一株产海藻糖合酶假单胞菌菌株的分离及鉴定
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摘要
由薄层层析法检测试验发现,从昆明磷矿粉中分离出的一株真细菌,其粗酶液能将麦芽糖异构化为海藻糖。通过显微观察发现,该菌株为革兰氏阴性菌,无色、杆状、具有鞭毛;菌体直径大小0.6~0.8μm,长度约1.4~2.0μm;胞内没有聚-β-羟丁酸(PHB)包含体;最佳生长温度为30℃,最适生长pH为7.0。经16S rRNA序列比对、DNA(G+C)mol%和DNA-DNA杂交等分子生物学技术以及生理生化特性检测后,认定该菌株为假单胞菌属(Pseudomonas sp.)一种,将其命名为BIE-1。该菌株增加了假单胞菌属菌种多样性,为海藻糖生物合成真细菌菌株提供了新的选择。
A eubacterium was isolated from ground phosphate rock in Kunming, the detection results of thin layer chromatography(TLC) showed that the strain crude enzyme liquid could iosmerize maltose heterogeneous into trehalose. Through microscopic observation, the strain was gram-negative bacterium, colourless, rod-shaped, with a flagellum, 0.6-0.8 μm in diameter and about 1.4-2.0 μm in length. No accumulation of poly-β-hydroxybutyrate(PHB) granules as inclusion bodies was observed. The optimal growth temperature of the strain was 30 ℃ and the optimal pH was 7.0. The strain was identified as Pseudomonas sp. and named BIE-1 by biological techniques(including 16 S rRNA sequence analysis, DNA(G+C) mol%,DNA-DNA hybridization, etc.) and physiological-biochemical characteristic. It increased the species diversity of Pseudomonas sp., which provided new choice for the trehalose biosynthesis by eubacteria.
A eubacterium was isolated from ground phosphate rock in Kunming, the detection results of thin layer chromatography(TLC) showed that the strain crude enzyme liquid could iosmerize maltose heterogeneous into trehalose. Through microscopic observation, the strain was gram-negative bacterium, colourless, rod-shaped, with a flagellum, 0.6-0.8 μm in diameter and about 1.4-2.0 μm in length. No accumulation of poly-β-hydroxybutyrate(PHB) granules as inclusion bodies was observed. The optimal growth temperature of the strain was 30 ℃ and the optimal pH was 7.0. The strain was identified as Pseudomonas sp. and named BIE-1 by biological techniques(including 16 S rRNA sequence analysis, DNA(G+C) mol%,DNA-DNA hybridization, etc.) and physiological-biochemical characteristic. It increased the species diversity of Pseudomonas sp., which provided new choice for the trehalose biosynthesis by eubacteria.
引文
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[18]WANG T F,LIU H J,DAI K,et al.Expression of trehalose synthase gene from Pseudomonas putida P06 in Pichia pastoris[J].Pakistan JPharm Sci,2014,27(3 Suppl):659-662.
[2]FEOFILOVA E P,USOVB A I,MYSYAKINA I S,et al.Trehalose chemical structure,biological functions,and practical application[J]Microbiology,2014,83(3):184-194.
[3]朱玥明,张峻,邢来君,等.海藻糖合酶的分子生物学研究进展[J]微生物学报,2009,49(1):6-12.
[4]NISHIMOTO T,NAKANO M,NAKADA T,et al.Purification and prop erties of a novel enzyme,trehalose synthase,from Pimelobacter sp.R48[J].Biosci Biotechnol Biochem,1996,60(4):640-644.
[5]李忠奎.海藻糖合酶基因在毕赤酵母中的克隆和表达[D].济南:齐鲁工业大学,2014.
[6]黄日波.海藻糖[M].北京:化学工业出版社,2010:25-30.
[7]刘德海,权淑静,解复红,等.海藻糖合成酶产生菌筛选、鉴定及其产酶特性[J].中国酿造,2016,35(9):95-100.
[8]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:56-70.
[9]崔昌浩,田晶,徐龙权.气相色谱法在检测细胞脂肪酸及菌种鉴定中的应用[J].大连轻工业学院学报,2007,26(2):104-107.
[10]韩福芹,胡琳,徐迪诚.微生物的醌类测定[J].哈尔滨商业大学学报:自然科学版,2001,11(2):97-98,101.
[11]李琳,李槿年,余为一.细菌染色体DNA G+C mol%含量测定方法研究进展[J].动物医学进展,2003,24(1):50-52.
[12]TAMURA K,PETERSON D,PETERSON N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Mol Biol Evolut,2011,28(10):2731-2739.
[13]XIN Y H,ZHANG D C,LIU H C,et al.Pseudomonas tuomuerensis sp.nov.,isolated from a bird's nest[J].Int J Syst Evol Micr,2009,59(1):139-143.
[14]高云.海洋假单胞菌来源的新型海藻糖合成酶的基因克隆、表达及性质研究[D].上海:第二军医大学,2013.
[15]GAO Y,XI Y,LU XL,et al.Cloning,expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp.P8005[J].World J Microbiol Biotechnol,2013,29(11):2195-2206.
[16]薛鸿毅.恶臭假单胞菌海藻糖合酶基因在大肠杆菌中的表达[D].济南:齐鲁工业大学,2012.
[17]WANG T,JIA S,DAI K,et al.Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose[J].Can J Microbiol,2014,60(9):599-604.
[18]WANG T F,LIU H J,DAI K,et al.Expression of trehalose synthase gene from Pseudomonas putida P06 in Pichia pastoris[J].Pakistan JPharm Sci,2014,27(3 Suppl):659-662.