High-level synthesis of endochitinase ChiA74 in Escherichia coli K12 and its promising potential for use in biotechnology J. Cristóbal Castañeda-Ramírez & Norma M. de la Fuente-Salcido & Rubén Salcedo-Hernández & Fabiola León-Galván & Dennis K. Bideshi & J. Eleazar Barboza-Corona Received: 9 September 2012 / Accepted: 24 January 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013 Abstract In the present study, we expressed the chiA74 gene of Bacillus thuringiensis in Escherichia coli K12 and demon- strated that the active ChiA74 enzyme was produced at a high level in this strain. The ChiA74 enzymatic activity (in units per milliliter) was approximately 500 % greater in E. coli K12 when compared to that produced in E. coli DH5α. Moreover, we showed that, when using our protocol, ChiA74 preparations obtained from recombinant E. coli K12 did not contain live bacteria, although transformable DNA (erm, bla genes) was detected. Nucleic acids were subsequently easily eliminated when samples were treated with magnesium. Importantly, ChiA74 was secreted by E. coli K12 and the active enzyme was shown to generate chitin-derived oligosaccharides (C- OGS) with degrees of polymerization of 2, 3, 4, 5, and 6. From an applied perspective, the C-OGS showed activity against various pathogenic bacteria. In addition, we demon- strated that ChiA74 was not toxic to Hek 293 and 3T3 L1 cells, i.e., the enzyme did not induce apoptosis or affect normal cellular cycle and also did not produce abnormal changes in cell morphology. The potential biotechnological use of producing endochitinase of B. thuringiensis in a microorgan- ism recognized as safe (i.e., E. coli K12) is discussed. Introduction Various strains of Bacillus thuringiensis are among the most important insecticidal bacteria used worldwide as biopesticides in agriculture and mosquito vector abatement programs. These strains synthesize a plethora of crystal toxins (Cry) active against many orders of insects (Federici 2005). B. thuringiensis also secretes chitinases that enhance the toxicity of Cry proteins (Wiwat et al. 2000). Importantly, from an applied perspective, chitinases could also be used to generate chitin-derived oligo- saccharides (C-OGS) for use as natural food preservatives or for pharmacologic purposes (Aam et al. 2010; Barboza-Corona et al. 2012; Wang et al. 2007). However, regarding the latter, the applied use of chitinases derived from B. thuringiensis in food and medicinal industries is currently impractical because this species has not attained the generally recognized as safe (GRAS) nor qualified presumption of safe (QPS) status. Moreover, it is unlikely that enzymes synthesized directly by this microorganism would be approved for practical use as many strains are known to harbor cytotoxin and enterotoxin genes (Swiecicka et al. 2006) whose putative products could pose a potential health risk to consumers (Olempska-Beer et al. 2006; Pariza and Johnson 2001). Recently, we have successfully isolated endochitinase ChiA74, native to a Mexican strain of B. thuringiensis subsp. kenyae, following heterologous expression in Escherichia coli DH5α and showed that the enzyme hydrolyzed chitin to pro- duce C-OGS (Barboza-Corona et al. 2003; Ortiz-Rodríguez et al. 2010). Although E. coli DH5α has been routinely used in research laboratories, it does not have the GRAS or QPS status, as does E. coli K12 (Olempska-Beer et al. 2006) that is used routinely for the commercial production of recombinant pro- teins, for example, chymosin, an enzyme used to make cheese J. C. Castañeda-Ramírez : N. M. de la Fuente-Salcido : R. Salcedo-Hernández : F. León-Galván : J. E. Barboza-Corona (*) Division of Life Sciences, Food Department, University of Guanajuato Campus Irapuato-Salamanca, Irapuato, Guanajuato 36500, México e-mail: josebar@ugto.mx N. M. de la Fuente-Salcido School of Biological Sciences, Autonomous University of Coahuila, Torreón, Coahuila 27440, México D. K. Bideshi Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA 92504, USA D. K. Bideshi Department of Entomology, University of California, Riverside, Riverside, CA 92521, USA Folia Microbiol DOI 10.1007/s12223-013-0229-7