印染厂活性污泥宏基因组文库中脱色酶的克隆及其脱色机理的研究

Textile waste water is really intractable because of its high chromaticity, high chemical oxygen demand and the poor biodegradability. Biological treatments are frequently used to treat the textile waste water, which the decolorizing enzymes degrade chromophore to reduce the chromaticity of textile waste water and improve its biodegradability. Screening of high-performance decolorizing microorganisms and decolorizing enzymes is critical to the biological treatment of textile waste water, therefore, metagenomics approach was applied in this study and a metagenomic library of textile active sludge was constructed previously to investigate its genetic resource of decolorizing enzymes. And six active malachite green decolorizing clones were isolated by the function-driven screening. In this study, these malachite green decolorizing clones will be further identified the decolorizing enzymes and investigated their decolorizing activities, enzymatic characteristics and elucidated their decolorizing mechanism, by applying the molecular cloning, bioinformatics analysis, protein expression, purification, and biochemical assay integrated. Metagenomics-based screening of decolorizing genetic resource applied in this study will effectively mining the textile active sludge environmental DNA which including a vast of uncultured microorganism, and identify numerous novel decolorizing enzymes for the biological treatment of textile waste water.

印染废水的色度高、化学需氧量大、是一类较难处理的工业废水。目前主要应用低成本且环境友好的生物法处理含有高浓度染料的印染废水，通过脱色酶特异降解染料的发色基团，显著降低印染废水的色度进而改善其可生化处理性；而生物法处理印染废水的核心是高效脱色菌或脱色酶的筛选。为了深入挖掘印染厂活性淤泥环境DNA（包括大量的未培养微生物DNA）中的脱色酶基因资源，申请人前期以浙江某印染厂污水处理系统中的活性淤泥为研究材料构建了宏基因组文库，并基于活性筛选策略得到6个孔雀石绿阳性脱色克隆。本项目基于这些孔雀石绿脱色克隆，综合运用分子克隆、生物信息学、蛋白表达纯化、体外酶学等技术手段，进一步克隆相应的脱色酶基因并对其脱色活性、酶学特征及脱色的分子机理进行深入研究。本项目基于宏基因组技术对活性污泥中大量未培养微生物资源进行挖掘，将有效地拓展新的脱色酶基因资源，为生物法处理印染废水提供更多高效的脱色酶样本。

印染废水中的染料因其对环境和人体健康的潜在威胁而备受关注。近年来，生物脱色法处理染料污染物展现出了广阔的前景；在此背景下，本研究利用印染工厂污水处理系统的活性污泥构建了宏基因组文库，从中挖掘功能性脱色酶。获得了6个不同文库粘粒能使三苯基甲烷类染料孔雀石绿(MG)脱色，经基因亚克隆和测序证明这些不同文库粘粒中都保守存在着相同的脱色酶基因，说明该基因在染料废水处理系统中广泛存在，预示其可能存在不同菌株中水平转移的情况。该脱色酶基因编码一个膜转运蛋白，生物信息学分析表明该蛋白属于小型多药抗性蛋白家族(SMR family)，将其命名为tmt基因(triphenylmethane transporter gene)。为研究tmt的功能，将其克隆到大肠杆菌中进行表达，结果表明tmt能够明显提高宿主对孔雀石绿的脱色率，同时还能使宿主对结晶紫、孔雀石绿、亮绿等三苯甲烷染料的耐受性提高4-8倍，对环丙沙星、美罗培南和利福平等抗生素的抗性提高2倍。对Tmt中8个保守氨基酸位点进行了定点突变，研究不同突变株对染料脱色及包括染料在内的小分子物质耐受性的影响，结果表明，大部分位点的突变显著降低了宿主对三苯甲烷类染料及其它小分子物质的耐受性，但V51、A75和V100位点的突变会增强对结晶紫、孔雀石绿或美罗培南的耐受。当添加离子泵抑制剂奥美拉唑时会显著抑制Tmt对小分子物质的转运活性，使宿主对这些小分子的抗性恢复到对照水平。上述结果表明Tmt是一种外排蛋白，与三苯基甲烷染料及众多环境中残留抗生素的耐受性显著相关，该蛋白能够有效增强宿主对极端环境如印染污水处理系统环境的适应能力，从而帮助对印染污水的生物法处理。本研究还揭示了与染料分子转运密切相关的几个关键氨基酸位点，为后续对该蛋白的进一步改造及应用创造了条件。