Department of Biological Science and Biotechnology, Hannam University, 1646 Yooseong-daero, Yooseong-gu, Daejon 34054, South Korea.
Department of Biological Science and Biotechnology, Hannam University, 1646 Yooseong-daero, Yooseong-gu, Daejon 34054, South Korea.
Enzyme Microb Technol. 2020 May;136:109492. doi: 10.1016/j.enzmictec.2019.109492. Epub 2020 Jan 11.
To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.
为了阐明由细菌和昆虫结构域组成的重组杂合几丁质酶的功能改变，我们分别从细菌物种苏云金芽孢杆菌（BtChi）和鳞翅目昆虫物种菜蛾（MbChi）的几丁质酶编码 cDNA 中克隆了组成结构域，交换了一个物种的信号肽（LSP）-催化域（CD）-连接区（LR）（LCL）与另一个物种的几丁质结合域（ChBD），并确认和分析了重组杂合几丁质酶在转化的大肠杆菌菌株中的功能表达及其几丁质酶活性。两个重组 cDNA 中的每一个，即 MbChi 的 LCL 与 BtChi 的 ChBD 相连（MbLCL-BtChBD）和 BtChi 的 LCL 与 MbChi 的 ChBD 相连（BtLCL-MbChBD），都成功地引入并在大肠杆菌 BL21 菌株中表达。虽然两种杂合酶都通过 SDS-PAGE 和 Western blot 检测到表达，但引入的基因对两种转化大肠杆菌菌株的几丁质代谢的影响似乎有很大的不同。BtLCL-MbChBD 不仅显著增加了细胞增殖率、细胞外和细胞内几丁质酶活性，还增加了细胞内氨基葡萄糖和 N-乙酰氨基葡萄糖水平，而 MbLCL-BtChBD 在三个测试对象中的表现与转化的每个菌株中的两种天然几丁质酶的表现大致相同，表明细菌 CD 的 TIM 桶结构与特征性的六个半胱氨酸残基和包括保守的六个富含半胱氨酸的区域（6C）的昆虫 ChBD2 的组合增强了酶分子与几丁质化合物的结合，从而提高了细菌 CD 的催化效率。
