栉孔扇贝（Chlamys farreri）酚氧化酶的分离纯化及特性研究
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摘要
前期研究发现，细菌多糖刺激和病毒感染均可导致栉孔扇贝（Chlamysfarreri）血细胞中酚氧化酶活力急剧升高，表明酚氧化酶是栉孔扇贝免疫应答系统中最为敏感高效的因子之一（Xing et al.,2008；Lin et al.,2011）。本论文从栉孔扇贝血细胞中分离纯化出酚氧化酶，分析了该酶的生化及酶学特性；研究了酚氧化酶体外氧化产物的抗菌活性；分析比较了栉孔扇贝酚氧化酶与海湾扇贝（Argopecten irradians）、菲律宾蛤仔（Ruditapes philippinarum）、皱纹盘鲍（Haliotis discus hannai）、毛蚶（Scapharca subcrenata）酚氧化酶在生化及酶学特性上的差异，为栉孔扇贝酚氧化酶免疫机制的研究积累了数据。
（1）栉孔扇贝酚氧化酶的分离纯化及生化和酶学特性研究。采用线性连续梯度非变性电泳（native-PAGE）结合凝胶过滤层析的方法从栉孔扇贝血细胞中分离纯化出酚氧化酶，研究了该酶的底物特异性、动力学参数以及温度、pH、二价金属离子和抑制剂对酶活的影响。结果显示：提纯的酚氧化酶在native-PAGE中分子量为576kDa，SDS-PAGE中分子量为53kDa，表明该酶是由一种亚基组成的寡聚体；酚氧化酶对L-DOPA、邻苯二酚、多巴胺和对苯二酚的米氏常数Km分别为0.61mmol/L、0.20mmol/L、0.88mmol/L和0.83mmol/L，表明该酶是一种漆酶型的酚氧化酶，其对邻苯二酚的亲和力最高，对多巴胺的亲和力最低，对L-DOPA的亲和力高于对苯二酚；酚氧化酶在30℃和pH8.0时具有最高酶活力；酚氧化酶活力受到Ca2+和Mg2+的强烈激活，但受到Fe2+的显著抑制；酚氧化酶活力不受硫代尿素和叠氮钠的影响，但受到抗坏血酸、亚硫酸钠、柠檬酸、半胱氨酸、二乙基二硫代氨基甲酸钠（DETC）、乙二胺四乙酸二钠（EDTA）的强烈抑制，表明该酶是一种含铜的金属酶。
（2）栉孔扇贝酚氧化酶体外氧化产物的抗菌活性研究。通过观察和测定提纯的栉孔扇贝酚氧化酶与底物处理后的细菌生长情况，研究了栉孔扇贝酚氧化酶体外氧化产物对溶藻胶弧菌Vibrio alginolyticus、副溶血弧菌Vibrioparahaemolyticus、杀鲑气单胞菌Aeromonas salmonicida、爱德华氏菌Edwardsiellatarda、停乳链球菌Streptococcus dysgadysgalactiae、海豚链球菌Streptococcus iniae和溶壁微球菌Micrococcus lysodeikticus的抗菌活性。细菌生长曲线测定结果显示：酚氧化酶自身不能抑制细菌生长，酚氧化酶与L-DOPA或多巴胺的混合物可抑制细菌生长，表明是酚氧化酶的氧化产物而不是酚氧化酶具有抗菌活性；使用L-DOPA作底物时，酚氧化酶氧化产物仅对杀鲑气单胞菌显示出明显的抑制作用，而使用多巴胺作为底物时，酚氧化酶氧化产物对革兰氏阴性的溶藻胶弧菌、副溶血弧菌和杀鲑气单胞菌均表现出强烈的抑制作用，表明以多巴胺为底物获得的酚氧化酶氧化产物比以L-DOPA为底物获得的氧化产物具有更强的细胞毒性；酚氧化酶的体外氧化产物对革兰氏阳性的停乳链球菌、海豚链球菌和溶壁微球菌无抑制作用，表明与链球菌和微球菌相比，栉孔扇贝酚氧化酶氧化产物对弧菌和气单胞菌细胞成分的毒性作用更大；酚氧化酶氧化产物对同为革兰氏阴性的爱德华氏菌无抑制作用，分析原因可能是爱德华氏菌分泌的胞外产物中含有一些具有抗氧化活力的多糖，这些多糖抑制了酚氧化酶的氧化反应。显微镜检测结果显示：经过与栉孔扇贝酚氧化酶和多巴胺的共孵育后，溶藻胶弧菌、副溶血弧菌和杀鲑气单胞菌的菌体透明度降低，多数菌体丧失了运动能力，细菌聚集成团，被黑色素所覆盖。
（3）栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍、毛蚶酚氧化酶的生化及酶学特性比较研究。采用线性连续梯度非变性电泳结合邻苯二酚发色方法从海湾扇贝、菲律宾蛤仔、皱纹盘鲍、毛蚶的血细胞中分离出酚氧化酶，分析比较了栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍、毛蚶酚氧化酶在底物特异性、动力学参数以及金属离子和抑制剂对酶活的影响上的差异。结果显示：在检测到的酚氧化酶种类上，栉孔扇贝与海湾扇贝、菲律宾蛤仔、皱纹盘鲍相同，在血细胞中仅检测到一种酚氧化酶，而从毛蚶的血细胞中则检测出4种酚氧化酶；在分子量上，栉孔扇贝酚氧化酶（576kDa）与海湾扇贝（555kDa）和菲律宾蛤仔（563kDa）中的酚氧化酶较为接近，而与皱纹盘鲍（228kDa）和毛蚶（391kDa、206kDa、174kDa、未知分子量，分别命名为S391PO、S206PO、S174PO和SuPO）中的酚氧化酶差异较大；在底物特异性上，栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍和毛蚶中的酚氧化酶相似，均可以氧化邻二酚类底物和对二酚类底物，为漆酶型的酚氧化酶；在动力学参数上，栉孔扇贝酚氧化酶对邻苯二酚、L-DOPA、对苯二酚、多巴胺4种底物的Km值与海湾扇贝酚氧化酶比较接近，表明这两种酶在催化能力上相近；在二价金属离子对酶活的影响上，栉孔扇贝与其他4种贝类的酚氧化酶的活力均对二价金属离子极为敏感，都受到Fe2+的显著抑制，但Ca2+和Mg2+都介导的酚氧化酶活力增强现象只在栉孔扇贝和海湾扇贝中存在，综合比较得出，栉孔扇贝酚氧化酶的二价金属离子影响结果与海湾扇贝酚氧化酶较为接近，表明这两种酚氧化酶在蛋白结构上可能具有很高的相似性；在抑制剂对酶活的影响上，栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍和毛蚶中的酚氧化酶都受到EDTA和DETC的强烈抑制，均为含铜的金属酶，但栉孔扇贝酚氧化酶与海湾扇贝酚氧化酶以及毛蚶中的S206PO和SuPO较为相似，其活力均不受硫代尿素和叠氮钠的影响。
Previous studies showed that in the scallop Chlamys farreri, bacterialpolysaccharides challenge or viral infection caused a rapid and dramatic increase ofphenoloxidase (PO) activity in hemocytes, indicating that PO is one of the mostsensitive and effective factors in the immune responses of C. farreri (Xing et al.,2008;Lin et al.,2011). In this dissertation, PO of the scallop C. farreri was purified fromhemocytes and characterized biochemically and enzymatically, then the antibacterialactivity of C. farreri PO reaction products generated in vitro was characterized, finally,the difference among C. farreri PO and POs from Argopecten irradians, Ruditapesphilippinarum, Haliotis discus hannai and Scapharca subcrenata on biochemical andenzymatic characteristics were analyzed.
(I) Phenoloxidase in the scallop C. farreri: purification, biochemical andenzymatic characterization. PO of the scallop C. farreri was purified fromhemocytes using linear-gradient native PAGE combined with gel permeation columnchromatography, and characterized based on substrate specificity, kinetic parameters,and the effects of temperature, pH, divalent metal ions, and inhibitors on enzymaticactivity. The results show that purified PO had a molecular mass of576kDa in nativePAGE and53kDa in SDS-PAGE, suggesting that C. farreri PO is an aggregatecomposed of one subunit. The analysis of the kinetics indicated that the Kmvalues ofPO for L-DOPA, catechol, dopamine and hydroquinone were0.61mmol/L,0.20mmol/L,0.88mmol/L and0.83mmol/L, respectively, which suggests that C. farreriPO was a laccase-type phenoloxidase, showing the highest affinity for catechol, thelowest affinity for dopamine, and a higher affity for L-DOPA than hydroquinone. ThePO had optimal activities at30oC and a pH of8.0, and its activity was greatlyenhanced by Ca2+and Mg2+, and inhibited by Fe2+. In addition, the PO activity wasinhibited by sodium sulfite, ascorbic acid, sodium diethyldithiocarbamate (DETC),cysteine, citric acid, and ethylenediamine tetraacetic acid disodium (EDTA), which suggests that C. farreri PO was a copper-containing metalloenzyme.
(II) Antibacterial activity of C. farreri PO reaction products generated in vitro.Antibacterial activity of C. farreri PO reaction products against Vibrio alginolyticus, V.parahaemolyticus, Aeromonas salmonicida, Edwardsiella tarda, Streptococcusdysgadysgalactiae, Streptococcus iniae and Micrococcus lysodeikticus wascharacterized by observation and determination of bacterial growth after treatingbacteria with purified PO and substrates. The results show that the mixture of PO andsubstrates inhibited bacterial growth, while PO solely had no effect on bacterialgrowth, illustrating that it is PO reaction products not PO have the antibacterialactivity. L-DOPA-derived compounds only showed inhibition in A. salmonicida,while dopamine-derived compounds showed strong inhibitions in the Gram-negativeV. alginolyticus, V. parahaemolyticus and A. salmonicida, indicating thatdopamine-derived compounds are more cytotoxic than L-DOPA-derived compounds.No significant inhibition was found in Gram-positive S. dysgadysgalactiae, S. iniaeand M. lysodeikticus, suggesting that PO reaction compounds are more harmful to thecell components of Vibrio and Aeromonas than those of Streptococcus andMicrococcus. Besides, PO reaction products showed no inhibition in E. tarda, whichwas also Gram-negative, suggesting that the extracellular polysaccharides with strongantioxidant activities, produced by Edwardsiella, might inhibit the PO reaction.Microscopic observation showed that after incubation with PO and dopamine, V.alginolyticus, V. parahaemolyticus and A. salmonicida lacked transparency andmobility, and often appeared as aggregates covered with black pigments.
(III) Comparison of C. farreri PO and POs from A. irradians, R. philippinarum,H. discus hannai and S. subcrenata based on biochemical and enzymaticcharacteristics. POs of A. irradians, R. philippinarum, H. discus hannai and S.subcrenata were isolated from hemocytes using linear-gradient native-PAGEcombined with catechol staining, then C. farreri PO was compared and analyzed withthe isolated POs based on substrate specificity, kinetic parameters and the effects ofdivalent metal ions and inhibitors on enzymatic activity. The results of isolation showthat C. farreri was similar to A. irradians, R. philippinarum and H. discus hannai, in which only one PO was detected in hemocytes, and different from S. subcrenata, inwhich four POs were detected. In terms of molecular mass in native-PAGE, C. farreriPO (576kDa) was similar to POs in A.irradians (555kDa) and R. philippinarum(563kDa), and differed greatly from POs in H. discus hannai (228kDa) and S.subcrenata (391,206,174kDa and an unknown one, which were named as S391PO,S206PO, S174PO and SuPO respectively). As far as substrate specificity is concerned,C. farreri PO was similar to POs from A. irradians, R. philippinarum, H. discushannai and S. subcrenata, which could oxidize o-diphenols and p-dipehnols, werelaccase-type phenoloxidase. Kinetic analysis indicated that the Kmvalues of C. farreriPO for L-DOPA, catechol, dopamine and hydroquinone were more similar to that of A.irradians PO, suggesting that the catalytic capability is parallel between the two POs.Metal ions test showed that C. farreri PO and POs from A. irradians, R.philippinarum, H. discus hannai and S. subcrenata were all sensitive to divalent metalions, and all inhibited by Fe2+, both Ca2+-and Mg2+-mediated enhancement of POactivity were only found in C. farreri and A. irradians, and in general the results ofmetal ions test in C. farreri PO are very similar to that in A. irradians PO, whichsuggests that there might be very high similarity on protein structure between the twoPOs. Besides, the strong inhibition of EDTA and DETC indicating that C. farreri POand POs from A. irradians, R. philippinarum, H. discus hannai and S. subcrenatawere all copper-containing metalloenzyme, however, C. farreri PO was more similarto A. irradians PO, S206PO and SuPO, which were not inhibited by thiourea andsodium azide.
（1）栉孔扇贝酚氧化酶的分离纯化及生化和酶学特性研究。采用线性连续梯度非变性电泳（native-PAGE）结合凝胶过滤层析的方法从栉孔扇贝血细胞中分离纯化出酚氧化酶，研究了该酶的底物特异性、动力学参数以及温度、pH、二价金属离子和抑制剂对酶活的影响。结果显示：提纯的酚氧化酶在native-PAGE中分子量为576kDa，SDS-PAGE中分子量为53kDa，表明该酶是由一种亚基组成的寡聚体；酚氧化酶对L-DOPA、邻苯二酚、多巴胺和对苯二酚的米氏常数Km分别为0.61mmol/L、0.20mmol/L、0.88mmol/L和0.83mmol/L，表明该酶是一种漆酶型的酚氧化酶，其对邻苯二酚的亲和力最高，对多巴胺的亲和力最低，对L-DOPA的亲和力高于对苯二酚；酚氧化酶在30℃和pH8.0时具有最高酶活力；酚氧化酶活力受到Ca2+和Mg2+的强烈激活，但受到Fe2+的显著抑制；酚氧化酶活力不受硫代尿素和叠氮钠的影响，但受到抗坏血酸、亚硫酸钠、柠檬酸、半胱氨酸、二乙基二硫代氨基甲酸钠（DETC）、乙二胺四乙酸二钠（EDTA）的强烈抑制，表明该酶是一种含铜的金属酶。
（2）栉孔扇贝酚氧化酶体外氧化产物的抗菌活性研究。通过观察和测定提纯的栉孔扇贝酚氧化酶与底物处理后的细菌生长情况，研究了栉孔扇贝酚氧化酶体外氧化产物对溶藻胶弧菌Vibrio alginolyticus、副溶血弧菌Vibrioparahaemolyticus、杀鲑气单胞菌Aeromonas salmonicida、爱德华氏菌Edwardsiellatarda、停乳链球菌Streptococcus dysgadysgalactiae、海豚链球菌Streptococcus iniae和溶壁微球菌Micrococcus lysodeikticus的抗菌活性。细菌生长曲线测定结果显示：酚氧化酶自身不能抑制细菌生长，酚氧化酶与L-DOPA或多巴胺的混合物可抑制细菌生长，表明是酚氧化酶的氧化产物而不是酚氧化酶具有抗菌活性；使用L-DOPA作底物时，酚氧化酶氧化产物仅对杀鲑气单胞菌显示出明显的抑制作用，而使用多巴胺作为底物时，酚氧化酶氧化产物对革兰氏阴性的溶藻胶弧菌、副溶血弧菌和杀鲑气单胞菌均表现出强烈的抑制作用，表明以多巴胺为底物获得的酚氧化酶氧化产物比以L-DOPA为底物获得的氧化产物具有更强的细胞毒性；酚氧化酶的体外氧化产物对革兰氏阳性的停乳链球菌、海豚链球菌和溶壁微球菌无抑制作用，表明与链球菌和微球菌相比，栉孔扇贝酚氧化酶氧化产物对弧菌和气单胞菌细胞成分的毒性作用更大；酚氧化酶氧化产物对同为革兰氏阴性的爱德华氏菌无抑制作用，分析原因可能是爱德华氏菌分泌的胞外产物中含有一些具有抗氧化活力的多糖，这些多糖抑制了酚氧化酶的氧化反应。显微镜检测结果显示：经过与栉孔扇贝酚氧化酶和多巴胺的共孵育后，溶藻胶弧菌、副溶血弧菌和杀鲑气单胞菌的菌体透明度降低，多数菌体丧失了运动能力，细菌聚集成团，被黑色素所覆盖。
（3）栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍、毛蚶酚氧化酶的生化及酶学特性比较研究。采用线性连续梯度非变性电泳结合邻苯二酚发色方法从海湾扇贝、菲律宾蛤仔、皱纹盘鲍、毛蚶的血细胞中分离出酚氧化酶，分析比较了栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍、毛蚶酚氧化酶在底物特异性、动力学参数以及金属离子和抑制剂对酶活的影响上的差异。结果显示：在检测到的酚氧化酶种类上，栉孔扇贝与海湾扇贝、菲律宾蛤仔、皱纹盘鲍相同，在血细胞中仅检测到一种酚氧化酶，而从毛蚶的血细胞中则检测出4种酚氧化酶；在分子量上，栉孔扇贝酚氧化酶（576kDa）与海湾扇贝（555kDa）和菲律宾蛤仔（563kDa）中的酚氧化酶较为接近，而与皱纹盘鲍（228kDa）和毛蚶（391kDa、206kDa、174kDa、未知分子量，分别命名为S391PO、S206PO、S174PO和SuPO）中的酚氧化酶差异较大；在底物特异性上，栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍和毛蚶中的酚氧化酶相似，均可以氧化邻二酚类底物和对二酚类底物，为漆酶型的酚氧化酶；在动力学参数上，栉孔扇贝酚氧化酶对邻苯二酚、L-DOPA、对苯二酚、多巴胺4种底物的Km值与海湾扇贝酚氧化酶比较接近，表明这两种酶在催化能力上相近；在二价金属离子对酶活的影响上，栉孔扇贝与其他4种贝类的酚氧化酶的活力均对二价金属离子极为敏感，都受到Fe2+的显著抑制，但Ca2+和Mg2+都介导的酚氧化酶活力增强现象只在栉孔扇贝和海湾扇贝中存在，综合比较得出，栉孔扇贝酚氧化酶的二价金属离子影响结果与海湾扇贝酚氧化酶较为接近，表明这两种酚氧化酶在蛋白结构上可能具有很高的相似性；在抑制剂对酶活的影响上，栉孔扇贝酚氧化酶与海湾扇贝、菲律宾蛤仔、皱纹盘鲍和毛蚶中的酚氧化酶都受到EDTA和DETC的强烈抑制，均为含铜的金属酶，但栉孔扇贝酚氧化酶与海湾扇贝酚氧化酶以及毛蚶中的S206PO和SuPO较为相似，其活力均不受硫代尿素和叠氮钠的影响。
Previous studies showed that in the scallop Chlamys farreri, bacterialpolysaccharides challenge or viral infection caused a rapid and dramatic increase ofphenoloxidase (PO) activity in hemocytes, indicating that PO is one of the mostsensitive and effective factors in the immune responses of C. farreri (Xing et al.,2008;Lin et al.,2011). In this dissertation, PO of the scallop C. farreri was purified fromhemocytes and characterized biochemically and enzymatically, then the antibacterialactivity of C. farreri PO reaction products generated in vitro was characterized, finally,the difference among C. farreri PO and POs from Argopecten irradians, Ruditapesphilippinarum, Haliotis discus hannai and Scapharca subcrenata on biochemical andenzymatic characteristics were analyzed.
(I) Phenoloxidase in the scallop C. farreri: purification, biochemical andenzymatic characterization. PO of the scallop C. farreri was purified fromhemocytes using linear-gradient native PAGE combined with gel permeation columnchromatography, and characterized based on substrate specificity, kinetic parameters,and the effects of temperature, pH, divalent metal ions, and inhibitors on enzymaticactivity. The results show that purified PO had a molecular mass of576kDa in nativePAGE and53kDa in SDS-PAGE, suggesting that C. farreri PO is an aggregatecomposed of one subunit. The analysis of the kinetics indicated that the Kmvalues ofPO for L-DOPA, catechol, dopamine and hydroquinone were0.61mmol/L,0.20mmol/L,0.88mmol/L and0.83mmol/L, respectively, which suggests that C. farreriPO was a laccase-type phenoloxidase, showing the highest affinity for catechol, thelowest affinity for dopamine, and a higher affity for L-DOPA than hydroquinone. ThePO had optimal activities at30oC and a pH of8.0, and its activity was greatlyenhanced by Ca2+and Mg2+, and inhibited by Fe2+. In addition, the PO activity wasinhibited by sodium sulfite, ascorbic acid, sodium diethyldithiocarbamate (DETC),cysteine, citric acid, and ethylenediamine tetraacetic acid disodium (EDTA), which suggests that C. farreri PO was a copper-containing metalloenzyme.
(II) Antibacterial activity of C. farreri PO reaction products generated in vitro.Antibacterial activity of C. farreri PO reaction products against Vibrio alginolyticus, V.parahaemolyticus, Aeromonas salmonicida, Edwardsiella tarda, Streptococcusdysgadysgalactiae, Streptococcus iniae and Micrococcus lysodeikticus wascharacterized by observation and determination of bacterial growth after treatingbacteria with purified PO and substrates. The results show that the mixture of PO andsubstrates inhibited bacterial growth, while PO solely had no effect on bacterialgrowth, illustrating that it is PO reaction products not PO have the antibacterialactivity. L-DOPA-derived compounds only showed inhibition in A. salmonicida,while dopamine-derived compounds showed strong inhibitions in the Gram-negativeV. alginolyticus, V. parahaemolyticus and A. salmonicida, indicating thatdopamine-derived compounds are more cytotoxic than L-DOPA-derived compounds.No significant inhibition was found in Gram-positive S. dysgadysgalactiae, S. iniaeand M. lysodeikticus, suggesting that PO reaction compounds are more harmful to thecell components of Vibrio and Aeromonas than those of Streptococcus andMicrococcus. Besides, PO reaction products showed no inhibition in E. tarda, whichwas also Gram-negative, suggesting that the extracellular polysaccharides with strongantioxidant activities, produced by Edwardsiella, might inhibit the PO reaction.Microscopic observation showed that after incubation with PO and dopamine, V.alginolyticus, V. parahaemolyticus and A. salmonicida lacked transparency andmobility, and often appeared as aggregates covered with black pigments.
(III) Comparison of C. farreri PO and POs from A. irradians, R. philippinarum,H. discus hannai and S. subcrenata based on biochemical and enzymaticcharacteristics. POs of A. irradians, R. philippinarum, H. discus hannai and S.subcrenata were isolated from hemocytes using linear-gradient native-PAGEcombined with catechol staining, then C. farreri PO was compared and analyzed withthe isolated POs based on substrate specificity, kinetic parameters and the effects ofdivalent metal ions and inhibitors on enzymatic activity. The results of isolation showthat C. farreri was similar to A. irradians, R. philippinarum and H. discus hannai, in which only one PO was detected in hemocytes, and different from S. subcrenata, inwhich four POs were detected. In terms of molecular mass in native-PAGE, C. farreriPO (576kDa) was similar to POs in A.irradians (555kDa) and R. philippinarum(563kDa), and differed greatly from POs in H. discus hannai (228kDa) and S.subcrenata (391,206,174kDa and an unknown one, which were named as S391PO,S206PO, S174PO and SuPO respectively). As far as substrate specificity is concerned,C. farreri PO was similar to POs from A. irradians, R. philippinarum, H. discushannai and S. subcrenata, which could oxidize o-diphenols and p-dipehnols, werelaccase-type phenoloxidase. Kinetic analysis indicated that the Kmvalues of C. farreriPO for L-DOPA, catechol, dopamine and hydroquinone were more similar to that of A.irradians PO, suggesting that the catalytic capability is parallel between the two POs.Metal ions test showed that C. farreri PO and POs from A. irradians, R.philippinarum, H. discus hannai and S. subcrenata were all sensitive to divalent metalions, and all inhibited by Fe2+, both Ca2+-and Mg2+-mediated enhancement of POactivity were only found in C. farreri and A. irradians, and in general the results ofmetal ions test in C. farreri PO are very similar to that in A. irradians PO, whichsuggests that there might be very high similarity on protein structure between the twoPOs. Besides, the strong inhibition of EDTA and DETC indicating that C. farreri POand POs from A. irradians, R. philippinarum, H. discus hannai and S. subcrenatawere all copper-containing metalloenzyme, however, C. farreri PO was more similarto A. irradians PO, S206PO and SuPO, which were not inhibited by thiourea andsodium azide.
引文
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