化工学报
化工學報
화공학보
CIESC Jorunal
2015年
9期
3669-3677
,共9页
王小艳%樊艳爽%韩蓓佳%冯旭东%李春
王小豔%樊豔爽%韓蓓佳%馮旭東%李春
왕소염%번염상%한배가%풍욱동%리춘
N-糖基化%β-葡萄糖醛酸苷酶%热稳定性%分子模拟%动力学
N-糖基化%β-葡萄糖醛痠苷酶%熱穩定性%分子模擬%動力學
N-당기화%β-포도당철산감매%열은정성%분자모의%동역학
N-glycosylation%β-glucuronidase%thermostability%molecular simulation%kinetics
以 N-糖基化提高毕赤酵母重组表达 β-葡萄糖醛酸苷酶(PGUS-P)的热稳定性为目的,在 PGUS-P 模拟结构分析的基础上,半理性方法设计并通过定点突变引入具有 EAS(enhanced aromatic sequence)序列特征的新 N-糖基化位点,经毕赤酵母重组表达后,获得了 3 个新糖基化的突变酶 PGUS-P-26、PGUS-P-35 和 PGUS-P-259.反应动力学分析表明,与原始 PGUS-P 相比,突变酶 PGUS-P-26、PGUS-P-35 和 PGUS-P-259 催化甘草酸水解的Km 变小,Kcat/Km 增加,表明其对底物甘草酸的亲和力和催化效率均得到提高.热稳定性分析表明,PGUS-P-35和 PGUS-P-259 的热稳定性得到改善,在 65℃下保温 90 min,其热稳定性相对 PGUS-P 分别提高了 13%和 11%.研究表明在蛋白质的合适位点引入糖基修饰对提高蛋白的热稳定性具有促进作用.
以 N-糖基化提高畢赤酵母重組錶達 β-葡萄糖醛痠苷酶(PGUS-P)的熱穩定性為目的,在 PGUS-P 模擬結構分析的基礎上,半理性方法設計併通過定點突變引入具有 EAS(enhanced aromatic sequence)序列特徵的新 N-糖基化位點,經畢赤酵母重組錶達後,穫得瞭 3 箇新糖基化的突變酶 PGUS-P-26、PGUS-P-35 和 PGUS-P-259.反應動力學分析錶明,與原始 PGUS-P 相比,突變酶 PGUS-P-26、PGUS-P-35 和 PGUS-P-259 催化甘草痠水解的Km 變小,Kcat/Km 增加,錶明其對底物甘草痠的親和力和催化效率均得到提高.熱穩定性分析錶明,PGUS-P-35和 PGUS-P-259 的熱穩定性得到改善,在 65℃下保溫 90 min,其熱穩定性相對 PGUS-P 分彆提高瞭 13%和 11%.研究錶明在蛋白質的閤適位點引入糖基脩飾對提高蛋白的熱穩定性具有促進作用.
이 N-당기화제고필적효모중조표체 β-포도당철산감매(PGUS-P)적열은정성위목적,재 PGUS-P 모의결구분석적기출상,반이성방법설계병통과정점돌변인입구유 EAS(enhanced aromatic sequence)서렬특정적신 N-당기화위점,경필적효모중조표체후,획득료 3 개신당기화적돌변매 PGUS-P-26、PGUS-P-35 화 PGUS-P-259.반응동역학분석표명,여원시 PGUS-P 상비,돌변매 PGUS-P-26、PGUS-P-35 화 PGUS-P-259 최화감초산수해적Km 변소,Kcat/Km 증가,표명기대저물감초산적친화력화최화효솔균득도제고.열은정성분석표명,PGUS-P-35화 PGUS-P-259 적열은정성득도개선,재 65℃하보온 90 min,기열은정성상대 PGUS-P 분별제고료 13%화 11%.연구표명재단백질적합괄위점인입당기수식대제고단백적열은정성구유촉진작용.
To improve the thermostability of recombinant β-glucuronidase expressed in Pichia pastoris (PGUS-P) by N-glycosylation, new N-glycosylation sites were semi-rationally designed according to the simulated structure of PGUS-P. Three new N-glycosylation sites with EAS (enhanced aromatic sequence) were introduced by site-specific mutagenesis. After expression in Pichia pastoris, three mutant enzymes with new N-glycosylation were obtained, named as PGUS-P-26, PGUS-P-35 and PGUS-P-259. The kinetic analysis indicated that Vmax of PGUS-P-35 was improved from 111.25 μmol·(L·min)?1 to 120.48 μmol·(L·min)?1 and all of the three mutant enzymes showed a greater affinity and catalytic efficiency towards substrate glycyrrhizin compared to PGUS-P. The thermostability of PGUS-P-35 and PGUS-P-259 at 65℃ increased by 13% and 11% compared with that of PGUS-P, respectively. This study demonstrated that the introduction of N-glycosylation at the suitable region of enzyme could increase its thermostability.
