Shuimoliuyun (Talk | contribs) |
Shuimoliuyun (Talk | contribs) |
||
Line 458: | Line 458: | ||
ceaS2 enzyme is the most important enzyme in our entire acrylic acid synthesis pathway, but the activity of wild type is not high. So it is exceedingly necessary to modify it on the basis of the "part" level to improve its catalytic reactivity. We used the AEMD platform to conduct the mutational design for ceaS2 enzyme in order to figure out a more accurate scheme of mutation, which can also exert great beneficial impact on the later experiments. <br> | ceaS2 enzyme is the most important enzyme in our entire acrylic acid synthesis pathway, but the activity of wild type is not high. So it is exceedingly necessary to modify it on the basis of the "part" level to improve its catalytic reactivity. We used the AEMD platform to conduct the mutational design for ceaS2 enzyme in order to figure out a more accurate scheme of mutation, which can also exert great beneficial impact on the later experiments. <br> | ||
We have totally identified XX mutational sites, and its point mutation transformation. The experimental results show that there are XX sites, where the enzyme activity gets boosted, after the transformation. Compared to wild type ceaS2 enzyme, the highest activity has increased by XX times, whose effect is obviously noticeable. This also demonstrates the ability of this designing platform. </h4> | We have totally identified XX mutational sites, and its point mutation transformation. The experimental results show that there are XX sites, where the enzyme activity gets boosted, after the transformation. Compared to wild type ceaS2 enzyme, the highest activity has increased by XX times, whose effect is obviously noticeable. This also demonstrates the ability of this designing platform. </h4> | ||
− | <h3>Introduction</h3> | + | <h3 style="text-align:center">Introduction</h3> |
<h4>Enzyme engineering has been extensively used to optimize biocatalysts in industrial biotechnology since most of enzymes in nature prefer to organisms adaptation but not industrial production (Alvizo, et al., 2014; Ma, et al., 2009; Savile, et al., 2010). Traditionally, optimized enzymes were obtained by random site-directed or saturated mutagenesis such as Error Prone PCR, DNA shuffling and so on (Kabumoto, et al., 2009; Qi, et al., 2009; Reetz and Carballeira, 2007; Yep, et al., 2008). Due to the immense possibility of sequence mutation at amino acids level, it is a time-consuming and low efficiency task to obtain a high efficient biocatalyst by random mutation. <br> | <h4>Enzyme engineering has been extensively used to optimize biocatalysts in industrial biotechnology since most of enzymes in nature prefer to organisms adaptation but not industrial production (Alvizo, et al., 2014; Ma, et al., 2009; Savile, et al., 2010). Traditionally, optimized enzymes were obtained by random site-directed or saturated mutagenesis such as Error Prone PCR, DNA shuffling and so on (Kabumoto, et al., 2009; Qi, et al., 2009; Reetz and Carballeira, 2007; Yep, et al., 2008). Due to the immense possibility of sequence mutation at amino acids level, it is a time-consuming and low efficiency task to obtain a high efficient biocatalyst by random mutation. <br> | ||
With the availability of an increasing number of protein structural and biochemical data, rational design of enzymatic mutation has become more and more popular (Bloom, et al., 2005; Chica, et al., 2005; Kiss, et al., 2013; Li, et al., 2012; Steiner and Schwab, 2012). Many strategies have been used to obtain evolutionary information, catalytic sites and substrate channels by integrating sequence and structural features of enzymes. Previous studies have developed many effective computational tools for enzyme engineering, such as the enzyme design software Rosetta (Leaver-Fay, et al., 2011) and stability design software Foldx (Van, et al., 2011) and so on (Table S2). However, most of them only focus on one feature, like the thermo-stability based on the known PDB structure, and often request professional backgrounds in protein structure, biochemistry, bioinformatics and so on. | With the availability of an increasing number of protein structural and biochemical data, rational design of enzymatic mutation has become more and more popular (Bloom, et al., 2005; Chica, et al., 2005; Kiss, et al., 2013; Li, et al., 2012; Steiner and Schwab, 2012). Many strategies have been used to obtain evolutionary information, catalytic sites and substrate channels by integrating sequence and structural features of enzymes. Previous studies have developed many effective computational tools for enzyme engineering, such as the enzyme design software Rosetta (Leaver-Fay, et al., 2011) and stability design software Foldx (Van, et al., 2011) and so on (Table S2). However, most of them only focus on one feature, like the thermo-stability based on the known PDB structure, and often request professional backgrounds in protein structure, biochemistry, bioinformatics and so on. | ||
</h4> | </h4> | ||
− | <h3>What is | + | <h3 style="text-align:center">What is AEMD?</h3> |
+ | |||
<h4>AEMD is a web-based pipeline, which integrates several approaches together for enzyme stability, selectivity and activity engineering. This pipeline can generate comprehensive reports, which include the recommended mutation for improving enzyme catalytic property. Specifically, users can get the recommended mutation only inputting sequence information of target enzymes, which is very useful in the situation without professional knowledge and the known protein structure, since AEMD contains a functional module that can automatically predict structure of the target enzyme based on the known structures in Protein Data Bank (PDB).<br> | <h4>AEMD is a web-based pipeline, which integrates several approaches together for enzyme stability, selectivity and activity engineering. This pipeline can generate comprehensive reports, which include the recommended mutation for improving enzyme catalytic property. Specifically, users can get the recommended mutation only inputting sequence information of target enzymes, which is very useful in the situation without professional knowledge and the known protein structure, since AEMD contains a functional module that can automatically predict structure of the target enzyme based on the known structures in Protein Data Bank (PDB).<br> | ||
AEMD-Web provides a web interface, enabling users to conveniently predict mutants which could improve the stability, selectivity and activity of enzymes. Users can obtain the suggestion of mutations for almost all enzyme even without protein structure. In the future, we will construct a comprehensive enzymatic mutant database and integrate new computing technology, to improve the efficiency of enzyme engineering in industrial biotechnology. </h4> | AEMD-Web provides a web interface, enabling users to conveniently predict mutants which could improve the stability, selectivity and activity of enzymes. Users can obtain the suggestion of mutations for almost all enzyme even without protein structure. In the future, we will construct a comprehensive enzymatic mutant database and integrate new computing technology, to improve the efficiency of enzyme engineering in industrial biotechnology. </h4> | ||
Line 471: | Line 472: | ||
<h4>Fig.1 Workflow of the Stability analysis (A), Selectivity analysis (B) and Activity analysis (C).The blue color rectangle blocks represent the inputs of sequence or PDB file, and the output of recommended mutation sites. The green and gray color rectangle blocks represent the evolution- and energy-based analysis process, respectively. The yellow color diamond blocks represent the use of other softwares and approaches. The processes were shown in Supplementary methods【click here】in more detail.</h4> | <h4>Fig.1 Workflow of the Stability analysis (A), Selectivity analysis (B) and Activity analysis (C).The blue color rectangle blocks represent the inputs of sequence or PDB file, and the output of recommended mutation sites. The green and gray color rectangle blocks represent the evolution- and energy-based analysis process, respectively. The yellow color diamond blocks represent the use of other softwares and approaches. The processes were shown in Supplementary methods【click here】in more detail.</h4> | ||
− | <h3>Process</h3> | + | <h3 style="text-align:center">Process</h3> |
+ | |||
<h4>This time we utilized AEMD's Stability mode (click here for AEMD user's guide) to screen for mutational sites that benefit the ceaS2 enzyme activity.<br> | <h4>This time we utilized AEMD's Stability mode (click here for AEMD user's guide) to screen for mutational sites that benefit the ceaS2 enzyme activity.<br> | ||
Because of the complexity of enzyme catalysis, it’s difficult to predict point mutation improving protein activity accurately. How AEMD work?<br> | Because of the complexity of enzyme catalysis, it’s difficult to predict point mutation improving protein activity accurately. How AEMD work?<br> |
Revision as of 16:04, 1 November 2017