کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9602417 | 1156 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals
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کلمات کلیدی
FMNBM3P450NADPHflavin adenine dinucleotideCyPSDSNADP+ - NADP +CPR - احیای قلبی ریویFAD - بدEnzyme immobilization - بی حرکت سازی آنزیمDirected evolution - تکامل هدایتBiotransformation - زیستدگرگونیsodium dodecyl sulphate - سدیم دودسیل سولفاتCytochrome P450 - سیتوکروم پی۴۵۰Substrate recognition - شناسایی بسترflavin mononucleotide - فلاون مونونوکلئوتیدBMP - مدیریت فرایند کسب و کار
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals](/preview/png/9602417.png)
چکیده انگلیسی
The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and É-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the β2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 °C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 °C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biomolecular Engineering - Volume 22, Issues 1â3, June 2005, Pages 81-88
Journal: Biomolecular Engineering - Volume 22, Issues 1â3, June 2005, Pages 81-88
نویسندگان
Irene Axarli, Ariadne Prigipaki, Nikolaos E. Labrou,