A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

International Journal of Molecular Sciences, vol. 22, pp. 3041

Abstract

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

Authors 5

1. Gheorghița Menghiu Aachen Institute for Biology VII

   RWTH Aachen University · West University of Timişoara

   Affiliation as printed

   Advanced Environmental Research Laboratories, Department of Biology–Chemistry, West University of Timisoara, Oituz 4, 300086 Timisoara, Romania

   Institute for Biology VII, Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

2. Vasile Ostafe

   West University of Timişoara

   Affiliation as printed

   Advanced Environmental Research Laboratories, Department of Biology–Chemistry, West University of Timisoara, Oituz 4, 300086 Timisoara, Romania

3. Radivoje Prodanović

   University of Belgrade

   Affiliation as printed

   Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia

4. Rainer Fischer Aachen Institute for Biology VII

   RWTH Aachen University · Purdue University West Lafayette

   Affiliation as printed

   Departments of Biological Sciences and Chemistry, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA

   Institute for Biology VII, Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

5. Raluca Ostafe corresponding Aachen Institute for Biology VII

   RWTH Aachen University · Purdue University West Lafayette

   Affiliation as printed

   Institute for Biology VII, Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

   Purdue Institute of Inflammation, Immunology and Infectious Disease, Molecular Evolution, Protein Engineering and Production, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA