【作者】 王志新；

【导师】 廖祥儒；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 博士

【摘要】 漆酶(Laccase,EC 1.10.3.2)是一种含铜的多酚氧化酶,在环境污染物的降解与脱毒、纸浆的生物制造与漂白、染料的脱色与脱毒、木质素及其衍生物的降解与转化以及食品性状的改良等领域具有广阔的应用价值和潜力。本论文从自然界筛选到一株产漆酶的白腐菌,并对其进行了鉴定,研究了其发酵条件、产酶机理、酶的分离纯化和性质以及在染料脱色和降解中的作用,主要结果如下所示:利用平板显色特征和产漆酶能力测定,筛选到一株产漆酶的白腐菌株SYBC-L1,经形态学和分子生物学鉴定,确认该菌株属于担子菌纲、多孔菌科、密孔菌属、血红密孔菌,命名为血红密孔菌Pycnoporus sanguineus SYBC-L1。其18S和5.8S rDNA的GenBank登录号分别为EU888830和EU888831。对P. sanguineus SYBC-L1的液态发酵产漆酶条件进行了优化,利用一些工农业副产物,如麸皮和豆粕作碳氮源,首先通过单因素法初步优化了其发酵培养基和培养条件,再经过响应面设计法对培养基组分进一步优化,得到的优化培养基为:葡萄糖50 g/L,麸皮52.5 g/L,豆粕15 g/L,NH4H2PO4 1.0 g/L,CuSO4·5H2O 2.0 mmol/L,KH2PO4 0.5 mmol/L,NaCl 17 mmol/L,阿魏酸26.1μmol/L,初始pH 4.0。优化后菌株的漆酶产量为61.25 U/ml,为优化前基础培养基的87倍。根据其发酵过程数据分别建立了菌体生长、漆酶合成和底物消耗动力学模型。在此基础上进行7 L发酵罐扩大培养,第10 d漆酶产量为55 U/ml。对P. sanguineus SYBC-L1在环境污染物——水葫芦的综合利用方面进行了研究,以水葫芦为固态发酵基质,采用响应面法优化了P. sanguineus SYBC-L1的固态发酵产漆酶条件。优化后的培养基为:水葫芦25.1%,木屑13.9%,CuSO4·5H2O 1.5 mmol/L,没食子酸40μmol/L,起始含水量65%,初始pH 6.0。菌株优化后的漆酶活力为32.02 U/g干基,约为优化前的4.5倍。这种发酵方法不仅提高了水葫芦的综合利用价值,而且有利于水葫芦生物质的进一步利用。对P. sanguineus SYBC-L1在漆酶发酵过程中的生理特性进行了研究,发现菌体在漆酶高产时氧化水平提高,丙二醛、H2O2、抗坏血酸含量以及SOD、CAT活力明显上升,这表明菌体在漆酶合成时受到了一定的氧化胁迫作用。采用Fenton试剂对P. sanguineus SYBC-L1发酵产漆酶进行氧化胁迫试验,结果显示H2O2 : Fe2+比例为10:1时可明显促进漆酶的产生,推测漆酶的产生可能与活性氧诱导的基因表达有关。经过硫酸铵分级盐析、DEAE-cellulose阴离子交换层析和Sephadex G-100分子筛层析,分离纯化得到2个漆酶同工酶LacI和LacII,SDS-PAGE显示均为单体蛋白,分子量分别为58.89和63.07 kDa,糖含量分别为36.92%和13.58%,且二者在330 nm处均有特征吸收峰。LacI和LacII的最适反应pH值位于酸性范围,反应温度较宽,高温和低温条件均具有较好的催化活性,且酸碱稳定性和热稳定性良好。SDS、NaN3、β-巯基乙醇和KI对LacI和LacII具有抑制作用,而EDTA、Na+和低浓度的Cu2+、Mg2+及Zn2+稍有促进作用。LacI和LacII的催化底物广泛,ABTS是其最适宜的底物。以ABTS为底物时,Km值分别为0.0166和0.0435 mmol/L,kcat/Km值分别为19,640.36和31,172.64 S-1·mM-1。经UPLC/MS/MS鉴定及氨基酸序列分析,LacI和LacII均属于密孔菌属漆酶。对P. sanguineus SYBC-L1漆酶在工业染料的脱色和降解方面进行了研究,结果显示蒽醌类染料是漆酶的反应底物,可直接被漆酶降解,偶氮类染料在漆酶介体存在的情况下可被降解。小分子介体的添加有助于提高染料的脱色效率,RBBR、酸性红1和活性黑5在添加丁香醛连氮后,反应20 min,脱色率分别为94.91%、90.87%和72.15%;酸性蓝129和活性蓝4在HBT介体存在的情况下脱色60 min,其脱色率分别为81.73%和92.18%。利用高效液相色谱对染料降解后的产物进行分析,发现有降解中间产物生成,并且经过植物毒理试验表明,这些降解物的毒性大大降低,对小麦发芽率完全没有抑制,仅对胚芽和根芽的长度稍有影响。更多还原

【Abstract】 Laccase (EC1.10.3.2, benzenediol: oxygen oxidoreductase) is a oxidoreductase belonging to the multinuclear copper-containing oxidase and can be investigated for a variety of applications including detoxification of harmful substances, bleaching of paper pulp, decolorization of textile dyes, transformation of lignins or their derivatives and improvement of food properties. In this study, a new laccase-producing strain was isolated and identified. The fermentation medium and condition for laccase production were optimized. The physiological characteristics of the strain for laccase production were also investigated. Laccases produced by Pycnoporus sanguineus SYBC-L1 were purified and characterized. The applications in decolotization and degradation of textile dyes were studied. The results were as follows:A white-rot fungus SYBC-L1, which produced laccase, was isolated using PDA screening medium. According to morphological and molecular biological identification, the strain was belonged to Pycnoporus sanguineus, located in phylum Basidiomycota, order Polyporales, family Polyporaceae, genus Pycnoporus and designated as Pycnoporus sanguineus SYBC-L1. The GenBank accession numbers of the 18S and 5.8S rDNA gene sequence were EU888830 and EU888831, respectively.The fermentation process for laccase production by P. sanguineus SYBC-L1 in submerged fermentation was studied by using some industrial and agricultural by-products. The fermentation medium and conditions were preliminarily optimized by one-factor-at-a-time method, followed by further optimization via response surface methodology. The obtained optimal parameters were glucose 50 g/L, wheat bran 52.5 g/L, soybean meal powder 15 g/L, NH4H2PO4 1.0 g/L, CuSO4·5H2O 2.0 mmol/L, KH2PO4 0.5 mmol/L, NaCl 17 mmol/L, ferulaic acid 26.1μmol/L, initial medium pH 4.0. Under the optimal fermentation condition, the maximum laccase activity reached 61.25 U/ml, which was approximately 87 folds than that in the basal medium. The kinetic models of submerged fermentation for mycelial growth, laccase production and substrate consumption were developed based on the experimental results. Based on previous results, the fermentation process was enlarged in 7 L bioreactor and the laccase yield was 55 U/ml after cultivation for 10 d.The utilization of an environmental waster of water hyacinth by P. sanguineus SYBC-L1 was investigated. The fermentation medium were optimized by response surface methodology using water hyacinth as the substrate. The optimal medium were water hyacinth 25.1%, sawdust 13.9%, CuSO4·5H2O 1.5 mmol/L, gallic acid 40μmol/L, initial moisture 65%, initial medium pH 6.0. Under the optimal fermentation condition, the maximum laccase activity reached 32.02 U/g dry substrate, which was approximately 4.5 folds than that in the basal medium. This strategy not only increased the value of water hyacinth’s utilization, but also contributed to the further use of water hyacinth.The physiological characteristics of P. sanguineus SYBC-L1 during the laccase producing process were investigated. It was found that when the yield of laccase was high, the oxidation level was relatively high, the contents of malondialdehyde, H2O2 and ascorbic acid were increased and the activities of SOD and CAT were enhanced, indicating that the strain was under oxidative stress during the laccase production period. The oxidation stress study on P. sanguineus SYBC-L1 was performed by using Fenton reagent, the result revealed that H2O2 and Fe2+ with the ratio of 10:1 could significantly promote the laccase production. It was supposed that laccase secretion might be regulated by reactive oxygen, which could induce the gene expression for laccase production.Two laccase isozymes (LacI and LacII) were purified by using (NH4)2SO4 fractionation, DEAE-cellulose anion-exchange chromatography and Sephadex G-100 filtration. SDS-PAGE showed that both LacI and LacII were monomeric proteins with the molecular mass of 55.89 and 63.07 kDa, respectively. LacI and LacII respectively contained 36.92% and 13.58% carbohydrates. The UV–vis spectrum of the purified laccases both showed a broad shoulder near 330 nm. The optimal pH values were observed in the acidic region and the catalytic temperatures were broad. LacI and LacII both showed not only high catalytic activity at high or low temperature, but also good stabilities toward pH and temperature. LacI and LacII were strongly inhibited by SDS, NaN3,β-mercaptoethanol and KI, but slightly enhanced by EDTA and metal ions of Na+, Cu2+, Mg2+ and Zn2+ at low concentration. LacI and LacII showed a wide range of substrates and the best substrates were both found to be ABTS. The Km values of LacI and LacII was 0.0166 and 0.0435 mmol/L and the catalytic efficiencies was 19640.36 and 31172.64 S-1·mM-1, respectively, for ABTS as substrate. Through UPLC/MS/MS and protein sequences analysis, LacI and LacII were showed to be identical with laccases from genus Pycnoporus.The decolorization and degradation of dyes by P. sanguineus SYBC-L1 laccase were invistegated. Anthraquinone dyes were the laccase substrates and could be directly degraded by P. sanguineus SYBC-L1 laccases, however, azo dyes were not the laccase substrates and could be degraded in the presence of some redox mediators. The decolorization rates could be enhanced by adding some small molecular weight redox mediators. The decolorization rates of RBBR, acid red 1 and reactive black 5 were 94.91%, 90.87% and 72.15%, respectively, after 20 min incubation by adding syringaldehyde, while in the presence of HBT, the decolorization rates of acid blue 129 and reactive blue 4 were 81.73% and 92.18%, respectively, after 60 min incubation. The biodegradations were monitored by HPLC and the results showed some degradation products were generated in the degradation process. Phytotoxicity study revealed that the biodegraded products had no toxicity on wheat germination, but only slightly inhibited on the length of the plumule and radical.更多还原