title: High-level synthesis of endochitinase ChiA74 in Escherichia coli K12 and its promising potential for use in biotechnology.
abstract: In the present study, we expressed the chiA74 gene of Bacillus thuringiensis in Escherichia coli K12 and demonstrated 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 demonstrated 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 microorganism recognized as safe (i.e., E. coli K12) is discussed.
authors: Division of Life Sciences, Food Department, University of Guanajuato Campus Irapuato-Salamanca, Irapuato, Guanajuato, 36500, México.
authors: J Cristóbal
authors: JC
authors: Castañeda-Ramírez
authors: Norma M
authors: NM
authors: de la Fuente-Salcido
authors: Rubén
authors: R
authors: Salcedo-Hernández
authors: Fabiola
authors: F
authors: León-Galván
authors: Dennis K
authors: DK
authors: Bideshi
authors: J Eleazar
authors: JE
authors: Barboza-Corona
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002688:Chitinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000201:enzymology / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D002688:Chitinases
references: J Appl Microbiol. 2008 Nov;105(5):1511-20
references: 19146488
references: Antonie Van Leeuwenhoek. 2009 Jun;96(1):31-42
references: 19337851
references: Appetite. 2007 May;48(3):333-7
references: 17137678
references: Lett Appl Microbiol. 2010 Aug;51(2):184-90
references: 20557451
references: J Invertebr Pathol. 2005 May;89(1):30-8
references: 16039303
references: Appl Environ Microbiol. 2003 Feb;69(2):1023-9
references: 12571025
references: Annu Rev Physiol. 2011;73:479-501
references: 21054166
references: Nucleic Acids Res. 1990 Jun 25;18(12):3660
references: 2163525
references: Regul Toxicol Pharmacol. 2006 Jul;45(2):144-158
references: 16769167
references: Biochem Biophys Res Commun. 2007 May 25;357(1):26-31
references: 17400187
references: J Invertebr Pathol. 2000 Nov;76(4):270-7
references: 11112372
references: Biotechnology (N Y). 1991 Apr;9(4):349-51
references: 1367006
references: Nat Protoc. 2006;1(3):1458-61
references: 17406435
references: J Microbiol Biotechnol. 2010 Feb;20(2):311-8
references: 20208434
references: Regul Toxicol Pharmacol. 2001 Apr;33(2):173-86
references: 11350200
references: J Exp Med. 2009 May 11;206(5):1149-66
references: 19414556
references: Microb Ecol. 2006 Oct;52(3):544-51
references: 16944343
references: Int J Food Microbiol. 2002 Mar 25;74(1-2):65-72
references: 11929171
references: J Biotechnol. 2008 Jan 1;133(1):50-7
references: 17950946
references: Mar Drugs. 2010 Apr 27;8(5):1482-517
references: 20559485
references: PLoS One. 2011;6(9):e24304
references: 21931677