变色栓菌发酵产漆酶的过程优化
发布时间：2018-11-11 20:50
【摘要】：漆酶(苯二醇:氧氧化还原酶,EC 1.10.3.2)是一种含铜的多酚氧化酶,是蓝色多铜氧化酶家族的重要成员,由于其具有催化多种底物的氧化能力,已经引起食品、造纸、环保、纺织及生物传感器等各种工业应用的重点关注。为了能满足漆酶日益增长的市场需求,本论文开展了变色栓菌产漆酶发酵过程优化的研究,建立了优化漆酶发酵的玉米浆及法尼醇添加策略,并进一步在通气搅拌式发酵罐中进行了变色栓菌漆酶发酵优化策略的放大实验,为后续漆酶的工业化生产奠定基础。玉米浆的添加明显促进了变色栓菌发酵产漆酶,在优化的玉米浆添加浓度为20 g L-1的条件下,发酵液漆酶活力在第5天达到633.3 UL-1,比对照提高了 96.3%。玉米浆中的酚类物质经鉴别有5种,分别是香草酸、咖啡酸、对香豆酸、阿魏酸和芥子酸,其中咖啡酸及对香豆酸对变色栓菌的产酶没有影响,香草酸及阿魏酸是有效的产酶促进剂,芥子酸能抑制变色栓菌的产酶,而香草酸和阿魏酸二者具有共同促进漆酶生产的作用。添加真菌信号分子法尼醇具有显著促进变色栓菌发酵产漆酶的作用,在发酵开始时添加60 μM的法尼醇,发酵周期6天,发酵液漆酶活力达到629.3 UL-1,比对照提高92.3%。对菌丝形态观察后发现,添加法尼醇能形成许多超支化的结构。法尼醇的添加能引起变色栓菌胞内H202含量的增加,第1天时胞内H202含量是对照的2.7倍,H202含量的增加引起细胞防御机制的启动,漆酶在细胞抵御氧化胁迫中起着关键的作用。对变色栓菌漆酶lac基因及rhoA基因进行定量分析,添加法尼醇后lac基因表达量比对照要高,第3天时lac基因表达量最高是对照的2.26倍,基因表达量的增加提高了漆酶的合成能力;与菌丝形态有关的rhoA基因表达量也因法尼醇的添加而提高,第3天时最高rhoA表达量是对照的4.4倍,rhoA基因的超高表达导致菌丝的超支化,从而促进了漆酶的分泌,采用法尼醇的优化策略,在5 L和2000 L通气搅拌式发酵罐中完成变色栓菌发酵产漆酶的放大。在5 L发酵罐中法尼醇的优化变色栓菌发酵漆酶水平为704.5 U L-1,比未加法尼醇的提高105.6%,比摇瓶中的产量629.3 U L-1高11.9%。在2000 L发酵罐中法尼醇优化变色栓菌发酵漆酶水平为543.6 U L-1,比未加法尼醇的对照组提高53%。发酵结束,发酵液经过滤浓缩可制备粗酶液。经盐析-柱层析分离纯化及磁性分离均能制得纯酶,但磁性分离更具优势及工业应用的价值。漆酶纯酶的最适反应pH为4.0,最适反应温度25℃,在40℃仍然具有较高活性,10 mM的Cu2+能够提高漆酶活性10%。漆酶能够降解多种酚类物质,其降解最适pH条件为4.0-5.0,而最适反应温度:4-甲氧基酚和2,4-二氯酚为35℃,苯酚为25℃。漆酶能够降解多种染料,所降解染料的最适降解pH和温度为5.0和40℃,染料脱色速率的大小主要受染料结构及性状的影响。
[Abstract]:Laccase (Phenediol: oxygen Oxidoreductase, EC 1.10.3.2) is a copper-containing polyphenol oxidase and an important member of the blue polycopper oxidase family. Paper, environmental protection, textile and biosensor and other industrial applications focus on. In order to meet the increasing market demand of laccase, this paper studied the optimization of laccase fermentation process by Color-changing suppository bacteria, and established the strategy of cornmilk and farnesol adding to optimize laccase fermentation. Furthermore, the optimization strategy of laccase fermentation of Color-changing suppository was carried out in the aerated agitation fermenter, which laid a foundation for the subsequent industrial production of laccase. The addition of corn pulp significantly promoted the laccase production by Color-changing Thrombidium. The laccase activity of the fermentation broth reached 633.3 UL-1, on the 5th day, and the laccase activity of the fermentation broth increased by 96.3g 路L ~ (-1) compared with the control at the optimized concentration of 20 g 路L ~ (-1). The phenolic substances in corn pulp were identified as vanillic acid, caffeic acid, p-coumaric acid, ferulic acid and erucic acid. Vanillic acid and ferulic acid are effective promoters for producing laccase. Erucic acid can inhibit the production of laccase, while vanillic acid and ferulic acid can promote laccase production together. The addition of fungous signal molecule farnesol could significantly promote the laccase production of Color-changing Thrombium. At the beginning of fermentation, 60 渭 M farnesol was added. The fermentation period was 6 days, and the laccase activity of the fermentation broth reached 629.3 UL-1,. 92.3% higher than the control. After observation of mycelium morphology, it was found that many hyperbranched structures could be formed by adding farnesol. The addition of farnesol could increase the intracellular H202 content of Colotropic suppositories. On the first day, the intracellular H202 content was 2.7 times higher than that of the control, and the increase of H202 content led to the initiation of the cell defense mechanism. Laccase plays a key role in cell resistance to oxidative stress. The quantitative analysis of laccase lac gene and rhoA gene in Color-changing suppositories showed that the expression of lac gene was higher than that of the control, and the highest expression of lac gene was 2.26 times higher than that of the control on the 3rd day. The ability of laccase synthesis was improved with the increase of gene expression. The expression of rhoA gene related to mycelium morphology was also increased by the addition of farnesol. The highest expression of rhoA was 4.4 times higher than that of the control on the third day. The hyperbranched expression of rhoA gene led to hyphal hyperbranching, which promoted the secretion of laccase. The fermentation of laccase was amplified in 5 L and 2000 L aerated agitation fermenter with farnesol. The optimal laccase level of farnesol in 5 L fermenter was 704.5 U L -1, 105.6% higher than that without farnesol, and 11.9 U L ~ (-1) higher than that of shaking flask (629.3 U L ~ (-1). In a 2000 L fermenter, the laccase level of fermentative laccase of the strain was 543.6 U L -1, which was 53% higher than that of the control group without farnesol. At the end of fermentation, the fermentation broth can be filtrated and concentrated to produce the crude enzyme solution. The purified enzyme can be obtained by salting-column chromatography and magnetic separation, but magnetic separation has more advantages and industrial application value. The optimum reaction temperature of laccase was 4.0, and the optimum reaction temperature was 25 鈩，
本文编号：2326095
[Abstract]:Laccase (Phenediol: oxygen Oxidoreductase, EC 1.10.3.2) is a copper-containing polyphenol oxidase and an important member of the blue polycopper oxidase family. Paper, environmental protection, textile and biosensor and other industrial applications focus on. In order to meet the increasing market demand of laccase, this paper studied the optimization of laccase fermentation process by Color-changing suppository bacteria, and established the strategy of cornmilk and farnesol adding to optimize laccase fermentation. Furthermore, the optimization strategy of laccase fermentation of Color-changing suppository was carried out in the aerated agitation fermenter, which laid a foundation for the subsequent industrial production of laccase. The addition of corn pulp significantly promoted the laccase production by Color-changing Thrombidium. The laccase activity of the fermentation broth reached 633.3 UL-1, on the 5th day, and the laccase activity of the fermentation broth increased by 96.3g 路L ~ (-1) compared with the control at the optimized concentration of 20 g 路L ~ (-1). The phenolic substances in corn pulp were identified as vanillic acid, caffeic acid, p-coumaric acid, ferulic acid and erucic acid. Vanillic acid and ferulic acid are effective promoters for producing laccase. Erucic acid can inhibit the production of laccase, while vanillic acid and ferulic acid can promote laccase production together. The addition of fungous signal molecule farnesol could significantly promote the laccase production of Color-changing Thrombium. At the beginning of fermentation, 60 渭 M farnesol was added. The fermentation period was 6 days, and the laccase activity of the fermentation broth reached 629.3 UL-1,. 92.3% higher than the control. After observation of mycelium morphology, it was found that many hyperbranched structures could be formed by adding farnesol. The addition of farnesol could increase the intracellular H202 content of Colotropic suppositories. On the first day, the intracellular H202 content was 2.7 times higher than that of the control, and the increase of H202 content led to the initiation of the cell defense mechanism. Laccase plays a key role in cell resistance to oxidative stress. The quantitative analysis of laccase lac gene and rhoA gene in Color-changing suppositories showed that the expression of lac gene was higher than that of the control, and the highest expression of lac gene was 2.26 times higher than that of the control on the 3rd day. The ability of laccase synthesis was improved with the increase of gene expression. The expression of rhoA gene related to mycelium morphology was also increased by the addition of farnesol. The highest expression of rhoA was 4.4 times higher than that of the control on the third day. The hyperbranched expression of rhoA gene led to hyphal hyperbranching, which promoted the secretion of laccase. The fermentation of laccase was amplified in 5 L and 2000 L aerated agitation fermenter with farnesol. The optimal laccase level of farnesol in 5 L fermenter was 704.5 U L -1, 105.6% higher than that without farnesol, and 11.9 U L ~ (-1) higher than that of shaking flask (629.3 U L ~ (-1). In a 2000 L fermenter, the laccase level of fermentative laccase of the strain was 543.6 U L -1, which was 53% higher than that of the control group without farnesol. At the end of fermentation, the fermentation broth can be filtrated and concentrated to produce the crude enzyme solution. The purified enzyme can be obtained by salting-column chromatography and magnetic separation, but magnetic separation has more advantages and industrial application value. The optimum reaction temperature of laccase was 4.0, and the optimum reaction temperature was 25 鈩，
本文编号：2326095
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