海洋细菌DL-6几丁质酶和几丁质结合蛋白的生化性质与功能研究
发布时间：2018-10-19 15:42
【摘要】：几丁质是海洋环境中含量最丰富,自然界中仅次于纤维素的第二大生物质资源。微生物几丁质酶(chitinase, chi, EC3.2.1.14)降解体系可协同作用几丁质制备高附加值几丁寡糖、几丁单糖及其衍生物等,具有增强免疫力、抗肿瘤、抗菌、降血压和降低胆固醇等生物活性,在医药、农业、工业和食品等领域具有巨大的应用潜力。海洋具有低温、高盐、低光照及寡营养等极端环境,海洋微生物为了适应海洋环境,进化了与之相适应的特点,其基因组中蕴含有价值的基因资源。因此,研究产几丁质酶系海洋微生物及其分泌几丁质酶系的生化性质及几丁质降解机制等有助于阐明海洋环境几丁质降解代谢途径,开发利用新的几丁质酶资源。本论文以胶体几丁质为唯一碳源,从大连渤海湾底泥样品中分离到高产低温几丁质酶的海洋细菌。菌株形态特征结合16SrDNA系统发育分析,鉴定该菌株属于假交替单胞菌属,命名为Pseudoalteromonas sp.DL-6。酶谱分析与荧光底物酶活性检测推测Pseudoalteromonas sp.DL-6分泌不同作用类型的几丁质酶系。通过PCR技术成功克隆Pseudoalteromonas sp.DL-6的两个几丁质酶基因chiA和chiC,一个几丁质结合蛋白基因CBP58。构建了原核表达载体pET28a-ChiA、 pET28a-ChiC与pET23b-CBP58,在大肠杆菌中实现这些基因的可溶性表达。重组蛋白ChiA、CBP58和ChiC通过Ni-NTA亲和层析,1L培养基上清液中分别纯化获得33.74mg ChiA、19.04 mg ChiC和11.90mg CBP58, ChiA和ChiC的纯化倍数和回收率为7.32和7.18及63.25%和79.37%。酶学性质研究表明几丁质酶ChiA和ChiC具有适冷特性,最适作用温度分别为20℃和30℃,在4℃以胶体几丁质为底物的Kcat/Km分别为0.56和1.83。ChiC表现出较强的耐盐特性,在5 M NaCl下仍保持最高酶活的60%以上。除几丁质外,ChiA和ChiC还可以降解Chitosan和Avicel等,具有广泛的底物适应性。酶解产物分析表明ChiA为随机作用底物释放聚合度2-6几丁寡糖的内切几丁质酶,ChiC为持续作用底物生成几丁二糖的外切几丁质酶。扫描电子显微镜观察表明CBP58对几丁质多糖链产生剥离和松解等破坏作用。底物结合实验表明CBP58对α-chitin和collodial chitin结合能力最强,β-chitin及其nano-whiskers次之,对icel具有较弱结合能力。海洋耐冷细菌Pseudoalteromonas sp.DL-6几丁质酶系统通过协同降解机制作用几丁质,不仅为生物转化生产几丁寡糖提供了新途径,也有助于进一步阐明海洋环境几丁质代谢机制。
[Abstract]:Chitin is the second largest biomass resource in the marine environment after cellulose. Microbial chitinase (chitinase, chi, EC3.2.1.14) degradation system can co-act chitin to prepare high value-added chitin oligosaccharide, chitin monosaccharide and its derivatives, which have the biological activities of enhancing immunity, anti-tumor, antibacterial, lowering blood pressure and lowering cholesterol, etc. It has great application potential in medicine, agriculture, industry and food. The ocean has the extreme environment of low temperature, high salt, low light and oligonutrition. In order to adapt to the marine environment, marine microorganisms have evolved the characteristics of adapting to the marine environment, and the genome contains valuable genetic resources. Therefore, it is helpful to clarify the metabolic pathway of chitinase degradation and exploit new chitinase resources by studying the biochemical properties of chitinase system and the mechanism of chitin degradation. In this paper, the marine bacteria with high low temperature chitinase were isolated from the sediments of Bohai Bay, Dalian, with colloidal chitin as the sole carbon source. The morphological characteristics of the strain combined with the analysis of 16SrDNA phylogeny showed that the strain belonged to Pseudomonas, named Pseudoalteromonas sp.DL-6.. Enzyme spectrum analysis and fluorescence substrate enzyme activity test speculated that Pseudoalteromonas sp.DL-6 secreted chitinases of different types. Cloning of two chitinase genes (chiA) from Pseudoalteromonas sp.DL-6 and a chitin binding protein gene CBP58. from chiC, by PCR technique Prokaryotic expression vectors pET28a-ChiA, pET28a-ChiC and pET23b-CBP58, were constructed to express these genes in E. coli. The purification times and recoveries of 33.74mg ChiA,19.04 mg ChiC and 11.90mg CBP58, ChiA and ChiC were 7.32, 7.18, 63.25% and 79.37%, respectively, in the supernatant of 1L medium by Ni-NTA affinity chromatography. The enzymatic properties of chitinase ChiA and ChiC were studied. The optimum reaction temperature was 20 鈩，
本文编号：2281574
[Abstract]:Chitin is the second largest biomass resource in the marine environment after cellulose. Microbial chitinase (chitinase, chi, EC3.2.1.14) degradation system can co-act chitin to prepare high value-added chitin oligosaccharide, chitin monosaccharide and its derivatives, which have the biological activities of enhancing immunity, anti-tumor, antibacterial, lowering blood pressure and lowering cholesterol, etc. It has great application potential in medicine, agriculture, industry and food. The ocean has the extreme environment of low temperature, high salt, low light and oligonutrition. In order to adapt to the marine environment, marine microorganisms have evolved the characteristics of adapting to the marine environment, and the genome contains valuable genetic resources. Therefore, it is helpful to clarify the metabolic pathway of chitinase degradation and exploit new chitinase resources by studying the biochemical properties of chitinase system and the mechanism of chitin degradation. In this paper, the marine bacteria with high low temperature chitinase were isolated from the sediments of Bohai Bay, Dalian, with colloidal chitin as the sole carbon source. The morphological characteristics of the strain combined with the analysis of 16SrDNA phylogeny showed that the strain belonged to Pseudomonas, named Pseudoalteromonas sp.DL-6.. Enzyme spectrum analysis and fluorescence substrate enzyme activity test speculated that Pseudoalteromonas sp.DL-6 secreted chitinases of different types. Cloning of two chitinase genes (chiA) from Pseudoalteromonas sp.DL-6 and a chitin binding protein gene CBP58. from chiC, by PCR technique Prokaryotic expression vectors pET28a-ChiA, pET28a-ChiC and pET23b-CBP58, were constructed to express these genes in E. coli. The purification times and recoveries of 33.74mg ChiA,19.04 mg ChiC and 11.90mg CBP58, ChiA and ChiC were 7.32, 7.18, 63.25% and 79.37%, respectively, in the supernatant of 1L medium by Ni-NTA affinity chromatography. The enzymatic properties of chitinase ChiA and ChiC were studied. The optimum reaction temperature was 20 鈩，
本文编号：2281574
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