【摘要】：幾丁質(zhì)是海洋環(huán)境中含量最豐富,自然界中僅次于纖維素的第二大生物質(zhì)資源。微生物幾丁質(zhì)酶(chitinase, chi, EC3.2.1.14)降解體系可協(xié)同作用幾丁質(zhì)制備高附加值幾丁寡糖、幾丁單糖及其衍生物等,具有增強(qiáng)免疫力、抗腫瘤、抗菌、降血壓和降低膽固醇等生物活性,在醫(yī)藥、農(nóng)業(yè)、工業(yè)和食品等領(lǐng)域具有巨大的應(yīng)用潛力。海洋具有低溫、高鹽、低光照及寡營養(yǎng)等極端環(huán)境,海洋微生物為了適應(yīng)海洋環(huán)境,進(jìn)化了與之相適應(yīng)的特點(diǎn),其基因組中蘊(yùn)含有價(jià)值的基因資源。因此,研究產(chǎn)幾丁質(zhì)酶系海洋微生物及其分泌幾丁質(zhì)酶系的生化性質(zhì)及幾丁質(zhì)降解機(jī)制等有助于闡明海洋環(huán)境幾丁質(zhì)降解代謝途徑,開發(fā)利用新的幾丁質(zhì)酶資源。本論文以膠體幾丁質(zhì)為唯一碳源,從大連渤海灣底泥樣品中分離到高產(chǎn)低溫幾丁質(zhì)酶的海洋細(xì)菌。菌株形態(tài)特征結(jié)合16SrDNA系統(tǒng)發(fā)育分析,鑒定該菌株屬于假交替單胞菌屬,命名為Pseudoalteromonas sp.DL-6。酶譜分析與熒光底物酶活性檢測推測Pseudoalteromonas sp.DL-6分泌不同作用類型的幾丁質(zhì)酶系。通過PCR技術(shù)成功克隆Pseudoalteromonas sp.DL-6的兩個(gè)幾丁質(zhì)酶基因chiA和chiC,一個(gè)幾丁質(zhì)結(jié)合蛋白基因CBP58。構(gòu)建了原核表達(dá)載體pET28a-ChiA、 pET28a-ChiC與pET23b-CBP58,在大腸桿菌中實(shí)現(xiàn)這些基因的可溶性表達(dá)。重組蛋白ChiA、CBP58和ChiC通過Ni-NTA親和層析,1L培養(yǎng)基上清液中分別純化獲得33.74mg ChiA、19.04 mg ChiC和11.90mg CBP58, ChiA和ChiC的純化倍數(shù)和回收率為7.32和7.18及63.25%和79.37%。酶學(xué)性質(zhì)研究表明幾丁質(zhì)酶ChiA和ChiC具有適冷特性,最適作用溫度分別為20℃和30℃,在4℃以膠體幾丁質(zhì)為底物的Kcat/Km分別為0.56和1.83。ChiC表現(xiàn)出較強(qiáng)的耐鹽特性,在5 M NaCl下仍保持最高酶活的60%以上。除幾丁質(zhì)外,ChiA和ChiC還可以降解Chitosan和Avicel等,具有廣泛的底物適應(yīng)性。酶解產(chǎn)物分析表明ChiA為隨機(jī)作用底物釋放聚合度2-6幾丁寡糖的內(nèi)切幾丁質(zhì)酶,ChiC為持續(xù)作用底物生成幾丁二糖的外切幾丁質(zhì)酶。掃描電子顯微鏡觀察表明CBP58對幾丁質(zhì)多糖鏈產(chǎn)生剝離和松解等破壞作用。底物結(jié)合實(shí)驗(yàn)表明CBP58對α-chitin和collodial chitin結(jié)合能力最強(qiáng),β-chitin及其nano-whiskers次之,對icel具有較弱結(jié)合能力。海洋耐冷細(xì)菌Pseudoalteromonas sp.DL-6幾丁質(zhì)酶系統(tǒng)通過協(xié)同降解機(jī)制作用幾丁質(zhì),不僅為生物轉(zhuǎn)化生產(chǎn)幾丁寡糖提供了新途徑,也有助于進(jìn)一步闡明海洋環(huán)境幾丁質(zhì)代謝機(jī)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，

本文編號：2281574