干旱区生态环境知识资源中心(Arid): Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt

Arid

DOI	10.1186/s43141-022-00312-9
	Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt
	Ali, Safaa M.; Soliman, Nadia A.; Abdal-Aziz, Samia Abd Allah; Abdel-Fattah, Yasser R.
通讯作者	Ali, SM (corresponding author)，City Sci Res & Technol Applicat SRTA City, Alexandria 21934, Egypt.
来源期刊	JOURNAL OF GENETIC ENGINEERING AND BIOTECHNOLOGY
ISSN	2090-5920
出版年	2022
卷号	20 期号:1
英文摘要	Background: 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. Results: Total soil-DNA was successfully extracted, and then digested by different restriction enzymes. Purified fragments ranged similar to 200-6500 bp were ligated and were cloned into plasmid cloning vector (pUC19) by using Escherichia coli DH5a (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 mu/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 celISNSY was expressed under lacz promoter at 1 mM of isopropyl beta-d-1-thiogalactopyranoside (IPTG), giving 21 mu/ml cellulase after similar to 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 similar to 59 kDa appeared in the induced sample. The maximum enzyme activity of crude cellSNSY was observed at 45 degrees 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. Conclusions: 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 degrees C and for alkaline pH 8.5. These features make it an ample and viable for many applications.
类型	Article
语种	英语
开放获取类型	Green Published, gold
收录类别	ESCI
WOS记录号	WOS:000752912900001
WOS关键词	CARBOXYMETHYL CELLULASE ; MICROBIAL CELLULASES ; ENZYME ; OPTIMIZATION ; BACTERIA ; DNA ; IDENTIFICATION ; PURIFICATION ; ESTERASE
WOS类目	Biotechnology & Applied Microbiology ; Environmental Sciences ; Toxicology
WOS研究方向	Biotechnology & Applied Microbiology ; Environmental Sciences & Ecology ; Toxicology
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/376969
作者单位	[Ali, Safaa M.; Abdal-Aziz, Samia Abd Allah] Genet Engn & Biotechnol Res Inst GEBRI, Nucle Acid Res Dept, City Sci Res & Technol Applicat, Alexandria, Egypt; [Ali, Safaa M.] City Sci Res & Technol Applicat SRTA City, Alexandria 21934, Egypt; [Soliman, Nadia A.; Abdel-Fattah, Yasser R.] City Sci Res & Technol Applicat SRTA City, Genet Engn & Biotechnol Res Inst GEBRI, Bioproc Dev Dept, Alexandria, Egypt
推荐引用方式 GB/T 7714	Ali, Safaa M.,Soliman, Nadia A.,Abdal-Aziz, Samia Abd Allah,et al. Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt[J],2022,20(1).
APA	Ali, Safaa M.,Soliman, Nadia A.,Abdal-Aziz, Samia Abd Allah,&Abdel-Fattah, Yasser R..(2022).Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt.JOURNAL OF GENETIC ENGINEERING AND BIOTECHNOLOGY,20(1).
MLA	Ali, Safaa M.,et al."Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt".JOURNAL OF GENETIC ENGINEERING AND BIOTECHNOLOGY 20.1(2022).