【作者】 王越；

【导师】 张苓花；

【作者基本信息】 大连海事大学 ， 环境科学， 2011， 博士

【摘要】 环形氨基酸Ectoine (1,4,5,6-四氢-2-甲基-4-嘧啶羧酸)是由嗜盐菌在高渗透压诱导下合成的一种具有两性离子特性的渗透压补偿性溶质,对蛋白质、核酸、细胞等在逆环境下具有保护作用。本论文以植酸酶和脂肪酶为酶蛋白,从发酵制备、对酶的保护应用和作用机理等方面对Ectoine进行了研究。从盐池淤泥中筛选了一株中度嗜盐菌B2。细胞抽提物中经1 H-NMR鉴定含有Ectoine。菌株经16S rDNA鉴定属于盐单胞菌属(Halomonas sp.)。在含2mol/L NaCl的谷氨酸单钠培养基中发酵合成Ectoine最大量达到3.5g/L。通过与其他补偿性溶质比较,考察了Ectoine对植酸酶(PHYAⅡ)的热稳定性保护作用。研究了Ectoine的添加量、保护方式及对PHYAⅡ高温水解的影响。实验结果表明,Ectoine作为PHYAⅡ的热稳定性保护剂,用量最少,保护作用好。利用PHYAⅡ失活热力学和动力学分析,结合紫外和荧光光谱技术,推测Ectoine提高了酶热稳定性的作用机理。结果表明,Ectoine可以提高酶分子ΔU*、ΔH*、ΔG*和t1/2,降低失活速率常数k；荧光、紫外吸收光谱表明Ectoine对酶热处理后的肽链伸展不利。因此推测Ectoine的添加主要导致酶内部或酶与外界水介质的氢键发生变化,从而有利于酶热稳定性增加。以转酯合成生物柴油和酯化合成油酸乙酯为反应体系,考察Ectoine对Novozym435和Aspergillus oryzae DM-01全细胞脂肪酶在有机相中酶活性的影响。结果表明,作为底物的甲醇和乙醇在一定浓度下对脂肪酶具有抑制作用。添加适量Ectoine，在脂肪酶一次或反复使用中,均对脂肪酶有显著保护作用。和同质量的其他补偿性溶质比较,Ectoine保护效果最好。添加过量Ectoine则会抑制酶活性。利用傅立叶变换红外光谱技术(FTIR),结合酶催化动力学分析,探讨Ectoine提高脂肪酶在甲(乙)醇和油酸中活性的保护机理。对Novozym435的红外原始图谱和二级结构分析表明,适量Ectoine会降低甲醇对酶的氢键缔合作用,通过增加β-折叠结构来提高酶稳定性,酶催化动力学也表明适量Ectoine会减小甲醇对酶的亲和力。对A. oryzae DM-01脂肪酶红外光谱分析表明,油酸和无水乙醇都对酶构象有影响,其中油酸使酶的分子内氢键明显减弱,对酶构象影响较大。添加Ectoine,使酶的二级结构含量变化趋势减小,会更接近自然状态的酶。更多还原

【Abstract】 The cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), one of the representative compatible solutes with the characteristic of zwitterions, accumulates in halophilic and halotolerant bacteria. Previous studies indicated that ectoine functions on proteins, nucleic acids and cells as a stabilizer against some adverse conditions. The produciton of ectoine and its bioprotective application and mechanism were studied in the paper using phytase and lipase as model protein.A moderately halophilic bacterium B2 was isolated from silt in salt lake.’H-NMR analysis was used to identify ectoine in cell extract. Based on the analysis of 16S rDNA, strain was identified as Halomonas (Halomonas sp.). When B2 was cultivated in MG medium with 2 mol/L NaCl, the maximum ectoine concentration was 3.5 g/LThe protective effects of ectoine on thermal stability of phytase (PHYAⅡwas investigated by comparing with other compatible solutes. The concentration, protective mode and effect on pyrohydrolysis of ectoine were studied. The result showed that ectoine had a highest thermoprotection efficiency for PHYAⅡas thermalstability protective additive.According the analyses of thermodynamics and kinetics of heat inactivation of PHYA II, combining with UV and fluorescence spectra, the mechanism of improving enzyme thermal stability by ectoine was speculated. The result indicated that the addition of ectoine improved△U*,△H*,△G* and t1/2, as well as reduced constant k of inactivation rate. The fluorescence and UV spectra showed that ectoine was disadvantaged for stretching of peptide chain after enzyme heat treatment. Therefore, it was suggested that addition of ectoine mainly led to change of hydrogen bond either inside enzyme or between enzyme and water in media, whereas benefit for increasing of the thermal stability of enzymes.Effect of ectoine on Novozym435 and whole cell lipase from Aspergillus oryzae DM-01 were investigated using transesterification for biodiesel and esterification for aethylis oleas as model reactions in organic media. The result indicated lipase would be inhibited by excess metnanol (alcohol) as substrates. Ectoine exhibited significant protection on enzymatic activity in once or reuse. Compared with other compatible solutes, ectoine showed the best protection. However, excess ectoine would result in inhibitting enzymatic activity.Using Fourier transform infrared spectroscopy (FTIR), combined with enzymatic kinetic analysis, the mechanism of improving activities of lipase in methanol (ethanol) and oleic acid by additine of Ectoine was investgated. Original infrared spectra and secondary structure analysis of Novozym435 showed that optimal ectoine could reduce the hydrogen bond association of methanol on enzyme and increase stability of enzyme by increasingβ-sheet structure. In addition, enzyme catalysis kinetics also shows that supplementation with an appropriate amount of ectoine led to a decrease in the affinity of lipase for methanol. The infrared spectrum of A. oryzae DM-01 lipase showed that oleic acid and absolute ethanol had an effect on enzyme conformation, in which oleic acid significantly decreased intramolecular hydrogen bonding to impact on enzyme conformation on greater degree. Addition of ectoine reduced the change trend of secondary structure, which closed to the natural conformation of the enzyme.更多还原