煤炭是一种不可再生的碳氢资源，其组成结构复杂，对其直接进行能源利用不仅对环境造成严重污染，使人类健康受到极大危害，而且造成这种资源的极大浪费。因此，基于煤碳氢结构特点的高效转化利用技术开发具有十分重要现实意义。煤的生物转化是煤炭高效转化的重要途径之一。但是传统的煤炭生物转化存在产物难分离，转化周期过长的缺点。因此，本论文将基因工程技术和磁性固定化酶技术相结合用于煤的生物转化研究，以实现煤的高效转化，提高溶煤产物分离效率，同时实现溶煤酶的重复利用，降低转化成本的根本目标。 采用共沉淀法合成了Fe3O4，用Stöber法合成Fe3O4@SiO2粒子，利用硅烷化偶联试剂在磁性粒子表面接枝螯合配体（IDA），进一步与Ni2+螯合，从而得到表面富含Ni2+的磁性固定化酶载体。采用TEM、XRD、FTIR对其进行了结构表征。结果表明：所制备的磁性载体具有以Fe3O4为核的核壳结构，载体表面被SiO包覆后成功接枝IDA；VSM分析表明，载体具有超顺磁性能，饱和磁化强度为18.09 emu/mg；用UV-Vis对表面官能团定量分析，探讨不同合成条件对表面环氧基和IDA含量的影响，结果表明，IDA功能化的磁性载体受pH、反应时间和IDA的用量影响较大，在pH=7.3，浓度为8 mg/mL的IDA溶液中反应12 h可制得表面IDA含量为180.33 μmol/mg的磁性载体Fe3O4@SiO2@GPTMS-IDA。 利用基因工程技术在毕赤酵母GS115中表达获得His标签融合的木质素过氧化物酶（Lip），利用Ni2+与His标签的亲和结合原理，制备基于His-IDA亲和结合的磁性固定化木质素酶。采用DNA测序、PCR、琼脂糖凝胶电泳和SDS-PAGE电泳等方法进行了分析。结果表明：利用pPIC ZαA质粒成功构建含有His标签的pPIC ZαA-LipH8重组质粒，测序表明目的基因序列与数据库一致，没有发生突变；电泳分析表明成功表达了目的基因，获得带有His标签的木质素过氧化物酶；IDA功能化的磁性载体可以与带有His标签的木质素过氧化物酶特异性结合，成功制备基于IDA-His亲和的磁性固定化酶。 用固定化酶对神府煤进行转化，探讨不同因素对酶促反应的影响。结果表明，该固定化酶与神府煤的反应受H2O2浓度、pH、温度、酶用量、煤的氧化程度、反应时间的影响较大，该反应进行2天即可将神府煤转化18.2%；固定化酶与神府煤作用3次后其活性仍保持原来的82.8%，可以实现溶煤酶在煤炭生物转化中的重复利用。对产物进行了FTIR和UV-Vis分析，推测产物结构主要为被-C=O、-CH3、-OH、-OCH3、Ar-OOCCH3等基团取代的芳香类化合物。

Coal is a non-renewable hydrocarbon resource whose composition is complex, and its direct energy use not only causes serious pollution, but also influence human health and causes a great waste of resources. The development of coal efficient transformation and utilization technology has great significance. Coal biotransformation is one of the important ways to efficiently convert coal. However, the traditional coal biotransformation is difficult to separate products, and the transformation period is too long. Therefore, this paper combines genetic engineering and magnetic immobilized enzyme technology to study biotransformation of coal to achieve efficient conversion of coal, and developed to simplify separation of coal products, while achieving reuse of coal-soluble enzymes and reducing the conversion cost. Fe3O4 was prepared by coprecipitation method, and Fe3O4@SiO2 particles were synthesized by Stöber method. The magnetic particles was modified by silanization coupling reagent to graft chelating ligand (IDA) on the surface, and further chelated with Ni2+ to obtain Ni2+ -based magnetic immobilized enzyme carrier. The results of TEM, XRD and FTIR show that the prepared magnetic carrier has a core-shell structure with Fe3O4 as the nucleus. The surface of the carrier is covered by SiO ,and IDA is successfully grafted. The VSM analysis showed that the carrier had superparamagnetic properties and the saturation magnetization was 18.09 emu/mg. The surface functional groups were quantitatively analyzed by UV-Vis to investigate the effects of different synthesis conditions on surface epoxy and IDA contents, The results show that IDA functionalized magnetic carrier is influenced by pH, reaction time and IDA content. The magnetic carrier Fe3O4@SiO2@GPTMS-IDA, whose surface IDA content is of 180.33 μmol / mg, can be prepared by reacting for 12 h in pH 7.3 and 8 mg/mL IDA. His tagged lignin peroxidase was obtained using genetic engineering technique, and expressed in Pichia pastoris GS115. The immobilized Lignin peroxidase (Lip) based on His-IDA affinity was prepared based on Ni2+ and His tag affinity binding. The results show that: recombinant plasmid pPIC ZαA-LipH8 containing the His tag was successfully constructed. SDS-PAGE results showed that the target gene was successfully expressed and the His tagged lignin peroxidase was obtained. IDA functionalized magnetic carrier could specifically bind to lignin peroxidase with His tag, so magnetic immobilized enzyme was successfully prepared. The immobilized enzyme was used to transform Shenfu coal and explore the effect of different factors on enzymatic reaction. The results showed that immobilized enzyme biotransforming Shenfu coal reaction was influenced by H2O2 concentration, pH, temperature, amount of enzyme, degree of coal oxidation and reaction time. The reaction was carried out for 2 days, and transformation rate was 18.2% The im5mobilized enzyme remained 82.8% activity after three times action, and could realize the reuse in coal biotransformation. FTIR and UV-Vis analysis suggest that the product structure mainly contain -C=O, -CH3, -OH, -OCH3, Ar-OOCCH3 and other groups to replace the aromatic compounds.