Functional Display of an Amoebic Chitinase in Escherichia coli Expressing the Catalytic Domain of EhCHT1 on the Bacteria
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Functional Display of an Amoebic Chitinase in Escherichia coli Expressing the Catalytic Domain of EhCHT1 on the Bacterial Cell Surface Ricardo Torres-Bañaga 1 & Rosa E. Mares-Alejandre 1 & Celina Terán-Ramírez 1 & Ana L. Estrada-González 1 & Patricia L.A. Muñoz-Muñoz 1 & Samuel G. Meléndez-López 1 & Ignacio A. Rivero 2 & Marco A. Ramos-Ibarra 1 Received: 20 March 2020 / Accepted: 16 July 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Poor solubility is the main drawback of the direct industrial exploitation of chitin, the second most abundant biopolymer after cellulose. Chemical methods are conventional to solubilize chitin from natural sources. Enzymatic hydrolysis of soluble chitinous substrates is a promising approach to obtain value-added byproducts, such as N-acetylglucosamine units or low molecular weight chito-oligomers. Protein display on the bacterial membrane remains attractive to produce active enzymes anchored to a biological surface. The Lpp-OmpA system, a gene fusion of the Lpp signal sequence with the OmpA transmembrane region, represents the traditional system for targeting enzymes to the E. coli surface. EhCHT1, the amoebic chitinase, exhibits an efficient endochitinolytic activity and significant biochemical features, such as stability over a wide range of pH values. Using an extended Lpp-OmpA system as a protein carrier, we engineered E. coli to express the catalytic domain of EhCHT1 on the surface and assess the endochitinase activity as a trait. Engineered bacteria showed a consistent hydrolytic rate over a typical substrate, suggesting that the displayed enzyme has operational stability. This study supports the potential of biomembrane-associated biocatalysts as a reliable technology for the hydrolysis of soluble chitinous substrates. Keywords Amoebic chitinase . Endochitinase . Escherichia coli . Bacterial surface display . Membrane-associated biocatalyst
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003389-5) contains supplementary material, which is available to authorized users.
* Marco A. Ramos-Ibarra [email protected] Extended author information available on the last page of the article
Applied Biochemistry and Biotechnology
Introduction Chitin is a linear homopolysaccharide composed of N-acetylglucosamine units linked by β-(1,4) bonds. It is the second foremost abundant natural polymer (after cellulose), representing the major component of numerous protective structures, such as the exoskeleton of arthropods and the cell wall of fungi. As a biological polymer, it has features with potential medical and pharmaceutical applications (i.e., biocompatible and biodegradable). Despite the latter, poor solubility represents the main drawback of its direct industrial exploitation [1–4]. Produced by numerous organisms, chitinases are enzymes that degrade chitin by hydrolyzing internal O-glycosidic β-(1,4) bonds (ENZYME entry: EC 3.2.1.14, https://www. expasy.org/), and the name refers to endo- an
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