【作者】 董学卫；

【导师】 朱启忠；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 论文首先从液体培养体系出发,探索不同培养条件对白毒鹅膏菌分泌漆酶的影响。我们筛选出白毒鹅膏菌的综合产酶培养基为（g/L）:马铃薯200 g/L、麸皮20 g/L、KH₂PO₄ 3g/L、MgSO₄ 1.5g/L、V_B10.01 g/L、酵母膏5 g/L。其最佳pH值范围为5.0-5.4,转速为120 rpm,温度为25℃,装液量为50 mL。按此条件进行培养,漆酶从第5d酶活力迅速提升,第8d达产酶高峰,随后产酶能力下降。实验通过盐析、透析、DEAE-纤维素DE52离子交换层析和Sephedex G-100凝胶过滤层析等步骤,分离纯化得到电泳纯的漆酶。以邻联甲苯胺作为底物,最终漆酶被纯化了22.03倍,活力回收率为22.02%。通过SDS-聚丙烯酰胺凝胶电泳检测,漆酶的相对分子量为63kDa。该酶具有较宽的pH值稳定性,在pH 3.6到5.2范围内能够较好地保持酶活力;该漆酶最适反应温度为20℃,在20℃到60℃范围内能够稳定的保持较长时间,但在70℃时保持5 min,且能保持20%的活力。β-巯基乙醇或L-半胱氨酸是该酶的高效抑制剂,但是EDTA或DMSO对其抑制作用不是很强。白毒鹅膏菌漆酶催化反应符合Miehaelis-Menten动力学规律。在20℃,pH 4.6的醋酸缓冲溶液中,漆酶以邻联甲苯胺作为底物时,米氏常数为66.7μmol/L。潜在的抑制剂测试中,L-半胱氨酸及2-巯基乙醇在所试浓度范围内均完全抑制漆酶的活力,SDS高浓度时对漆酶的活力高度抑制,EDTA和DMSO在所试浓度范围内对漆酶的活力微弱抑制。利用白毒鹅膏菌漆酶可以较为彻底的降解6种染料,底物范围较广,在降解过程中,要持续通氧气（振荡）。直接黑G降解的最适pH值为5.2,而中性深黄GL降解的最适pH值为3.5。两种染料与白毒鹅膏菌漆酶作用的最适温度均为50℃。底物染料浓度为20mg/mL时,酶活力超过10 U/mL后,进一步提高酶活力对r_D基本无影响,中性深黄GL总脱色率维持在80%左右,直接黑G约维持在60%。采用海藻酸钠包埋法和海藻酸钠-壳聚糖包埋-交联法固定化漆酶。探讨了固定化条件、固定化漆酶及游离酶的酶学性质。结果表明,包埋法和包埋-交联法固定化漆酶的最佳条件分别为海藻酸钠浓度3%、CaCl₂浓度1.5%和海藻酸钠浓度2%、CaCl₂浓度2%、壳聚糖浓度1.5%、戊二醛浓度1%。两种固定化漆酶的最适pH和最适温度相同,分别为5.0和30℃,游离酶为4.6和20℃。将固定化酶应用在偶氮染料的脱色中,包埋法脱色效果接近游离酶并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。与包埋法相比,包埋-交联法固定化漆酶机械强度更强,操作稳定性更好,使得固定化酶的重复利用率更高,但应用于染料脱色时,由于壳聚糖的吸附作用使得漆酶对于染料的降解作用降低,因此制备应用于染料降解的固定化漆酶时还应对辅料进行优化。更多还原

【Abstract】 Firstly,In the liquid culture system,the influence on the production of laccase by Amanita verna in different fermentation conditions has been studied.Through a series of orthogonal experiments,effects of carbon source,nitrogen source and other factors on laccase production from strain Amanita verna were studied.The liquid medium used for laccase production contained（per liter）200g potato extract with 20g dextrose, 5g yeast extract,20g bran,3g KH₂PO₄,1.5g MgSO₄,pH5.0-5.4.Shaking cultivation was performed at 25℃on a rotary shaker at 120 rpm.Under the optimized fermentation process,the laccase production by strain Amanita verna was increased from the 5th day,reached the highest production on the 8th day.The laccase was purified by a three-step procedure involving salting out,ion exchange,size exclusion chromatography.A typical procedure provided 22.03-fold purification,with a yield of 22.02%,using o-tolidine as substrate.The molecular weight of the purified laccase was 63 kDa as estimated by 12%（w/v）SDS-PAGE gel.The enzyme’s pH optimum for o-tolidine was 4.6 and optimal activity was at 20℃.After pre-incubation at different pH values for 3.5h and different temperatures for 70 min,more than 60%of the initial laccase activity was retained between pH 3.6 to 5.2 and a relative activity of 19.3%was observed at 60℃.The Km value of laccase for o-Tolidine was 66.7μmol/L at pH4.6.The effect of different chemicals on laccase activity was also tested.This laccase was strongly inhibited by L-cysteine,β-mercaptoethanol even at a low concentration,and slightly inhibited by EDTA.The combination of thermotolerance with low pH optima for aromatic compounds substrates suggests that the laccase possesses potential for use in biotechnological applications.The laccase from Amanita verna could degradate 6 dyes efficiently under keep on ventilating condition.The laccase showed maximal decolorization rates of both dyes at 50℃and optimal decolorization rates at pH 5.5 and 3.5 for direct black G and neutral dark yellow GL,respectively.The higher the pollutant primary concentration and laccase activity,the higher decoloration rate,when the primary concentration and laccase activity were below 20mg/L and 10 U/mL,respectively.The decoloration rate of direct black G was 80%,while it was 60%to neutral dark yellow GL.Laccase was immobilized by the sodium alginate entrapment method and the sodium alginate-chitosan entrapment-crosslinking method.The immobilization conditions and the characterizations of the immobilized and free laccase were investigated.The results showed that the optimal conditions of immobilized laccase by the entrapment method and the entrapment-crosslinking method were 3%sodium alginate,1.5%CaCl₂ and 2%sodium alginate,2%CaCl₂,1.5%chitosan,1% glutaradehyde,respectively.These two kinds of immobilized laccase exhibited the same optimal pH value and optimal action temperature which were 5.0 and 30℃, however the free enzyme’s optimal pH value and optimal action temperature were 4.6 and 20℃.The ability of decolourizing azo dyes in shaky situation by sodium alginate-immobilized laccase approached to the free enzyme,and there was no loss of enzyme activity during 2 repeated batch reactions.Compare with entrapment method, entrapment-crosslinking method has a better mechanical strength and operation stability,but the decolorization rate was low because of chitosan’ adsorption,so we should optimize accessories of entrapment-crosslinking immobilized laccase used for dye degradation.更多还原