拟青霉漆酶发酵调控、分离纯化及性质的研究
|
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。
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.
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。
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.
引文
1. Malmstrom B G, Reinhammar B, Vanngard T. The state of copper in stellacyanin and laccase from the lacquer tree Rhus vernicifera [J]. Biochimica et Biophysica Acta, 1970, 205(1):48-57
2. Alexandre G, Zhulin I B. Laccases are widespread in bacteria [J]. Trends in Biotechnology, 2000, 18:41-42
3. Galhaup C, Wagner H, Hinterstoisser B, etc. Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens [J]. Enzyme and Microbial Technology, 2002, 30(4):529-536
4. Gnanamani A, Jayaprakashvel M, Arulmani M, etc. Effect of inducers and culturing processes on laccase synthesis in Phanerochaete chrysosporium NCIM 1197 and the constitutive expression of laccase isozymes [J]. Enzyme and Microbial Technology, 2006, 38:1017-1021
5. Yatsu J, Asano T. Cuticle laccase of the silkworm, Bombyx mori: Purification, gene identification and presence of its inactive precursor in the cuticle [J]. Insect Biochemistry and Molecular Biology, 2009, 39(4):254-262
6. Zhang H, Zhang Y, Huang F, etc. Purification and characterization of a thermostable laccase with unique oxidative characteristics from Trametes hirsuta [J]. Biotechnology Letters, 2009, 31(6):837-843
7. Tong P G, Hong Y Z, Xiao Y Z, etc. High production of laccase by a new basidiomycete, Trametes sp. [J]. Biotechnology Letters, 2007, 29(2):295-301
8. Liu L H, Lin Z W, Zheng T, etc. Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969 [J]. Enzyme and Microbial Technology, 2009, 44(6-7):426-433
9. Stajic M, Persky L, Friesem D, etc. Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species [J]. Enzyme and Microbial Technology, 2006, 38(1-2):65-73
10. Pozdnyakova N N, Turkovskaya O V, Yudina E N, etc. Yellow laccase from the fungus Pleurotus ostreatus D1: Purification and characterization [J]. Applied Biochemistry and Microbiology, 2006, 42(1):56-61
11. Ryu S H, Lee A Y, Kim M. Molecular characteristics of two laccase from the basidiomycete fungus Polyporus brumalis [J]. Journal of Microbiology, 2008, 46(1):62-69
12. Makela M R, Hilden K S, Hakala T K, etc. Expression and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown on wood [J]. Current Genetics,2006, 50(5):323-333
13. Baldrian P. Fungal laccases - occurrence and properties [J]. FEMS Microbiology Reviews, 2006, 30(2):215-242
14. Alcalde M. Laccase: biological functions, molecular structure and industrial applications[M] [J]. Heidelberg: Springer, 2007. 2461-2476
15. Leontievsky A A, Vares T, Lankinen P, etc. Blue and yellow laccases of ligninolytic fungi [J]. FEMS Microbiology Letters, 1997, 156:9-14
16. Leontievsky A, Myasoedova N, Pozdnyakova N, etc. 'Yellow' laccase of Panus tigrinus oxidizes non-phenolic substrates without electron-transfer mediators [J]. FEBS Letters, 1997, 413(3):446-448
17. Palmieri G, Giardina P, Bianco C, etc. A novel white laccase from Pleurotus ostreatus [J]. The Journal of Biological Chemistry, 1997, 272(50):31301-31307
18.杨秀清,赵晓霞,赵永福,等.一种pH稳定的黄色漆酶的快速纯化和性质特征[J].微生物学通报, 2009, 36(2):299-308
19. Sahay R, Yadav R S S, Yadav K D S. Purification and Characterization of Laccase Secreted by L-lividus [J]. Applied Biochemistry and Biotechnology, 2009, 157(2):311-320
20. Singh G, Capalash N, Goel R, etc. A pH-stable laccase from alkali-tolerant gamma-proteobacterium JB: Purification, characterization and indigo carmine degradation [J]. Enzyme and Microbial Technology, 2007, 41:794-799
21. Martin C, Pecyna M, Kellner H, etc. Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene [J]. Applied Microbiology and Biotechnology, 2007, 77(3):613-624
22. Ullrich R, Huong L M, Dung N L, etc. Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization [J]. Applied Microbiology and Biotechnology, 2005, 67(3):357-363
23. Zouari-Mechichi H, Mechichi T, Dhouib A, etc. Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme [J]. Enzyme and Microbial Technology, 2006, 39(1):141-148
24. Jung H C, Xu F, Li K C. Purification and characterization of laccase from wood-degrading fungus Trichophyton rubrum LKY-7 [J]. Enzyme and Microbial Technology, 2002, 30(2):161-168
25. Sadhasivam S, Savitha S, Swaminathan K, etc. Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1 [J]. Process Biochemistry, 2008, 43(7):736-742
26. Shleev S V, Morozova O, Nikitina O, etc. Comparison of physico-chemical characteristics of four laccases from different basidiomycetes [J]. Biochimie, 2004, 86(9-10):693-703
27. Niladevi K N, Jacob N, Prema P. Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: Purification and characterization [J]. Process Biochemistry, 2008, 43(6):654-660
28. Minussi R C, Pastore G M, Duran N. Potential applications of laccase in the food industry [J]. Trends in Food Science & Technology, 2002, 13(6-7):205-216
29. Rodríguez Couto S, Toca Herrera J L. Industrial and biotechnological applications of laccases: a review [J]. Biotechnology Advances, 2006, 24(5):500-513
30. Liu N, Shi S, Gao Y, etc. Fiber modification of kraft pulp with laccase in presence of methyl syringate [J]. Enzyme and Microbial Technology, 2009, 44:89-95
31. Leonowicz A, Cho N S, Luterek J, etc. Fungal laccase: properties and activity on lignin [J]. Journal of Basic Microbiology, 2001, 41(3-4):185-227
32. Kalme S, Jadhav S, Jadhav M, etc. Textile dye degrading laccase from Pseudomonas desmolyticum NCIM 2112 [J]. Enzyme and Microbial Technology, 2009, 44:65-71
33. Tavares A P M, Cristovao R O, Loureiro J M, etc. Optimisation of reactive textile dyes degradation by laccase-mediator system [J]. Journal of Chemical Technology and Biotechnology, 2008, 83(12):1609-1615
34. Yaropolov A I, Skorobogatko O V, Vartanov S S, etc. Laccase: properties, catalytic mechanism, and applicability [J]. Applied Biochemistry and Biotechnology, 1994, 49(3):257-280
35. Riva S. Laccases: blue enzymes for green chemistry [J]. Trends in Biotechnology, 2006, 24(5):219-226
36. Witayakran S, Ragauskas A J. Synthetic Applications of Laccase in Green Chemistry [J]. Advanced Synthesis & Catalysis, 2009, 351(9):1187-1209
37. Fukuda T, Uchida H, Takashima Y, etc. Degradation of bisphenol A by purified laccase from Trametes villosa [J]. Biochemical and Biophysical Research Communications, 2001, 284(3):704-706
38. Blanquez P, Sarra M, Vicent T. Development of a continuous process to adapt the textile wastewater treatment by fungi to industrial conditions [J]. Process Biochemistry, 2008, 43(1):1-7
39. Chefetz B, Kerem Z, Chen Y, etc. Isolation and partial characterization of laccase from a thermophilic composted municipal solid waste [J]. Soil Biology and Biochemistry, 1998,
30(8-9):1091-1098
40. Trovaslet M, Enaud E, Guiavarc'h Y, etc. Potential of a Pycnoporus sanguineus laccase inbioremediation of wastewater and kinetic activation in the presence of an anthraquinonic acid dye [J]. Enzyme and Microbial Technology, 2007, 41(3):368-376
41. Minussi R C, Miranda M A, Silva J A, etc. Purification, characterization and application of laccase from Trametes versicolor for colour and phenolic removal of olive mill wastewater in the presence of 1-hydroxybenzotriazole [J]. African Journal of Biotechnology, 2007, 6(10):1248-1254
42. Leonowicz A, Rogalski J, Jaszek M, etc. Cooperation of fungal laccase and glucose 1-oxidase in transformation of Bj?rkman lignin and some phenolic compounds [J]. Holzforschung, 1999, 53(4):376-380
43. Schlosser D, Grey R, Fritsche W. Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood [J]. Applied Microbiology and Biotechnology, 1997, 47(4):412-418
44. Linden R M, Schilling B C, Germann U A, etc. Regulation of laccase synthesis in induced Neurospora crassa cultures [J]. Current Genetics, 1991, 19(5):375-381
45. Bose S, Mazumder S, Mukherjee M. Laccase production by the white-rot fungus Termitomyces clypeatus [J]. Journal of Basic Microbiology, 2007, 47(2):127-131
46. Palmieri G, Giardina P, Bianco C, etc. Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus [J]. Applied and Environmental Microbiology, 2000, 66(3):920-924
47. Valagkova V, Baldrian P. Estimation of bound and free fractions of lignocellulose-degrading enzymes of wood-rotting fungi Pleurotus ostreatus, Trametes versicolor and Piptoporus betulinus [J]. Research in Microbiology, 2006, 157(2):119-124
48. Svobodova K, Majcherczyk A, Novotny C, etc. Implication of mycelium-associated laccase from Irpex lacteus in the decolorization of synthetic dyes [J]. Bioresource Technology, 2008, 99(3):463-471
49. Waterman S R, Hacham M, Panepinto J, etc. Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice [J]. Infection and Immunity, 2007, 75(2):714-722
50. H?lker U, Dohse J, H?fer M. Extracellular Laccase in Ascomycetes Trichoderma atroviride and Trichoderma harzianum [J]. Folia Microbiologica, 2002, 47(2):423-427
51. Sharma K K, Kuhad R C. Laccase: enzyme revisited and function redefined [J]. Indian Journal of Microbiology, 2008, 48(3):309-316
52. Eggert C, Temp U, Dean J F, etc. Laccase-mediated formation of the phenoxazinone derivative, cinnabarinic acid [J]. FEBS Letters, 1995, 376(3):202-206
53. Crowe J D, Olsson S. Induction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluorescens strains and a range of chemical treatments [J]. Applied and Environmental Microbiology, 2001, 67(5):2088-2094
54. Nagai M, Kawata M, Watanabe H, etc. Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies [J]. Microbiology, 2003, 149:2455-2462
55. Hall M, Scott T, Sugumaran M, etc. Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning [J]. Proceedings of the National Academy of Sciences, 1995, 92(17):7764-7768
56. Hao J J, Song F Q, Huang F, etc. Production of laccase by a newly isolated deuteromycete fungus Pestalotiopsis sp. and its decolorization of azo dye [J]. Journal of Industrial Microbiology & Biotechnology, 2007, 34(3):233-240
57. Kluczek-Turpeinen B, Tuomela M, Hatakka A. Lignin degradation in a compost environment by the deuteromycete Paecilomyces inflatus [J]. Applied Microbiology and Biotechnology, 2003, 61(4):374-379
58. Liang Z Q, Han Y F, Chu H L. A new thermotolerant Paecilomyces species which produces laccase and a biform sporogenous structure [J]. Fungal Divers, 2007, 27:95-102
59. Dong J L, Zhang Y W, Zhang R H, etc. Influence of culture conditions on laccase production and isozyme patterns in the white-rot fungus Trametes gallica [J]. Journal of Basic Microbiology, 2005, 45(3):190-198
60. Jonsson L J, Saloheimo M, Penttila M. Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of Icc1 and expression in Pichia pastoris [J]. Current Genetics, 1997, 32(6):425-430
61.董佳里.粗毛栓菌漆酶的生化和分子生物学研究[D]: [博士学位论文].四川:四川大学生物科学学院, 2004
62. Tellez-Jurado A, Arana-Cuenca A, Becerra A E G, etc. Expression of a heterologous laccase by Aspergillus niger cultured by solid-state and submerged fermentations [J]. Enzyme and Microbial Technology, 2006, 38(5):665-669
63. Jolivalt C, Madzak C, Brault A, etc. Expression of laccaseⅢb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications [J]. Applied Microbiology and Biotechnology, 2005, 66(4):450-456
64. Guo M, Lu F P, Pu J, etc. Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica [J]. Applied Microbiology and Biotechnology, 2005, 69(2):178-183
65. Kiiskinen L L, Kruus K, Bailey M, etc. Expression of Melanocarpus albomyces laccase inTrichoderma reesei and characterization of the purified enzyme [J]. Microbiology, 2004,
150:3065-3074
66. Liu W, Chao Y, Liu S, etc. Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris [J]. Applied Microbiology and Biotechnology, 2003, 63(2):174-181
67. Bulter T, Alcalde M, Sieber V, etc. Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution [J]. Applied and Environmental Microbiology, 2003, 69(8):987-995
68. Hong Y Z, Xiao Y Z, Zhou H M, etc. Expression of a laccase cDNA from Trametes sp. AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation [J]. FEMS Microbiology Letters, 2006, 258(1):96-101
69. Yaver D S, Golightly E J. Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family [J]. Gene, 1996, 181(1-2):95-102
70. Litvintseva A P, Henson J M. Cloning, characterization, and transcription of three laccase genes from Gaeumannomyces graminis var. tritici, the take-all fungus [J]. Applied and Environmental Microbiology, 2002, 68(3):1305-1311
71. Hong F, Meinander N Q, Jonsson L J. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris [J]. Biotechnology and Bioengineering, 2002, 79(4):438-449
72. Fonseca M I, Shimizu E, Zapata P D, etc. Laccase-producing ability and the inducing effect of copper on laccase production of white rot fungi native from Misiones (Argentina) [J]. Enzyme and Microbial Technology, 2009, doi:10.1016/j.enzmictec.2009.07.003
73. Bettin F, Montanari Q, Calloni R, etc. Production of laccases in submerged process by Pleurotus sajor-caju PS-2001 in relation to carbon and organic nitrogen sources, antifoams and Tween 80 [J]. Journal of Industrial Microbiology & Biotechnology, 2009, 36(1):1-9
74. Elisashvili V, Kachlishvili E. Physiological regulation of laccase and manganese peroxidase production by white-rot basidiomycetes [J]. Journal of Biotechnology, 2009, 144(1):37-42
75. Dekker R F H, Barbosa A M, Giese E C, etc. Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05 [J]. International Microbiology, 2007,
10(3):177-185
76. D'Souza-Ticlo D, Verma A K, Mathew M, etc. Effect of nutrient nitrogen on laccase production, its isozyme pattern and effluent decolorization by the fungus NIOCC #2a,isolated from mangrove wood [J]. Indian Journal of Marine Sciences, 2006, 35(4):364-372
77. Baldrian P. Increase of laccase activity during interspecific interactions of white-rot fungi [J]. FEMS Microbiology Ecology, 2004, 50(3):245-253
78. Rodríguez Couto S, Toca-Herrera J L. Laccase production at reactor scale by filamentous fungi [J]. Biotechnology Advances, 2007, 25(6):558-569
79. Hess J, Leitner C, Galhaup C, etc. Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor [J]. Applied Biochemistry and Biotechnology, 2002, 98:229-241
80. Ryan D R, Leukes W D, Burton S G. Fungal bioremediation of phenolic wastewaters in an airlift reactor [J]. Biotechnology Progress, 2005, 21(4):1068-1074
81. Olivieri G, Marzocchella A, Salatino P, etc. Olive mill wastewater remediation by means of Pleurotus ostreatus [J]. Biochemical Engineering Journal, 2006, 31(3):180-187
82. Rodríguez Couto S, Rodríguez A, Paterson R R M, etc. Laccase activity from the fungus Trametes hirsuta using an air-lift bioreactor [J]. Letters in Applied Microbiology, 2006, 42(6):612-616
83. Rivela I, Rodríguez Couto S, Sanromán A. Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor [J]. Biotechnology Letters, 2000, 22(18):1443-1447
84. Rodríguez Couto R, Sanroman M A, Hofer D, etc. Production of laccase by Trametes hirsuta grown in an immersion bioreactor and its application in the decolorization of dyes from a leather factory [J]. Engineering in Life Sciences, 2004, 4(3):233-238
85. Hoshida H, Fujita T, Murata K, etc. Copper-dependent production of a Pycnoporus coccineus extracellular laccase in Aspergillus oryzae and Saccharomyces cerevisiae [J]. Bioscience, Biotechnology, and Biochemistry, 2005, 69(6):1090-1097
86. Skorobogatko O V, Stepanova E V, Gavrilova V P, etc. Effects of inducers on the synthesis of extracellular laccase by Coriolus hirsutus, a basidial fungus [J]. Applied Biochemistry and Microbiology, 1996, 32(5):473-476
87. Arora D S, Gill P K. Effects of various media and supplements on laccase production by some white rot fungi [J]. Bioresource Technology, 2001, 77(1):89-91
88. Malhotra K, Sharma P, Capalash N. Copper and dyes enhance laccase production inγ-proteobacterium JB [J]. Biotechnology Letters, 2004, 26(13):1047-1050
89. Wong Y X, Yu J. Laccase-catalyzed decolorization of synthetic dyes [J]. Water Research, 1999, 33(16):3512-3520
90. Pazarlioglu N K, Sariisik M, Telefoncu A. Laccase: production by Trametes versicolor andapplication to denim washing [J]. Process Biochemistry, 2005, 40(5):1673-1678
91. Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Analytical Chemistry, 1959, 31(3):426-428
92. Widsten P, Kandelbauer A. Laccase applications in the forest products industry: A review [J]. Enzyme and Microbial Technology, 2008, 42(4):293-307
93.林剑伟,阙友雄,陈天生,等.核糖体DNA的内转录间隔区序列标记在真菌分类鉴定中的应用[J].生物技术通讯, 2007, 18(2):292-294
94.袁长婷.核糖体RNA基因间隔区ITS及IGS在真菌分子生物学鉴定和分型中的应用[D]: [博士学位论文].上海:第二军医大学皮肤病真菌病学(专业), 2001
95.张志华,洪葵.核酸序列直接分析在真菌鉴定方面的应用[J].华南热带农业大学学报, 2006, 12(2):39-43
96. Tanaka E, Ito-Kuwa S, Nakamura K, etc. Comparisons of the laccase gene among serotypes and melanin-deficient variants of Cryptococcus neoformans [J]. Microbiology and Immunology, 2005, 49(3):209-217
97. Ronne H. Glucose repression in fungi [J]. Trends in Genetics, 1995, 11(1):12-17
98. Mayer A M, Staples R C. Laccase: new functions for an old enzyme [J]. Phytochemistry, 2002, 60(6):551-565
99. Liang Z Q, Han Y F, Chu H L. Studies on the genus Paecilomyces in China.Ⅰ[J]. Fungal Divers, 2005, 20:83-101
100. Xiao Y Z, Chen Q, Hang J, etc. Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2 [J]. Mycologia, 2004, 96(1):26-35
101. Mohandass C, Knutson K, Ragauskas A J. Laccase treatment of recycled blue dyed paper: physical properties and fiber charge [J]. Journal of Industrial Microbiology & Biotechnology, 2008, 35:1103-1108
102. Galhaup C, Goller S, Peterbauer C K, etc. Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions [J]. Microbiology, 2002, 148:2159-2169
103. Zhao J, Kwan H S. Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes [J]. Applied and Environmental Microbiology, 1999, 65(11):4908-4913
104. Labbe S, Thieled D J. Copper ion inducible and repressible promoter systems in yeast [J]. Methods Enzymol, 1999, 306:144-153
105.余增亮,邱励俭,霍裕平.离子注入生物效应及育种研究进展[J].安徽农学院学报,1991, 18(4):251-257
106. Su C X, Zhou W, Fan Y H, etc. Mutation breeding of chitosanase-producing strain Bacillus sp. S65 by low-energy ion implantation [J]. Journal of Industrial Microbiology & Biotechnology, 2006, 33(12):1037-1042
107. Liu Z Q, Zhang J F, Zheng Y G, etc. Improvement of astasanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation [J]. Journal of Applied Microbiology, 2008, 104:861-872
108.虞龙,许安,王纪,等.低能离子注入在Vc高产菌株选育中的应用[J].激光生物学报, 1999, 8(3):217-220
109. Chen Y, Lin Z X, Zou Z Y, etc. High yield antibiotic producing mutants of Streptomyces erythreus induced by low energy ion implantation [J]. Nuclear Instruments & Methods in Physics Research, Section B, 1998, 140:341-348
110.宋道军,姚建铭,邵春林,等.离子注入微生物产生“马鞍型”存活曲线的可能作用机制[J].核技术, 1999, 22(3):129-132
111.诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社, 1994
112. Ben Younes S, Mechichi T, Sayadi S. Purification and characterization of the laccase secreted by the white rot fungus Perenniporia tephropora and its role in the decolourization of synthetic dyes [J]. Journal of Applied Microbiology, 2007, 102(4):1033-1042
113. Chernykh A, Myasoedova N, Kolomytseva M, etc. Laccase isoforms with unusual properties from the basidiomycete Steccherinum ochraceum strain 1833 [J]. Journal of Applied Microbiology, 2008, 105(6):2065-2075
114. Bayramoglu G, Yilmaz M, Arica M Y. Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes [J]. Bioprocess and Biosystems Engineering, 2009, doi: 1007/s00449-009-0345-6
115. Xavier A, Tavares A P M, Agapito M S M, etc. Sequential batch reactor for eucalypt kraft pulp effluent treatment with Trametes versicolor [J]. Journal of Chemical Technology and Biotechnology, 2008, 83(12):1602-1608
116. Ryan S, Schnitzhofer W, Tzanov T, etc. An acid-stable laccase from Sclerotium rolfsii with potential for wool dye decolourization [J]. Enzyme and Microbial Technology, 2003, 33(6):766-774
117. Gu S B, Li S C, Feng H Y, etc. A novel approach to microbial breeding: low-energy ion implantation [J]. Applied Microbiology and Biotechnology, 2008, 78:201-209
118.刘晓秋,张国青,钱世钧.氟氏链霉菌离子束注入突变谱的分析[J].微生物学报,2007, 47(2):265-269
119. Tavares A P M, Coelho M A Z, Coutinho J A P, etc. Laccase improvement in submerged cultivation: induced production and kinetic modelling [J]. Journal of Chemical Technology and Biotechnology, 2005, 80(6):669-676
120. D'Souza D T, Tiwari R, Sah A K, etc. Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes [J]. Enzyme and Microbial Technology, 2006, 38(3-4):504-511
121. D'Souza T M, Merritt C S, Reddy C A. Lignin-modifying enzymes of the white rot basidiomycetes Ganoderma lucidum [J]. Applied and Environmental Microbiology, 1999, 65:5407-5313
122. Kapich A N, Prior B A, Botha A, etc. Effect of lignocellulose-containing substrate on production of ligninolytic peroxidasees in submerged cultures of Phanerochaete chrysosporium ME-446 [J]. Enzyme and Microbial Technology, 2004, 24:187-195
123. Michniewicz A, Ullrich R, Ledakowicz S, etc. The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties [J]. Applied Microbiology and Biotechnology, 2006, 69(6):682-688
124. De Souza C G M, Tychanowicz G K, De Souza D F, etc. Production of laccase isoforms by Pleurotus pulmonarius in response to presence of phenolic and aromatic compounds [J]. Journal of Basic Microbiology, 2004, 44(2):129-136
125. Binz T, Canevascini G. Purification and paritial characterization of the extracellular laccase from Ophiostoma novo-ulmi [J]. Current Microbiology, 1997, 35:278-281
126. Quaratino D, Federici F, Petruccioli M, etc. Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79 [J]. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2007, 91(1):57-69
127. Zhu X D, Gibbons J, Garcia-Rivera J, etc. Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor [J]. Infection and Immunity, 2001, 69(9):5589-5596
128. Chen R R. Permeability issues in whole-cell bioprocesses and cellular membrane engineering [J]. Applied Microbiology and Biotechnology, 2007, 74(4):730-738
129. Tanaka T, Eguchi S, Aoki T, etc. Production of laccase by membrane-surface liquid culture of Trametes versicolor using a poly (L-lactic acid) membrane [J]. Biochemical Engineering Journal, 2007, 33(2):188-191
130. Tellez-Tellez M, Fernandez F J, Montiel-Gonzalez A M, etc. Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation [J]. Applied Microbiology and Biotechnology, 2008, 81(4):675-679
131. Wang J W, Wu J H, Huang W Y, etc. Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon [J]. Bioresource Technology, 2006, 97(5):786-789
132. Staszczak M. The role of the ubiquitin-proteasome system in the response of the ligninolytic fungus Trametes versicolor to nitrogen deprivation [J]. Fungal Genetics and Biology, 2008, 45(3):328-337
133. Cabaleiro D R, Rodríguez Couto S, Sanroman A, etc. Comparison between the protease production ability of ligninolytic fungi cultivated in solid state media [J]. Process Biochemistry, 2002, 37(9):1017-1023
134. Mtui G, Nakamura Y. Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam [J]. Biotechnology Letters, 2002, 24(21):1743-1747
135. Wang Y C, Xue W, Sims A H, etc. Isolation of four pepsin-like protease genes from Aspergillus niger and analysis of the effect of disruptions on heterologous laccase expression [J]. Fungal Genetics and Biology, 2008, 45(1):17-27
136. Staszczak M, Zdunek E, Leonowicz A. Studies on the role of proteases in the white-rot fungus Trametes versicolor: Effect of PMSF and chloroquine on ligninolytic enzymes activity [J]. Journal of Basic Microbiology, 2000, 40(1):51-63
137. Meza J C, Auria R, Lomascolo A, etc. Role of ethanol on growth, laccase production and protease activity in Pycnoporus cinnabarinus ss3 [J]. Enzyme and Microbial Technology, 2007, 41(1-2):162-168
138. B?ckle B, Martínez M J, Guillén F, etc. Mechanism of peroxidase inactivation in liquid cultures of the ligninolytic fungus Pleurotus pulmonarius [J]. Applied and Environmental Microbiology, 1999, 65(3):923-928
139. Palmieri G, Bianco C, Cennamo G, etc. Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus [J]. Applied and Environmental Microbiology, 2001, 67(6):2754-2759
140. Germann U A, Muller G, Hunziker P E, etc. Characterization of two allelic forms of Neurospora crassa laccase. Amino- and carboxyl-terminal processing of a precursor [J]. The Journal of Biological Chemistry, 1988, 263(2):885-896
141. Kim M S, Baek M J, Lee M H, etc. A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia Larvae [J]. The Journal of Biological Chemistry, 2002, 277:39999-40004
142. Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Analytical Biochemistry,1976, 72(1):248-254
143.汪家政,范明.蛋白质技术手册[M].北京:科学出版社, 2001
144. Min K L, Kim Y H, Kim Y W, etc. Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis [J]. Archives of Biochemistry and Biophysics, 2001, 392(2):279-286
145. Khan A, Williams K, Molloy M P, etc. Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels [J]. Protein Expression and Purification, 2003, 32(2):210-220
146. Bonants P J, Fitters P F, Thijs H, etc. A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs [J]. Microbiology, 1995, 141:775-784
2. Alexandre G, Zhulin I B. Laccases are widespread in bacteria [J]. Trends in Biotechnology, 2000, 18:41-42
3. Galhaup C, Wagner H, Hinterstoisser B, etc. Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens [J]. Enzyme and Microbial Technology, 2002, 30(4):529-536
4. Gnanamani A, Jayaprakashvel M, Arulmani M, etc. Effect of inducers and culturing processes on laccase synthesis in Phanerochaete chrysosporium NCIM 1197 and the constitutive expression of laccase isozymes [J]. Enzyme and Microbial Technology, 2006, 38:1017-1021
5. Yatsu J, Asano T. Cuticle laccase of the silkworm, Bombyx mori: Purification, gene identification and presence of its inactive precursor in the cuticle [J]. Insect Biochemistry and Molecular Biology, 2009, 39(4):254-262
6. Zhang H, Zhang Y, Huang F, etc. Purification and characterization of a thermostable laccase with unique oxidative characteristics from Trametes hirsuta [J]. Biotechnology Letters, 2009, 31(6):837-843
7. Tong P G, Hong Y Z, Xiao Y Z, etc. High production of laccase by a new basidiomycete, Trametes sp. [J]. Biotechnology Letters, 2007, 29(2):295-301
8. Liu L H, Lin Z W, Zheng T, etc. Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969 [J]. Enzyme and Microbial Technology, 2009, 44(6-7):426-433
9. Stajic M, Persky L, Friesem D, etc. Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species [J]. Enzyme and Microbial Technology, 2006, 38(1-2):65-73
10. Pozdnyakova N N, Turkovskaya O V, Yudina E N, etc. Yellow laccase from the fungus Pleurotus ostreatus D1: Purification and characterization [J]. Applied Biochemistry and Microbiology, 2006, 42(1):56-61
11. Ryu S H, Lee A Y, Kim M. Molecular characteristics of two laccase from the basidiomycete fungus Polyporus brumalis [J]. Journal of Microbiology, 2008, 46(1):62-69
12. Makela M R, Hilden K S, Hakala T K, etc. Expression and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown on wood [J]. Current Genetics,2006, 50(5):323-333
13. Baldrian P. Fungal laccases - occurrence and properties [J]. FEMS Microbiology Reviews, 2006, 30(2):215-242
14. Alcalde M. Laccase: biological functions, molecular structure and industrial applications[M] [J]. Heidelberg: Springer, 2007. 2461-2476
15. Leontievsky A A, Vares T, Lankinen P, etc. Blue and yellow laccases of ligninolytic fungi [J]. FEMS Microbiology Letters, 1997, 156:9-14
16. Leontievsky A, Myasoedova N, Pozdnyakova N, etc. 'Yellow' laccase of Panus tigrinus oxidizes non-phenolic substrates without electron-transfer mediators [J]. FEBS Letters, 1997, 413(3):446-448
17. Palmieri G, Giardina P, Bianco C, etc. A novel white laccase from Pleurotus ostreatus [J]. The Journal of Biological Chemistry, 1997, 272(50):31301-31307
18.杨秀清,赵晓霞,赵永福,等.一种pH稳定的黄色漆酶的快速纯化和性质特征[J].微生物学通报, 2009, 36(2):299-308
19. Sahay R, Yadav R S S, Yadav K D S. Purification and Characterization of Laccase Secreted by L-lividus [J]. Applied Biochemistry and Biotechnology, 2009, 157(2):311-320
20. Singh G, Capalash N, Goel R, etc. A pH-stable laccase from alkali-tolerant gamma-proteobacterium JB: Purification, characterization and indigo carmine degradation [J]. Enzyme and Microbial Technology, 2007, 41:794-799
21. Martin C, Pecyna M, Kellner H, etc. Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene [J]. Applied Microbiology and Biotechnology, 2007, 77(3):613-624
22. Ullrich R, Huong L M, Dung N L, etc. Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization [J]. Applied Microbiology and Biotechnology, 2005, 67(3):357-363
23. Zouari-Mechichi H, Mechichi T, Dhouib A, etc. Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme [J]. Enzyme and Microbial Technology, 2006, 39(1):141-148
24. Jung H C, Xu F, Li K C. Purification and characterization of laccase from wood-degrading fungus Trichophyton rubrum LKY-7 [J]. Enzyme and Microbial Technology, 2002, 30(2):161-168
25. Sadhasivam S, Savitha S, Swaminathan K, etc. Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1 [J]. Process Biochemistry, 2008, 43(7):736-742
26. Shleev S V, Morozova O, Nikitina O, etc. Comparison of physico-chemical characteristics of four laccases from different basidiomycetes [J]. Biochimie, 2004, 86(9-10):693-703
27. Niladevi K N, Jacob N, Prema P. Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: Purification and characterization [J]. Process Biochemistry, 2008, 43(6):654-660
28. Minussi R C, Pastore G M, Duran N. Potential applications of laccase in the food industry [J]. Trends in Food Science & Technology, 2002, 13(6-7):205-216
29. Rodríguez Couto S, Toca Herrera J L. Industrial and biotechnological applications of laccases: a review [J]. Biotechnology Advances, 2006, 24(5):500-513
30. Liu N, Shi S, Gao Y, etc. Fiber modification of kraft pulp with laccase in presence of methyl syringate [J]. Enzyme and Microbial Technology, 2009, 44:89-95
31. Leonowicz A, Cho N S, Luterek J, etc. Fungal laccase: properties and activity on lignin [J]. Journal of Basic Microbiology, 2001, 41(3-4):185-227
32. Kalme S, Jadhav S, Jadhav M, etc. Textile dye degrading laccase from Pseudomonas desmolyticum NCIM 2112 [J]. Enzyme and Microbial Technology, 2009, 44:65-71
33. Tavares A P M, Cristovao R O, Loureiro J M, etc. Optimisation of reactive textile dyes degradation by laccase-mediator system [J]. Journal of Chemical Technology and Biotechnology, 2008, 83(12):1609-1615
34. Yaropolov A I, Skorobogatko O V, Vartanov S S, etc. Laccase: properties, catalytic mechanism, and applicability [J]. Applied Biochemistry and Biotechnology, 1994, 49(3):257-280
35. Riva S. Laccases: blue enzymes for green chemistry [J]. Trends in Biotechnology, 2006, 24(5):219-226
36. Witayakran S, Ragauskas A J. Synthetic Applications of Laccase in Green Chemistry [J]. Advanced Synthesis & Catalysis, 2009, 351(9):1187-1209
37. Fukuda T, Uchida H, Takashima Y, etc. Degradation of bisphenol A by purified laccase from Trametes villosa [J]. Biochemical and Biophysical Research Communications, 2001, 284(3):704-706
38. Blanquez P, Sarra M, Vicent T. Development of a continuous process to adapt the textile wastewater treatment by fungi to industrial conditions [J]. Process Biochemistry, 2008, 43(1):1-7
39. Chefetz B, Kerem Z, Chen Y, etc. Isolation and partial characterization of laccase from a thermophilic composted municipal solid waste [J]. Soil Biology and Biochemistry, 1998,
30(8-9):1091-1098
40. Trovaslet M, Enaud E, Guiavarc'h Y, etc. Potential of a Pycnoporus sanguineus laccase inbioremediation of wastewater and kinetic activation in the presence of an anthraquinonic acid dye [J]. Enzyme and Microbial Technology, 2007, 41(3):368-376
41. Minussi R C, Miranda M A, Silva J A, etc. Purification, characterization and application of laccase from Trametes versicolor for colour and phenolic removal of olive mill wastewater in the presence of 1-hydroxybenzotriazole [J]. African Journal of Biotechnology, 2007, 6(10):1248-1254
42. Leonowicz A, Rogalski J, Jaszek M, etc. Cooperation of fungal laccase and glucose 1-oxidase in transformation of Bj?rkman lignin and some phenolic compounds [J]. Holzforschung, 1999, 53(4):376-380
43. Schlosser D, Grey R, Fritsche W. Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood [J]. Applied Microbiology and Biotechnology, 1997, 47(4):412-418
44. Linden R M, Schilling B C, Germann U A, etc. Regulation of laccase synthesis in induced Neurospora crassa cultures [J]. Current Genetics, 1991, 19(5):375-381
45. Bose S, Mazumder S, Mukherjee M. Laccase production by the white-rot fungus Termitomyces clypeatus [J]. Journal of Basic Microbiology, 2007, 47(2):127-131
46. Palmieri G, Giardina P, Bianco C, etc. Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus [J]. Applied and Environmental Microbiology, 2000, 66(3):920-924
47. Valagkova V, Baldrian P. Estimation of bound and free fractions of lignocellulose-degrading enzymes of wood-rotting fungi Pleurotus ostreatus, Trametes versicolor and Piptoporus betulinus [J]. Research in Microbiology, 2006, 157(2):119-124
48. Svobodova K, Majcherczyk A, Novotny C, etc. Implication of mycelium-associated laccase from Irpex lacteus in the decolorization of synthetic dyes [J]. Bioresource Technology, 2008, 99(3):463-471
49. Waterman S R, Hacham M, Panepinto J, etc. Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice [J]. Infection and Immunity, 2007, 75(2):714-722
50. H?lker U, Dohse J, H?fer M. Extracellular Laccase in Ascomycetes Trichoderma atroviride and Trichoderma harzianum [J]. Folia Microbiologica, 2002, 47(2):423-427
51. Sharma K K, Kuhad R C. Laccase: enzyme revisited and function redefined [J]. Indian Journal of Microbiology, 2008, 48(3):309-316
52. Eggert C, Temp U, Dean J F, etc. Laccase-mediated formation of the phenoxazinone derivative, cinnabarinic acid [J]. FEBS Letters, 1995, 376(3):202-206
53. Crowe J D, Olsson S. Induction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluorescens strains and a range of chemical treatments [J]. Applied and Environmental Microbiology, 2001, 67(5):2088-2094
54. Nagai M, Kawata M, Watanabe H, etc. Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies [J]. Microbiology, 2003, 149:2455-2462
55. Hall M, Scott T, Sugumaran M, etc. Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning [J]. Proceedings of the National Academy of Sciences, 1995, 92(17):7764-7768
56. Hao J J, Song F Q, Huang F, etc. Production of laccase by a newly isolated deuteromycete fungus Pestalotiopsis sp. and its decolorization of azo dye [J]. Journal of Industrial Microbiology & Biotechnology, 2007, 34(3):233-240
57. Kluczek-Turpeinen B, Tuomela M, Hatakka A. Lignin degradation in a compost environment by the deuteromycete Paecilomyces inflatus [J]. Applied Microbiology and Biotechnology, 2003, 61(4):374-379
58. Liang Z Q, Han Y F, Chu H L. A new thermotolerant Paecilomyces species which produces laccase and a biform sporogenous structure [J]. Fungal Divers, 2007, 27:95-102
59. Dong J L, Zhang Y W, Zhang R H, etc. Influence of culture conditions on laccase production and isozyme patterns in the white-rot fungus Trametes gallica [J]. Journal of Basic Microbiology, 2005, 45(3):190-198
60. Jonsson L J, Saloheimo M, Penttila M. Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of Icc1 and expression in Pichia pastoris [J]. Current Genetics, 1997, 32(6):425-430
61.董佳里.粗毛栓菌漆酶的生化和分子生物学研究[D]: [博士学位论文].四川:四川大学生物科学学院, 2004
62. Tellez-Jurado A, Arana-Cuenca A, Becerra A E G, etc. Expression of a heterologous laccase by Aspergillus niger cultured by solid-state and submerged fermentations [J]. Enzyme and Microbial Technology, 2006, 38(5):665-669
63. Jolivalt C, Madzak C, Brault A, etc. Expression of laccaseⅢb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications [J]. Applied Microbiology and Biotechnology, 2005, 66(4):450-456
64. Guo M, Lu F P, Pu J, etc. Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica [J]. Applied Microbiology and Biotechnology, 2005, 69(2):178-183
65. Kiiskinen L L, Kruus K, Bailey M, etc. Expression of Melanocarpus albomyces laccase inTrichoderma reesei and characterization of the purified enzyme [J]. Microbiology, 2004,
150:3065-3074
66. Liu W, Chao Y, Liu S, etc. Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris [J]. Applied Microbiology and Biotechnology, 2003, 63(2):174-181
67. Bulter T, Alcalde M, Sieber V, etc. Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution [J]. Applied and Environmental Microbiology, 2003, 69(8):987-995
68. Hong Y Z, Xiao Y Z, Zhou H M, etc. Expression of a laccase cDNA from Trametes sp. AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation [J]. FEMS Microbiology Letters, 2006, 258(1):96-101
69. Yaver D S, Golightly E J. Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family [J]. Gene, 1996, 181(1-2):95-102
70. Litvintseva A P, Henson J M. Cloning, characterization, and transcription of three laccase genes from Gaeumannomyces graminis var. tritici, the take-all fungus [J]. Applied and Environmental Microbiology, 2002, 68(3):1305-1311
71. Hong F, Meinander N Q, Jonsson L J. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris [J]. Biotechnology and Bioengineering, 2002, 79(4):438-449
72. Fonseca M I, Shimizu E, Zapata P D, etc. Laccase-producing ability and the inducing effect of copper on laccase production of white rot fungi native from Misiones (Argentina) [J]. Enzyme and Microbial Technology, 2009, doi:10.1016/j.enzmictec.2009.07.003
73. Bettin F, Montanari Q, Calloni R, etc. Production of laccases in submerged process by Pleurotus sajor-caju PS-2001 in relation to carbon and organic nitrogen sources, antifoams and Tween 80 [J]. Journal of Industrial Microbiology & Biotechnology, 2009, 36(1):1-9
74. Elisashvili V, Kachlishvili E. Physiological regulation of laccase and manganese peroxidase production by white-rot basidiomycetes [J]. Journal of Biotechnology, 2009, 144(1):37-42
75. Dekker R F H, Barbosa A M, Giese E C, etc. Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05 [J]. International Microbiology, 2007,
10(3):177-185
76. D'Souza-Ticlo D, Verma A K, Mathew M, etc. Effect of nutrient nitrogen on laccase production, its isozyme pattern and effluent decolorization by the fungus NIOCC #2a,isolated from mangrove wood [J]. Indian Journal of Marine Sciences, 2006, 35(4):364-372
77. Baldrian P. Increase of laccase activity during interspecific interactions of white-rot fungi [J]. FEMS Microbiology Ecology, 2004, 50(3):245-253
78. Rodríguez Couto S, Toca-Herrera J L. Laccase production at reactor scale by filamentous fungi [J]. Biotechnology Advances, 2007, 25(6):558-569
79. Hess J, Leitner C, Galhaup C, etc. Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor [J]. Applied Biochemistry and Biotechnology, 2002, 98:229-241
80. Ryan D R, Leukes W D, Burton S G. Fungal bioremediation of phenolic wastewaters in an airlift reactor [J]. Biotechnology Progress, 2005, 21(4):1068-1074
81. Olivieri G, Marzocchella A, Salatino P, etc. Olive mill wastewater remediation by means of Pleurotus ostreatus [J]. Biochemical Engineering Journal, 2006, 31(3):180-187
82. Rodríguez Couto S, Rodríguez A, Paterson R R M, etc. Laccase activity from the fungus Trametes hirsuta using an air-lift bioreactor [J]. Letters in Applied Microbiology, 2006, 42(6):612-616
83. Rivela I, Rodríguez Couto S, Sanromán A. Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor [J]. Biotechnology Letters, 2000, 22(18):1443-1447
84. Rodríguez Couto R, Sanroman M A, Hofer D, etc. Production of laccase by Trametes hirsuta grown in an immersion bioreactor and its application in the decolorization of dyes from a leather factory [J]. Engineering in Life Sciences, 2004, 4(3):233-238
85. Hoshida H, Fujita T, Murata K, etc. Copper-dependent production of a Pycnoporus coccineus extracellular laccase in Aspergillus oryzae and Saccharomyces cerevisiae [J]. Bioscience, Biotechnology, and Biochemistry, 2005, 69(6):1090-1097
86. Skorobogatko O V, Stepanova E V, Gavrilova V P, etc. Effects of inducers on the synthesis of extracellular laccase by Coriolus hirsutus, a basidial fungus [J]. Applied Biochemistry and Microbiology, 1996, 32(5):473-476
87. Arora D S, Gill P K. Effects of various media and supplements on laccase production by some white rot fungi [J]. Bioresource Technology, 2001, 77(1):89-91
88. Malhotra K, Sharma P, Capalash N. Copper and dyes enhance laccase production inγ-proteobacterium JB [J]. Biotechnology Letters, 2004, 26(13):1047-1050
89. Wong Y X, Yu J. Laccase-catalyzed decolorization of synthetic dyes [J]. Water Research, 1999, 33(16):3512-3520
90. Pazarlioglu N K, Sariisik M, Telefoncu A. Laccase: production by Trametes versicolor andapplication to denim washing [J]. Process Biochemistry, 2005, 40(5):1673-1678
91. Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Analytical Chemistry, 1959, 31(3):426-428
92. Widsten P, Kandelbauer A. Laccase applications in the forest products industry: A review [J]. Enzyme and Microbial Technology, 2008, 42(4):293-307
93.林剑伟,阙友雄,陈天生,等.核糖体DNA的内转录间隔区序列标记在真菌分类鉴定中的应用[J].生物技术通讯, 2007, 18(2):292-294
94.袁长婷.核糖体RNA基因间隔区ITS及IGS在真菌分子生物学鉴定和分型中的应用[D]: [博士学位论文].上海:第二军医大学皮肤病真菌病学(专业), 2001
95.张志华,洪葵.核酸序列直接分析在真菌鉴定方面的应用[J].华南热带农业大学学报, 2006, 12(2):39-43
96. Tanaka E, Ito-Kuwa S, Nakamura K, etc. Comparisons of the laccase gene among serotypes and melanin-deficient variants of Cryptococcus neoformans [J]. Microbiology and Immunology, 2005, 49(3):209-217
97. Ronne H. Glucose repression in fungi [J]. Trends in Genetics, 1995, 11(1):12-17
98. Mayer A M, Staples R C. Laccase: new functions for an old enzyme [J]. Phytochemistry, 2002, 60(6):551-565
99. Liang Z Q, Han Y F, Chu H L. Studies on the genus Paecilomyces in China.Ⅰ[J]. Fungal Divers, 2005, 20:83-101
100. Xiao Y Z, Chen Q, Hang J, etc. Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2 [J]. Mycologia, 2004, 96(1):26-35
101. Mohandass C, Knutson K, Ragauskas A J. Laccase treatment of recycled blue dyed paper: physical properties and fiber charge [J]. Journal of Industrial Microbiology & Biotechnology, 2008, 35:1103-1108
102. Galhaup C, Goller S, Peterbauer C K, etc. Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions [J]. Microbiology, 2002, 148:2159-2169
103. Zhao J, Kwan H S. Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes [J]. Applied and Environmental Microbiology, 1999, 65(11):4908-4913
104. Labbe S, Thieled D J. Copper ion inducible and repressible promoter systems in yeast [J]. Methods Enzymol, 1999, 306:144-153
105.余增亮,邱励俭,霍裕平.离子注入生物效应及育种研究进展[J].安徽农学院学报,1991, 18(4):251-257
106. Su C X, Zhou W, Fan Y H, etc. Mutation breeding of chitosanase-producing strain Bacillus sp. S65 by low-energy ion implantation [J]. Journal of Industrial Microbiology & Biotechnology, 2006, 33(12):1037-1042
107. Liu Z Q, Zhang J F, Zheng Y G, etc. Improvement of astasanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation [J]. Journal of Applied Microbiology, 2008, 104:861-872
108.虞龙,许安,王纪,等.低能离子注入在Vc高产菌株选育中的应用[J].激光生物学报, 1999, 8(3):217-220
109. Chen Y, Lin Z X, Zou Z Y, etc. High yield antibiotic producing mutants of Streptomyces erythreus induced by low energy ion implantation [J]. Nuclear Instruments & Methods in Physics Research, Section B, 1998, 140:341-348
110.宋道军,姚建铭,邵春林,等.离子注入微生物产生“马鞍型”存活曲线的可能作用机制[J].核技术, 1999, 22(3):129-132
111.诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社, 1994
112. Ben Younes S, Mechichi T, Sayadi S. Purification and characterization of the laccase secreted by the white rot fungus Perenniporia tephropora and its role in the decolourization of synthetic dyes [J]. Journal of Applied Microbiology, 2007, 102(4):1033-1042
113. Chernykh A, Myasoedova N, Kolomytseva M, etc. Laccase isoforms with unusual properties from the basidiomycete Steccherinum ochraceum strain 1833 [J]. Journal of Applied Microbiology, 2008, 105(6):2065-2075
114. Bayramoglu G, Yilmaz M, Arica M Y. Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes [J]. Bioprocess and Biosystems Engineering, 2009, doi: 1007/s00449-009-0345-6
115. Xavier A, Tavares A P M, Agapito M S M, etc. Sequential batch reactor for eucalypt kraft pulp effluent treatment with Trametes versicolor [J]. Journal of Chemical Technology and Biotechnology, 2008, 83(12):1602-1608
116. Ryan S, Schnitzhofer W, Tzanov T, etc. An acid-stable laccase from Sclerotium rolfsii with potential for wool dye decolourization [J]. Enzyme and Microbial Technology, 2003, 33(6):766-774
117. Gu S B, Li S C, Feng H Y, etc. A novel approach to microbial breeding: low-energy ion implantation [J]. Applied Microbiology and Biotechnology, 2008, 78:201-209
118.刘晓秋,张国青,钱世钧.氟氏链霉菌离子束注入突变谱的分析[J].微生物学报,2007, 47(2):265-269
119. Tavares A P M, Coelho M A Z, Coutinho J A P, etc. Laccase improvement in submerged cultivation: induced production and kinetic modelling [J]. Journal of Chemical Technology and Biotechnology, 2005, 80(6):669-676
120. D'Souza D T, Tiwari R, Sah A K, etc. Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes [J]. Enzyme and Microbial Technology, 2006, 38(3-4):504-511
121. D'Souza T M, Merritt C S, Reddy C A. Lignin-modifying enzymes of the white rot basidiomycetes Ganoderma lucidum [J]. Applied and Environmental Microbiology, 1999, 65:5407-5313
122. Kapich A N, Prior B A, Botha A, etc. Effect of lignocellulose-containing substrate on production of ligninolytic peroxidasees in submerged cultures of Phanerochaete chrysosporium ME-446 [J]. Enzyme and Microbial Technology, 2004, 24:187-195
123. Michniewicz A, Ullrich R, Ledakowicz S, etc. The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties [J]. Applied Microbiology and Biotechnology, 2006, 69(6):682-688
124. De Souza C G M, Tychanowicz G K, De Souza D F, etc. Production of laccase isoforms by Pleurotus pulmonarius in response to presence of phenolic and aromatic compounds [J]. Journal of Basic Microbiology, 2004, 44(2):129-136
125. Binz T, Canevascini G. Purification and paritial characterization of the extracellular laccase from Ophiostoma novo-ulmi [J]. Current Microbiology, 1997, 35:278-281
126. Quaratino D, Federici F, Petruccioli M, etc. Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79 [J]. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2007, 91(1):57-69
127. Zhu X D, Gibbons J, Garcia-Rivera J, etc. Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor [J]. Infection and Immunity, 2001, 69(9):5589-5596
128. Chen R R. Permeability issues in whole-cell bioprocesses and cellular membrane engineering [J]. Applied Microbiology and Biotechnology, 2007, 74(4):730-738
129. Tanaka T, Eguchi S, Aoki T, etc. Production of laccase by membrane-surface liquid culture of Trametes versicolor using a poly (L-lactic acid) membrane [J]. Biochemical Engineering Journal, 2007, 33(2):188-191
130. Tellez-Tellez M, Fernandez F J, Montiel-Gonzalez A M, etc. Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation [J]. Applied Microbiology and Biotechnology, 2008, 81(4):675-679
131. Wang J W, Wu J H, Huang W Y, etc. Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon [J]. Bioresource Technology, 2006, 97(5):786-789
132. Staszczak M. The role of the ubiquitin-proteasome system in the response of the ligninolytic fungus Trametes versicolor to nitrogen deprivation [J]. Fungal Genetics and Biology, 2008, 45(3):328-337
133. Cabaleiro D R, Rodríguez Couto S, Sanroman A, etc. Comparison between the protease production ability of ligninolytic fungi cultivated in solid state media [J]. Process Biochemistry, 2002, 37(9):1017-1023
134. Mtui G, Nakamura Y. Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam [J]. Biotechnology Letters, 2002, 24(21):1743-1747
135. Wang Y C, Xue W, Sims A H, etc. Isolation of four pepsin-like protease genes from Aspergillus niger and analysis of the effect of disruptions on heterologous laccase expression [J]. Fungal Genetics and Biology, 2008, 45(1):17-27
136. Staszczak M, Zdunek E, Leonowicz A. Studies on the role of proteases in the white-rot fungus Trametes versicolor: Effect of PMSF and chloroquine on ligninolytic enzymes activity [J]. Journal of Basic Microbiology, 2000, 40(1):51-63
137. Meza J C, Auria R, Lomascolo A, etc. Role of ethanol on growth, laccase production and protease activity in Pycnoporus cinnabarinus ss3 [J]. Enzyme and Microbial Technology, 2007, 41(1-2):162-168
138. B?ckle B, Martínez M J, Guillén F, etc. Mechanism of peroxidase inactivation in liquid cultures of the ligninolytic fungus Pleurotus pulmonarius [J]. Applied and Environmental Microbiology, 1999, 65(3):923-928
139. Palmieri G, Bianco C, Cennamo G, etc. Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus [J]. Applied and Environmental Microbiology, 2001, 67(6):2754-2759
140. Germann U A, Muller G, Hunziker P E, etc. Characterization of two allelic forms of Neurospora crassa laccase. Amino- and carboxyl-terminal processing of a precursor [J]. The Journal of Biological Chemistry, 1988, 263(2):885-896
141. Kim M S, Baek M J, Lee M H, etc. A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia Larvae [J]. The Journal of Biological Chemistry, 2002, 277:39999-40004
142. Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Analytical Biochemistry,1976, 72(1):248-254
143.汪家政,范明.蛋白质技术手册[M].北京:科学出版社, 2001
144. Min K L, Kim Y H, Kim Y W, etc. Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis [J]. Archives of Biochemistry and Biophysics, 2001, 392(2):279-286
145. Khan A, Williams K, Molloy M P, etc. Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels [J]. Protein Expression and Purification, 2003, 32(2):210-220
146. Bonants P J, Fitters P F, Thijs H, etc. A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs [J]. Microbiology, 1995, 141:775-784