基于脂肪酶活性状态下构象的差异性分析预测突变热点并构建能高效水解胆固醇酯的脂肪酶突变体

The cholesterol ester content in the TMP pulp is positively correlated with the content of pitch deposition. However, most the available commercial lipases don't display the hydrolysis activity to various cholesterol esters, which decreases the lipases' application effect to control pitch problems in the pulp and paper field. In this project, a lipase from Burkholderia sp. ZYB002 is to be redesigned to improve its' hydrolysis activity to cholesterol ester. The Burkholderia sp. ZYB002 lipase was developed by the project applicant group and displayed the excellent potential to control the pitch problem. The following experiments will be carried out, 1) The 3D structure of the lipase from Burkholderia sp. ZYB002 will be modeled by homologous alignment; 2) Various conformation in activated state will be modeled by MD, including the lipase' conformation on the substrate-water interface, the conformation of enzyme-substrate complex at transition state, et al.; 3) the difference will be analyzed among various conformations; 4) the potential mutation hotspots will be predicted; 5) the mutation gene library will be constructed and screened; The following prospective results could be attained once the project will be approved, 1) the lipase mutants with high-level hydrolysis activity to cholesterol ester could be selected from the mutation gene library; 2) the molecular mechanism of the lipase mutants with cholesterol esterase activity could be clarified; 3) the novel composite lipase displayed lipase activity and cholesterol esterase activity could be developed; 4) a novel technology applied in the pulp and paper field could be invented.

TMP纸浆中胆固醇酯含量的高低直接影响到纸浆树脂沉积量的多少。目前酶法控制TMP纸浆树脂障碍工艺中使用的商品化脂肪酶大都缺乏水解胆固醇酯的活性，降低了脂肪酶控制TMP纸浆树脂障碍的效果。本课题以拥有自主知识产权、在酶法控制TMP纸浆树脂障碍工艺中具有良好应用前景的Burkholderia sp.ZYB002脂肪酶作为原始出发材料，通过同源建模和分子动力学模拟技术，分别构建脂肪酶水解三酰甘油酯及胆固醇酯时，在界面激活及形成酶-底物过渡态复合物过程中，脂肪酶分子在活性状态下的各种构象；比较各种构象之间的差异性，预测影响脂肪酶水解胆固醇酯活性的关键氨基酸残基（突变热点）。构建针对突变热点的小型紧凑型突变基因文库，快速筛选出能高效水解胆固醇酯的脂肪酶突变体；阐明脂肪酶突变体高效水解胆固醇酯的分子机制；制备出少量脂肪酶突变体酶制剂，并与野生型脂肪酶复配，优化复合脂肪酶控制TMP纸浆树脂障碍工艺。

TMP纸浆树脂是一种包含胆固醇酯在内的复杂混合物。纸浆树脂的沉积是影响纸机运行效率和纸张质量的重要因素。酶法控制纸浆树脂障碍是一种绿色的生产加工工艺，具有节能环保等优点。开发满足该生产工艺需要的酶制剂具有重要的经济意义和市场价值。针对该工艺的生产特点，本项目进行了下列研究工作：..(1) 分析测试了Burkholderia cepacia脂肪酶LipA(BCL)、Bacillus subtilis脂肪酶LipA（BSL）、Ophiostoma piceae胆固醇酯酶(OPE)和Mycobacterium smegmatis的酰基转移酶（MsAcT）等降解树脂模拟底物的能力，发现：BSL和BCL可以高效水解聚ε-己内酯；而OPE可以高效水解poly(ethyl acrylate)和polyvinyl acetate。..(2) 成功构建了酯解酶酶活提高五倍多的BCL突变体（LipA-Tyr175Phe）；热稳定性显著性提高的BCL突变体（LipA-F221D/N125E, T5012提高了11.1℃，t1/2提高了约50.57倍)；构建了脂肪酶LipA可溶性表达提高了70.3倍的重组菌株。..(3)制备出催化效率显著提高的固定化酶制剂。..(4)开发出高效控油的酶法-原位化学氧化偶联工艺，不仅可有效控制纸浆树脂障碍，并同步实现对纸浆的漂白和对纸浆纤维的修饰。..上述酶法控制纸浆树脂障碍工艺用酶及其工艺的开发，有助于酶法生产工艺的推广。