CAS OpenIR
变色栓菌产漆酶和多糖的发酵过程调控
王科峰
Subtype博士
Thesis Advisor刘春朝
2017-01
Degree Grantor中国科学院研究生院
Degree Name博士
Degree Discipline生物化工
Keyword漆酶,法尼醇,酪醇,胞外多糖,胞内糖肽
Abstract

变色栓菌,又名云芝,是一种极具应用价值的蘑菇真菌,其资源的开发利用越来越受到重视。在液态发酵培养中,变色栓菌主要产生两种代谢产物:漆酶和多糖。漆酶属于蓝色多铜氧化酶,被称为绿色环保型催化剂,在环境保护、纺织工业、生物检测、有机合成等领域都有广泛的应用前景。多糖又分为胞外多糖和胞内糖肽。胞外多糖和胞内糖肽均属于β-D-葡聚糖,具有抗肿瘤、抗氧化、抗病毒、免疫调节、降血糖、保肝护肝等生理活性,在食品和医药等行业具有巨大的应用潜力。然而,目前漆酶和多糖的生产主要存在产率低、成本高的问题,这大大限制了漆酶和多糖的大规模应用。针对上述存在的问题,本论文首先利用芳香族酚类化合物——香草酸促进变色栓菌发酵产漆酶,并用磁性介孔氧化硅纳米颗粒直接从发酵液中捕获漆酶,制备磁性漆酶催化剂,探究其催化氧化HMF的能力;然后利用真菌群体感应信号分子——法尼醇调控变色栓菌的菌丝形态和刺激漆酶的生物合成,通过同时促进漆酶合成与分泌进一步提高漆酶产量;利用法尼醇调控菌丝形态促进漆酶分泌的作用,探究法尼醇对变色栓菌胞外多糖生产的影响,并对生产的胞外多糖进行理化性质定性和抗氧化、抗肿瘤活性分析;另外,利用真菌群体感应信号分子——酪醇能够促进DNA复制的特性,调控变色栓菌的细胞生长和胞内糖肽的生产,并对生产的胞内糖肽进行理化性质定性和抗肿瘤活性分析。取得的主要结果如下:在变色栓菌发酵培养中,80.0 mg/L香草酸在发酵第2天添加到培养体系中,发酵6天后,漆酶产量和蛋白含量分别达到581.8 U/L和96.7 mg/L,与对照组相比分别提高了80.4% 和46.1%。用5 L搅拌式发酵罐进行放大培养,漆酶产量和蛋白含量分别达到740.2 U/L和130.0 mg/L,相比揺瓶培养分别提高了27.2% 和34.4%。随后用磁性介孔氧化硅纳米颗粒从发酵液中直接捕获漆酶,制备出磁性漆酶催化剂。以TEMPO为介体,该磁性漆酶催化剂具有催化氧化HMF生成FDCA的能力。在最佳反应条件(pH 5.5,35℃,10.0 mg磁性漆酶催化剂,24.0 mM TEMPO)下,磁性漆酶催化剂-TEMPO系统可以催化HMF转化生成90.2% 的FDCA,重复使用6次后,磁性漆酶催化剂仍保持84.8%的初始催化活性,表现出良好的稳定性和可重用性。为了进一步提高变色栓菌的漆酶发酵水平和生产强度,探究了法尼醇对变色栓菌的菌丝形态和漆酶生产的影响。法尼醇能够通过调控变色栓菌的菌丝形态和生理状态,显著促进漆酶生产。在最佳诱导浓度(4.0 mM)作用下,发酵6天后胞外漆酶产量和蛋白含量分别达到2189.2 U/L和145.0 mg/L,与对照组相比分别提高了5.8倍和1.2倍。在5 L发酵罐放大培养中,发酵液中漆酶产量和蛋白含量分别为3064.8 U/L和196.5 mg/L,相比揺瓶培养分别提高了40.0% 和35.5%。凝胶电泳显示,法尼醇主要促进三个漆酶同工酶的生物合成,漆酶在变色栓菌分泌的胞外蛋白体系中的含量显著提高,这有利于漆酶下游的分离纯化。由于法尼醇处理使变色栓菌细胞处于高水平氧化应激的生理状态,导致漆酶基因表达水平显著提高,从而促进大量漆酶的生物合成。同时,法尼醇通过调控菌丝形态发生相关基因的表达,使变色栓菌形成了菌丝较短、顶端膨大的超支化形态,这种菌丝形态能够显著促进胞内漆酶的分泌。进一步研究了法尼醇对变色栓菌胞外多糖生产的调控,0.8 mM法尼醇在发酵第2天添加到培养体系中,发酵9天后胞外多糖产量达到2.56 g/L,相比对照组提高了1.7倍。在5 L发酵罐进行放大培养,胞外多糖产量达到3.20 g/L,相比揺瓶培养提高了25.0%。法尼醇通过促进多糖生物合成和调节菌丝形态,显著提高了胞外多糖产量,且生产的胞外多糖含有更多的糖醛酸、葡聚糖和高分子量多糖组分(134 kDa, 85.0%),这些理化性质使其具有更高的抗氧化和抗肿瘤活性。酪醇能够促进二型态真菌DNA复制,利用此特性刺激变色栓菌的细胞生长和胞内糖肽的生产。在最佳诱导浓度(5.0 mM)作用下,变色栓菌胞内糖肽产量达到390.0 mg/L,与对照组相比提高了95.0%,发酵周期缩短了1/3。在5 L发酵罐放大培养中,胞内糖肽产量达到450.0 mg/L,相比揺瓶培养提高了15.4%。酪醇通过促进变色栓菌的细胞生长和胞内糖肽的生物合成来提高胞内糖肽产量,且生产的胞内糖肽具有更高的蛋白质和葡聚糖含量,这使其具有更强的抗肿瘤活性。胞内糖肽的抗肿瘤机制与胞外多糖相同,均是通过阻滞细胞周期(捕获S期细胞)和诱导细胞凋亡来抑制肿瘤细胞增殖。;Trametes versicolor (known as Yunzhi in China) is one of the most popular mushroom fungi with great application values. In submerged cultures, T. versicolor mainly produces two products including laccase and polysaccharides. Laccase belonging to the blue multicopper oxidase is called green and environmental friendly catalyst. It has wide application prospect in environmental protection, textile industry, biological detection and organic synthesis. The polysaccharides consist of extracellular polysaccharide (EPS) and intracellular protein-polysaccharide (IPS). EPS and IPS belonging to β-D-glucans have gained wide attention for their various biological functions including antitumor, antioxidant, antiviral, immunomodulating, antidiabetic and hepatoprotective effects. They have great application potential in food and medicine industries. Due to the increasing demand for laccase and polysaccharides, the enhanced production of laccase and polysaccharides gains great interest. Therefore, vanillic acid as an aromatic phenolic compound was used to improve laccase production. The laccase in fermentation broth was directly captured by magnetic mesoporous silica nanoparticles for fabricating magnetic laccase catalyst. The catalytic oxidation of HMF was investigated by the magnetic laccase catalyst with TEMPO as the mediator. Farnesol as a fungal quorum sensing signal molecule was applied to regulate T. versicolor morphology and stimulate the laccase biosynthesis and secretion, which further increased laccase yield. In addition, EPS production stimulated by farnesol was also studied. The physicochemical properties of EPS from farnesol-induced cultures were characterized, and its antioxidant and antitumor activities were evaluated. T. versicolor cell growth and IPS production was regulated by fungal quorum sensing signal molecule-tyrosol. The physicochemical properties of IPS from tyrosol-induced cultures were characterized, and its antitumor activity was also evaluated. An efficient strategy for laccase production in T. versicolor submerged cultures was developed using vanillic acid as the inducer. The optimized vanillic acid treatment strategy consisted of exposing 2-day-old mycelia cultures to 80.0 mg/L vanillic acid. After 6 days, laccase activity of 581.8 U/L and protein concentration of 96.7 mg/L were obtained, which represented 1.80-fold and 1.46-fold increase compared to control cultures, respectively. In 5-L aerated stirred bioreactor, the maximal laccase activity and protein concentration reached 740.2 U/L and 130.0 mg/L that were increased by 27.2% and 34.4% compared with those in culture flask, respectively. The magnetic laccase catalyst was prepared by directly capturing the laccase from fermentation broth using magnetic mesoporous silica nanoparticles. With TEMPO as the mediator, it has the remarkable capability of oxidizing HMF to FDCA. Under the optimal reaction conditions (pH 5.5, 35℃, 10.0 mg magnetic laccase catalyst, 24.0 mM TEMPO), 90.2% FDCA was obtained after 96 h of reaction. Furthermore, the magnetic laccase catalyst exhibited good recyclability and stability, maintaining 84.8% of its original activity following 6 reuse cycles.In order to further improve the laccase productivity of T. versicolor, the effect of farnesol on mycelial morphology and laccase production was evaluated. Farnesol significantly promoted the laccase production by regulating mycelial morphology and physiological status of T. versicolor. Extracellular laccase activity and protein concentration reached a maximum of 2189.2 U/L and 145.0 mg/L in farnesol-induced cultures under optimal concentration (4.0 mM), which were 6.8-fold and 2.2-fold than those obtained in the control cultures, respectively. In 5-L bioreactor, the maximal laccase activity and protein concentration reached 3064.8 U/L and 196.5 mg/L that were increased by 40.0% and 35.5% compared with those in culture flask, respectively. SDS-PAGE and native-PAGE showed that farnesol mainly stimulated the biosynthesis of three laccase isoforms. Farnesol significantly enhanced laccase content in the secreted extracellular proteins, which was advantageous to the separation and purification of laccase in downstream processing engineering. Farnesol treatment resulted in a significant increase of oxidative stress level that significantly enhanced the expression of laccase genes for improving intracellular laccase biosynthesis. In addition, farnesol made T. versicolor develop into a hyperbranched morphology with short hyphae and bulbous tips by regulating several morphogenesis-related genes, which accelerated the secretion of intracellular laccase into culture medium.A novel strategy of exposing 2-day-old mycelia cultures to 0.8 mM farnesol was also developed to stimulate EPS production in T. versicolor submerged cultures. After 9 days, EPS yield reached a maximum of 2.56 g/L that was 2.7-fold greater than that of control cultures. In 5-L bioreactor, a peak value of 3.20 g/L was achived that was increased by 25.0% compared with that in culture flask. Farensol significantly increased EPS production by promoting polysaccharide biosynthesis and regulating mycelial morphology. EPS from farnesol-induced cultures (EPS-F) contained more uronic acid, glucan and high molecular weight polysaccharide fraction (134 kDa, 85.0%). These physicochemical properties led to strong antioxidant and antitumor activities of EPS-F.T. versicolor cell growth and IPS production was stimulated by tyrosol that had the ability to promote DNA replication in dimorphic fungi. IPS yield was increased to 390.0 mg/L in tyrosol-induced cultures under optimal concentration (5.0 mM), which was 1.95-fold higher than that of control cultures. Moreover, the fermentation period was shortened by one-third. In 5-L bioreactor, IPS production achieved a maximum value of 450.0 mg/L that was increased by 15.4% compared with that in culture flask. Tyrosol significantly improved IPS production by promoting cell growth and stimulating IPS biosynthesis in T. versicolor submerged cultures. The IPS from tyrosol-induced cultures (IPS-T) contained higher contents of protein and glucan, which led to its strong antitumor activity. The anti-proliferative mechanism was identified as cell cycle arrest with cell accumulation in S phase and increase in apoptosis, which was the same as that of EPS-F.

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26826
Collection中国科学院过程工程研究所
研究所(批量导入)
Recommended Citation
GB/T 7714
王科峰. 变色栓菌产漆酶和多糖的发酵过程调控[D]. 中国科学院研究生院,2017.
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