IOCAS-IR  > 海洋生物分类与系统演化实验室
一株深海细菌新属的鉴定及Cellulophaga sp.C.2.1菌株降解卡拉胶相关酶的克隆表达
秦芳
Subtype硕士
Thesis Advisor张德超
2021-05-18
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name工程硕士
Keyword海洋细菌 新属 卡拉胶酶 异源表达
Abstract

海洋因其独特的环境,孕育了无数具有特殊代谢方式和特殊代谢产物的微生物。海洋细菌有多种不同的类型,其中以革兰氏阴性杆菌居多,常见的有假单胞菌属、微球菌属、芽孢杆菌属等十几个属。由于现有研究条件等限制,绝大多数微生物至今无法在实验室中获得纯培养,这更加限制了人们对海洋极端环境中的微生物的研究和认识。近年来,关于海洋微生物的研究越来越多,因为们不仅具有极高的应用价值,而且在研究生物的起源与进化中的地位也十分重要。

微生物资源的开发和利用需要建立在分类和鉴定等基础性的工作上。本研究从西太平洋卡罗琳海山的海水中分离到菌株KN1116T,采用多相分类的手段对该菌株进行了分类鉴定。该菌株细胞革兰氏染色呈阴性,菌落为黄色、严格需氧,在电镜下观察细胞呈细长的杆状。基于16S rRNA基因序列的系统发育分析表明,KN1116T菌株与Chryseoglobus属有较高的亲缘关系,与Chryseoglobus frigidaquae EC11T16S rRNA基因序列同源性最高(98.5%)。主要的细胞脂肪酸是anteiso-C15:0iso-C16:0。菌株KN1116T的呼吸醌主要包括甲基萘醌MK-12MK-11MK-10MK-13。极性脂成分主要为二磷脂酰甘油、磷脂酰甘油,此外还有6种未知糖脂、2种未知磷脂和1种未知极性脂。菌株KN1116T的胞壁肽聚糖类型为B1β,其特征二氨基酸为24-二氨基丁酸(DAB)。基因组特征分析结果表明,KN1116T菌株基因组大小为2.7MbpG+C含量为69.4%。根据表型特征、化学成分特征、系统发育特征和基因组学特征,提出KN1116T菌株是微杆菌科(Microbacteriaceae)一个新属的新种,建议命名为Marinisubtilis pacificus gen. nov ., sp. nov .。其模式株为KN1116T=CGMCC 1.17143T=KCTC 49299T)。

卡拉胶是一种分布广泛的海藻多糖,用卡拉胶酶制备的卡拉胶寡糖的多种药用活性已被证实明显优于其他降解方法获得的卡拉胶寡糖,因此卡拉胶降解酶认为是生产卡拉胶寡糖最强有力的工具。本研究从海洋红藻表面分离得到一株具有显著降解卡拉胶活性的细菌C.2.1,经形态观察和16S rDNA序列比对分析,鉴定该菌株为Cellulophaga并将其命名为Cellulophaga sp.C.2.1。结合全基因组数据和NCBI数据库中收录的其他已发表的卡拉胶酶基因序列,找到2个与降解卡拉胶能力相关的基因糖苷水解酶16家族基因C.2.1-2202930bpι-卡拉胶酶基因C.2.1-12191476bp),对这2个卡拉胶降解酶基因进行了重组载体构建及异源表达,经纯化后获得的具有降解卡拉胶活性的重组蛋白大小分别为κ-CgsA2202 34.8 KDaι-CgsA1219 53.6 KDa。同时对2种酶的酶学性质进行了研究,结果表明,两种卡拉胶酶的最适反应温度为40 30 最适反应pH值分别为4.58.5,在底物浓度为0.1%时酶活力受盐浓度影响不大,大多数金属离子对两种酶的活性均有抑制作用,仅有Ca2+κ-CgsA2202的活性具有轻微的促进两种酶对3种常见卡拉胶降解作用

Other Abstract

Because of its unique environment, the ocean nurtures numerous microorganisms with special metabolic patterns and special metabolic products. The marine bacteria have many different types, of which the Gram-negative bacilli are the most common, including Pseudomonas spp., Micrococcus spp., Bacillus spp., and a dozen other genera. Due to existing research conditions and other limitations, the majority of microorganisms cannot be obtained in pure culture in the laboratory so far, which further limits the research and understanding of microorganisms in extreme marine environments. In recent years, an increasing number of studies have been conducted on marine microorganisms because they are not only of great application, but also play an important role in the study of the origin and evolution of organisms.

Basical research such as classification and identification is a prerequisite for the development and utilization of microbial resources. In this study, strain KN1116T was isolated from the waters of the Caroline Seamount in the western Pacific Ocean. It was taxonomically identified by means of a polyphasic classification. It was a Gram-stain-positive, yellow, aerobic, slender rod-shaped bacterial strain. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain KN1116T was related to the genus Chryseoglobus and had highest 16S rRNA gene sequence identity with Chryseoglobus frigidaquae EC11T (98.5%). The predominant cellular fatty acids were anteiso-C15:0 and iso-C16:0. The quinone system for strain KN1116T comprised menaquinone MK-12, MK-11, MK-10 and MK-13. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, six unknown glycolipids, two unidentified phospholipids and one unknown polar lipid. The cell-wall peptidoglycan of strain KN1116T was of the type B1β, containing 2,4-diaminobutyric acid (DAB) as the diamino acid. Genome sequencing revealed the strain KN1116T had a genome size of 2.7 Mbp and a G+C content of 69.4%. Based on phenotypic, chemotaxonomic, phylogenetic and genomic data, strain KN1116T represents a novel species of a novel genus of the family Microbacteriaceae, for which the name Marinisubtilis pacificus gen. nov., sp. nov. is proposed. The type strain of Marinisubtilis pacificus is KN1116T (=CGMCC 1.17143T = KCTC 49299T).

Carrageenan is a widely distributed algal polysaccharide, whose degradation product, carrageenan oligosaccharide, has been confirmed to have various medicinal activities. Carrageenan degrading enzymes are the most powerful tools for the production of carrageenan oligosaccharides. Strain C.2.1 which could hydrolyze carrageenan was isolated from coastal algae samples around Qingdao city. Strain C.2.1 was identified as Cellulophaga sp. C.2.1 by morphology observation and 16S rDNA sequencing. This genus Cellulophaga is one of the major genera for carrageenase. Based on genome data and the carrageenase gene sequences included in the NCBI database, two genes related to carrageenan degrading enzyme were found: glycoside hydrolase family 16 gene C.2.1-2202 (930bp) and ι-carrageenase gene C.2.1-1219 (1476bp). The recombinant vectors were constructed and heterologously expressed for these two carrageenan degrading enzyme genes, and the sizes of the recombinant proteins with carrageenan degrading activity obtained after purification were κ-CgsA2202 34.8 KDa and ι-CgsA1219 53.6 KDa, respectively. The enzymatic properties of the two enzymes were also investigated, and the results showed that the optimum reaction temperatures of the two carrageenan enzymes were 40 and 30 , and the optimum reaction pH was 4.5 and 8.5, respectively. The enzyme activity was not greatly affected by the salt concentration at a substrate concentration of 0.1%. Most metal ions inhibited the activity of both enzymes, and only Ca2+ slightly promoted the activity of κ-CgsA2202, and both enzymes degraded the three common carrageenans.

MOST Discipline Catalogue工学::生物工程
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/170703
Collection海洋生物分类与系统演化实验室
Recommended Citation
GB/T 7714
秦芳. 一株深海细菌新属的鉴定及Cellulophaga sp.C.2.1菌株降解卡拉胶相关酶的克隆表达[D]. 中国科学院海洋研究所. 中国科学院大学,2021.
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