Ali Safaa M, Soliman Nadia A, Abdal-Aziz Samia Abd Allah, Abdel-Fattah Yasser R
Nucleic Acid Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological applications, Alexandria, Egypt.
Present address: City of Scientific Research and Technological Applications (SRTA-City),, New Burg El-Arab City, Universities and Research Institutes Zone, Alexandria, Post 21934, Egypt.
J Genet Eng Biotechnol. 2022 Feb 8;20(1):20. doi: 10.1186/s43141-022-00312-9.
Wadi El Natrun microorganisms have been considered as a new resource for natural products due to its extreme condition of salinity and alkalinity. Therefore, this study was devoted to generate metagemic library from soils collected from such an extreme environment in order to clone a novel cellulase for physique industrial applications.
Total soil-DNA was successfully extracted, and then digested by different restriction enzymes. Purified fragments ranged ~ 200-6500 bp were ligated and were cloned into plasmid cloning vector (pUC19) by using Escherichia coli DH5α (E. coli) host cells. A constructed metagenomic library composed of 270 clones was screened on carboxymethylcellulose (CMC) agar plate where the active clones had been characterized by the formation of the yellowish halo zone. Thereafter, clone 1 was selected as the most active as being based on cellulase activity quantification (19 μ/ml). Plasmid related to clone 1 encoded cellSNSY gene of approximately 1.5 kb was subjected to molecular characterization; the obtained partial sequence of 861 bps encoded 287 amino acids showing 76% similarity to the endoglucanase gene of Bacillus amyloliquefaciens. The recombinant cellSNSY was expressed under lacz promoter at 1 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG), giving 21 μ/ml cellulase after ~ 27 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an activity staining of the recombinant cellSNSY which revealed an active band with a molecular mass ~ 59 kDa appeared in the induced sample. The maximum enzyme activity of crude cellSNSY was observed at 45 °C and for a pH of 8.5. Interestingly, the enzyme activity was slightly inhibited by ethylenediamine tetraacetic acid (EDTA) and methanol. It showed high resistance to the tested heavy metals and the surfactant which ordered Zn> (SDS,Fe)>Mn>Cu.
This study established an easy and a skillful way to clone/express a new found cellulase gene(s) under lacZ promoter. The isolated recombinant cellSNSY showed 76% similarity to endoglucanase gene, and the enzyme showed tolerance to the mostly tested agents including heavy metals, surfactant, solvents, and EDTA. Additionally, the studied recombinant showed a high stability up to 55 °C and for alkaline pH 8.5. These features make it an ample and viable for many applications.
由于纳特龙湖微生物所处的极端盐碱环境，它们被视为天然产物的新来源。因此，本研究致力于从这种极端环境采集的土壤中构建宏基因组文库，以克隆一种用于工业应用的新型纤维素酶。
成功提取了土壤总DNA，然后用不同的限制性内切酶进行消化。将大小约为200 - 6500 bp的纯化片段进行连接，并使用大肠杆菌DH5α宿主细胞克隆到质粒克隆载体（pUC19）中。在羧甲基纤维素（CMC）琼脂平板上筛选由270个克隆组成的宏基因组文库，活性克隆通过形成淡黄色晕圈来表征。此后，基于纤维素酶活性定量（19 μ/ml），选择克隆1作为活性最高的克隆。对与克隆1相关的编码约1.5 kb的cellSNSY基因的质粒进行分子表征；获得的861 bp的部分序列编码287个氨基酸，与解淀粉芽孢杆菌的内切葡聚糖酶基因显示出76%的相似性。重组cellSNSY在1 mM异丙基β - D - 1 - 硫代半乳糖苷（IPTG）存在下于lacz启动子下表达，约27小时后产生21 μ/ml的纤维素酶。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和重组cellSNSY的活性染色显示，在诱导样品中出现了一条分子量约为59 kDa的活性条带。粗制cellSNSY的最大酶活性在45°C和pH 8.5时观察到。有趣的是，该酶活性受到乙二胺四乙酸（EDTA）和甲醇的轻微抑制。它对所测试的重金属和表面活性剂表现出高抗性，抗性顺序为Zn >（SDS，Fe）> Mn > Cu。
本研究建立了一种简便且熟练的方法，可在lacZ启动子下克隆/表达新发现的纤维素酶基因。分离的重组cellSNSY与内切葡聚糖酶基因显示出76%的相似性，该酶对包括重金属、表面活性剂、溶剂和EDTA在内的大多数测试试剂表现出耐受性。此外，所研究的重组体在高达55°C和碱性pH 8.5时表现出高稳定性。这些特性使其在许多应用中具有充足性和可行性。
