Bioscience Evidence 2024, Vol.14, No.3, 131-142 http://bioscipublisher.com/index.php/be 136 which are critical for the pretreatment and conversion of lignocellulosic materials (Xing et al., 2012). Fungi-derived lignocellulolytic enzymes have also been highlighted for their high catalytic activity and stability, making them suitable for industrial applications in biofuel production (Saldarriaga-Hernández et al., 2020). 5.2 Industrial biocatalysts Directed evolution has been instrumental in developing biocatalysts for pharmaceutical synthesis. Enzymes tailored through directed evolution have been used to create novel enzyme functions and improve existing ones, thereby enhancing the efficiency of pharmaceutical production processes (Zhao et al., 2002). The combination of directed evolution and rational design has accelerated the development of biocatalysts, making them more effective for synthesizing pharmaceutical compounds. Advances in computational design have also contributed to the engineering of enzymes with improved reactivity and substrate specificity, further supporting their application in pharmaceutical synthesis (Planas-Iglesias et al., 2021). In the food industry, directed evolution has been employed to enhance the properties of enzymes used in various processes. For example, enzymes have been engineered to improve their stability, activity, and efficiency, making them more suitable for food production applications. The use of biocatalysts in the food industry has been driven by the need for environmentally friendly and cost-effective alternatives to traditional chemical processes (Porter et al., 2016). Computational techniques have also played a role in optimizing enzyme properties for food industry applications, ensuring that they perform effectively in non-native environments (Planas-Iglesias et al., 2021). 5.3 Environmental bioremediation: degradation of pollutants Directed evolution has also been applied to develop enzymes for environmental bioremediation, particularly in the degradation of pollutants. Enzymes engineered through directed evolution have been shown to catalyze a wide variety of chemical reactions, making them suitable for bioremediation applications (Porter et al., 2016). The development of new catalytic activities in enzymes has expanded their potential for degrading environmental pollutants, offering a sustainable and efficient solution for bioremediation (Chen and Arnold, 2020). Advances in protein engineering have further enhanced the catalytic properties of enzymes, enabling them to function effectively in diverse environmental conditions (Planas-Iglesias et al., 2021). 6 Advances in Enzyme Engineering 6.1 Protein engineering techniques Protein engineering has evolved significantly, leveraging both rational design and directed evolution to enhance enzyme catalytic efficiency. Rational design requires detailed knowledge of enzyme structure and function relationships, allowing for targeted modifications to improve performance. On the other hand, directed evolution mimics natural selection through iterative cycles of mutagenesis and screening, enabling the discovery of enzyme variants with enhanced or novel activities without prior structural knowledge (Kaur and Sharma, 2006; Zeymer and Hilvert, 2018). Recent advancements in ultrahigh-throughput screening (uHTS) have further accelerated this process by allowing the rapid evaluation of vast libraries of enzyme variants, thus overcoming traditional bottlenecks in the screening process (Markel et al., 2019). Additionally, machine learning has been integrated into directed evolution workflows to predict beneficial mutations and navigate the sequence space more efficiently, reducing experimental efforts and enhancing the quality of evolved enzymes (Wu et al., 2019; Zhou, 2024). 6.2 Directed evolution success stories: examples of significantly improved catalytic efficiency Directed evolution has yielded numerous success stories in enhancing enzyme catalytic efficiency. For instance, the evolution of the Kemp eliminase HG3 to HG4 demonstrated significant improvements in catalytic efficiency, with key mutations leading to a more pre-organized and rigidified active site (Broom et al., 2020). Another notable example is the engineering of cytochrome P450 monooxygenases for regio- and stereoselective steroid hydroxylation, where directed evolution combined with mutability landscaping and molecular dynamics simulations achieved high levels of selectivity and activity (Acevedo-Rocha et al., 2018). Furthermore, directed evolution has been instrumental in creating enzymes with new-to-nature activities, such as the stereodivergent synthesis of enantiomeric products in carbene Si–H insertion reactions, showcasing the potential of this approach
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