【作者】 张岩；

【导师】 田健； 李俊华；

【作者基本信息】 河北农业大学 ， 工程硕士（专业学位）， 2020， 硕士

【摘要】 几丁质作为目前已知天然可再生产物中含量仅低于纤维素位列第二的多糖类物质,其降解后生成的寡糖在现代食品工业、农业,生物医学等众多行业均具有重要的价值。几丁质酶作为一种特异性降解几丁质的糖苷水解酶,能识别并有效断裂β-1,4-糖苷键,从而实现通过大分子多糖生产寡糖产品。本研究针对目前行业中几丁质酶热稳定性较差以及酶活力偏低等问题,通过在海洋宏基因组文库中筛选出9个几丁质酶基因,然后根据酶活力和蛋白表达量,对最优的几丁质酶Chi304进行酶学性质检测,并通过基因共识设计单点突变,成功提高其最适温度及稳定性。具体结果如下:1.通过生物信息学手段在海洋宏基因组文库中筛出9个最有潜力的几丁质酶基因,经过密码子优化后使用EcoRI、XhoI将基因构建在pET-30a(+)载体上,并在大肠杆菌BL21中表达蛋白,通过DNS法酶活力检测和SDS-PAGE电泳分析,最终确定Chi304具有较高酶活力和蛋白表达量,分子量大小为71kDa。2.进一步测定Chi304的酶学基本性质,发现该酶在85℃和pH值9.0的环境中能够发挥最大酶活力,且在90℃下保温1h剩余酶活仍高达50%,80℃处理6h活力仍可保持约100%,具有极高热稳定性。弱碱性环境中处理1 h对酶活力基本没有影响,是目前发现的碱性条件下热稳定性最好的酶。但Chi304对金属离子比较敏感,常见二价金属离子无法促进其活力提高,其中Cd2+、Fe2+严重抑制了其活性。Chi304动力学参数Km,kcat 和 Vmax 分别为 0.48±0.07 mg·mL-1、764.60±19.42 min-1、4.92μmol·min-1·mL-1。转化常数kcat/Km为 1579.75±268.60 mL·mg-1·s-1。3.通过TLC、HPLC、SEM和MALDI-TOF检测产物研究Chi304底物降解机理发现,Chi304同时具有外切型、内切型几丁质酶活力及N-乙酰葡萄糖苷酶活力,可将几丁质分解为稀疏多孔结构,产物主要为(GlcNAc)2和少量GlcNAc、(GlcNAc)3。另外降解产物对黄单胞菌和枯草芽孢杆菌的生长具有明显抑制作用。4.通过基因共识(Consensus)对Chi304进行单点突变,结果发现10个突变位点中421位天冬氨酸(D)突变为谷氨酸(E)后,最适温度增加至90℃;在90℃下热处理30 min其剩余酶活仍高于Chi304,kcat/Km转化常数相比野生型提高30%。而酶催化活力、最适pH值和pH值稳定性并没有明显改变,这在一定程度上也说明基因共识设计主要有助于蛋白分子的稳定性提高。本研究利用生物信息学方法在海洋宏基因组中定向筛选到热稳定性极佳的几丁质酶基因,并通过基因共识设计成功提高了该酶最适温度及温度稳定性。从而为今后几丁质酶筛选提供了新思路,并为Chi304在今后食品工业中的应用奠定了基础。更多还原

【Abstract】 Chitin,as the second most abundant polysaccharide in the known natural renewable products,is only lower than cellulose.Oligosaccharides produced by the degradation of chitin have important value in the modern food industry,agriculture,biomedicine,and many other industries.As a specific glycoside hydrolase that degrades chitin,chitinase can recognize and effectively break the β-1,4-glycoside bond,thus realizing the production of oligosaccharide products by polysaccharides.In this study,aiming at the poor thermal stability and low enzyme activity of chitinase in the current industry,nine chitinase genes were screened out from marine metagenomic libraries,and then the best property of chitinase Chi304 was tested about its enzymatic properties,and some single point mutations were designed by genetic consensus to successfully improve optimal temperature and stability of chitinase.The results are as follows:1.Nine of most potential chitinase genes were screened out from marine metagenomic libraries by bioinformatics.After codon optimization,the genes were constructed on the pET-30a(+)vector using EcoRI and XhoI,and the proteins were expressed in E.coli BL21.Through DNS enzyme activity detection and SDS-PAGE electrophoresis analysis,it was finally determined that Chi304 had high enzyme activity and protein expression.The molecular weight of Chi304 is 71 kDa.2.After further testing,the optimum temperature and pH value of chi304 were 85℃and 9.0,respectively.It was worth noting that the residual enzyme activity remains 50%after heat treatment at 90℃ for 1 h and 100%after treatment at 80℃ for 6 h.This indicates that chi304 has very high thermal stability.Treatment in a weakly alkaline environment for 1 hour will hardly affect the activity of the enzyme.At present,Chi304 is the enzyme with the best thermal stability under alkaline conditions.However,Chi304 is sensitive to metal ions,and ordinary divalent metal ions cannot promote its activity.For example,Cd2+and Fe2+severely inhibit its activity.The kinetic parameters Km,Kcat,and Vmax of Chi304 were 0.48±0.07 mg·mL-1、764.60±19.42 min-1、4.92 μmol·min-1·mL-1,respectively.The conversion constant kcat/Km value was 1579.75±268.60 mL·mg-1·s-1.3.Through the detection of TLC,HPLC,SEM,and MALDI-TOF,the degradation mechanism of Chi304 on the substrate was studied,and it was found that Chi304 has both exo-and endo-chitinase activity and N-acetylglucosidase activity,which can reduce chitin Decompose into a sparse porous structure.The products are mainly(GlcNAc)2 and a small amount of GlcNAc,(GlcNAc)3.In addition,the product has a significant inhibitory effect on the growth of Xanthomonas and Bacillus subtilis.4.The Chi304 single point mutation was constructed by consensus design.It was found that the optimum temperature of D421E rose to 90℃ among the 10 mutants.After heat treatment at 90℃ for 30 minutes,the residual enzyme activity was still higher than Chi304,and the kcat/Km value is 30%higher than that of the wild type.However,the catalytic activity of the enzyme,the optimal pH value,and pH stability did not change significantly,which also indicated that the consensus design mainly helps to improve the stability of proteins.In this study,bioinformatics methods were used to screen the chitinase genes with excellent thermal stability in the marine genome.The optimal temperature and temperature stability of chitinase were successfully improved through consensus design.This provides a new idea for future chitinase screening and lays the foundation for the future application of Chi304 in the food industry.更多还原