【作者】 张宽朝；

【导师】 林毅；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2009， 硕士

【摘要】 霍山石斛(Dendrobium huoshanense C. Z. Tang et S. J. Cheng)产于安徽省霍山县,是国家重点保护的传统名贵药用植物;其富含石斛多糖和生物碱等多种生物活性成分,具有益胃生津、滋阴清热、抗衰老、提高机体免疫力等功效。但由于霍山石斛中生物碱含量普遍较低,无法满足市场的需求,因此,加强霍山石斛次生产物合成调控,提高生物碱含量,促进药用品质改善,成为霍山石斛研究中的重点。苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL,EC4.3.1.5) ,是苯丙烷类代谢途径的关键酶和限速酶,其主要催化L-苯丙氨酸脱氨生成反式肉桂酸,可为多种酚类、生物碱、类黄酮等终产物提供前体物质。已有研究表明石斛生物碱含量与PAL活性有关,PAL可能是石斛属植物生物碱合成的关键因素之一。研究霍山石斛PAL可以为进一步研究PAL在生物碱调控中的作用机理提供基础,还可以为常规育种或生物技术育种创造高含量生物碱的霍山石斛提供基础。本研究以霍山石斛组织培养试管苗为材料,进行了PAL的分离纯化、基本酶学性质和外界影响因子的研究。主要研究结果如下:采用35%-75%饱和硫酸铵分级沉淀、透析袋脱盐、PEG20000浓缩、DE-52阴离子交换层析和Sephadex G-150凝胶层析对PAL进行分离纯化,得到了分离纯化的PAL,蛋白质得率为1.29%,酶的得率为10.38%,纯化倍数为8。样品于SDS-聚丙烯酞胺凝胶电泳检验为单一蛋白带,说明得到了PAL纯品。SDS-PAGE测量该酶亚基分子量时,考马斯亮蓝R-250染色后凝胶上仅有一条带,说明该酶各亚基大小相等;由该带的相对迁移率和由标准蛋白质迁移率所得的标准曲线得到该酶的亚基分子量约为50KD。因大多数植物PAL由4个相同大小的亚基构成,推测该酶的分子量约为200KD。纯化后的霍山石斛PAL,于210-340nm波长下进行波长扫描发现,在220nm有肽键的强吸收,说明可能有共轭体系的存在;在280nm附近处(284nm)有吸收峰,说明酶分子中存在含苯基侧链的氨基酸残基。PAL反应进程曲线表明,在3h以内,反应产物与反应时间基本成线性关系。PAL在硼酸缓冲液中最适pH值为pH8.4,在pH8.2-8.6之间有较高的稳定性,用pH4.0和pH10.0的缓冲液处理,均会使酶分子结构发生变化,导致酶活性降低。PAL最适反应温度是45℃,而且具有较好的耐热性,60℃处理20min其残存活性仍保持在50%以上。随着底物L-苯丙氨酸浓度的逐渐增大,酶的活性也逐渐增大,最适底物浓度为2mmol/L。以D-苯丙氨酸、L-酪氨酸代替L-苯丙氨酸作为霍山石斛PAL反应底物,L-Phe作为反应底物与D-Phe及L-Tyr作为反应底物对PAL活性影响的差异达到极显著水平,差异达到1%极显著水平。而随着酶液浓度的增加,反应速度也随之增加,当酶液量达到一定时,反应速度突然加快,当达到更高浓度之后反应速度将减缓。用Lineweaver-Burk法进行双倒数作图,计算得两个Km值分别为:Km1为0.4425×10-3mol/L,Km2为0.847×10-4mol/L,贮存实验证明该酶最好在-20℃下用甘油贮存。重金属AgNO3,HgCl2都能不同程度地抑制酶活性,且两者对PAL活性的抑制均达到极显著水平。EDTA、EDTA-Na2对PAL都有较强的抑制作用,但与对照相比,EDTA-Na2处理差异不显著,而EDTA差异极显著。AlCl3、ZnSO4、MnSO4、MgSO4都不同程度地促进PAL的活性,但与对照相比差异均未达到极显著,最佳处理效果为AlCl3>ZnSO4>MgSO4>MnSO4;在供试浓度范围内,最佳处理浓度为AlCl3 1mmol/L、ZnSO4 1mmol/L、MgSO4 2mmol/L、MnSO4 1mmol/L。而CuSO4、CaCl2(0.5mmol/L除外)都不同程度地抑制酶的活性,与对照相比CuSO4差异极显著,CaCl2处理差异不显著。Al3+盐不同形态对PAL酶活性影响不同,AlCl3、Al2(S04)3均能促进PAL酶活性升高,且与对照相比,均达5%显著水平,但AlCl3的促进作用优于Al2(S04)3;Al(N03)3处理则抑制PAL活性,但与对照相比,未达显著水平。L-酪氨酸、L-组氨酸、L-胱氨酸、L-半胱氨酸、D-苯丙氨酸均是霍山石斛PAL的抑制剂,与对照相比均达到极显著水平,而且L-胱氨酸抑制程度随抑制剂浓度增加而增强。更多还原

【Abstract】 The herb of Dendrobium huoshanense is a kind of precious traditional Chinese medicinal materials in Anhui Province and is protected by government for its medicinal value.Because it has affluent amylose and alkaloids,it can diminish inflammation,clear the sight and improve the immunity of human body.But because of its low alkaloids content,it has been unable to meet the needs of the market.So strengthening the secondary metabolites’s synthesize control,increasing alkaloids content and accelerating the improvement of medicinal value become emphases in Dendrobium’s study.Phenylalanine ammonia-lyase (PAL,EC4.3.1.5) mostly catalyzes the spontaneous nonoxidative deamination of L-phenylalanine(L-Phe) to trans-Cinnamic acid, which provides precursors for alkaloids,phenolics,flavanoids and isoflavanoids.PAL is the key enzyme and rate-limit enzyme in phenylpropanoid pathway.Some researches indicated that the differentiation of Dendrobium’s alkaloids content related to PAL activity.PAL may be the key factor for the synthesize of Dendrobium’s alkaloids.Study on D. huoshanense PAL and its properties can interpret the function of PAL in synthesize of alkaloids,and can lay the base for artificially creating higher alkaloids content D. huoshanense by routine breeding or genetic engineering.In this research,PAL was isolated and purified from D. huoshanense and the characterization of the PAL and its outer factors was studied.The main results were as followes:1.After series isolating and purifying process of precipitation between 35% and 75% ammonium sulfate saturation, dialysis, concentration by PEG20000,DE-52 anion- exchange chromatography and Sephadex G-150 filtration column,the recovery of protein was 1.29%,and that of PAL was 10.38%, and the purification multiple was 8.2.The molecular weight of the subunit was measured by SDS-PAGE, and one protein band was observed, which proved that the PAL was purified to homogeneity.The molecular weight of the subunit was measured was 50KD by the Rf of the subunit according to the standard line obtained from the Rf of the standard proteins. And it was reported that PAL was made up of four subunits in the same size,so it was estimated that the molecular weight of D. huoshanense PAL was about 200 KD.3.PAL,which was purified,had strong absorbance at 220nm and 284nm by UV, speculating that it has conjugate structure and contain amino acid groups of phenyl-containing. 4.The chart of PAL reaction showed that the output and reaction time was linear in 3h.Its optimum pH was 8.4 in boracic acid buffer and PAL can remain stable in pH8.2-8.6. After being treated with pH4.0 and pH10.0 buffer,the PAL activities fell because of the molecular structure of enzyme changed.The optimum temperature of PAL was 45℃and the PAL can be able to bear higher temperature,its activity remained about 50% under 20 min at 60℃.Along with the augment of L-Phe’s concentration,the activity of PAL was increased gradually,and the optimum substrate concentration was 2mmol/L. When L-Phe was replaced by D-Phe or L-Tyr as substance on PAL activity,the effects between L-Phe and D-Phe or L-Tyr were extremely significant.While along with the augment of PAL’s concentration,the reaction rate was increased gradually,and when achieving stated concentration the reaction rate quickened abruptly but was slowdown after achieving a higher concentration.By the Lineweaver-Burk plot for Km,there are two Km values:Km1=0.4425×10-3mol/L, Km2= 0.847×10-4mol/L.PAL had better be reserved in low temperature with glycerol.5.Heavy metal salts AgNO3 and HgCl2 all inhibited PAL activities in different degree,and compared with contral the effects were extremely significant. EDTA and EDTA-Na2 inhibited PAL activities,but compared with contral the effect of EDTA was extremely significant while EDTA-Na2 was not. AlCl3,ZnSO4,MnSO4 and MgSO4 enhanced the PAL activities in diferent degree and these effect were not obvious compared with contral.The effect ranked was AlCl3>ZnSO4>MgSO4>MnSO4 and the most effective concentrations in the present experiment were as followed: AlCl3 1mmol/L,ZnSO4 1mmol/L,MgSO4 2mmol/L and MnSO4 1mmol/L.However, CuSO4 and CaCl2 (except 0.5mmol/L) inhibited the PAL activities.Compared with contral the effect of CuSO4 was extremely significant but was not obvious for diferent concentrations CaCl2. The effects of different Al3+ salts on PAL activities were different. AlCl3 and Al2(S04)3 enhanced the PAL activities and the effects were significant, while AlCl3’effects were higher than Al2(S04)3’.Al(N03)3 inhibited PAL activities, and the effect was not obvious.6.L-Tyr,L-His,L-cystine,L-Cys and D-Phe were all PAL’inhibitor,and the effects were extremely significant compared with contral.And the results also showed that the inhibitory effects increased with the rise in the concentration of the L-cystine.更多还原