【作者】 刘志钰；

【导师】 堵国成；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 博士

【摘要】 漆酶(p-diphenol:dioxygen oxidoreductase; EC 1.10.3.2)属于一种含铜的多酚氧化酶,可以催化氧化多种酚类、氨基酚类、多胺类化合物、木质素和金属有机化合物等。由于其广泛的底物特异性,漆酶在食品、纺织、造纸、化工和环境等行业中的应用已吸引了越来越多的关注。然而,多数漆酶发酵周期较长、产酶效率较低,限制了漆酶的工业化应用。国内外对真菌漆酶的研究历史较长,但绝大多数研究仍处在摇瓶发酵水平。同时,对真菌漆酶在细胞中的分布、功能及分泌机制等尚不完全清楚。大量漆酶生产菌仍有待开发研究,以加强对真菌漆酶的生理功能、分泌特性及分泌机制的了解,为解决当前漆酶发酵过程中存在的发酵周期长、漆酶活性较低等问题奠定基础。本研究从土壤中筛选获得一株产漆酶的拟青霉菌株,应用诱变育种提高产漆酶能力并获得优良的漆酶酶学特性,同时,探讨了漆酶的分泌特性和发酵调控方法等,并对拟青霉漆酶进行分离纯化及酶学性质研究。其主要研究内容及结果如下:1.通过平板初筛和摇瓶复筛,从各种富含木质素的样品中筛选得到一株产漆酶菌株S2。根据菌株的培养特征、菌落和孢子形态并结合ITS rDNA片段序列比对以及进化树分析,将该菌株鉴定为拟青霉属(Paecilomyces sp.),并命名为Paecilomyces sp. WSH-L07。其最适产酶条件为:初始pH 6.5,装液量100 ml/250 ml摇瓶,30℃,150 r/min振荡培养。在最适产酶条件下,培养8天,漆酶酶活达到820 U/l。在Paecilomyces sp. WSH-L07漆酶发酵培养过程中,只有添加铜离子后才能检测到Paecilomyces sp. WSH-L07漆酶酶活。当以CuSO4和亚甲基蓝为复合诱导物时,可进一步提高Paecilomyces sp. WSH-L07漆酶酶活。铜离子和亚甲基蓝的最适添加浓度分别为50和20μmol/l,最佳添加时间分别为24和12 h,在此条件下,Paecilomyces sp. WSH-L07漆酶酶活可达1650 U/l。2.以Paecilomyces sp. WSH-L07为出发菌株,经低能氮离子注入后,筛选得到一株遗传性能稳定的突变株S152,其产漆酶能力与野生菌株WSH-L07相比提高了3倍以上。通过比较野生菌株和突变菌株漆酶的酶学性质,发现两种来源的漆酶均以2,2’-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)(简称ABTS)和愈创木酚为底物时催化反应效率最高,最适反应pH分别为3.4和5.0,但突变株S152所分泌的漆酶具有更宽的活性pH范围;野生菌株所分泌漆酶最适反应温度为55℃,当温度高于55℃时快速失活,而来源于突变株的漆酶所分泌的漆酶最适反应温度保持在60~65℃间;在中性和碱性环境中,突变株所分泌的漆酶更加稳定,同时,热稳定性也有一定程度的改善;除NaN3外,野生菌株WSH-L07和突变株S152对金属离子和低浓度的抑制剂均具有良好的耐受性。漆酶酶谱分析表明两种来源的漆酶所分泌的同工酶种类和分泌比例相同,包括三条ABTS活性带(Lac 1,Lac 2及Lac 3)和两条愈创木酚活性带(Lac 1,Lac 3)。3.考察了不同碳氮源对Paecilomyces sp. S152漆酶分泌的影响。当以20 g/l的麦芽糖、10 g/l的大豆蛋白胨和1 g/l的酵母粉为碳氮源时,Paecilomyces sp. S152漆酶酶活最高,为5064 U/l。Paecilomyces sp. S152漆酶酶谱分析表明,碳氮源种类对Paecilomyces sp. S152漆酶同工酶种类无影响,但可调节不同同工酶的分泌比例。利用正交试验和多元线性回归,确定在突变菌株Paecilomyces sp. S152发酵培养过程中复合诱导物最适添加条件为:发酵11 h和12 h后,分别添加15.4μmol/l亚甲基蓝溶液和76μmol/l CuSO4溶液,在此条件下发酵7天后漆酶酶活为5730±150 U/l;在发酵过程中,亚甲基蓝可被Paecilomyces sp. S152漆酶有效降解。复合诱导物对Paecilomyces sp. S152漆酶分泌的促进作用推测与在恶劣环境下微生物的自我防御能力相关,亚甲基蓝对细胞的毒害作用促使细胞大量分泌漆酶以用于降解环境中的亚甲基蓝。在Paecilomyces sp. S152细胞内和细胞外均有漆酶分布,添加1.5 g/l的Tween 80可促进胞内漆酶的释放、提高胞外漆酶酶活、缩短发酵周期(6天),生产强度提高至946 U/l/d。4.根据不同培养温度下Paecilomyces sp. S152发酵过程和动力学分析,提出两阶段变温培养策略,即在培养初期控制温度35℃,发酵92 h后控制温度为30℃。结果表明,采用两阶段变温培养策略后,Paecilomyces sp. S152漆酶的比产酶速率在整个发酵阶段均保持在较高水平,漆酶酶活与在恒温30℃和35℃培养下相比,分别提高了45%和117%,同时,Paecilomyces sp. S152漆酶的生产强度和葡萄糖转换率也均得到改善。两阶段变温培养策略提高漆酶酶活的机理推测是:在发酵培养初期,采用较高的培养温度有利于细胞的生长代谢、降低底物抑制作用,同时改善了细胞通透性,促进胞内漆酶分泌至培养环境中;实验表明蛋白酶可能参与了Paecilomyces sp. S152漆酶蛋白的活化过程,在发酵培养中后期,降低培养温度后,蛋白酶酶活保持在较高水平,维持了漆酶蛋白的活化,使得漆酶酶活随培养时间继续上升。5.通过乙醇沉淀、Q-Sepharose HP阴离子交换色谱及Superdex 75凝胶过滤层析,获得电泳纯的Paecilomyces sp. S152漆酶同工酶Lac 1和Lac 3,比酶活分别为196.75和327.43 U/mg,纯化倍数分别为19.95和33.21,回收率分别为19%和27.66%。Lac 1和Lac 3表观分子量约为79 kDa。与常见蓝色漆酶不同,Lac 1和Lac 3不具有蓝铜吸收谱带,纯化后的蛋白呈浅黄色,与某些真菌所分泌的黄色漆酶性质相似。催化动力学分析表明,在以ABTS和愈创木酚为底物时,Lac 3与底物的亲和性和催化反应速率均优于Lac 1。同时,分离纯化得到电泳纯的Paecilomyces sp. S152蛋白酶,其分子量约为38 kDa。该蛋白酶属于丝氨酸蛋白酶家族,其活性可被丝氨酸蛋白酶抑制剂PMSF强烈抑制。在无细胞体系中,添加PMSF后,漆酶蛋白的活化作用减弱;同时,在发酵培养过程中,添加1 mmol/l PMSF导致蛋白酶酶活和漆酶酶活均显著下降。以上结果表明蛋白酶的存在有利于提高Paecilomyces sp. S152漆酶酶活,抑制蛋白酶活性,将阻碍漆酶蛋白的活化过程。更多还原

【Abstract】 Laccases (p-diphenol: dioxygen oxidoreductase; EC 1.10.3.2) belong to the group of copper-containing polyphenol oxidases, which catalyze the oxidation of a wide range of polyphenols, aminophenols, polyamines, lignin and some inorganic ions. Laccases have attracted increasing attention in the recent years due to their potential application in diverse industrial sectors such as in food, textile and pulp industries; however, industrial applications of laccases are usually hindered by their long fermentation period and low laccase yield. Although fungal laccase has been extensively investigated for decades, the production of laccase was still performed mostly in flask scale. To improve laccase productivity, screening of new laccase producing strains for further understanding of laccase location in cells, the function of laccase and the laccase-secretion properties were necessary.In this study, a novel laccase-producing strain was isolated from soil and was identified as Paecilomyces species. An novel approach of microbial breeding was employed to improve laccase production and enzyme properties. The laccase-secretion characteristics of the mutant and the laccase fermentation control strategies were investigated. Besides, the purification and properties of laccase from Paecilomyces species were also investigated. The main results are as follows:1. A novel laccase-producing strain was isolated from a lignin-containing sample by selective plates and flask fermentation, and the newly isolated strain was identified as Paecilomyces sp. WSH-L07 according to the morphological characteristics and analysis of internal transcribed spacer (ITS) rDNA gene sequences. The optimal fermentation parameters for enhanced laccase secretion were found to be: 250 ml flasks containing 100 ml medium (initial pH 6.5) and cultivated at 30°C and 150 r/min for 8 days. Under the optimal fermentation conditions, a maximal laccase activity of 820 U/l was obtained. Moreover, detectable laccase was only found in the copper-supplemented medium, suggesting copper was essential for laccase production by Paecilomyces sp. WSH-L07. Further enhanced laccase production was achieved by complex inducers methylene blue and CuSO4. The addition of 20μmol/l methylene blue and 50μmol/l CuSO4 after cultivation for 12 and 24 h, respectively, gave the maximum laccase production with an activity of 1650 U/l.2. Low-energy ion implantation was employed to breed laccase producing strain Paecilomyces sp. WSH-L07, and a genetically stable mutant S152 was obtained with four times laccase activity compared with that of the wild strain. The optimum substrate of both the wild and mutant laccases was 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and followed by guaiacol with optimal pH at 3.4 and 5.0, respectively, while the mutant laccase exhibited a broader active pH range. The mutant laccase had a higher optimal catalytic temperature (60oC to 65oC) than the wild one (55oC), and the wild laccase deactivated rapidly when temperature increased above 55oC. Furthermore, the mutant laccase was more stable under neutral and alkaline conditions. A thermostability experiment revealed that the mutant laccase was superior to wild laccase. Both the wild and mutant laccases were almost not affected by the tested metal ions, and mildly inhibited by SDS (0.5 mmol/l), EDTA (1 mmol/l) and DTT (0.5 mmol/l). The activities were almost completely inhibited by 0.1 mmol/l NaN3. The zymogram analysis of laccase from the mutant S152 indicates that the activity profiles obtained were similar to that from the wild strain with at least three ABTS-active bands (Lac 1, Lac 2 and Lac 3) but only two guaiacol-active bands (Lac 1 and Lac 3).3. The culture medium containing maltose (20 g/l), bean peptone (10 g/l) and yeast extract (1 g/l) was found to favor laccase production by Paecilomyces sp. S152 with an enzyme activity of 5064 U/l. The zymogram analysis indicated that nutrition conditions had no effect on the types of laccase isozymes, but regulated the secretion ratio among the different isozymes. The optimal addition concentration and addition time of complex inducers were determined by orthogonal experiment and multiple linear regressions. The maximum laccase activity of 5730±150 U/l was obtained in the fermentation broth, which was supplemented with 15.4μmol/l methylene blue and 76μmol/l copper sulphate after incubation for 11 and 12 h, respectively. Methylene blue was almost completely degraded during cultivation, suggesting methylene blue could be degraded efficiently by laccase from Paecilomyces sp. S152. The increasing laccase activity under the complex-induction condition may be due to the defensive effect of microorganism, which inducing the secretion of laccase to reduce the position of methylene blue. The intra- and extra- cellular location of laccases were detected in the cells of Paecilomyces sp. S152. To improve cell permeability, Tween 80 (1.5 g/l) was added into fermentation broth and an enhanced extracellular laccase activity was detected with the shortened fermentation period (6 days) and improved (946 U/l/d).4. Based on the kinetics of laccase production under different culture temperature, a two-stage temperature-shifted strategy was developed as follows: temperature was controlled at 35oC during the first 92 h of cultivation, and then switched to 30oC. By applying the strategy, a combination of the optimum laccase-excretion periods was achieved, and a succession of qp (specific laccase formation rate) peak time was observed, and thus, the laccase production was increased by 45% and 117% compared with that of 30oC and 35oC cultivation, respectively. Also, the increased laccase productivity and laccase yield to glucose were obtained by applying the temperature-shifted strategy. The possible mechanisms for enhanced laccase activity by applying the strategy were explained as: higher temperature was more favorable to improve cell growth and cell permeability, and avoid glucose repression in early period of cultivation; while a temperature-stress by reducing incubation temperature at later period of cultivation would induce the secretion of protease, which may be involved into the activation of pro-laccase, resulted in the enhancement of laccase activity.5. Laccase isozyme Lac 1 and Lac 3 were purified from the fermentation broth of Paecilomyces sp. S152 by ethanol precipitation, anion exchange (Q-Sepharose HP) and gel filtration chromatography (Superdex 75). The purified Lac 1 and Lac 3 exhibited a high specific activity of 196.75 and 327.43 U/mg for ABTS. The purification folds of Lac 1 and Lac 3 are 19.95 and 33.21, with the recovery yield of 19 and 27.66%, respectively. The molecular weight of Lac 1 and Lac 3 are both 79 kDa. Both enzymes were absence of blue copper absorption spectrum and exhibited yellow color, which were different from the wildly existed blue laccases and similar to several yellow laccases from other fungi. The value of Km and Kcat/Km on ABTS and guaiacol revealed that the substrate affinity and catalytic rate of Lac 3 were superior to that of Lac 1. Furthermore, a protease was purified from the fermentation broth of Paecilomyces sp. S152 and identified as serine protease which could be strongly inhibited by serine protease inhibitor PMSF. In the cell-free system, the activation of pro-laccase was inhibited in the presence of PMSF; addition of 1 mmol/l PMSF during fermentation resulted in a significant decrease of both protease and laccase activities. The results above suggested that the presence of protease could promote the activity of laccase and, while inhibition of protease activity would not favor for activation of pro-laccase.更多还原