胆红素氧化酶诱导产生、分离纯化及对染料降解的研究
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摘要
疣孢漆斑菌代谢产生的胞外胆红素氧化酶(EC.1.3.3.5)是一种含铜的多酚氧化酶,能氧化胆红素和多种漆酶底物,在环境保护领域有较大的应用潜力。本文对疣孢漆斑菌产胆红素氧化酶的发酵条件和胞外胆红素氧化酶的纯化及其对染料脱色等方面进行了研究。
探索疣孢漆斑菌的最佳培养条件为：发酵培养时间4天、培养温度26℃、培养基初始pH6.0、孢子液稀释度10-3、250 mL的三角瓶装100 mL发酵液、土豆汁浓度20%时菌体产酶最佳。采用单因素实验和正交实验相结合的方法,研究培养基中C源、N源、金属离子及诱导剂对疣孢漆斑菌产酶的影响。结果表明在产酶培养基中,葡萄糖和大豆蛋白胨为最适C源和N源,添加2 mmol/L铜离子和0.1 mmol/L的诱导剂均可以提高发酵液的酶活。正交实验得到的优化组合为：葡萄糖10g/L,大豆蛋白胨7.5 g/L,CuSO41 mmol/L,没食子酸0.1 mmol/L,优化后发酵液酶活提高了3倍。
经过硫酸铵盐析、透析、DEAE-Sepharose FF离子交换层析和Sephadex G-75凝胶过滤等一系列纯化步骤从350 mL粗酶液中得到2.43 mg纯化的胆红素氧化酶,最终纯化倍数为3.12,总活力回收率为27.92%。由SDS-PAGE电泳图片计算得出纯酶的分子量为68.1kDa, Native PAGE电泳图片是单一条带,所得的纯酶具有较高的纯度。Native PAGE电泳中ABTS染色和胆红素染色说明纯酶可以氧化两种底物,靛红染色表明纯酶可以有效地使染料靛红脱色。纯酶的N端氨基酸序列和飞行质谱(MALDI-TOF-MS)的结果与已报道疣孢漆斑菌的N端氨基酸序列完全一致,N端氨基酸序列为VAQISPQYPMFTVPL。
纯酶用胆红素为底物测得最适反应pH7.5,最适反应温度为40℃；以ABTS为底物最适pH为3.4；纯酶25℃时在pH7.5～8.0的缓冲液中保温1 h较稳定；最适pH下纯酶在40℃以下保温1 h酶活较稳定。纯化后的胆红素氧化酶能催化氧化胆红素和ABTS两种底物,但对胆红素的亲和性较好,氧化速度也更快。1 mmol/L半胱氨酸、10 mmol/L的NaN3和1mmol/L二硫疏糖醇对胆红素氧化酶活力有强烈的抑制。大多数金属离子对酶反应具有抑制作用或影响效果不明显,但铜离子可以提高酶活力。
共培养体系表明疣孢漆斑菌可以降解多种染料。疣孢漆斑菌对染料靛红的降解过程是生物吸附(菌体吸附)和生物降解(酶降解)共同作用的结果,液体培养7天靛红的脱色率达到90%以上。初步纯化的胆红素氧化酶可以在30℃～50℃、pH5.5～9.5和靛红浓度在50mg/L～200 mg/L的条件下有效地降解靛红。初步纯化的胆红素氧化酶在1000 U/L的酶活下只需50 min就可以降解90%以上100 mg/L的染料靛红,显示出胆红素氧化酶在染料废水处理中具有广阔的应用前景。
Bilirubin oxidase (EC.1.3.3.5) produced by Myrothecium verrucaria is a multicopper oxidase. It can catalyze the oxidation of biliverdin and several laccase substrates, showing a promising potential in environmental applications. This paper was focused on fermentation process and purification of bilirubin oxidase form M. verrucaria and decolorization of dyes.
The result showed that the optimal culture conditions were as follow: cultured for 4days, culture temperature was 26℃, broth initial pH was pH6.0, spores liquid dilution was 10-3,250 mL Erlenmeyer flasks contained 100 mL,20% liquid potato culture medium. The effects of carbon source, nitrogen source, metal ions and inducers on enzyme production of M. verrucaria were investigated using one factor method and orthogonal design. The results showed that glucose and soya peptone were the optimal carbon source and nitrogen source, respectively. A higher enzyme activity was achieved with the addition of 2 mmol/L Cu2+ and 0.1 mmol/L of inducers. The orthogonal design experiment indicated that the optimal medium was:glucose 10 g/L, soya peptone 7.5 g/L, CuSO4 1 mmol/L, gallic acid 0.1 mmol/L. The enzyme activity was enhanced 3 times over the initial values after the fermentation medium was optimized using orthogonal design.
Furthermore, the bilirubin oxidase was purified using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The purification fold was 3.12 and recovery of total bilirubin oxidase activity was 27.92%. The molecular mass of the enzyme was 68.1 kDa as determined by SDS-PAGE, and single protein band appeared on the Native PAGE gel identifying the enzyme had been purified to electrophoretic homogeneity. The Native PAGE stained with ABTS and bilirubin showed the purified bilirubin oxidase was able to catalyze the oxidation of ABTS and bilirubin. The Native PAGE gel which was stained with indigo carmine, it further demonstrated purified BOD indeed had ability to degrade indigo carmine. The purified enzyme was identified by N-terminal amino acid sequence and MALDI-TOF-MS, homologies of up 100% were found with bilirubin oxidase from M. verrucaria.
The optimum reaction pH of the purified enzyme for bilirubin was 7.5 and maximum enzyme activity was at 40℃. The optimum pH of the enzyme reaction for ABTS was approximate to pH 3.4. The enzyme activity showed slight decrease after incubation at 25℃for 1 h at pH 7.5～8.0; the enzyme activity was stable after incubation for 1 h at temperature up to 40℃. The purified enzyme can catalyze the oxidation of bilirubin and ABTS, it catalytic efficiency for bilirubin was also higher than for ABTS. In addition the enzyme activity was strongly inhibited by 1 mmol/L L-cysteine,10 mmol/L sodium azide and 1 mmol/L DTT. Bilirubin oxidase activity was decreased by the addition of most metal ions. However, only the copper ion made the enzyme activity increased remarkably.
Co-culture system showed that M. verrucaria can degrade a variety of dyes. The biosorption and biodegradation of the dye were detected in the process of decolorization, finally more than 90% decolorization efficiency was achieved by incubating for 7 d at 26℃. Additionally, The partial purified bilirubin oxidase can efficiently decolorize indigo carmine at 30℃～50℃, pH5.5～9.5 and dye concentration of 50 mg/L-200 mg/L. The partial purified bilirubin oxidase was able to decolorize more than 90% of indigo carmine (100 mg/L) in only 50 min with an enzymatic activity of 1000 U/L, suggesting a broad application prospect of bilirubin oxidase in dye effluents decolorization.
探索疣孢漆斑菌的最佳培养条件为：发酵培养时间4天、培养温度26℃、培养基初始pH6.0、孢子液稀释度10-3、250 mL的三角瓶装100 mL发酵液、土豆汁浓度20%时菌体产酶最佳。采用单因素实验和正交实验相结合的方法,研究培养基中C源、N源、金属离子及诱导剂对疣孢漆斑菌产酶的影响。结果表明在产酶培养基中,葡萄糖和大豆蛋白胨为最适C源和N源,添加2 mmol/L铜离子和0.1 mmol/L的诱导剂均可以提高发酵液的酶活。正交实验得到的优化组合为：葡萄糖10g/L,大豆蛋白胨7.5 g/L,CuSO41 mmol/L,没食子酸0.1 mmol/L,优化后发酵液酶活提高了3倍。
经过硫酸铵盐析、透析、DEAE-Sepharose FF离子交换层析和Sephadex G-75凝胶过滤等一系列纯化步骤从350 mL粗酶液中得到2.43 mg纯化的胆红素氧化酶,最终纯化倍数为3.12,总活力回收率为27.92%。由SDS-PAGE电泳图片计算得出纯酶的分子量为68.1kDa, Native PAGE电泳图片是单一条带,所得的纯酶具有较高的纯度。Native PAGE电泳中ABTS染色和胆红素染色说明纯酶可以氧化两种底物,靛红染色表明纯酶可以有效地使染料靛红脱色。纯酶的N端氨基酸序列和飞行质谱(MALDI-TOF-MS)的结果与已报道疣孢漆斑菌的N端氨基酸序列完全一致,N端氨基酸序列为VAQISPQYPMFTVPL。
纯酶用胆红素为底物测得最适反应pH7.5,最适反应温度为40℃；以ABTS为底物最适pH为3.4；纯酶25℃时在pH7.5～8.0的缓冲液中保温1 h较稳定；最适pH下纯酶在40℃以下保温1 h酶活较稳定。纯化后的胆红素氧化酶能催化氧化胆红素和ABTS两种底物,但对胆红素的亲和性较好,氧化速度也更快。1 mmol/L半胱氨酸、10 mmol/L的NaN3和1mmol/L二硫疏糖醇对胆红素氧化酶活力有强烈的抑制。大多数金属离子对酶反应具有抑制作用或影响效果不明显,但铜离子可以提高酶活力。
共培养体系表明疣孢漆斑菌可以降解多种染料。疣孢漆斑菌对染料靛红的降解过程是生物吸附(菌体吸附)和生物降解(酶降解)共同作用的结果,液体培养7天靛红的脱色率达到90%以上。初步纯化的胆红素氧化酶可以在30℃～50℃、pH5.5～9.5和靛红浓度在50mg/L～200 mg/L的条件下有效地降解靛红。初步纯化的胆红素氧化酶在1000 U/L的酶活下只需50 min就可以降解90%以上100 mg/L的染料靛红,显示出胆红素氧化酶在染料废水处理中具有广阔的应用前景。
Bilirubin oxidase (EC.1.3.3.5) produced by Myrothecium verrucaria is a multicopper oxidase. It can catalyze the oxidation of biliverdin and several laccase substrates, showing a promising potential in environmental applications. This paper was focused on fermentation process and purification of bilirubin oxidase form M. verrucaria and decolorization of dyes.
The result showed that the optimal culture conditions were as follow: cultured for 4days, culture temperature was 26℃, broth initial pH was pH6.0, spores liquid dilution was 10-3,250 mL Erlenmeyer flasks contained 100 mL,20% liquid potato culture medium. The effects of carbon source, nitrogen source, metal ions and inducers on enzyme production of M. verrucaria were investigated using one factor method and orthogonal design. The results showed that glucose and soya peptone were the optimal carbon source and nitrogen source, respectively. A higher enzyme activity was achieved with the addition of 2 mmol/L Cu2+ and 0.1 mmol/L of inducers. The orthogonal design experiment indicated that the optimal medium was:glucose 10 g/L, soya peptone 7.5 g/L, CuSO4 1 mmol/L, gallic acid 0.1 mmol/L. The enzyme activity was enhanced 3 times over the initial values after the fermentation medium was optimized using orthogonal design.
Furthermore, the bilirubin oxidase was purified using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The purification fold was 3.12 and recovery of total bilirubin oxidase activity was 27.92%. The molecular mass of the enzyme was 68.1 kDa as determined by SDS-PAGE, and single protein band appeared on the Native PAGE gel identifying the enzyme had been purified to electrophoretic homogeneity. The Native PAGE stained with ABTS and bilirubin showed the purified bilirubin oxidase was able to catalyze the oxidation of ABTS and bilirubin. The Native PAGE gel which was stained with indigo carmine, it further demonstrated purified BOD indeed had ability to degrade indigo carmine. The purified enzyme was identified by N-terminal amino acid sequence and MALDI-TOF-MS, homologies of up 100% were found with bilirubin oxidase from M. verrucaria.
The optimum reaction pH of the purified enzyme for bilirubin was 7.5 and maximum enzyme activity was at 40℃. The optimum pH of the enzyme reaction for ABTS was approximate to pH 3.4. The enzyme activity showed slight decrease after incubation at 25℃for 1 h at pH 7.5～8.0; the enzyme activity was stable after incubation for 1 h at temperature up to 40℃. The purified enzyme can catalyze the oxidation of bilirubin and ABTS, it catalytic efficiency for bilirubin was also higher than for ABTS. In addition the enzyme activity was strongly inhibited by 1 mmol/L L-cysteine,10 mmol/L sodium azide and 1 mmol/L DTT. Bilirubin oxidase activity was decreased by the addition of most metal ions. However, only the copper ion made the enzyme activity increased remarkably.
Co-culture system showed that M. verrucaria can degrade a variety of dyes. The biosorption and biodegradation of the dye were detected in the process of decolorization, finally more than 90% decolorization efficiency was achieved by incubating for 7 d at 26℃. Additionally, The partial purified bilirubin oxidase can efficiently decolorize indigo carmine at 30℃～50℃, pH5.5～9.5 and dye concentration of 50 mg/L-200 mg/L. The partial purified bilirubin oxidase was able to decolorize more than 90% of indigo carmine (100 mg/L) in only 50 min with an enzymatic activity of 1000 U/L, suggesting a broad application prospect of bilirubin oxidase in dye effluents decolorization.
引文
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[2]徐贵祥.一种潜在的治疗严量黄疽的药物胆红素氧化酶.国外医学药学分册,1991,18(3)：164～167
[3]曾容珍.新生儿黄疸防治研究新动态.国外医学妇幼保健分册,1996,7(1)：1～3
[4]李春元,李梓.新生儿高胆红素血症的治疗.制剂分册,2002：165～169
[5]Solomon EI., Sundaram UM, Machonkin TE. Multicopper oxidases and oxygenases. Chem. Rev.,1996,96:2563～2605
[6]宋克征.胆红素及检测方法研究的进展.国外医学临床生物化学与检验学分册,2002,23(5)：283～284
[7]杨帆,杨力.胆红素氧化酶法测定血清胆红素的方法学评价.当代医师杂志,1997,2(10)：23～24
[8]Hoegger PJ, Kilaru S, James TY, et al. Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBSJ,2006,273:2308～2326
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