Cold-adapted Laccase Production By Pycnoporus Sp. SYBC-L10 And Its Applications In The Degradation Of Antibiotics

Degradation of antibiotic residues has always been an urgent environmental problem.Biological methods show great potential in this field because of their low toxicity and low consumption.However,low removal efficiency and incomplete removal of biological process are the key technical difficulties affecting its industrial application.In this study,an extracellular fungal laccase(Lac-Q)with robust cold-adapted and thermostable characteristics from Pycnoporus sp.SYBC-L10 was purified and characterized.In addition,the ability of Lac-Q-mediator systems to remove tetracycline antibiotics(TCs)and sulfonamide antibiotics(SAs)under various conditions was also evaluated.The antimicrobial activity of TCs and SAs before and after laccase-mediated oxidation was evaluated.The proposed transformation pathways of the oxytetracycline(OTC)and sulfamethoxazole(SMX)in the Lac-Q-mediator systems were proposed.Finally,the medium and culture condition for Lac-Q production by Pycnoporus sp.SYBC-L10 in submerged fermentation were optimized.The primary findings of this study were as follows:(1)Laccase(Lac-Q)secreted by Pycnoporus sp.SYBC-L10 was purified using ammonium sulfate precipitation and a Hi Trap Q HP anion exchange chromatography column.Lac-Q with an apparent molecular weight of about 60kDa.After purification,the specific activity of the laccase from Pycnoporus sp.SYBC-L10 increased 16.83-fold,from 57.92U·mg^-1 to 1032.56U·mg^-1,using ABTS as a substrate with yield of 46.32%.The N-terminal amino acid sequence of Lac-Q was found to be AIGPVADLTLTNAAV.The optimum pH of Lac-Q was 3.0 for the ABTS and DMP.The study of the pH stability revealed that Lac-Q was stable from pH 3.0 to pH 10.0 at room temperature,especially at pH 6.0.The optimum temperature of Lac-Q was 70.0?for the ABTS and DMP.Remarkably,Lac-Q showed about51.0%of the maximal activity incubation at 0.0?,implying that Lac-Q has a strong tolerance to cold conditions.Lac-Q retained 108.9%,106.7%,106.5%,and 68.0%of its original activity after a 3.0 h incubation at 40.0?,50.0?,60.0?,and 70.0?,respectively.Fe²⁺and Fe³⁺exhibited a strong inhibitory effect on the Lac-Q activity.However,Al³⁺showed a strong activation effect on the Lac-Q activity.Lac-Q was slightly inhibited by L-Cysteine and Dithiothreitol and strongly inhibited by NaCl,NaF and NaN₃.The K_m values of Lac-Q with ABTS and DMP were 0.0191 and 0.0712mmol·L^-1,respectively.In addition,the catalytic efficiencies(k_cat/K_m)varied from 3012.92 L·s^-1mmol^-1 for DMP up to 60536.13 L·s^-1mmol^-1 for ABTS.The CD spectrum show that Lac-Q is 10.6%?-helix,36.5%?-sheet,21.3%?-turn,and 31.6%random coils.The presence of Cu²⁺and Al³⁺significantly altered the CD spectra.(2)The effect of mediators on removal of tetracycline(TC)or OTC by Lac-Q-mediated oxidation was evaluated.Results shown that the Lac-Q–ABTS system showed the highest degradation rates.In addition,the ability of a Lac-Q-mediator system to remove TC and OTC under various conditions was also evaluated.The presence of Al³⁺,Cu²⁺,and Fe³⁺accelerated the removal rate of TC and OTC by the Lac-Q–ABTS system and the presence of the Mn²⁺ion inhibited the removal rate of TC and OTC by the Lac-Q–ABTS system.Lac-Q at 10.0U·m L^-1 coupled with 1.0mmol·L^-1 ABTS degraded 100%of the 50.0 mg·L^-1 TC or OTC after 5.0 min with a static incubation at 0.0?(pH 6.0).After treatment with the Lac-Q–ABTS system,the growth inhibition of the Bacillus altitudinis SYBC hb4(B.altitudinis)and Escherichia coli BL21(E.coli)by TC and OTC was completely lost.The present report is the first time TCs'antimicrobial activity has been efficiently eliminated at0.0?(pH 6.0).The findings of this study demonstrate novel areas of application for the use of the laccase–ABTS system for removing TCs and reducing their toxicity at low temperatures.The antimicrobial activity of TC against B.altitudinis and E.coli was shown to be significantly reduced after treatment with either the laccase–ABTS system,the laccase–SA system,or the laccase–HBT system,while was no significant change after treatment with Lac-Q–3-HAA system and methanol control.Finally,seven transformation products(TPs)of OTC(namely TP445,TP431,TP413,TP399,TP381,TP367,and TP351)were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS.A possible degradation pathway of the OTC in the Lac-Q–ABTS system including deamination,demethylation,and dehydration was proposed.The rapidly removal of amino and hydroxyl groups in OTC was the primary reason for the reduction in the antimicrobial activity of OTC.(3)The effect of mediators on removal of SMX or sulfadimethoxine(SDM)by Lac-Q-mediated oxidation was evaluated.Results shown that the Lac-Q–SA system showed the highest degradation rates.In addition,the ability of a Lac-Q-mediator system to remove SMX and SDM under various conditions was also evaluated.The Al³⁺,Cu²⁺,and Fe³⁺ions remarkably promoted the removal of SMX and SDM by the Lac-Q–SA system,and the Mn²⁺ion strongly decelerated the removal of SMX and SDM by the Lac-Q–SA system.The mixture of SMX and the TPs still retained high antimicrobial activity against B.altitudinis and E.coli after treatment with the Lac-Q–SA system.The antimicrobial activity of SMX against B.altitudinis was shown to be absolutely eliminated after treatment with the Lac-Q–ABTS system and significantly reduced after treatment with the Lac-Q–3-HAA system,while was no significant change after treatment with Lac-Q–SA system,Lac-Q–HBT system,and methanol control.During SMX oxidation by the Lac-Q–ABTS system,six TPs were detected,namely TP192,TP190,TP102,TP101,TP99,and TP95.A possible degradation pathway of the SMX in the Lac-Q–ABTS system was proposed.The elimination of SO₂ was the primary reason for the reduction in the antimicrobial activity of SMX.(4)The optimized medium for laccase production by Pycnoporus sp.SYBC-L10 in submerged fermentation were:15.0g·L^-1 glucose,5.0g·L^-1 fructose,2.0g·L^-1 diammonium hydrogen citrate,6.0g·L^-1 soybean meal,0.375g·L^-1 CuSO₄·5H₂O,0.5mmol·L^-1 guaiacol,0.05%Tween-80,0.5g·L^-1 KCl,1.0g·L^-1 KH₂PO₄,0.5g·L^-1 Mg SO₄·7H₂O,1.0g·L^-1 yeast extract,0.01g·L^-1 Mn SO₄·H₂O.The optimized culture condition was:initial pH 4.0,seed age60.0 h,10.0%of inoculation concentration,and incubated at 30.0?with shaking at 200r·min^-1.After 7 d of shaking flask fermentation in the optimized medium and optimized culture condition,the dry weight of thallus reached 3.26g·L^-1,the highest laccase activity was 66.81U·m L^-1 and the concentration of 3-hydroxyanthranilic acid(3-HAA)was 0.10mmol·L^-1.