Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli

BMC Biotechnology

Marina Duarte Pinto Lobo²  Fredy Davi Albuquerque Silva²  Patrícia Gadelha de Castro Landim²  Paloma Ribeiro da Cruz¹  Thaís Lima de Brito¹  Suelen Carneiro de Medeiros¹  José Tadeu Abreu Oliveira²  Ilka Maria Vasconcelos²  Humberto D’Muniz Pereira³  Thalles Barbosa Grangeiro¹ 

[1] Departamento de Biologia, UFC, Laboratório de Genética Molecular, Fortaleza, CE, Brazil

[2] Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, CE CEP 60.455-970, Brazil

[3] Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP CEP 13.566-590, Brazil

关键词: Secretion;    Heterologous;    Chitinase;    Chitin-binding domain;    Signal peptide;   

Others  :  1123167
DOI  :  10.1186/1472-6750-13-46

 received in 2012-10-25, accepted in 2013-05-17,  发布年份 2013

【 摘 要 】

Background

Chromobacterium violaceum is a free-living β-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the β-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum.

Results

The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-β-D-N,N’-diacetylchitobiose and p-nitrophenyl-β-D-N,N’,N”-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60°C for 30 min.

Conclusions

A C. violaceum chitinase was expressed in E. coli and purified by affinity chromatography on a chitin matrix. The secretion of the recombinant protein into the culture medium was directed by its native signal peptide. The mature enzyme was able to hydrolyze colloidal chitin and synthetic substrates. This newly identified signal peptide is a promising secretion factor that should be further investigated in future studies, aiming to demonstrate its usefulness as an alternative tool for the extracellular production of recombinant proteins in E. coli.

【 授权许可】

   
2013 Lobo et al.; licensee BioMed Central Ltd.

【 图 表 】

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