Department of Botany and Microbiology, Faculty of Science, Alexandria University, 1 Baghdad Street-Moharam Bek, Alexandria, 21568, Egypt.
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, P.O.Box 832, 163 Horreya Avenue, Chatby, Alexandria, 21526, Egypt.
Mol Biol Rep. 2022 Feb;49(2):951-969. doi: 10.1007/s11033-021-06914-9. Epub 2021 Nov 13.
Using in silico sequence analyses, the present study aims to clone and express the gene-encoding sequence of a GH19 chitinase from Enterobacter sp. in Escherichia coli.
The putative open reading frame of a GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM®-T and pET-28a (+) vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, GenBank accession no.: MK533791.2) was translated to a chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). The in silico protein sequence analysis of chiRAM revealed a class I GH19 chitinase: an N-terminus signal peptide (Met-Ala), a catalytic domain (Val-Glu and the catalytic triad Glu, Glu, and Ser), a proline-rich hinge region (Pro -Pro), a polycystic kidney disease protein motif (Gly -Ser ), a C-terminus chitin-binding domain (Ala- Glu), and conserved class I motifs (NYNY and AQETGG). A three-dimensional model was constructed by LOMETS MODELLER of PDB template: 2dkvA (class I chitinase of Oryza sativa L. japonica). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72 kDa; SDS-PAGE) in 1.0 mM IPTG induced E. coli BL21 (DE3) Rosetta strain at room temperature 18 h after induction. Optimized expression yielded active chiRAM with 1.974 ± 0.0002 U/mL, on shrimp colloidal chitin (SCC), in induced E. coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified a band of 72 kDa in the soluble fraction with a 52.3% coverage sequence exclusive to the GH19 chitinase of Enterobacter cloacae (WP_063869339.1).
Although chiRAM of Enterobacter sp. was successfully cloned and expressed in E. coli with appreciable chitinase activity, future studies should focus on minimizing IBs to facilitate chiRAM purification and characterization.
本研究旨在通过计算机序列分析，从肠杆菌属中克隆和表达 GH19 几丁质酶的基因编码序列，并在大肠杆菌中进行表达。
使用简并引物，分别将肠杆菌属 EGY1 菌株 GH19 几丁质酶的假定开放阅读框克隆并表达到 pGEM®-T 和 pET-28a（+）载体中。分离的核苷酸序列（1821bp，GenBank 登录号：MK533791.2）被翻译为 chiRAM 蛋白（606 个氨基酸，UniProt 登录号：A0A4D6J2L9）。chiRAM 的计算机蛋白质序列分析表明，它是一种 I 类 GH19 几丁质酶：一个 N 端信号肽（Met-Ala）、一个催化结构域（Val-Glu 和催化三联体 Glu、Glu 和 Ser）、一个富含脯氨酸的铰链区（Pro-Pro）、一个多囊肾病蛋白基序（Gly-Ser）、一个 C 端几丁质结合结构域（Ala-Glu）和保守的 I 类基序（NYNY 和 AQETGG）。通过 LOMETS MODELLER 基于 PDB 模板：2dkvA（水稻的 I 类几丁质酶）构建了三维模型。在诱导后 18 小时，在 1.0mM IPTG 诱导的大肠杆菌 BL21（DE3）Rosetta 菌株中，以包涵体（IBs）形式（~72kDa；SDS-PAGE）过量表达重组 chiRAM。在 SB 培养基中生长的诱导大肠杆菌 BL21（DE3）Rosetta 细胞中，用虾胶体几丁质（SCC）测定，优化表达得到了具有 1.974±0.0002U/mL 活性的 chiRAM。LC-MS/MS 在可溶性部分鉴定到 72kDa 的条带，其序列覆盖率为 52.3%，仅为阴沟肠杆菌 GH19 几丁质酶（WP_063869339.1）所特有。
尽管肠杆菌属的 chiRAM 已成功在大肠杆菌中克隆和表达，并具有相当的几丁质酶活性，但未来的研究应集中于最小化 IBs，以方便 chiRAM 的纯化和表征。
