Kinetic data from different acetoacetyl-CoA reductases

These folders contain experimental records, calculation datasheets and scripts to simulate or find best-fitted kinetic parameters from certain enzymatically catalyzed reactions. These reactions were catalyzed by four engineered enzymes derived from the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator. The DNA and amino acid sequences of these enzymes are provided. It is also provided the DNA sequence map of the vector where the amino acid encoding sequences for these engineered enzymes were inserted. Inside the folder named "kinetic data" it is also possible to find a TXT file with a detailed description of the content. The experimental conditions of the enzymatic assays is carefully explained. Briefly, this folder contains (1) the raw experimental data recorded with the software Gen 5 (Biotek). (2) It is also possible to find Microsoft Excel datasheets with the records of raw absorbance in time together with initial substrates, initial enzyme and product concentrations in time (reaction progress curves). Most of these progress curves were analyzed with the software DYNAFIT (Biokin) to obtain the kinetic parameters. The progress curves highlighted in red in the Microsoft Excel files were not considered for the statistical analysis due to experimental errors. The progress curves highlighted in yellow in the Microsoft Excel files were considered after the elimination of some outlier points. (3) Scripts enabling two kind of statistical analyses with DYNAFIT were included: scripts to make model discrimination analyses of the different reaction progress curves obtained in every single experiment, and scripts to calculate the confidence intervals for the kinetic parameters using a Monte Carlo approach. We included the scripting manual of DYNAFIT to understand the synthaxis of these scripts .(4) It is included two MATLAB scripts to make a comparison between the kinetic parameters obtained using the Michaelis-Menten model and the true kinetic parameters of a BiBi reversible reaction. (5) Finally, we included DYNAFIT, MATLAB and Microsoft Excel files enabling the calculation of the relative use of NADH over NADPH by one of the engineered enzymes, and also to calculate the metabolic flux capacity of this enzyme.

本数据集包含实验记录、计算数据表以及模拟或寻找特定酶促反应最佳拟合动力学参数的脚本。这些反应由来自嗜铁菌噬菌体phaB1基因编码的乙酰乙酰辅酶A还原酶的四种工程酶催化。提供了这些酶的DNA和氨基酸序列。同时，还提供了包含工程酶编码序列的载体DNA序列图谱。在名为“动力学数据”的文件夹内，您还可以找到一份详细描述内容的TXT文件。酶促分析实验条件被细致阐述。简要而言，该文件夹包含以下内容：（1）使用Gen 5（Biotek）软件记录的原始实验数据。（2）您还可以找到记录初始底物、初始酶和产物浓度随时间变化的Microsoft Excel数据表（反应进程曲线）。大多数进程曲线均使用DYNAFIT（Biokin）软件进行分析，以获取动力学参数。在Microsoft Excel文件中用红色突出显示的进程曲线因实验误差而未纳入统计分析。（3）包含两种统计分析的DYNAFIT脚本：用于对每个单独实验中获得的反应进程曲线进行模型区分分析的脚本，以及使用蒙特卡洛方法计算动力学参数置信区间的脚本。我们还包括了DYNAFIT脚本的编写手册，以便理解这些脚本的合成。（4）包含两个MATLAB脚本，用于比较使用米氏-门腾模型获得的动力学参数与BiBi可逆反应的真实动力学参数。（5）最后，我们还包含了DYNAFIT、MATLAB和Microsoft Excel文件，这些文件能够计算某工程酶相对于NADPH的NADH相对利用率，以及计算该酶的代谢通量能力。