聚丙烯酰胺在石油开采、水处理等领域有广泛应用，被称为“百业助剂”；其单体丙烯酰胺的合成生产也随之得到关注。微生物腈水合酶生物催化法凭借底物转化率高、环境友好等优势，已成为我国丙烯酰胺的主流生产工艺。然而，反应中的强放热效应和强极性有机溶剂产物却易促使腈水合酶失活，同步提升腈水合酶的活性和稳定性是亟需解决的实际工业问题。 本论文致力于利用红色红球菌（Rhodococcus ruber）中的分子伴侣实现腈水合酶活性和稳定性的同步提升。作为工业上丙烯酰胺的生产菌株，红球菌具有良好的有机溶剂稳定性。通过对其进行基因组和转录组分析，挖掘到GroES和GroEL2两种分子伴侣作为主要研究对象，同时以文献报道的耐溶剂分子伴侣Methanocaldococcus jannaschii rTHS 为参考对照。 通过分子伴侣的胞外热激实验（70℃/90℃），GroEL2体现出优秀的自身热稳定性和对其他蛋白的热保护性，效果与对照分子伴侣rTHS基本相当。进一步地，以GroEL2分子伴侣为代表，进行腈水合酶NHase与GroEL2在大肠杆菌中的共表达研究。采用同一启动子独立表达或各自启动子独立表达方案，GroEL2不能正常表达，且腈水合酶酶活损失严重。但将GroEL2与腈水合酶的α亚基C末端融合表达后，融合酶NHase-GroEL2的酶活较对照NHase提高63.6%。50℃热激15 min后，融合酶的表观酶活是对照的3.9倍；10% (v/v) 丙烯酰胺浸泡20 min后，融合酶的表观酶活是对照的2.1倍。融合表达方案被证明是本体系中最有效的共表达方案。 在此基础上，以同样的方式将各分子伴侣（GroES、GroEL2和rTHS）与天然腈水合酶NHase、蛋白质工程改造酶SB167、SB167-133-410和SB167-23-307在大肠杆菌中融合表达。其中， NHase-GroEL2仍具有最高的酶活和相对较好的稳定性，成为目前本课题组构建的、性能最佳的腈水合酶表达大肠杆菌。通过对NHase-分子伴侣融合大肠杆菌进行表观酶学参数和热失活常数的测定，GroEL2再次体现了其提升酶活性和稳定性的明显优势。 在光滑型红球菌R. ruber S-TH3中，通过强启动子Pami-150的调控实现了融合酶NHase-GroEL2的质粒表达。30% (v/v) 丙烯酰胺浸泡20 min后，改造后红球菌的酶活保留比例较对照提高了15.6%，体现了一定的工业应用前景。
Polyacryamide （PAM） is widely used in many fields such as enhanced oil recovery and waste water treatment. Therefore, the production of acrylamide （AM）, the monomer of PAM, is getting more and more attention. Due to the high conversion rate and the eco-friendly characteristic, the biocatalysis by nitrile hydratase has become the dominant AM production technology in China. However, the highly exothermic reaction and strong polar organic solvent product tend to accelerate the deactivation of nitrile hydratase in pratical production. Co-enhancing nitrile hydratase’s activity and stability is the urgent problem to be solved. This study tried to use moleculer chaperone from Rhodococcus ruber to achieve this goal. As the strain used in actual production, R. ruber shows pretty good AM tolerance. Hence, the genome and transcriptome analyses of R. ruber were performed, through which GroES and GroEL2 were identified. Methanocaldococcus jannaschii rTHS, a recommened chaperone in literature, was also used as the reference. Heat shock experiments in vitro （70℃/90℃） revealed that GroEL2 had outstanding self-thermostability and thermo-stabilization effect on other proteins, similar with rTHS. Further, as the representative of three chaperones, GroEL2 was co-expressed with nitrile hydratase NHase in E.coli. The monocistronic expression method by one promoter or the bicistronic expression method by separate promoters both seriously repaired NHase activity. When GroEL2 was fused to the C-terminal of NHase α subunite, the activity of NHase-GroEL2 chimera was increased by 63.6% compared with initial NHase, and the residual activity after heat shock （50℃, 15 min） and AM immersion （10% v/v, 20 min） was also increased by 2.9 and 1.1 folds, respectively. Fusion expression was proved to be the most effective strategy in this study. Based on these findings, three chaperones studied in this research （GroES, GroEL2 and rTHS） were also fused with original NHase, engineered nitrile hydratase SB167, SB167-133-410 and SB167-243-307 in the same way. NHase-GroEL2 still highlighted itself for the highest activity and relatively good stability, thus becoming the most well-performed nitrile hydratase expressing E.coli in this lab. The comparion of apparent enzymatic parameters and heat inactivation constants of NHase-chaperone chimeras proved GroEL2’s advantage on enhancing enzyme activity and stability again. Chimera NHase-GroEL2 was then introduced to smooth type R.ruber S-TH3 by plasmid pNV18.1, and expressed under the regulation of strong promoter Pami-150. Compared to the control, recombinant strain R.ruber S-TH3/Pami-150-NHase-GroEL2 showed 15.6% higher residual activity ratio after AM immersion （30% v/v, 20 min）, which demonstrated its potential in future industrial application.
