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来自内生真菌菌株kj831197.1的木聚糖酶基因的增强生产、克隆及表达:揭示其对植物病原真菌的体外抗真菌活性

Enhanced Production, Cloning, and Expression of a Xylanase Gene from Endophytic Fungal Strain kj831197.1: Unveiling the In Vitro Anti-Fungal Activity against Phytopathogenic Fungi.

作者信息

Ellatif Sawsan Abd, Abdel Razik Elsayed S, Al-Surhanee Ameena A, Al-Sarraj Faisal, Daigham Ghadir E, Mahfouz Amira Y

机构信息

Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications, New Borg El-Arab 21934, Egypt.

Plant Protection and Biomolecular Diagnosis Department, Arid Lands Cultivation Research Institute, City for Scientific Research and Technology Applications, New Borg El-Arab 21934, Egypt.

出版信息

J Fungi (Basel). 2022 Apr 25;8(5):447. doi: 10.3390/jof8050447.

Abstract

sp. is extensively applied as a beneficial fungus for the management of plant diseases, plant growth promotion, induced resistance, and plays an important role in global sustainable agriculture. This study aimed to enhance the production of microbial xylanase in high titer from the endophytic fungus kj831197.1, and the cloning of xylanase genes in DH5α using a pUC19 vector. A combination of glucose, 0.1 mM, Tween 80 with lactose, and 2 mM galactose combined with malt extract boostedthe enzyme production. Xylanase production was maximized at a pH of 5.0, temp. of 30 °C, and agitation of 150 rpm in the presence of malt extract and bagasse as the best nitrogen source and waste, respectively, using submerged fermentation. The molecular weight of highly purified xylanase was 32 KDa, identified using SDS-PAGE. The xylanase gene of kj831197.1 was screened in fungal DNA using definite primers specified in the gene bank database. The identified region was excised using restriction enzymes HindIII and EcoRI and cloned into a pUC19 plasmid vector. Optimization of fermentation conditions improved xylanase production about 23.9-fold.The antifungal efficacy of xylanase toward different phytopathogenic fungi was determined. The highest inhibition was against sp., and This study offered an economical, simple, and efficient method using kj831197.1 for the production of the xylanase enzyme via the submerged fermentation method.

摘要

某菌种作为一种有益真菌,在植物病害防治、促进植物生长、诱导抗性方面得到广泛应用,在全球可持续农业中发挥着重要作用。本研究旨在提高内生真菌kj831197.1高产微生物木聚糖酶的产量,并使用pUC19载体在DH5α中克隆木聚糖酶基因。葡萄糖、0.1 mM吐温80与乳糖的组合,以及2 mM半乳糖与麦芽提取物的组合提高了酶的产量。在麦芽提取物和甘蔗渣分别作为最佳氮源和废弃物存在的情况下,采用深层发酵,在pH值为5.0、温度为30℃、搅拌速度为150 rpm时,木聚糖酶产量达到最大值。使用SDS-PAGE鉴定,高度纯化的木聚糖酶分子量为32 kDa。使用基因库数据库中指定的特定引物在真菌DNA中筛选kj831197.1的木聚糖酶基因。使用限制性内切酶HindIII和EcoRI切除鉴定出的区域,并克隆到pUC19质粒载体中。发酵条件的优化使木聚糖酶产量提高了约23.9倍。测定了木聚糖酶对不同植物病原真菌的抗真菌效果。对某菌种的抑制作用最强,本研究提供了一种经济、简单且高效的方法,利用kj831197.1通过深层发酵法生产木聚糖酶。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/32c6/9144407/126d9f9fb7d2/jof-08-00447-g001.jpg

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