氟铃脲对斜纹夜蛾酚氧化酶活性的影响

【目的】探讨氟铃脲对斜纹夜蛾Spodoptera litura酚氧化酶活性的影响.【方法】应用酶动力学法,在确定斜纹夜蛾酚氧化酶最适反应条件基础上,测定氟铃脲与酚氧化酶直接作用及氟铃脲处理幼虫后酚氧化酶的活性.【结果】以邻苯二酚为底物,斜纹夜蛾酚氧化酶的最适pH 6.5,最适温度30℃.氟铃脲没有和酚氧化酶结构中的铜离子结合,而是直接与酶蛋白发生作用.氟铃脲浓度低于1 153 mg/L对离体酚氧化酶有激活作用,在138 mg/L激活作用最大,浓度高于1 153 mg/L则产生抑制作用.用46 mg/L和92 mg/L亚致死剂量氟铃脲连续处理5龄幼虫不同时间后,酚氧化酶活性均显著提高,且高浓度的激活作用大于低浓度的激活作用.进一步测定药剂处理幼虫24,48和72 h的血淋巴、表皮和头部酚氧化酶活性,随着氟铃脲处理时间延长,两个剂量的氟铃脲对血淋巴、表皮及头部酚氧化酶的激活作用逐渐增大,氟铃脲对血淋巴酚氧化酶的激活作用最大,表皮次之,头部最低.氟铃脲处理幼虫后,其预蛹和蛹酚氧化酶的活性也显著提高,表现出一定的后效应.【结论】一定浓度范围内,氟铃脲对斜纹夜蛾酚氧化酶具有激活作用.     

中国对虾（Penaeus chinensis）酚氧化酶的分离纯化及其部分生物化学性质

以中国对虾 (Penaeuschinensis)的血淋巴为材料 ,利用凝胶过滤和离子交换等方法 ,对酚氧化酶 (phe noloxidase ,E .C .1 .1 0 .3.1 )进行了分离纯化和生物化学性质研究。实验发现 ,酚氧化酶原 (prophenoloxidase)的分子量为 87.5kD左右 ,在实验操作过程中极易发生降解或自身降解 ,变成有活性的大小约 77kD的酚氧化酶 ,1 0g/LSDS可使该酶原全部被激活而成为有活性的酚氧化酶。以L 二羟苯丙氨酸 (L DOPA)为特异性底物对酚氧化酶活性进行研究发现 ,其最适pH值为 6 .0左右 ,最适温度为 4 0℃ ,Km 值约为 1 .99mmol/L。利用多种氧化酶抑制剂对酚氧化酶纯化样品的酶活性进行研究 ,发现抗坏血酸 (ascorbicacid)、半胱氨酸 (cysteine)和二硫苏糖醇(dithiothreitol)对酚氧化酶活性具有很强的抑制作用 ,硫脲 (thiourea)对酚氧化酶活性也具有较强的抑制作用 ,而酚氧化酶对苯甲酸 (benzoicacid)、柠檬酸 (citricacid)和亚硫酸钠 (sodiumsulfite)不敏感 ,而且该酶对酪氨酸等单酚还具有高特异性的酚氧化酶活性 ,表明它可能是一种酪氨酸酶型的酚氧化酶 ;此外 ,该酶对EDTA和金属离子非常敏感 ,其活性能被Cu2 强烈抑制 ,被Mg2 强烈激活 ,表明该酶很可能是一种金属酶 (metalloenzyme)     

花椒叶浸提液对土壤微生物数量和土壤酶活性的影响

通过用花椒叶浸提液浇灌盆栽花椒幼苗,研究浸提液对土壤酶和土壤微生物的影响.结果表明, 花椒叶浸提液使根际土中细菌、真菌和放线菌数量以及微生物总数均有不同程度的减少,根际土中真菌和放线菌的数量变化呈降-升-降-升的趋势.20、60和80 g·L^-1浓度的叶浸提液使非根际土中细菌的数量显著增加21.59%、107.55%和8.62%,而40 g·L-1浓度的叶浸提液则使非根际土中细菌数量显著降低22.56%.叶浸提液使根际土蛋白酶、蔗糖酶和酸性磷酸酶活性明显低于非根际土相应的酶活性,而过氧化氢酶和多酚氧化酶活性则显著升高.土壤的蛋白酶活性与蔗糖酶活性呈显著正相关,与土壤放线菌数量呈显著负相关;多酚氧化酶活性与蔗糖酶活性呈显著负相关,与细菌、真菌、放线菌以及微生物总数呈显著正相关;放线菌只与蛋白酶、多酚氧化酶、蔗糖酶3种酶活性及真菌呈显著相关,与过氧化氢酶、酸性磷酸酶以及细菌和微生物总数的相关性均不显著.     

荔枝(Litchi chinensis)果皮多酚氧化酶的部分纯化及性质

催化荔枝果皮褐变反应的多酚氧化酶,自新鲜果皮提取后,经丙酮沉淀和硫酸铵分级,酶活性增高8.2倍。该酶催化氧化邻位二元酚和三元酚的化合物,但不氧化一元酚。在40℃以下,该酶在1小时内活性保持不变,55℃以上,活性迅速下降。酶活性的最适温度为35℃。 以焦棓酚、D,L-3,4-二羟基苯丙氨酸或邻苯二酚作底物时,其Km值分别为1.85、2.5和5.0mM;Vmax值分别为41.62×10~3、0.85×10~3和0.24×10~3酶单位/分钟/毫克蛋白。 该酶强烈地受亚硫酸氢钠和二乙基二硫代氨基甲酸钠所抑制。二乙基二硫代氨基甲酸钠是荔枝果皮多酚氧化酶的非竞争性抑制剂,其Ki值为0.05mM。     

氮添加对湿地松林土壤水解酶和氧化酶活性的影响

通过3个水平野外氮添加控制试验(0、40、120 kg N·hm^-2·a^-1),研究氮添加对亚热带湿地松林土壤水解酶和氧化酶活性的影响.结果表明: 氮添加显著抑制了土壤有机质中碳、氮、磷水解酶和氧化酶的活性,导致β-1,4-葡糖苷酶(BG)、纤维素二糖水解酶(CBH)、β-1,4-乙酰基-葡糖胺糖苷酶(NAG)、过氧化物酶(PER)活性下降16.5%～51.1%,并且高水平氮添加对酶活性抑制效果更明显;氮添加导致α-1,4-葡糖苷酶(aG)、β-1,4-木糖苷酶(BX)、酸性磷酸酶(AP)、多酚氧化酶(PPO)活性降低14.5%～38.6%,不同水平氮添加处理间差异不显著.土壤酶活性存在明显的季节性差异,BG、NAG、BX、CBH、AP、PPO活性表现为3月>6月>10月,aG、PER活性表现为10月>3月>6月.多数土壤水解酶和氧化酶与pH呈显著正相关,与NO₃^--N含量呈显著负相关,表明氮添加导致pH降低和土壤中硝化作用增强,抑制了土壤水解酶和氧化酶活性.氮添加不利于亚热带土壤有机质的矿化和周转,并且随着氮添加量的增加,效果更明显.     

保幼激素类似物及蜕皮甾类对亚洲玉米螟幼虫酚氧化酶活性的影响

分别用1 μg/头、0.1 μg/头和0.01 μg/头浓度的保幼激素类似物methoprene（蒙五一五）体外处理亚洲玉米螟5龄幼虫,测定幼虫体壁组织、血清和血细胞溶离物中酚氧化酶的活性。结果表明： 1 μg/头 methoprene处理组和0.1 μg/头处理组幼虫体壁组织中酚氧化酶活性与对照组相比有显著提高（P<0.01）,血清和血细胞溶离物中酚氧化酶活性也显著上升（P<0.01）。将含有20-羟基蜕皮酮的人工饲料饲喂亚洲玉米螟5龄幼虫,处理组幼虫体壁组织的酚氧化酶活性下降(P<0.05),血清和血细胞溶离物中的酚氧化酶活性均低于对照组 （P<0.01）。这些结果表明methoprene可以诱导亚洲玉米螟5龄幼虫体内酚氧化酶活性的上升,而20-羟基蜕皮酮则抑制了酚氧化酶的活性。     

一株地衣芽孢杆菌产凝乳酶酶学性质研究

目的：对地衣芽孢杆菌所产凝乳酶的酶学特性进行研究。方法：在不同的温度、pH值、底物浓度、不同金属离子等条件下测定地衣芽孢杆菌产凝乳酶相对酶活力。结果：地衣芽孢杆菌所产凝乳酶最适凝乳温度为70℃;40℃以上热处理后凝乳活性有不同程度的损失,75℃热处理10min后凝乳酶活性丧失;pH值为5～8时凝乳活性随乳pH值的降低而增强,pH值为7时凝乳酶最稳定;Ca2+ 、Fe2+、Fe3+、Mn2+、Mg2+、Al3+对酶活性有一定的促进作用;Li2+、Na+、Cu2+、Zn2+对酶活性有抑制作用;底物浓度最适为250 g/L、Ca2+的最适浓度为0.014 g/L。     

铜对三叶草-土壤酶系统的影响

通过盆栽实验研究了重金属Cu污染对植物(三叶草)-土壤酶(脲酶、蔗糖酶、过氧化氢酶和多酚氧化酶)系统的影响.结果表明,随着Cu浓度增加,脲酶、蔗糖酶、过氧化氢酶和多酚氧化酶活性均逐渐减小,与Cu浓度有高度相关性,蔗糖酶>多酚氧化酶>脲酶>过氧化氢酶.在处理浓度不变情况下,酶活性随时间而变化,且呈现低Cu浓度(<00 mg·kg^-1)时4种酶活性均有所上升,而Cu浓度增高(00～3 000 mg·kg^-1)时各酶活性逐渐下降的趋势.统计分析表明,在每一梯度浓度上,4种酶在Ⅰ、Ⅱ、Ⅲ组内均存在显著差异性(P<0.01),与植物受重金属Cu污染时的生长情况一致.随着Cu浓度增加,土壤pH值逐渐下降,而电导率上升;同一Cu浓度下的pH值和电导率均随时间呈缓慢上升趋势,统计分析显示,二者在Ⅰ、Ⅱ、Ⅲ组内均存在显著差异性(P<0.01).土壤pH值和电导率与4种土壤酶活性有高度相关性,多酚氧化酶>蔗糖酶>过氧化氢酶>脲酶.这4种酶同时可作为检测土壤环境质量的指标.     

模拟氮沉降对温带森林土壤酶活性的影响

森林土壤酶作为土壤中最活跃组分,能影响生态系统的物质循环过程,其活性能快速反映氮沉降对土壤环境的变化。以北京地带性植被辽东栎林为研究对象,利用模拟氮沉降方法,原位设计低氮(50 kg N hm~(-2)a~(-1),N50)、高氮(150 kg N hm~(-2)a~(-1),N150)两个施氮水平,每个施氮水平设置Na NO_3、(NH_4)_2SO_4、NH4NO_33个不同的施氮类型,另设置空白对照(0 kg N hm~(-2)a~(-1),N0)。从时间格局上研究不同氮素化学形态和剂量对温带森林土壤6种酶(脲酶、酸性磷酸酶、碱性磷酸酶、β-葡萄糖苷酶、多酚氧化酶和过氧化氢酶)活性的影响。结果表明:在氮形态和水平的交互作用下,NH4NO3-N处理的脲酶活性显著高出NO~-_3-N处理的24.20%(N50),NH~+_4-N处理对酸性磷酸酶活性的影响显著高出NO~-_3-N处理的13.82%(N150);在NH~+_4-N和NH_4NO_3-N处理中,N50水平下的脲酶活性分别高出N0处理的38.90%和24.20%,差异显著。对无氮形态和水平交互作用的酶活性分析得出,不同的施氮水平,对碱性磷酸酶和多酚氧化酶的酶活性有显著促进作用,碱性磷酸酶活性在N50和N150处理下分别比N0高20.2%和11.5%,N50和N150处理对多酚氧化酶活性的促进作用分别比N0处理高64.3%和41.8%,差异显著(P0.05);NH~+_4-N处理对β-葡萄糖苷酶活性具有显著促进作用(P0.05),不同的施氮形态,对碱性磷酸酶、多酚氧化酶和过氧化氢酶的酶活性无显著影响。6种酶活性均呈现了显著的时间变化,氮添加对森林土壤酶活性的时间分异规律没有显著影响。此外,土壤微生物量碳、硝态氮和铵态氮含量与酶活性具有显著相关性(P0.05)。以上结果表明,氮添加通过改变森林土壤的环境因子,影响了土壤中的水解酶和氧化酶活性,进而改变了土壤有机碳库和养分循环。     

藏东南森林土壤微生物群落结构与土壤酶活性随海拔梯度的变化

【目的】针对青藏高原藏东南地区色季拉山不同海拔森林土壤,探讨微生物群落与土壤酶活性之间的联系以及受控因子。【方法】利用微生物细胞膜磷脂(PLFA)方法研究土壤微生物群落结构随海拔变化情况,分析土壤葡萄糖苷酶、酚氧化酶、蛋白酶、L-天冬酰胺酶、脲酶和酸性磷酸酶活性以及土壤理化性质随海拔的变化趋势。【结果】土壤理化性质和生化指标随海拔增高没有显著变化,如水分含量、有机碳、全氮、碳氮比、pH、无机氮和硝态氮,土壤葡萄糖苷酶、酚氧化酶、蛋白酶、L-天冬酰胺酶和酸性磷酸酶活性等;然而,微生物丰度呈现中峰优势分布规律,细菌、真菌、革兰氏阳性菌、革兰氏阴性菌和放线菌含量在海拔3 900 m和4 000 m处生物量显著高于低海拔和更高海拔。皮尔森相关性分析表明土壤pH是影响微生物群落结构的主要因子,但海拔梯度上的温度变化与微生物群落结构和酶活性不存在显著相关性;同时,有机碳、全氮、水溶性有机碳和水溶性有机氮和pH等理化指标与土壤酶活性显著相关。【结论】在藏东南色季拉山森林生态系统,海拔梯度对土壤微生物群落结构影响较大,土壤理化指标与生物特征对海拔梯度的响应较弱。     

马尾松和苦槠林根际土壤矿化和根系分解CO2释放的温度敏感性

以中亚热带马尾松林和苦槠林为对象,原位收集根际和非根际土壤、树木不同生态功能的根系,开展15 ℃、25 ℃、35 ℃和45 ℃恒温培养模拟试验,采用密闭气室碱液吸收法测定53 d内CO₂释放的动态变化.结果表明: 两种森林类型不同温度下土壤矿化CO₂释放速率的根际效应介于1.12～3.09,且培养前期高于培养后期;15 ℃下马尾松林和苦槠林差异不显著,25 ℃和35 ℃下前者低于后者,45 ℃下则相反.不同培养温度下两树种吸收根分解的CO₂释放速率均高于过渡根和贮存根,且马尾松均低于苦槠.两种森林类型CO₂释放的Q₁₀值均为土壤(1.21～1.83)显著高于根系(0.96～1.36).两种森林类型土壤矿化CO₂释放的Q₁₀值差异不显著,而马尾松根系分解CO₂释放的Q₁₀值高于苦槠.推断全球变暖导致的土壤矿化CO2释放的增量将远远高于根系分解,且马尾松林高于苦槠林;地带性顶极群落应对气候变化的抵抗力强于先锋树种群落.     

ABTS assay of phenol oxidase activity in soil

Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 degrees C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 microl of a 0.1 M ABTS solution.     

ABTS assay of phenol oxidase activity in soil

Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2′-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 °C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 μl of a 0.1 M ABTS solution.     

Interactive effects of wildfire and permafrost on microbial communities and soil processes in an Alaskan black spruce forest

Boreal forests contain significant quantities of soil carbon that may be oxidized to CO₂ given future increases in climate warming and wildfire behavior. At the ecosystem scale, decomposition and heterotrophic respiration are strongly controlled by temperature and moisture, but we questioned whether changes in microbial biomass, activity, or community structure induced by fire might also affect these processes. We particularly wanted to understand whether postfire reductions in microbial biomass could affect rates of decomposition. Additionally, we compared the short‐term effects of wildfire to the long‐term effects of climate warming and permafrost decline. We compared soil microbial communities between control and recently burned soils that were located in areas with and without permafrost near Delta Junction, AK. In addition to soil physical variables, we quantified changes in microbial biomass, fungal biomass, fungal community composition, and C cycling processes (phenol oxidase enzyme activity, lignin decomposition, and microbial respiration). Five years following fire, organic surface horizons had lower microbial biomass, fungal biomass, and dissolved organic carbon (DOC) concentrations compared with control soils. Reductions in soil fungi were associated with reductions in phenol oxidase activity and lignin decomposition. Effects of wildfire on microbial biomass and activity in the mineral soil were minor. Microbial community composition was affected by wildfire, but the effect was greater in nonpermafrost soils. Although the presence of permafrost increased soil moisture contents, effects on microbial biomass and activity were limited to mineral soils that showed lower fungal biomass but higher activity compared with soils without permafrost. Fungal abundance and moisture were strong predictors of phenol oxidase enzyme activity in soil. Phenol oxidase enzyme activity, in turn, was linearly related to both ¹³C lignin decomposition and microbial respiration in incubation studies. Taken together, these results indicate that reductions in fungal biomass in postfire soils and lower soil moisture in nonpermafrost soils reduced the potential of soil heterotrophs to decompose soil carbon. Although in the field increased rates of microbial respiration can be observed in postfire soils due to warmer soil conditions, reductions in fungal biomass and activity may limit rates of decomposition.     

Purification and characteristics of an enzyme with both bilirubin oxidase and laccase activities from mycelium of the basidiomycete <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

A homogenous enzyme with both bilirubin oxidase and laccase activities was isolated from a submerged culture of the basidiomycete Pleurotus ostreatus mycelium and characterized. The yield of the enzyme was 127 μg/g dry biomass of the mycelium. The specific activity of the enzyme was 21 and 261 U/mg to bilirubin and to a laccase substrate ABTS, respectively. The intracellular phenol oxidase from the P. ostreatus mycelium was identified as bilirubin oxidase with the amino acid sequence highly homologous to that of the pox2 gene-encoded product. The enzyme displayed the maximal laccase activity at 50–55°C to all substrates examined, whereas the pH optimum was substrate-dependent and changed from 3.0 for ABTS to 7.0 for syringaldazine and guaiacol. The enzyme maintained catalytic activity within a broad pH range but was inactivated at pH 4.0. The enzyme was thermostable but very sensitive to metal chelating inhibitors. Trypan Blue (5 mg/liter) was completely decolorizated upon 3 h of incubation with the bilirubin oxidase (20 mU/ml) at room temperature.     

Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver

Modifications of the analytical method to determine L-gulono-gamma-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-gamma-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-gamma-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-gamma-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-gamma-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-gamma-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-gamma-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.     

中国成熟天然林土壤有机碳垂直分异特征

以分布在中国不同气候区的131个成熟天然林土壤为研究对象,测定不同土层(0～10、10～20、20～30、30～50和50～100 cm)土壤有机碳(SOC)密度,分析其与气象因子、土壤性质的关系,研究天然林SOC垂直分布特征及其影响机理。结果表明: 温带针叶林、温带落叶阔叶林、亚热带落叶阔叶林和亚热带常绿阔叶林0～30 cm土层SOC密度均随土壤深度增加而降低。在0～100 cm土层,SOC密度地带性分异明显,温带针叶林SOC密度显著高于温带落叶阔叶林,亚热带常绿阔叶林SOC密度显著高于亚热带落叶阔叶林。SOC密度与土壤黏粒、年降水量以及地上净初级生产力呈显著正相关,与土壤pH和年均温呈显著负相关。年降水量与年均温调节天然林SOC输入与输出,土壤pH与黏粒影响天然林SOC积累,对成熟的天然针叶林与常绿阔叶林进行有效保护,有利于增加我国森林土壤碳库。     

Stoichiometry of soil enzyme activity at global scale

Extracellular enzymes are the proximate agents of organic matter decomposition and measures of these activities can be used as indicators of microbial nutrient demand. We conducted a global-scale meta-analysis of the seven-most widely measured soil enzyme activities, using data from 40 ecosystems. The activities of beta-1,4-glucosidase, cellobiohydrolase, beta-1,4-N-acetylglucosaminidase and phosphatase g(-1) soil increased with organic matter concentration; leucine aminopeptidase, phenol oxidase and peroxidase activities showed no relationship. All activities were significantly related to soil pH. Specific activities, i.e. activity g(-1) soil organic matter, also varied in relation to soil pH for all enzymes. Relationships with mean annual temperature (MAT) and precipitation (MAP) were generally weak. For hydrolases, ratios of specific C, N and P acquisition activities converged on 1 : 1 : 1 but across ecosystems, the ratio of C : P acquisition was inversely related to MAP and MAT while the ratio of C : N acquisition increased with MAP. Oxidative activities were more variable than hydrolytic activities and increased with soil pH. Our analyses indicate that the enzymatic potential for hydrolyzing the labile components of soil organic matter is tied to substrate availability, soil pH and the stoichiometry of microbial nutrient demand. The enzymatic potential for oxidizing the recalcitrant fractions of soil organic material, which is a proximate control on soil organic matter accumulation, is most strongly related to soil pH. These trends provide insight into the biogeochemical processes that create global patterns in ecological stoichiometry and organic matter storage.     

Biosensor for the determination of phenols based on cross-linked enzyme crystals (CLEC) of laccase

Cross-linked enzyme crystals (CLECs) are a versatile form of biocatalyst that can also be used for biosensor application. Laccase from Trametes versicolor (E.C.1.10.3.2) was crystallized, cross-linked and lyophilized with beta-cyclodextrin. The CLEC laccase was found to be highly active towards phenols like 2-amino phenol, guaiacol, catechol, pyrogallol, catechin and ABTS (non-phenolic). The CLEC laccase was embedded in 30% polyvinylpropylidone (PVP) gel and mounted into an electrode to make the sensor. The biosensor was used to detect the phenols in 50-1000 micromol concentration level. Phenols with lower molecular weight such as 2-amino phenol, catechol and pyrogallol gave a short response time where as the higher molecular weight substrates like catechin and ABTS had comparatively a long response time. The optimum pH of the analyte was 5.5-6.0 when catechol was used as substrate. The CLEC laccase retained good activity for over 3 months.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.