BMC Biotechnol. 2013 Jun 1;13:46. doi: 10.1186/1472-6750-13-46.
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.
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.
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.
粘质沙雷氏菌是一种生活在热带和亚热带地区的自由生活的β-变形菌。粘质沙雷氏菌 ATCC 12472 的基因组测序揭示了许多使其适应多种生态系统的基因。此外，还发现了一些在工业、医学和农业中有潜在应用的粘质沙雷氏菌基因，例如编码几丁质酶的基因。然而，ATCC 12472 菌株的几丁质酶基因尚未经过实验验证。几丁质酶（EC 3.2.1.14）水解几丁质中的β-（1,4）糖苷键，几丁质是节肢动物、软体动物和真菌中丰富的生物聚合物。这些酶作为潜在的生物防治剂对抗害虫和病原体具有巨大的生物技术兴趣。本工作旨在对粘质沙雷氏菌的一种几丁质酶进行实验验证。
粘质沙雷氏菌 ATCC 12472 的开放阅读框（ORF）CV2935 编码一种蛋白质（439 个残基），该蛋白质由信号肽、几丁质结合域、连接区和属于糖苷水解酶家族 18 的 C 端催化结构域组成。通过 PCR 扩增 ORF 并克隆到表达载体 pET303/CT-His 中。含有重组质粒的大肠杆菌 BL21(DE3)细胞的无细胞培养上清液中检测到高水平的几丁质酶活性，并在 IPTG 诱导下表达。通过几丁质基质亲和层析对分泌的重组蛋白进行纯化，通过变性聚丙烯酰胺凝胶电泳估计其表观分子量为 43.8 kDa。N 端测序证实了在重组产物的分泌过程中原信号肽的正确去除。该酶能够水解胶体几丁质和合成底物 p-硝基苯基-β-D-N,N'-二乙酰基壳二糖和 p-硝基苯基-β-D-N,N',N"-三乙酰基壳三糖。其最适 pH 值为 5.0，当在 60°C 加热 30 分钟时，该酶保留约 32%的活性。
在大肠杆菌中表达了粘质沙雷氏菌的几丁质酶，并通过几丁质基质亲和层析进行纯化。重组蛋白通过其天然信号肽分泌到培养基中。成熟酶能够水解胶体几丁质和合成底物。这种新鉴定的信号肽是一种很有前途的分泌因子，在未来的研究中应进一步研究，旨在证明其作为大肠杆菌中重组蛋白胞外生产的替代工具的有用性。
