کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
4752734 | 1416366 | 2017 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution
ترجمه فارسی عنوان
یک سیستم جریان مجهول برای اندازه گیری سینتیک آنزیمی ناهمگن با تفکیک زمان دوم
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
سینتیک حالت پیش قدم، مرحله فوران، سینتیک گذرا، بستر نامحلول سلبیو هیدرولاز، اندوگلیکوناز،
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
چکیده انگلیسی
Even though many enzyme processes occur at the interface of an insoluble substrate, these reactions are generally much less studied than homogenous enzyme reactions in the aqueous bulk. Interfacial (or heterogeneous) enzyme reactions involve several reaction steps, and the established experimental approach to elucidate multi-step reactions is transient (or pre steady-state) kinetics. A key requirement for pre steady-state measurements is good time resolution, and while this has been amply achieved in different commercial instruments, they are generally not applicable to precipitating suspensions of insoluble substrate. Perhaps for this reason, transient kinetics has rarely been reported for heterogeneous enzyme reactions. Here, we describe a quenched-flow system using peristaltic pumps and stirred substrate suspensions with a dead time below 100Â ms. The general performance was verified by alkali catalyzed hydrolysis of 2,4-dinitrophenyl acetate (DNPA), and the applicability to heterogeneous reactions was documented by two cellulases (Cel7A and Cel7B) acting on suspensions of microcrystalline cellulose (Avicel) at different loads up to 15Â g/l. The results showed distinctive differences between the two enzymes. In particular, we found that endo-lytic Cel7B combined very quickly with the substrate and reached the maximal activity within the dead-time of the instrument. Conversely, exo-lytic Cel7A showed a much slower initiation with maximal activity after 5-8Â s and a 10-fold lower turnover. We suggest that the instrument may provide an important tool in attempts to elucidate the mechanism of cellulases and other enzymes' action on insoluble substrate.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Enzyme and Microbial Technology - Volume 105, October 2017, Pages 45-50
Journal: Enzyme and Microbial Technology - Volume 105, October 2017, Pages 45-50
نویسندگان
Johan P. Olsen, Jeppe Kari, Kim Borch, Peter Westh,