antioxidant e hoʻomaopopo maikaʻi i ka hana antioxidant o nā laʻana i aʻo ʻia.78
ʻO kekahi o nā mea nui i loaʻa i kēia noiʻi, ʻo ia ka hōʻike ʻana o *Pleurotus ostreatus* laccase NRC 620 i ka hana kūpono ma 70°C a me ka pH 3.0. Ke hoʻohālikelike ʻia me nā laccase fungal ʻē aʻe i hoʻohana pinepine ʻia no ka hoʻomālamalama ʻana i ka wai, e like me *Trametes versicolor* a me *Ganoderma lucidum* laccases, hōʻike ʻo *P. ostreatus* NRC 620 i ke kūpaʻa wela kiʻekiʻe a me ka pH ʻakika. ʻO nā laccases mai *Trametes versicolor* a me *Ganoderma lucidum* e hōʻike pinepine i ka hana kūpono ma ka pae o 50-60°C a ma nā waiwai pH ma waena o 3.5 a me 5.0. Hiki i kēia ʻokoʻa ke kōkua i ka hoʻomaikaʻi ʻana i ka pono o ka hoʻomālamalama ʻana i ka wai, ʻoi aku hoʻi no nā wai ʻakika kahi e koʻikoʻi ai ke kūpaʻa ma nā waiwai pH haʻahaʻa. ʻO ke ʻano kū hoʻokahi o *P. Ke hoʻohālikelike ʻia me nā laccase fungal ʻē aʻe i aʻo ʻia, hōʻike ʻo *Pleurotus ostreatus* NRC 620 i ka hiki ke hana pono ma lalo o nā kūlana paʻakikī. ʻO kona mahana hana kūpono kiʻekiʻe e hōʻike ana i nā pono kūpono i nā noi ʻoihana, e like me ka wikiwiki o ka pane ʻana a me ka hoʻemi ʻana i ka haumia microbial. ʻO kona pH haʻahaʻa, kahi kūpono i ke ʻano acidic o nā wai he nui, he mea pono paha i nā kaʻina hana hoʻomālamalama wai. Hoʻāpono kēia mau hopena i ka ʻimi hou ʻana no ka noi nui, e hana ana i ka *Pleurotus ostreatus* NRC 620 i mea kūpono i nā kumu laccase fungal kuʻuna. Ke hoʻohālikelike ʻia me nā haʻawina ma mua, ua ʻike mākou ʻo ka mahana kūpono he 60°C a ʻo ka pH kūpono he 3.0. Ma hope o ka pane ʻana ma 60°C no 80 mau minuke, ua mālama ʻia ka *Ganoderma lucidum* laccase.46% o kāna hana.79 Wahi a Kurniawati lāua ʻo Nicelle80, Hōʻike nā enzymes *Ganoderma lucidum* i ke kūpaʻa maikaʻi loa a i ke kaulike ma 25°C a me nā waiwai pH mai 5.0 a 8.0, a me ke kūpaʻa ma pH 6.0 a me nā mahana mai 10 a 30°C. Ma kēia haʻawina, ua ʻike mākou ʻo ka pH kūpono a me ka mahana no ka hana enzyme no *Pleurotus ostreatus* he 3.0 a me 70°C, kēlā me kēia. Ma hope o ka hoʻomoʻa ʻana ma 40°C a me 50°C no ʻelua hola, ua mālama ka enzyme i 68.33% a me 59.61% o kāna hana, kēlā me kēia. Eia kekahi, ua hōʻike ʻo Pleurotus ostreatus NRC 620 laccase i ka hana kiʻekiʻe ma luna o kahi ākea ākea o ka mahana mai 50°C a 80°C, kokoke i ka hana kiʻekiʻe loa (69%–98%), me ka hana kiʻekiʻe loa i ʻike ʻia ma 70°C.
I ka hopena, ua hōʻike ka ʻōhiʻu laccase NRC620, i loaʻa ma lalo o nā kūlana paʻa, i ka hana kūpono a me ke kūpaʻa ma waena o nā ʻano pH a me nā kūlana mahana, e hōʻike ana i ke kūpaʻa kiʻekiʻe i hoʻohālikelike ʻia me nā kumu enzyme ʻē aʻe. ʻO ka hoʻohui ʻana o 10 mM MgSO₄ a me CuSO₄ i hoʻonui i ka hana enzyme ma kahi o 21% a me 35%, kēlā me kēia. I ka wā i hana ʻia ai i loko o ka wai ʻāpala, ua hoʻemi ka enzyme i ka pH a me ka viscosity, ʻoiai ua emi iki ka ʻike phenolic i ka wā e mālama ai.
Ua hōʻoia nā hopena i ka hiki o ka laccase i ka ʻoihana meaʻai, ʻoi aku hoʻi i ka wehewehe ʻana i nā mea inu. Ma ka wāwahi kūikawā ʻana i nā pūhui phenolic, ʻaʻole wale ka laccase e hōʻemi i ka turbidity a hoʻomaikaʻi i ka maopopo akā mālama pū i ka maikaʻi o nā wai huaʻai ma lalo o nā kūlana hana akahai. ʻAʻole e like me nā mea wehewehe kuʻuna e like me ka gelatin, bentonite, a me ka silica gel, ʻaʻole hana ka laccase i nā ʻōpala a wehe paha i nā ʻala ʻoluʻolu mai nā mea inu, e lilo ia i koho ʻoi aku ka maikaʻi o ke kaiapuni a me ka hoʻomau. Eia kekahi, i ka hoʻohālikelike ʻia me nā enzymes ʻē aʻe a me nā ʻano kānana, hāʻawi ʻo laccase i kahi hopena i kuhikuhi ʻia a kūpono hoʻi me ka ʻole o ka hoʻopilikia ʻana i ka maikaʻi o ka huahana.
ʻO Kyomuhimbo, HD lāua ʻo Brink, HG. Nā noi a me nā hoʻolālā hoʻopaʻa ʻana o nā laccases i loaʻa ke keleawe; he loiloi. Heliyon 9, e13156 (2023).
Ka manawa hoʻouna: Dec-15-2025