کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7560863 | 1491448 | 2013 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
MALDI-TOFPALDTNBSteric exclusionMatrix assisted laser desorption/ionization time of flight5,5′-dithiobis-(2-nitrobenzoic acid) - 5،5'-dithiobis- (2-nitrobenzoic acid)Site-directed mutagenesis - mutagenesis مواجه با سایتChemical modification - اصلاح شیمیاییStructural rigidity - سختی سازهLipase - لیپازwide type - نوع گسترده ایMolecular dynamic - پویایی مولکولی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5â²-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (dep) of l-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of l-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1834, Issue 12, December 2013, Pages 2494-2501
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1834, Issue 12, December 2013, Pages 2494-2501
نویسندگان
Chen Hui, Wu Jianping, Yang Lirong, Xu Gang,