Rapid micro-assays for amylolytic activities determination: customization and validation of the tests

Monika Borkowska,Wojciech Białas,Monika Kubiak,Ewelina Celińska

doi:10.1007/s00253-018-09610-0

Monika Borkowska, Wojciech Białas + Show 2 more

Open Access

https://doi.org/10.1007/s00253-018-09610-0

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Abstract

High-throughput function-based screening techniques remain the major bottleneck in the novel biocatalysts development pipeline. In the present study, we customized protocols for amylolytic activity determination (Somogyi-Nelson and starch-iodine tests) to micro-volume thermalcycler-based assays (linearity range 60–600 μM of reducing sugar, R2 = 0.9855; 0–2 mg/mL of starch, R2 = 0.9921, respectively). Exploitation of a thermalcycler enabled rapid and accurate temperature control, further reduction of reagents and samples volumes, and limited evaporation of the reaction mixtures, meeting several crucial requirements of an adequate enzymatic assay. In the optimized micro-volume Somogyi-Nelson protocol, we were able to reduce the time required for high-temperature heating sixfold (down to 5 min) and further increase sensitivity of the assay (tenfold), when compared to the previous MTP-based protocol. The optimized microassays have complementary scope of specificities: micro-starch-iodine test for endoglucanases, micro-Somogyi-Nelson test for exoglucanases. Due to rapid, micro-volume and high-throughput character, the methods can complement toolbox assisting development of novel biocatalysts and analysis of saccharides-containing samples.

Highlights

Significant effort is being pursued towards development of highly efficient, industrially relevant biocatalysts—either whole microbial cells or individual enzymes
It turned out that the previous protocols could be adopted to the new format with excellent linearity (R2 = 0.9921; p < 0.0001) in the desired range (8–80 μg starch/microassay), and the absorbance values below 2.0 (Fig. 1b; Table S1)
This study reports customization of standard macro-volume and MTP-based assays for amylolytic activity determination to the micro-volume format protocols employing a thermalcycler, and validation thereof

Summary

Introduction

Significant effort is being pursued towards development of highly efficient, industrially relevant biocatalysts—either whole microbial cells or individual enzymes. Amongst the most important advantages of the MTP (microtiter plate)-based screening, one can name higher accessibility to the required equipment, reagents and consumables, broad dynamic range allowing for detection of comparatively low improvements in the targeted enzyme function compared to other methods like colony screening assays (Kelly et al 2008), similarity to a miniature cuvette systems, allowing for customization of standard enzymatic assays to the MTP format Popularity of this approach is reflected by a number of regularly published new protocols describing customization of enzymatic assays to the MTP format, including industrially relevant enzymes such as lipases (Mustafa et al 2016) or glycosidases—by adopting glucose assay (Visvanathan et al 2016), Somogyi-Nelson method (Shao and Lin 2018), or 3,5-dinitrosalicylic acid (DNS) method (Goncalves et al 2010), and cellulolytic enzymes (Xiao et al 2005; King et al 2009)

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