کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
16802 | 42613 | 2016 | 9 صفحه PDF | دانلود رایگان |

• Aflatoxin-detoxifizym (ADTZ) has been successfully modified to get trypsin-resistance using a strategy in the rational molecular design based on computational chemistry method and enzymatic reaction theory.
• To overcome the challenge of a large amount of computation involved in the modification of ADTZ due to its big amount of sensitive sites (72 lys/arg residues) to trypsin coupled with taking “induced fit effect” into account, the strategy reported here is functional.
• The strategy of rational design includes: the calculations of the solvent accessibility of all the lys/arg residue sites which has been divided into external and internal sites. With performing the constrained protein–protein docking configuration, the result structure of trypsin-ADTZ would show the hot sites of lys/arg residue of ADTZ which was more exposure to trypsin. To limit the amount of mutations the opinions of that an idea that a mutant should maintain a similar structure with the wild type has been insisted.
The resistance of feed enzymes against proteases is crucial in livestock farming. In this study, the trypsin resistance of aflatoxin-detoxifizyme (ADTZ) is improved. ADTZ possesses 72 lys/arg residue sites, 45 of which are scattered on the outermost layers of the molecule (RSA ≧ 25%). These 45 lys/arg sites could be target sites for trypsin hydrolysis. By considering shape-matching (including physical and secondary bond interactions) and the “induced fit-effect”, we hypothesized that some of these lys/arg sites are vulnerable to trypsin. A protein–protein docking simulation method was used to avoid the massive computational requirements and to address the intricacy of selecting candidate sites, as candidate site selection is affected by space displacement. Optimal mutants (K244Q/K213C/K270T and R356E/K357T/R623C) were predicted by computational design with protein folding energy analysis and molecular dynamics simulations. A trypsin digestion assay was performed, and the mutants displayed much higher stability against trypsin hydrolysis compared to the native enzyme. Moreover, temperature- and pH-activity profiles revealed that the designed mutations did not affect the catalytic activity of the enzyme.
Journal: Enzyme and Microbial Technology - Volume 86, May 2016, Pages 84–92