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-  2016 

产L-酪氨酸花椒内生菌的分离鉴定

DOI: 10.13982/j.mfst.1673-9078.2016.2.011

Keywords: 花椒 内生菌 鉴定 耐寒短杆菌 L-酪氨酸
Zanthoxylum bungeanum endophyte identification Brevibacterium frigoritolerans L-tyrosine

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Abstract:

采用研磨法从汉源产红花椒果实中分离筛选出了6株内生菌,包括4株内生细菌和2株内生真菌。本文以其中一株内生细菌Z2为研究对象,对其进行了形态特征、生理生化特征及分子生物学特征鉴定。实验结果发现,Z2菌体短杆状,大小约为0.6~0.7 μm×1.5~2.5 μm,革兰氏阳性菌,无芽孢,无鞭毛。生理生化特征、16S rDNA基因序列及进化树的结果均表明,Z2为耐寒短杆菌(Brevibacterium frigoritolerans)。经过摇瓶液体发酵后,利用高效液相色谱法对其发酵液及未发酵培养基进行了对比分析,发现其中有新物质产生。通过Sephadex LH-20层析柱初步分离发酵液后,利用半制备型高效液相色谱仪对该物质所在组分进行进一步分离纯化,采用电喷雾电离质谱和核磁共振等方法对该物质进行结构解析,结果表明该物质为L-酪氨酸,得率为12.90 mg/L发酵液。
Six endophytic strains were isolated from the fruits of Zanthoxylum Bungeanum by grinding, and these included four strains of bacteria and two strains of fungi. The endophytic bacteria, Z2, was used as the research object in the present study, and its morphological, physiological, biochemical, and molecular biological characteristics were investigated. It was found that the strain Z2 was Gram-positive without spores and flagella, and was observed as a short rod of about 1.5~2.5 μm in length and 0.6~0.7 μm in width. According to the results on the physiological and biochemical characteristics, 16S rDNA gene sequence, and the phylogenetic tree, Z2 was identified as Brevibacterium frigoritolerans. During comparative analysis of the broth fermentated with Z2 and the broth without fermentation using high-performance liquid chromatography (HPLC), a new substance was found in the fermentation broth. This new substance was further purified by Sephadex LH-20 column chromatography and by semipreparative HPLC. Based on of the data from electrospray ionisation tandem mass spectrometry and nuclear magnetic resonance imaging, this substance was identified as L-tyrosine and its content was 12.90 mg/L in the fermented broth.

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