Skip to main content
SpringerLink
Log in

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

  • Methods and protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 μl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.

Key Points

• A frugal method was developed for directed enzyme evolution.

• Mutagenesis libraries were successfully screened on a paper platform.

• Smartphone imaging was efficiently used to measure enzyme activities.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Akyazi T, Basabe-Desmonts L, Benito-Lopez F (2018) Review on microfluidic paper-based analytical devices towards commercialisation. Anal Chim Acta 1001:1–17

    Article  CAS  Google Scholar 

  • Bachas-Daunert PG, Sellers ZP, Wei Y (2009) Detection of halogenated organic compounds using immobilized thermophilic dehalogenase. Anal Bioanal Chem 395:1173–1178

    Article  CAS  Google Scholar 

  • Bagheri N, Cinti S, Caratelli V, Massoud R, Saraji M, Moscone D, Arduini F (2019) A 96-well wax printed Prussian Blue paper for the visual determination of cholinesterase activity in human serum. Biosens Bioelectron 134:97–102

    Article  CAS  Google Scholar 

  • Bidmanova S, Kotlanova M, Rataj T, Damborsky J, Trtilek M, Prokop Z (2016) Fluorescence-based biosensor for monitoring of environmental pollutants: from concept to field application. Biosens Bioelectron 84:97–105

    Article  CAS  Google Scholar 

  • Bjork SM, Joensson HN (2019) Microfluidics for cell factory and bioprocess development. Curr Opin Biotechnol 55:95–102

    Article  CAS  Google Scholar 

  • Bogale FT, Gul I, Wang L, Deng J, Chen Y, Majerić-Elenkov M, Tang L (2020) Biodegradation of 1,2,3-trichloropropane to valuable (S)-2,3 DCP using a one-pot reaction system. Catalysts. https://doi.org/10.3390/catal10010003

  • Busin V, Burgess S, Shu W (2018) A hybrid paper-based microfluidic platform toward veterinary P-ELISA. Sensors Actuators B Chem 273:536–542

    Article  CAS  Google Scholar 

  • Chen B, Lim S, Kannan A, Alford SC, Sunden F, Herschlag D, Dimov IK, Baer TM, Cochran JR (2015) High-throughput analysis and protein engineering using microcapillary arrays. Nat Chem Biol 12:76–81

    Article  Google Scholar 

  • Debon A, Pott M, Obexer R, Green AP, Friedrich L, Griffiths AD, Hilvert D (2019) Ultrahigh-throughput screening enables efficient single-round oxidase remodelling. Nat Catal 2:740–747

    Article  CAS  Google Scholar 

  • Dekker RFH, Ling KY, Barbosa AM (2000) A simple method for monitoring chromatography column eluates for laccase activity during enzyme purification. Biotechnol Lett 22:105–108

    Article  CAS  Google Scholar 

  • Dias AA, Matos AJS, Fraga I, Sampaio A, Bezerra RMF (2017) An easy method for screening and detection of laccase activity. Open Biotechnol J 11:89–93

    Article  CAS  Google Scholar 

  • Fabritz S, Maaß F, Olga A, Tim H, Björn S, Harald K (2012) A sensitive method for rapid detection of alkyl halides and dehalogenase activity using a multistep enzyme assay. AMB Express 2:1–8. https://doi.org/10.1186/2191-0855-2-51

    Article  CAS  Google Scholar 

  • Fox RJ, Davis SC, Mundorff EC, Newman LM, Gavrilovic V, Ma SK, Chung LM, Ching C, Tam S, Muley S, Grate J, Gruber J, Whitman JC, Sheldon RA, Huisman GW (2007) Improving catalytic function by ProSAR-driven enzyme evolution. Nat Biotechnol 25:338–344

    Article  CAS  Google Scholar 

  • Gielen F, Hours R, Emond S, Fischlechner M, Schell U, Hollfelder F (2016) Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS). Proc Natl Acad Sci 113:E7383–E7389

    Article  CAS  Google Scholar 

  • Gul I, Bogale TF, Deng J, Wang L, Feng J, Tang L (2020a) A high-throughput screening assay for the directed evolution-guided discovery of halohydrin dehalogenase mutants for epoxide ring-opening reaction. J Biotechnol 311:19–24

    Article  CAS  Google Scholar 

  • Gul I, Bogale TF, Deng J, Chen Y, Fang R, Feng J, Tang L (2020b) Enzyme-based detection of epoxides using colorimetric assay integrated with smartphone imaging. Biotechnol Appl Biochem. https://doi.org/10.1002/bab.1898

  • Hasan S, Geissler D, Wink K, Hagen A, Heiland JJ, Belder D (2019) Fluorescence lifetime-activated droplet sorting in microfluidic chip systems. Lab Chip 19:403–409

    Article  CAS  Google Scholar 

  • Hol FJH, Whitesides GM, Dekker C (2019) Bacteria-in-paper, a versatile platform to study bacterial ecology. Ecol Lett. https://doi.org/10.1111/ele.13274

  • Ji D, Xu N, Liu Z, Shi Z, Low SS, Liu J, Cheng C, Zhu J, Zhang T, Xu H, Yu X, Liu Q (2019) Smartphone-based differential pulse amperometry system for real-time monitoring of levodopa with carbon nanotubes and gold nanoparticles modified screen-printing electrodes. Biosens Bioelectron 129:216–223

    Article  CAS  Google Scholar 

  • Koesdjojo MT, Pengpumkiat S, Wu Y, Boonloed A, Huynh D, Remcho TP, Remcho VT (2015) Cost effective paper-based colorimetric microfluidic devices and mobile phone camera readers for the classroom. J Chem Educ 92:737–741

    Article  CAS  Google Scholar 

  • Leferink NGH, Dunstan MS, Hollywood KA, Swainston N, Currin A, Jervis AJ, Takano E, Scrutton NS (2019) An automated pipeline for the screening of diverse monoterpene synthase libraries. Sci Rep 9:1–12. https://doi.org/10.1038/s41598-019-48452-2

    Article  CAS  Google Scholar 

  • Liao Q, Du X, Jiang W, Tong Y, Zhao Z, Fang R, Feng J, Tang L (2018) Cross-linked enzyme aggregates (CLEAs) of halohydrin dehalogenase from Agrobacterium radiobacter AD1: preparation, characterization and application as a biocatalyst. J Biotechnol 272-273:48–55

    Article  CAS  Google Scholar 

  • Lim JW, Shin KS, Moon J, Lee SK, Kim T (2016) A microfluidic platform for high-throughput screening of small mutant libraries. Anal Chem 88:5234–5242

    Article  CAS  Google Scholar 

  • Longwell CK, Labanieh L, Cochran JR (2017) High-throughput screening technologies for enzyme engineering. Curr Opin Biotechnol 48:196–202

    Article  CAS  Google Scholar 

  • Ma F, Chung MT, Yao Y, Nidetz R, Lee LM, Liu AP, Feng Y, Kurabayashi K, Yang GY (2018) Efficient molecular evolution to generate enantioselective enzymes using a dual-channel microfluidic droplet screening platform. Nat Commun 9:1–8. https://doi.org/10.1038/s41467-018-03492-6

    Article  CAS  Google Scholar 

  • Markel U, Essani KD, Besirlioglu V, Schiffels J, Streit WR, Schwaneberg U (2020) Advances in ultrahigh-throughput screening for directed enzyme evolution. Chem Soc Rev 49:233–262

    Article  CAS  Google Scholar 

  • Michelini E, Calabretta MM, Cevenini L, Lopreside A, Southworth T, Fontaine DM, Simoni P, Branchini BR, Roda A (2019) Smartphone-based multicolor bioluminescent 3D spheroid biosensors for monitoring inflammatory activity. Biosens Bioelectron 123:269–277

    Article  CAS  Google Scholar 

  • Nan-Wei W, Zhi-Qiang L, Feng X, Kai H, Ling-Jiao T, Yu-Guo Z (2015) An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols. Appl Microbiol Biotechnol 99:4019–4029

    Article  Google Scholar 

  • Natesan M, Wu SW, Chen CI, Jensen SMR, Karlovac N, Dyas BK, Mudanyali O, Ulrich RG (2019) A smartphone-based rapid telemonitoring system for ebola and Marburg disease surveillance. ACS Sens 4:61–68

    Article  CAS  Google Scholar 

  • Nevolova S, Manaskova E, Mazurenko S, Damborsky J, Prokop Z (2019) Development of fluorescent assay for monitoring of dehalogenase activity. Biotechnol J. https://doi.org/10.1002/biot.201800144

  • Noiphung J, Laiwattanapaisal W (2019) Multifunctional paper-based analytical device for in situ cultivation and screening of Escherichia coli infections. Sci Rep 9:1–10. https://doi.org/10.1038/s41598-018-38159-1

    Article  CAS  Google Scholar 

  • Noiphung J, Nguyen MP, Punyadeera C, Wan Y, Laiwattanapaisal W, Henry CS (2018) Development of paper-based analytical devices for minimizing the viscosity effect in human saliva. Theranostics 8:3797–3807

    Article  CAS  Google Scholar 

  • Ostafe R, Prodanovic R, Nazor J, Fischer R (2014) Ultra-high-throughput screening method for the directed evolution of glucose oxidase. Chem Biol 21:414–421

    Article  CAS  Google Scholar 

  • Pooja S, Jindal S, Bala K, Kumari K, Niharika N, Kaur J, Pandey G, Pandey R, Russell RJ, Oakeshott JG, Lal R (2014) Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay. Biodegradation 25:179–187

    Article  Google Scholar 

  • Sadler JC, Currin A, Kell DB (2018) Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting. Analyst 143:4747–4755

    Article  CAS  Google Scholar 

  • de Santana PC, Lourenço FR (2020) A smartphone-based bioassay for determining relative potency estimated from sigmoidal-response curves and respective measurement uncertainty. Microchem J 154:104626

    Article  Google Scholar 

  • Shen Y, Tran TT, Modha S, Tsutsui H, Mulchandani A (2019) A paper-based chemiresistive biosensor employing single-walled carbon nanotubes for low-cost, point-of-care detection. Biosens Bioelectron 130:367–373

    Article  CAS  Google Scholar 

  • Skopelitou K, Georgakis N, Efrose R, Flemetakis E, Labrou NE (2013) Sol–gel immobilization of haloalkane dehalogenase from Bradyrhizobium japonicum for the remediation of 1,2-dibromoethane. J Mol Catal B Enzym 97:5–11

    Article  CAS  Google Scholar 

  • Strong EB, Schultz SA, Martinez AW, Martinez NW (2019) Fabrication of miniaturized paper-based microfluidic devices (MicroPADs). Sci Rep 9:1–9. https://doi.org/10.1038/s41598-018-37029-0

    Article  CAS  Google Scholar 

  • Tang L, Li Y, Wang X (2010) A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries. J Biotechnol 147:164–168

    Article  CAS  Google Scholar 

  • Tang L, Zhu X, Zheng H, Jiang R, Majeric-Elenkov M (2012) Key residues for controlling enantioselectivity of Halohydrin dehalogenase from Arthrobacter sp. strain AD2, revealed by structure-guided directed evolution. Appl Environ Microbiol 78:2631–2637

    Article  CAS  Google Scholar 

  • Tang L, Zheng K, Liu Y, Zheng H, Wang H, Song C, Zhou H (2013) Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction. Biotechnol Appl Biochem 60:190–195

    Article  CAS  Google Scholar 

  • Thuo MM, Martinez RV, Lan WJ, Liu X, Barber J, Atkinson MBJ, Bandarage D, Bloch JF, Whitesides GM (2014) Fabrication of low-cost paper-based microfluidic devices by embossing or cut-and-stack methods. Chem Mater 26:4230–4237

    Article  CAS  Google Scholar 

  • Wang X, Ren L, Su Y, Ji Y, Liu Y, Li C, Li X, Zhang Y, Wang W, Hu Q, Han D, Xu J, Ma B (2017) Raman-activated droplet sorting (RADS) for label-free high-throughput screening of microalgal single-cells. Anal Chem 89:12569–12577

    Article  CAS  Google Scholar 

  • Wang Y, Liu Y, Liu W, Tang W, Shen L, Li Z, Fan M (2018) Quantification of combined color and shade changes in colorimetry and image analysis: water pH measurement as an example. Anal Methods 10:3059–3065

    Article  Google Scholar 

  • Wu N, Courtois F, Zhu Y, Oakeshott J, Easton C, Abell C (2010) Management of the diffusion of 4-methylumbelliferone across phases in microdroplet-based systems for in vitro protein evolution. Electrophoresis 31:3121–3128

    Article  CAS  Google Scholar 

  • Ye L, Yang C, Yu H (2018) From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution. Appl Microbiol Biotechnol 102:559–567

    Article  CAS  Google Scholar 

  • Zheng L, Cai G, Wang S, Liao M, Li Y, Lin J (2019) A microfluidic colorimetric biosensor for rapid detection of Escherichia coli O157:H7 using gold nanoparticle aggregation and smart phone imaging. Biosens Bioelectron 124-125:143–149

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 21673034) and was a subproject under the National Science and Technology Major Project on Water Pollution Prevention and Control (No. 2012ZX07203–003).

Author information

Authors and Affiliations

  1. School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China

    Ijaz Gul, Tadesse Fantaye Bogale, Yong Chen, Xian Yang, Ruiqin Fang, Juan Feng & Lixia Tang

  2. School of Computing Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, China

    Hui Gao

  3. Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, 610054, China

    Ruiqin Fang, Juan Feng, Hui Gao & Lixia Tang

Authors
  1. Ijaz Gul
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tadesse Fantaye Bogale
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xian Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ruiqin Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Juan Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hui Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lixia Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

IG, LT, RF, JF, and HG conceived and designed research. IG, TF, YC, and XY conducted experiments. IG and TF analyzed data. IG wrote the manuscript, LT reviewed and edited the manuscript. All of the authors read and approved the manuscript.

Corresponding author

Correspondence to Lixia Tang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals that were performed by any of the authors.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 689 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gul, I., Bogale, T.F., Chen, Y. et al. A paper-based whole-cell screening assay for directed evolution-driven enzyme engineering. Appl Microbiol Biotechnol 104, 6013–6022 (2020). https://doi.org/10.1007/s00253-020-10615-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-020-10615-x

Keywords

Search

Navigation