【作者】 张建；

【导师】 孙剑秋； 于慧敏；

【作者基本信息】 齐齐哈尔大学 ， 遗传学， 2012， 硕士

【摘要】 胆固醇氧化酶(ECI.1.3.6)能够氧化胆固醇生成胆甾-4-烯-3-酮，在胆固醇降解的第一步起关键作用。胆固醇氧化酶在食品加工、医疗检测、生物抗虫等方面的作用日益受到人们的重视，并且显示出巨大的应用潜力。针对目前胆固醇氧化酶酶活性低、异源表达效果差和应用潜力巨大的现实，本研究的重点集中于筛选具有高胆固醇氧化酶活性的产生菌。本实验从全国各地采集来的土壤样品51份，通过克隆显色法初步筛选和胆固醇氧化酶活性测定复筛，共获得108株胆固醇氧化酶阳性菌株，进而根据胆固醇氧化酶活性、表型特征等从中选择50株菌进行了多样性分析。利用16S rRNA基因的PCR扩增、测序及系统发育分析，这50株菌分别属于芽孢杆菌属(Bacillus)，假单胞菌属(Pseudomonas)，短杆菌属(Brevibacterium)，短小芽孢杆菌(Lysinibacillus)，节杆菌属(Arthrobacter)，红球菌属(Rhodococcus)，Vogesella属，类芽孢杆菌属(Psenibacillus)和类诺卡氏菌属(Nocardiodides)。其中菌株D48~T属于节杆菌属(Arthrobacter)，与Arthrobacter oryzae NBRC102055~T，Arthrobacter humicola NBRC102056~T，Arthrobacter alkaliphilus CCTCC AB206013~T，Arthrobacter niigatensis CCTCCAB206012~T和Arthrobacter defluvii DSM18782~T的同源性分别为97.74%，97.39%，97.39%，97.00%和96.99%；D94-1~T属于类诺卡氏菌属(Nocardioides)，与Nocardioidessalarius DSM18239~T, Nocardioides marinisabuli DSM18965~T, Nocardioides basaltis DSM22259T和Nocardioides dokdonensis KCTC19309~T的同源性分别为98.54%，98.30%，98.10%和97.76%；D75~T属于类芽孢杆菌属(Paenibacillus)，与Paenibacillus glycanilyticusNBRC16618~T, Paenibacillus xinjiangensis DSM16970T和Paenibacillus castanea DSM19417T同源性分别为99.25%，97.54%和97.21%，此三株菌可能为各属内的新种。对上述菌株进一步进行表型、生理生化、DNA-DNA杂交、细胞壁肽聚糖成分、细胞壁醌型以及细胞脂肪酸成分分析等，证明菌株D48~T为节杆菌属(Arthroacter)的一个新种，命名为Arthrobacter cupressi sp. nov.；菌株D94-1~T为类诺卡氏菌属(Nocardioides)的一个新种，命名为Nocardioides lianchengensis sp. nov.；D75~T为类芽孢杆菌属(Paenibacillus)的一个新种，命名为Paenibacillus catalpa sp. nov.。因此，本研究不仅丰富了胆固醇氧化酶产生菌的资源，还为胆固醇氧化酶的深入科学研究和实际应用奠定了理论基础。更多还原

【Abstract】 Cholesterol oxidase (ECI.1.3.6) catalyzes the first reaction of cholesterol catabolism togenerate cholest-4-en-3-one. Cholesterol oxidase is an enzyme of great commercial value andhas been used in biocatalysis for the production of a number of steroids, as an insecticidalprotein against boll weevil larvae and, in particular, as a diagnostic enzyme for determiningserum levels of cholesterol. There was a disadvantage in the research and application ofcholesterol oxidase that existing stains and recombinant of heterologous expression were allshowed low activity. For the attractive prospects, this study focused on the isolation andidentification of high cholesterol-oxidase-producing strains.In this study, a total of108cholesterol-oxidase-producing strains were firstly isolatedfrom51soil samples selected from all over the country using the colony staining and theactivities of cholesterol-oxidase method. Base on the enzyme activity and the phenotype, atotle of50strains were selected to analysis the diversity.By the polymerase chain reaction (PCR) amplification, sequencing and analysis of16SrRNA gene, the results permitted assignment these bacterial strains to seven genera, Bacillus,Pseudomonas, Brevibacterium, Lysinibacillus, Arthrobacter, Vogesella, Paenibacillus,Nocardiodides and Rhodococcus. The similiarity levels between strain D48~TandArthrobacter oryzae NBRC102055~T，Arthrobacter humicola NBRC102056T，Arthrobacteralkaliphilus CCTCC AB206013T，Arthrobacter niigatensis CCTCC AB206012~TandArthrobacter defluvii DSM18782~Twere97.74%，97.39%，97.39%，97.00%and96.99%,respectivily；between strain D94-1Tand Nocardioides salarius DSM18239~T, Nocardioidesmarinisabuli DSM18965~T, Nocardioides basaltis DSM22259~Tand Nocardioidesdokdonensis KCTC19309Twere98.54%，98.30%，98.10%and97.76%, respectivily；between the strain D75~Tand Paenibacillus glycanilyticus NBRC16618T, Paenibacillusxinjiangensis DSM16970~Tand Paenibacillus castanea DSM19417~Twere99.25%，97.54%and97.21%; These three strains would be novel species belong to corespons genus.On the basis of the evidence presented in this study, including the phynotype andchemotaxonomic properties, DNA-DNA hybrid, the cell-wall peptidoglycan type, thepredominant menaquinone, the major fatty acids and G+C mol%, strains D48~T, D94-1~TandD75~Trepresent a novel species of the genus Arthrobacter, Nocardioides and Paenibacillus, which the names Arthrobacter cupressi sp. nov., Nocardioides lianchengensis sp. nov. andPaenibacillus catalpa sp. nov., respectively.So, this study not only added the resources of cholesterol oxidase-producing strains, butalso laided foundation for the application of cholesterol oxidase.更多还原