白腐真菌共培养对磺胺二甲基嘧啶降解的影响

磺胺类抗生素是全国检出频率较高的一种抗菌药物,目前对磺胺类抗生素的生物降解方面研究较少,而生物法具有无二次污染、绿色环保的优点。因此本实验对杂色云芝和黄孢原毛平革菌共培养降解磺胺二甲基嘧啶进行研究。采用高效液相色谱(HPLC)和紫外分光光度计分别分析抗生素的浓度与酶活。结果显示,平板对峙实验中两菌种间菌丝呈僵持状,种间区域漆酶酶活始终高于其他区域。两种白腐真菌液体混配4 d时可以获得具有较高漆酶酶活的粗酶液,该粗酶液在48 h内对磺胺二甲基嘧啶的降解率为25.4%,明显高于杂色云芝纯培养粗酶液的降解率(9.6%)。在加入天然助氧剂和人工助氧剂2,2'-联氨双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(简称ABTS)后,混配粗酶液的降解率分别增至49%和93.1%。种间作用可以通过增加漆酶酶活以及产生助氧剂提升抗生素降解率,因此,共培养的白腐真菌可以作为抗生素污染生物治理的主要手段之一。     

碳源和氮源对毛栓菌菌丝体生长和漆酶分泌的影响

木质素是一种高度复杂的,由宋丙烷为基本结构单元构成的芳香族高分子化合物,是仅次于纤维素的第二大再生有机资源。木质素的结构使其难于降解,大多数生物不能直接利用,从而造成这一资源的巨大浪费,同时木质素的降解及其相关酶类在生物制浆和环境保护中具有重要意义。木质素生物降解酶主要有木素过氧化物酶。锰过氧化物酶、漆酶和多酚氧化酶等,国外已有黄泡原毛平革菌[1]和杂色云芝[2－6]的有关研究报道,但未见该菌的有关报道。国内周金燕[7]、秦小琼[8]等曾研究真菌漆酶。本文对毛栓菌产生漆酶的条件进行了探讨,以便为其在造纸和环…     

采用酶液活性测定法快速筛选及构建石油烃高效降解菌系

快速筛选复杂有机物降解微生物混合菌系,在污染物治理过程中具有重要的实践意义.本研究首次尝试利用MicroResp^TM技术分析微生物酶液活性的方法,快速标定高效降解菌及混合菌系的石油烃降解能力,并采用传统的摇瓶培养检测法予以验证.通过微生物胞内、胞外及混合酶液的活性分析,考察了不同酶系(胞外、胞内及混合酶液)、菌系对石油烃分子的降解情况.结果表明: 结合MicroResp^TM技术的酶液活性测定法能够快速检测石油烃代谢酶系的降解能力,其灵敏度好、通量高,与传统的菌株摇瓶培养方法的检测结果基本一致.其中,7株菌株的120种全组合菌系活性测定试验在12 h周期内1次完成.筛选周期由传统摇瓶培养所需的7 d缩短10倍以上.以酶活性测定结果为指导设计的复配菌系具有较高的降解效率,最高石油烃降解率达(56.1±1.6)%.表明本筛选方法精度高、通量高,可用于石油烃降解功能菌系的构建.     

微囊藻毒素降解菌的筛选、鉴定及其胞内粗酶液对藻毒素MCLR的降解

【目的】从巢湖底泥中分离筛选高效的藻毒素降解菌,并初步研究其胞内粗酶液降解藻毒素-LR(MC-LR)的特性,为水体中藻毒素污染的微生物治理提供有效的菌源与理论依据。【方法】利用富集驯化培养技术,以MC-LR为唯一碳源,分离筛选MC-LR降解菌,通过形态观察、生理生化实验及16S rRNA序列分析鉴定菌株,并考察其胞内粗酶液在不同条件下对MC-LR的降解特性。【结果】分离得到1株能高效降解MC-LR的菌株M6。分子鉴定结果表明,该菌株为蜡状芽胞杆菌(Bacillus cereus)。其降解MC-LR的活性物质为胞内酶,而且至少有3种酶参与了MC-LR的降解,它们是菌体本身的组织酶而非诱导酶。当反应体系pH值为8.0,胞内粗酶液浓度为404.9 mg/L,MC-LR的初始浓度为10 mg/L时降解率最高,16 h可达98.7%。【结论】分离出的MC-LR降解菌为蜡状芽胞杆菌,该菌株对MC-LR有较高的降解能力,并且酶促反应受到反应体系的pH值、胞内粗酶液浓度以及藻毒素初始浓度等因素的影响。     

木质素降解菌株的分离及其降解玉米秸秆过程中产酶特点

【目的】筛选高效降解木质素的菌株,并研究其以玉米秸秆为底物时木素降解酶活性。【方法】本研究以愈创木酚培养基和苯胺蓝培养基从吉林省不同经纬度的自然朽木及腐朽玉米秸秆土壤样品中分离、筛选得到高效降解木质素的菌株,并对其形态学鉴定,通过ITS序列分析构建系统发育树,分析菌株的分类地位。通过秸秆固体发酵过程产生的胞外木质素酶的活性分析,选出高效秸秆降解菌。【结果】筛选出1株高效降解秸秆的真菌,对其进行形态学特征和ITS序列分析,命名为白囊耙齿菌W2(Irpex lacteus W2)。该菌株在4–8 d内产生的锰过氧化物酶(Manganese peroxidase)呈上升趋势,并且在8 d达到峰值86.31 U/mL,与黄孢原毛平革菌(Phanerochaete chrysosporium)的最高酶活力45.86 U/mL相比,高出了88.20%(P0.01);该菌株的漆酶(Laccase)活力8 d时达到20.60 U/mL,比对照高40.76%(P0.05)。【结论】本研究分离到一株具有较强降解秸秆能力的真菌,初步鉴定为Irpex lacteus W2,具有较强的降解秸秆能力,其降解秸秆过程中产生较高的锰过氧化物酶与漆酶活力。     

锰离子Mn(Ⅱ)对糙皮侧耳木质素降解酶活性和原基形成的影响

木质素降解酶是糙皮侧耳Pleurotus ostreatus分解木质素的关键酶。本文以糙皮侧耳栽培菌株New831为试验材料,通过固体培养法和液体培养法测定了不同浓度锰离子Mn(II)对其菌丝生长、偶氮染料橙黄II降解率、木质素降解酶活性和原基形成的影响。结果表明,添加Mn(II)可促进糙皮侧耳菌丝生长,GP-Orange液体培养时的最适Mn(II)浓度为50μmol/L,GP-Urea-Orange固体培养时的最适Mn(II)浓度为70μmol/L;添加Mn(II)可加速橙黄II的降解,Mn(II)浓度为70μmol/L时,橙黄II降解率最高、脱色圈最大;漆酶和锰过氧化物酶活性均随Mn(II)浓度增加呈现"先升后降"的趋势,而木质素过氧化物酶活性则随Mn(II)浓度的增加而降低;添加Mn(II)可诱导原基形成,Mn(II)浓度为70μmol/L时,原基数量最多。此外,平板培养结果揭示木质素降解酶活性与菌丝生物量之间无相关性,其主要是在营养限制条件下表达。     

不同浓度外源乙酸钠对耐乙酸大肠埃希菌DA19代谢和关键酶酶活的影响

为研究外源乙酸钠对大肠埃希菌DA19生长代谢的影响,将该菌株在氮源限制基本培养基及添加不同浓度乙酸钠的氮源限制基本培养基中连续培养,测定稳态时生长代谢参数和胞内关键酶酶活。与MN培养基相比,葡萄糖比消耗速率和延胡索酸比生成速率随外源乙酸钠质量浓度增加而逐渐下降,丙酮酸比生成速率则随外源乙酸钠质量浓度增加而明显增加,而乙酸比生成速率则明显降低(除9 g/L乙酸钠外)。磷酸果糖激酶、异柠檬酸脱氢酶、异柠檬酸裂解酶、苹果酸脱氢酶、磷酸烯醇式丙酮酸羧化酶和乙酸激酶酶活随外源乙酸钠质量浓度增加而呈先下降后上升的趋势,而6-磷酸葡萄糖脱氢酶则随着外源乙酸钠质量浓度增加而逐渐降低。为了应对外源乙酸钠压力,大肠埃希菌DA19的生长代谢和中心代谢途径酶活都发生了明显改变。     

宏基因组来源耐Mn2+、热稳定细菌漆酶的分子克隆及酶学特性

【背景】漆酶和锰过氧化物酶(Manganese peroxidase,Mnp)是木质素降解的主要酶,二者有协同效应。Mnp活性依赖于Mn~(2+),而Mn~(2+)是大多数漆酶的抑制剂。【目的】获得耐Mn~(2+)的细菌漆酶用于木质素降解。【方法】构建许昌市某污水河污泥宏基因组文库,通过活性筛选获得细菌漆酶基因lac1542。使用大肠杆菌异源表达Lac1542,研究纯化后的重组蛋白酶学性质并进一步检测了含Lac1542复合酶系降解木质素能力。【结果】测序结果显示lac1542编码一个含513个氨基酸的蛋白。以ABTS为底物Lac1542最适反应pH为4.0,在pH 3.0-6.5范围内酶活性稳定。最适反应温度是75°C,在70°C以下酶活性稳定;100 mmol/L的Mn~(2+)仍能提高酶的活性。动力学参数研究发现,该酶的最适底物顺序为:ABTS丁香醛联氮儿茶酚2,6-DMP愈创木酚。Lac1542/Mnp复合酶系对木质素降解率为47.8%,比单独使用Mnp木质素降解率(22.4%)提高25.4%。Lac1542/Mnp/灰盖鬼伞过氧化物酶(Coprinus cinereus Peroxidase,CIP)复合酶系木质素降解高达71.5%,比Mnp/CIP酶系木质素降解率(48.9%)提高22.6%,加入Lac1542后的复合酶系能明显提高木质素的降解率。【结论】Lac1542的可溶性表达、耐受高浓度Mn~(2+)、热稳定性使得Lac1542可以替代一些经典的真菌漆酶应用于制浆、造纸、纤维素乙醇生产、染料脱色等工业。     

木材白腐菌分解木质素的酶系统-锰过氧化物酶、漆酶和木质素过氧化物酶催化分解木质素的机制

<正>近年来许多研究者进行了木材白腐菌分解木质素的酶系统对木质素的催化分解机制的研究。木材白腐菌在分解木质素的过程中会产生分解木质素的酶系统,氧化与分解木质素,这些酶系统主要包括细胞外过氧化物酶(锰过氧化物酶-MnP、木质素过氧化物酶-LiP)和细胞外酚氧化酶-漆酶(laccase)。在降解     

杂色云芝生物预处理对硬木和软木酶水解的影响

节能减排的生物预处理技术是促进木质纤维素酶水解转化乙醇的有效途径。本试验首次研究了白腐菌杂色云芝(Trametes vesicolor)生物预处理对柳木(Salix babylonica,硬木)和杉木(Cunninghamia lanceolata,软木)纤维素酶水解的影响及作用机制。结果显示生物预处理使硬木和软木的最终转化率分别增加4.78倍和4.02倍。通过研究酶与基质的相互作用发现,预处理后木材基质与酶亲和力的增强并不一定导致酶水解初始转化率的提高;但水解过程中转化速率的下降速度随着解吸附指数增加而降低,说明生物处理主要通过减少纤维素酶对基质的不可逆吸附,延缓水解过程中基质转化速率的急剧下降,从而提高水解效率。不可逆吸附的降低与预处理过程中木质素的部分降解与改性有一定关系。     

Induction of tolerance to oxidative stress in the green alga, Chlamydomonas reinhardtii, by abscisic acid

The effect of abscisic acid (ABA) on the tolerance to oxidative stress in a freshwater green alga, Chlamydomonas reinhardtii, was investigated. Exogenously added ABA enhanced the growth of this alga, which was observed under continuous illumination but not in the dark. The cells treated with ABA for 24 h showed tolerance to oxidative stress caused by exposure to paraquat or hydrogen peroxide. In the ABA‐treated cells, the activities of two antioxidant enzymes, catalase (CAT) and ascorbate peroxidase (APX), were significantly higher than those in the untreated control. The result suggests that ABA plays a role in the enhancement of tolerance to oxidative stress by increasing the activity of antioxidant enzymes.     

Paraquat-generated oxidative stress in rat liver induces heme oxygenase-1 and aminolevulinic acid synthase

The in vivo effect of the known herbicide, paraquat, on both hepatic oxidative stress and heme metabolism was studied. A marked increase in lipid peroxidation and a decrease in reduced glutathione (GSH) content were observed 1 h after paraquat administration. The activity of liver antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase was decreased 3 h after paraquat injection. Heme oxygenase-1 induction started 9 h after treatment, peaking at 15 h. delta-aminolevulinic acid synthase induction occurred once heme oxygenase had been enhanced, reaching its maximum (1.5-fold of control) at 16 h. delta-aminolevulinic acid dehydratase activity was 40% inhibited at 3 h showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of alpha-tocopherol (35 mmol/kg body weight) 2 h before paraquat treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels as well as heme oxygenase-1 and delta-aminolevulinic acid synthase induction. This study shows that oxidative stress produced by paraquat leads to an increase in delta-aminolevulinic acid synthase and heme oxygenase-1 activities, indicating that the herbicide affects both heme biosynthesis and degradation.     

Nitric oxide synthase like activity-dependent nitric oxide production protects against chilling-induced oxidative damage in Chorispora bungeana suspension cultured cells

In the present study, we used suspension cultured cells from Chorispora bungeana Fisch. and C.A. Mey to investigate whether nitric oxide (NO) is involved in the signaling pathway of chilling adaptive responses. Low temperatures at 4 °C or 0 °C induced ion leakage, lipid peroxidation and cell viability suppression, which were dramatically alleviated by exogenous application of NO donor sodium nitroprusside (SNP). The levels of reactive oxygen species (ROS) were obviously reduced, and the activities of antioxidant enzymes such as ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), glutathione reductase (GR, EC 1.6.4.2), peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) and the contents of ascorbic acid (AsA) and reduced glutathione (GSH) increased evidently in the presence of SNP under chilling stress. In addition, under low temperature conditions, treatment with NO scavenger PTIO or mammalian NO synthase (NOS) inhibitor l-NAME remarkably aggravated oxidative damage in the suspension cultures compared with that of chilling treatment alone. Moreover, measurements of NOS activity and NO production showed that both NOS activity and endogenous NO content increased markedly under chilling stress. The accumulation of NO was inhibited by l-NAME in chilling-treated cultures, indicating that most NO production under chilling may be generated from NOS-like activity. Collectively, these results suggest that chilling-induced NO accumulation can effectively protect against oxidative injury and that NOS like activity-dependent NO production might act as an antioxidant directly scavengering ROS or operate as a signal activating antioxidant defense under chilling stress, thus conferring an increased tolerance to chilling in C. bungeana suspension cultures.     

Paradoxically, prior acquisition of antioxidant activity enhances oxidative stress-induced cell death

Oxidative stress has been implicated in the induction of programmed cell death in a wide variety of organisms. Acquiring antioxidant capacity is thought to enhance the viability of cells challenged by a subsequent oxidative stress. Counter-intuitively, we show that in two phytoplankton species, Chlamydomonas reinhardtii and Peridinium gatunense , representing the green and red plastid lineages, oxidative stress induced cell death in cultures that already possessed high antioxidant activity but not in cells that exhibited low activity. Cell death of low antioxidant possessing cultures was markedly enhanced by the addition of dehydroascorbate, a product of ascorbate peroxidase (APX), but not of ascorbate or reduced glutathione, and was preceded by increased metacaspase expression and activity. These data suggested that the level of APX and its products, strongly upregulated by oxidative stress, serves as a possible surveillance signal, reporting that the cells already experienced an earlier oxidative stress. Our data presents a novel role of APX in antioxidant activity and response to oxidative stress in photosynthetic microorganisms. Elimination of cysts production by phytoplankton cells that were already damaged by oxidative stress (indicated by the rise in oxidized proteins) as the inoculum for the following year's population may be the evolutionary trigger for this phenomenon.     

外源一氧化氮提高白灵侧耳菌丝耐热性生化途径分析

为了探索外源一氧化氮（NO）提高食用菌菌丝体耐热性的生化途径,以白灵侧耳Pleurotus eryngii var. tuoliensis菌株CCMSSC 00489为材料,通过测定高温胁迫下外源添加硝普钠（sodium nitroprusside,SNP,NO供体）后,菌丝体内超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）和过氧化物酶（POD）等4个抗氧化酶活性的变化,研究外源NO在高温胁迫响应中对抗氧化酶的影响。试验表明,高温胁迫致使菌丝体内TBARS含量升高,膜脂过氧化加剧。在正常温度培养（CK）下,外源添加SNP无显著缓解膜脂过氧化的效果,而高温胁迫条件下缓解效果显著,高温胁迫6h和12h,TBARS含量较对照（未添加）分别下降31.5%和25%。研究表明,抗氧化酶类对外源NO的响应不同。在有外源添加SNP的高温胁迫条件下,菌丝体内的SOD、CAT和GR活性随处理时间的延长而显著增强,在处理72h达到最高,分别是对照（0h）的1.73、7.29和4.95倍。其中CAT是高温胁迫响应的主要抗氧化酶类,其活力可以mmol/L·min^-1·mg^-1 of protein计量,而其他种类的活力均仅以μmol/L·min^-1·mg^-1 of protein计量。在试验条件下,这些抗氧化酶类活性的提高与TBARS含量的降低相呼应,表明外源NO通过提高SOD、CAT、GR的活性降低高温胁迫下的活性氧水平,缓解其氧化损伤,提高菌丝体耐热性。POD活性在外源添加SNP的高温胁迫条件下显著降低。     

Oxidative stress decreases antioxidant enzyme activities in reaggregation cultures of rat brain cells

The brain has been suggested to be especially sensitive to damage by reactive oxygen species. In this study, we examined the effects of hyperoxic conditions on the activities and mRNA levels of antioxidant enzymes in reaggregation cultures of rat forebrain cells. Cultures were exposed to 80% oxygen for 12–60 h starting on Days 17 and 33 in culture. Superoxide dismutase activities and mRNA levels were not affected by hyperoxia, whereas catalase activity was slightly decreased after 24 h in 80% oxygen at Day 17. Glutathione peroxidase activity was markedly decreased already after 12 h of hyperoxia, and decreased activities of glutathione reductase and glucose-6-phosphate dehydrogenase were also noted. The glutathione peroxidase mRNA levels were increased in hyperoxic cultures at Day 17 but not at Day 33. These results suggest that the enzymatic defense mechanisms against reactive oxygen species in the brain are rather weak and deteriorate during oxidative stress but that a potential for compensatory upregulation exists at least during the first postnatal weeks.     

Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.     

Opposing roles for superoxide and nitric oxide in the NaCl stress-induced upregulation of antioxidant enzyme activity in cotton callus tissue

The roles of superoxide and NO in the NaCl-induced upregulation on antioxidant enzyme activity were investigated in NaCl-tolerant cotton calli. Both NaCl and paraquat treatments resulted in significant increases in superoxide production. The activities of ascorbate peroxidase (APX), catalase, glutathione reductase (GR), and peroxidase also increased significantly within 2 h after applying the stress. Pre-treatment with the superoxide scavenger, N-acetyl l-cysteine (NAC), completely removed the superoxide and inhibited the upregulation of antioxidant enzyme activity in the tissue treated with either NaCl or paraquat. NaCl stress also resulted in a significant increase in the NO level. Experiments were also carried out to measure antioxidant enzyme activity in cotton calli exposed to NO, the NO producer sodium nitroprusside (SNP), and the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) under different salt stress conditions. The direct addition of NO gas produced no change in the activities of catalase and GR and caused a significant decrease in APX activity when compared to the controls. When the calli was treated with SNP in the absence of NaCl stress, APX and GR activities decreased significantly and catalase activity was only slightly higher than the control. Treatment with SNP in the presence of NaCl stress resulted in a significant decrease in APX activity, and GR and APX activities were not significantly different from those observed in the NaCl treatment alone. In the presence of PTIO, the activities of all three enzymes increased in the presence or absence of NaCl stress. These results suggest that reactive oxygen species (ROS) such as superoxide radicals may serve as signal transduction molecules to switch “on” the early NaCl-induced up-regulation of antioxidant enzyme activity, while NO may play a role in switching “off” the response after other mechanisms in the cascade of events responsible for NaCl tolerance have been activated.