锗对霍山石斛类原球茎悬浮培养细胞生长和多糖合成及氧化还原态的影响

为解决霍山石斛类原球茎液体培养细胞生长缓慢和代谢水平低下的问题,研究了不同浓度的锗(GeO2)对霍山石斛类原球茎增殖、多糖合成及碳氮利用的影响,分析了类原球茎细胞内还原糖、可溶性蛋白质含量、抗氧化酶活性以及细胞氧化还原态的变化。结果表明,适当浓度的二氧化锗(4.0mg/L)显著促进霍山石斛类原球茎的增殖和多糖的积累,最大细胞干重为32.6g/L,最大多糖产量为3.78g/L;显著提高胞内还原糖和可溶性蛋白质含量,超氧化物歧化酶和过氧化氢酶的活性明显升高,而过氧化物酶的活性则有所降低;氧化还原态分析发现,二氧化锗处理的细胞内还原型谷胱甘肽/氧化型谷胱甘肽的值明显提高,谷胱甘肽还原酶活性升高。添加适量的二氧化锗有利于细胞生长和多糖合成。     

S-腺苷甲硫氨酸合成酶反应条件的优化

优化了重组毕赤酵母表达的S-腺苷甲硫氨酸合成酶催化L-甲硫氨酸(Met)和ATP合成 S-腺苷甲硫氨酸的条件,确定了该酶的最适酶活力检测条件为20mmol/L的L -Met,26mmol/ L的ATP,52mmol/L的MgCl2,300mmol/L的KCl,8mmol/L的还原型谷胱甘肽,100mmol/ L的Tris,反应液pH 8.5,35°C反应 1h,比活力达到23.84U/mg.该酶还可以催化以DL-Met代替L-Met为底物的S-腺苷甲硫氨酸合成反应,以降低生产成本.     

外源表油菜素内酯对NaHCO3胁迫下黄瓜幼苗生长及氧化还原平衡的影响

以‘津研四号’黄瓜为试材,以30 mmol·L^-1NaHCO_3模拟盐碱环境,采用水培法研究了0.2μmol·L^-1外源2,4表油菜素内酯(2,4-epibrassinolide,EBR)对盐碱胁迫下黄瓜幼苗生长和活性氧代谢的影响.结果表明:NaHCO_3胁迫显著诱导了叶片及根系中O2-·的产生和H_2O_2的积累,导致丙二醛含量和电解质渗透率提高.NaHCO_3胁迫下,超氧化物歧化酶、过氧化物酶、过氧化氢酶、抗坏血酸过氧化物酶、脱氢抗坏血酸还原酶、单脱氢抗坏血酸还原酶、谷胱甘肽还原酶活性及还原型抗坏血酸、还原型谷胱甘肽含量随胁迫时间延长呈现先升后降的趋势.外源EBR显著提高了NaHCO_3胁迫下黄瓜叶片和根系中抗氧化酶活性、抗氧化物质的含量以及As A/DHA(双脱氢抗坏血酸)和GSH/GSSG(氧化型谷胱甘肽)比值,维持了植株内的氧化还原平衡,降低了活性氧积累水平,缓解了膜脂过氧化,从而提高了黄瓜幼苗的盐碱耐受性.     

藜芦醇和吐温80对白腐菌产木质素降解酶的影响及在偶氮染料脱色中的作用

担子菌PM2在限氮液体培养下,分泌木质素过氧化物酶和锰过氧化物酶;藜芦醇、吐温 80的补充,提高了该菌锰过氧化物酶的产生,获得的最大锰过氧化物酶Mnp酶活为254.2u/L、190.2 u/L,分别是对照的3.4倍和2.5倍。选择三种偶氮染料,在染料体系下,进一步分析藜芦醇、吐温 80对担子菌PM2产过氧化物酶及染料脱色的影响。结果表明,担子菌PM2分泌的锰过氧化物酶Mnp与染料脱色有关,脱色程度受其分子结构特征影响;吐温80的补充,更有利于染料的脱色降解,48h后三种染料均可达到80%以上的脱色率。     

过量表达GST基因对盐胁迫下转基因拟南芥氧化损伤的影响

利用模式生物拟南芥作为实验材料,通过测定谷胱甘肽-抗坏血酸代谢相关酶（GST、GPX、APX、GR、DHAR、MDHAR）的活性和GSH、ASA、MDA含量以及生物量等来研究过量表达具有过氧化物酶活性的盐地碱蓬谷胱甘肽转移酶基因（GST基因）对盐胁迫下转基因拟南芥氧化损伤的影响。结果显示,转基因拟南芥比野生型具有较高的GST、GPX以及MDHAR酶活性;前者还具有较多的还原型谷胱甘肽和抗坏血酸,并且谷胱甘肽库氧化水平较野生型高。盐胁迫不但部分抑制了野生型拟南芥的生长,同时也导致了大量脂质过氧化物的积累;而盐胁迫对转基因拟南芥的生长抑制不明显,也没有较多的脂质过氧化物的积累。结果表明,过量表达盐地碱蓬谷胱甘肽转移酶基因提高．广转基因拟南芥依赖于还原型谷胱甘肽的过氧化物清除途径,同时有可能改变了GSH和ASA的代谢途径,这两方面的作用导致了转基因拟南芥氧化损伤的降低,使转基因拟南芥在盐胁迫下保持较好的生长态势。     

抗氧化系统在H2O2诱导的玉米幼苗耐热性形成中的作用

H2O2预处理可显著增强玉米幼苗的耐热性.H2O2预处理后,玉米幼苗抗氧化酶谷胱甘肽还原酶(GR)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)的活性及还原型抗氧化剂抗坏血酸(ASA)和谷胱甘肽(GSH)的水平显著提高,且H2O2预处理过的幼苗在高温处理期间及其后的恢复过程中均能保持相对较高的抗氧化酶活力和还原型/氧化型抗氧化剂比例.     

柱状田头菇菌丝对镉胁迫的抗氧化响应

研究了不同浓度Cd处理对柱状田头菇菌丝抗氧化酶及谷胱甘肽含量的影响.结果表明,在低浓度范围内随着Cd处理浓度的增加,菌丝抗氧化酶的活力上升,过氧化氢酶(CAT)与超氧化物歧化酶(SOD)的活性分别在Cd浓度为0.1和0.4mmol·L-1时达最大值;过氧化物酶(POD)、谷胱甘肽还原酶(GR)和脂氧合酶(LOX)的活性在Cd浓度为0.2mmol·L-1时达到峰值.而在高Cd浓度处理时,柱状田头菇菌丝抗氧化酶系(POD、CAT、SOD等)显著受到抑制.0.4~1.6mmol·L-1Cd处理可显著提高菌丝体内还原型谷胱甘肽(GSH)水平,却不影响氧化型谷胱甘肽(GSSG)含量.在整个试验过程中,均未检测到抗坏血酸及抗坏血酸过氧化物酶(APX)的活性.用聚丙烯酰胺凝胶电泳分析Cd胁迫下柱状田头菇菌丝抗氧化酶的同工酶谱发现,0.1~0.8mmol·L-1Cd处理可诱导过氧化物酶(POD)、酯酶(EST)和脂氧合酶(LOX)新同工酶的表达,提高组成型过氧化氢酶(CAT)、超氧化物歧化酶(SOD)同工酶的表达强度;1.6mmol·L-1Cd处理显著抑制POD、CAT、SOD等的表达.     

耐过氧化氢的锰过氧化物酶对三苯甲烷类染料的脱色

[目的]从一株白腐菌Trametes sp.SQ01中获得一种新型的锰过氧化物酶,探讨该酶的底物特异性和对过氧化氢的耐性,以及其对三苯甲烷类染料的脱色能力.[方法]通过丙酮沉淀和DEAE-cellulose 52柱层析法纯化锰过氧化物酶.利用UV-2010紫外可见分光光度法研究锰过氧化物酶对过氧化氢的耐性,同时,用紫外可见分光光度计对三苯甲烷类染料脱色效果进行分析.[结果]通过两步纯化,获得了均一性的锰过氧化物酶.该酶的最适pH和温度分别是4.5和70℃,在pH 3.0-8.0时,酶活相对稳定.该酶在二价锰离子存在下能够氧化2,6-二甲氧基苯酚、愈创木酚、2,2＇-连氮-双-(3-乙基苯并噻唑啉磺酸)和过氧化氢等化合物,同时也能作用二价锰离子.在与这些底物反应中,最适底物为过氧化氢(Km为3.7 tmmol/L).该酶具有抗过氧化氢漂白能力,锰过氧化物酶与高浓度的过氧化氢(2.5 mmol/L)作用60 min后仍能保持70％的活性.在所测试的染料中,锰过氧化物酶对结晶紫的脱色率最高达到65.8％.二价锰离子和过氧化氢对锰过氧化物酶脱色能力的影响进行研究,与孔雀绿相比,锰离子和过氧化氢对活性艳蓝脱色的影响很小.[结论]Trametes sp.SQ01锰过氧化物酶对过氧化氢的耐受性,以及对三苯甲烷类染料的高效脱色能力表明该酶在染料脱色降解方面有着广阔的应用前景.     

动物抗氧化系统中主要抗氧化酶基因的研究进展

抗氧化系统是机体清除体内多余的活性氧、保护自身免受氧化损伤的重要体系,其中超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶等起主要作用。本文将对动物抗氧化系统中,超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶基因的种类、分布、结构及表达进行综述。     

刺芹侧耳芳基醇氧化酶的分离纯化及其酶学性质

分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70kDa和4.2。以藜芦醇为底物,该酶最适反应pH为6.0,最适反应温度为70℃,金属离子Zn²⁺、Fe²⁺和Cu²⁺对芳基醇氧化酶的活性抑制作用明显,K_m和V_max分别为0.921mmol/L和80U/mg。     

Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase.

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).     

Soybean peroxidase as an effective bromination catalyst*

Soybean peroxidase (SBP), an acidic peroxidase isolated from the seed coat, has been shown to be an effective catalyst for the oxidation of a variety of organic compounds. In the present study, we demonstrate that SBP can catalyze halogenation reactions. In the presence of H(2)O(2), SBP catalyzed the oxidation of bromide and iodide but not chloride. Veratryl alcohol (3,4-dimethoxybenzyl alcohol) served as a useful substrate for SBP-catalyzed halogenations yielding the 6-bromo derivative. Halogenation of veratryl alcohol was optimal at pHs below 2.5 with rates of 2.4 μm/min, achieving complete conversions of 150-μm veratryl alcohol in 24 h. The enzyme showed essentially no brominating activity at pHs above 5.5. SBP-catalyzed bromination of veratryl alcohol proceeded with a maximum reaction velocity, (V(max))(apparent), of 5.8 x 10(-1) μm/min, a K(m) of 78 μm and a catalytic efficiency (k(cat)/K(m) of 1.37 x 10(5) M/min at pH 4.0, assuming all of the enzyme's active sites participate in the reaction. SBP also catalyzed the bromination of several other organic substrates including pyrazole to produce a single product 1-bromopyrazole, indole to yield both 5-bromoindole and 5-hydroxyindole, and the decarboxylative bromination of 3,4 dimethoxy-trans-cinnamic acid to trans-2-bromo-1-(3,4 dimethoxyphenyl)ethylene. A catalytic mechanism for SBP-catalyzed bromination has been proposed based on experimental results in this and related studies.     

Aromatic ring oxidation of vanillyl and veratryl alcohols by Lentinus edodes: possible artifacts in the lignin peroxidase and veratryl alcohol oxidase assays

The degradation pathway of vanillyl and veratryl alcohol by Lentinus edodes extracellular enzymes was studied. In both cases several products of side chain oxidation and aromatic ring cleavage were isolated and characterized. We have observed that the products from veratryl alcohol degradation by Lentinus edodes are quite different from those isolated from incubations with other white-rot fungi which have veraraldehyde as the major product, in fact, this compound is not produced as final metabolite in L. edodes incubations. This behaviour could explain the apparent absence of lignin peroxidase and veratryl alcohol oxidase activities in L. edodes cultures, since such activities are usually measured by monitoring veratraldehyde formation during the veratryl alcohol oxidation; thus, it is suggested that additional assay methods should be developed, with preferably direct observation of aromatic ring oxidation products.     

Steady-state and transient-state kinetic studies on the oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerocheate chrysosporium Burds

Catalysis of the H2O2-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H2O2 consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H2O2 (KM = 29 microM) and veratryl alcohol (KM = 72 microM) at pH 3.5. The magnitude of the turnover number varies from 2 to 3 s-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H2O2 reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK alpha approximately 3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH approximately 2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H2O2, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H2O2; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H2O2 and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates.     

Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium

Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production byPhanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.     

抗坏血酸对酵母蔗糖酶的激活动力学研究

采用甲苯自溶法从鲜酵母中提取了蔗糖酶,并用乙醇分级及ＤＥＡＥ－纤维素柱层析进行了纯化,用ＰＡＧ凝胶电泳作了纯度鉴定,在ｐＨ５．０,３０℃条件下进行了酶反应,用双倒数作图法测出其Ｋｍ＝２．１×１０－２ｍｏｌ／Ｌ,Ｖｍａｘ＝０．２６（每分钟的光密度值）．在此系统中,加入不同浓度的抗坏血酸（Ｖｉｔ．Ｃ）,发现其具有激活作用并存在量效关系．双倒数作图显示：酶的表观Ｖｍａｘ（Ｖｐ）随抗坏血酸浓度的增加而增大,但其表观Ｋｍ（Ｋｐ）不变（Ｋｐ＝Ｋｍ）．经实验结果分析,推论出抗坏血酸激活作用的酶促反应方程式,并推导出反应速度公式     

Synthesis of veratraldehyde from veratryl alcohol by lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electrochemical reactor

We report the synthesis of veratraldehyde from veratryl alcohol by Phanerochaete chrysosporium lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electroenzymatic reactor. The effects of operating parameters such as enzyme level, pH, and electrical potential on the efficiency of veratryl alcohol oxidation were investigated. Furthermore, we compared direct addition of hydrogen peroxide with electrogeneration of the material during enzymatic oxidation of veratryl alcohol. The electroenzymatic method using in situ-generated hydrogen peroxide was found to be effective for oxidation of veratryl alcohol by lignin peroxidase. The new method may be easily applied to biodegradation systems.     

Characterization of alkaliphilic laccase activity in the culture supernatant of Myrothecium verrucaria 24G-4 in comparison with bilirubin oxidase

An enzyme showing alkaliphilic laccase activity was purified from the culture supernatant of Myrothecium verrucaria 24G-4. The enzyme was highly stable under alkaline conditions, showed an optimum reaction pH of 9.0 for 4-aminoantipyrine/phenol coupling, and decolorized synthetic dyes under alkaline conditions. It showed structural and catalytic similarities with bilirubin oxidase, but preferably oxidized phenolic compounds. The enzyme catalyzed veratryl alcohol oxidation at pH 9.0 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a mediator, suggesting that the laccase mediator system functioned well under alkaline conditions.     

Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes

Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H(2)O(2) (2 mM) for optimum activity. The apparent K(m) values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H(2)O(2) were 37, 298, 1,001, 2,367 and 1,313 microM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.