A paper-based whole-cell screening assay for directed evolution-driven enzyme engineering

Gul, Ijaz; Bogale, Tadesse Fantaye; Chen, Yong; Yang, Xian; Fang, Rui-Qin; Feng, Jia‐Xun; Gao, Hui; Tang, Lixia

doi:10.1007/s00253-020-10615-x

Cited by 10 publications

(5 citation statements)

References 48 publications

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“…However, internal control for RPA could also add value to the system's reliability. Moreover, protein engineering approaches might be explored to introduce RPA and CRISPR variants that could work efficiently at room temperature (or lower) 43–45 . Clinical validation of a method is essential for practical implementation.…”

Section: Discussionmentioning

confidence: 99%

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CRISPR–Cas12‐based field‐deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome

Chen

Gul

Liu

et al. 2022

Journal of Medical Virology

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The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost‐effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR–Cas‐based system for naked‐eye detection of MPXV. The system harnesses the high selectivity of CRISPR–Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification. It can detect both the current circulating MPXV clade and the original clades. We reached a limit of detection (LoD) of 22.4 aM (13.5 copies/µl) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µl) in a two‐step assay, which is further reduced to 25 aM (15 copies/µl) in a one‐pot system. We compared our results with quantitative polymerase chain reaction and obtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample‐to‐answer time of 35 min.

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Section: Discussionmentioning

confidence: 99%

“…Moreover, protein engineering approaches might be explored to introduce RPA and CRISPR variants that could work efficiently at room temperature (or lower). [43][44][45] Clinical validation of a method is essential for practical implementation. Our study lacks clinical samples, necessitating the need for real-sample analysis by our method.…”

Section: Crispr-casmentioning

confidence: 99%

CRISPR–Cas12‐based field‐deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome

Chen

Gul

Liu

et al. 2022

Journal of Medical Virology

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“…One example of directed evolution in action is the dehalogenation of halohydrin dehalogenase, which uses less reagent and doesn't require pricey equipment. This technique shows the promise of digital image calorimetry in conjunction with paper-based whole-cell screening as a viable strategy for the identification of industrially significant enzymes [ 59 ].…”

Section: Strategies For Improving the Catalytic Properties Of Enzymes...mentioning

confidence: 99%

Current status and emerging frontiers in enzyme engineering: An industrial perspective

Ndochinwa,

Wang,

Amadi

et al. 2024

Heliyon

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“…Dehalogenation of the halohydrin dehalogenase is an example of using directed evolution, which reduces the consumption of reagents and eliminates the need for expensive instrumentation. This method demonstrates the potential for screening whole cells using paper-based whole-cell screening coupled with digital image calorimetry as a promising approach for the discovery of industrially important enzymes (40).…”

Section: Directed Evolutionmentioning

confidence: 99%

Enzyme engineering and its industrial applications

Amatto

Rosa-Garzon

Simões

et al. 2021

Biotech and App Biochem

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Recently, there has been an increase in the demand for enzymes with modified activity, specificity, and stability. Enzyme engineering is an important tool to meet the demand for enzymes adjusted to different industrial processes. Knowledge of the structure and function of enzymes guides the choice of the best strategy for engineering enzymes. Each enzyme engineering strategy, such as rational design, directed evolution, and semi-rational design, has specific applications, as well as limitations, which must be considered when choosing a suitable strategy. Engineered enzymes can be optimized for different industrial applications by choosing the appropriate strategy. This review features engineered enzymes that have been applied in food, animal feed, pharmaceuticals, medical applications, bioremediation, biofuels, and detergents.

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A paper-based whole-cell screening assay for directed evolution-driven enzyme engineering

Cited by 10 publications

References 48 publications

CRISPR–Cas12‐based field‐deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome

CRISPR–Cas12‐based field‐deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome

Current status and emerging frontiers in enzyme engineering: An industrial perspective

Enzyme engineering and its industrial applications

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