利用同源片段交换提高腈水合酶的热稳定性  

Enhancement of thermo-stability of nitrile hydratase by homologous-fragment swapping

作　　者：房月芹 崔文璟[2] 崔幼恬[2] 陈艳[3] 周哲敏[2] 

机构地区：[1]江南大学环境与土木工程学院,江苏无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [3]吉林大学军需科技学院,吉林长春130062

出　　处：《中国生物制品学杂志》2015年第3期276-280,284,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助(31070711)

摘　　要：目的通过计算机辅助半理性设计对热不稳定的恶臭假单胞菌Psedomonas putida NRRL-18668腈水合酶（EC4.2.1.84,nitrile hydratase,简称NHase）分子进行改造,提高酶催化的热稳定性和活性。方法以嗜热假诺卡菌Pseudonocardia thermophila JCM3095和睾丸酮丛毛单胞菌Comamonas testosterone 5-MGAM-4DNHase作为模板,利用位点靶向氨基酸重组（site-targeted amino acid recombination,STAR）软件和分子动力学模拟分析选择合适的同源靶片段,通过同源交换替换Pseudomonas putida NRRL-18668NHase相应的片段,构建7株分别含有3种不同来源片段的新型杂合NHase,检测其热稳定性及活性;圆二色光谱分析野生酶和杂合酶3AB的二级结构。结果 7株杂合NHase（1A、2B、2C、2BC、3AB、3AC、3ABC）经50℃热处理10 min后,与野生型NHase相比,热稳定性分别提高了1.5~3.5倍,其中杂合NHase 3AB的热稳定性提高了3.5倍,酶活力提高了1.4倍;野生酶和杂合酶3AB二级结构中α螺旋含量分别为（34.56±3.21）%和（36.88±1.41）%,,β折叠含量分别为（19.78±3.21）%和（18.69±1.74）%。结论通过利用热稳定性的同源片段来替换相应的热不稳定结构域这种理性设计的方法,成功将不耐热的NHase改造成耐热杂合NHase,同时提高了酶的比活力,并能维持NHase分子的基本二级结构不改变。Objective To improve the thermo-stability and catalytic activity of thermo-sensitive nitrile hydratase（NHase,EC 4. 2. 1. 84）of Psedomonas putida NRRL-18668 by molecular modification using computer-aided semi-rational design.Methods Homologous target fragments were screened by site-targeted amino acid recombination（STAR） software and molecular dynamic modeling using the NHases from Pseudonocardia thermophila JCM3095 and Comamonas testosterone 5-MGAM-4D as templates respectively. Seven chimeric NHases were generated by homologous fragment swapping on the corresponding fragment of NHase from Psedomonas putida NRRL-18668, and determined for thermo-stability and activity.The secondary structures of wild-type NHase and chimeric NHase 3AB were analyzed by circular dicroism（CD）spectrometry. Results Compared with that of wild-type NHase, the thermo-stabilities of seven chimeric NHases（1A, 2B,2C, 2BC, 3AB, 3AC and 3ABC） after treatment at 50 ℃ for 10 min increased by 1. 5 ~ 3. 5 folds. The thermo-stability and activity of NHase 3AB increased by 3. 5 and 1. 4 folds respectively. The α-helixe contents in secondary structures of wild-type NHase and chimeric NHase 3AB were（34. 56 ± 3. 21）% and（36. 88 ± 1. 41）%, while the β-sheet contents were（19. 78 ± 2. 14）% and（18. 69 ± 1. 74）%, respectively. Conclusion The thermo-sensitive NHase was rationally engineered to sets of thermo-stable chimeric enzyme using homologous fragment swapping by substituting themo-sensitive domains with homologous thermo-stable domain, of which the specific activity increased without alteration in secondary structure.

关 键 词：腈水合酶 稳定性 杂合酶 同源片段 分子改造 

分 类 号：Q559[生物学—生物化学]