pH-Dependence of Manganese (II) Oxidation Reaction by Novel Wild-Type and Mutants Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3) Enzymes

The goal of this study was to determine whether mutation of the Mn-binding site of wild-type recombinant Phlebia radiata manganese peroxidase 3 affected the pH-dependence kinetic parameters. pH range investigated was 2.5 – 12.0. The catalytic efficiency of the mutant enzymes at high and low pH in comparison to the wild-type was investigated using standard rPr-MnP3 protocol. Wild-type recombinant Phlebia radiata MnP3 enzyme showed optimal activity with Mn (II) as substrate at pH 5.0 and remained moderately active (approximately 40%) in the pH range of 6.0 - 9.0. The rPr-MnP3 mutants’ maximum activity ranged between 5.5 and 8.0. Wild-type and mutants rPr-MnP3 enzymes exhibited a similar pH profile with optimum pH of 3.0 for ABTS oxidation. Mutation has severely decreased the catalytic efficiency for Mn (II) oxidation at pH 5.0. The rPr-MnP3 enzymes showed enhanced affinity for Mn (II) at alkaline pH and a more alkaline range for catalysis than ever reported for any Manganese Peroxidase. This study reveals that at higher pH, rPr-MnP3 can function with alternative ligands in the Mn (II) site and does not have an absolutely obligate requirement for an all carboxylate ligand set. These results further strongly confirm that Mn2+ binding site is the only productive catalytic site for Mn (II) oxidation.

The Roles of pH in the Modification of Wild-Type Recombinant Phlebia radiata Manganese Peroxidase 3 Activities and Stability of Secondary Structures

This investigation is aimed at understanding the specific role of pH and calcium ions on the activity and stability of wild-type recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3). The pH-dependent cycle of reactions for rPr-MnP3 was evaluated by investigating time-dependent changes in the activity and electronic absorption spectrum of rPr-MnP3.The rPr-MnP3 had maximum efficacy (kcat/Km) for Mn (II) oxidation at pH 5.0 and 3.0 for oxidation of ABTS. Raising the pH of a solution of resting rPr-MnP3 from pH 6.7 (form XH) to pH 8.6 (form X−), a rapid alkaline transition occurs. Leaving the X− form of the enzyme at pH 8.6, it slowly becomes converted to a third form of the enzyme Y−, which returned to the original XH form of the enzyme at pH 6.7. Recovery of form XH from form Y− occurred through an intermediate Z form. The pH inactivation of rPr-MnP3 followed first-order kinetics. The rate of formation of XH from Z is pH-dependent and biphasic in nature, with measured rate constants (k) = 0.25 min−1, and half-life (T1/2) = 2.8 min. The pH-dependent properties observed may be indicative of a greater degree of conformational flexibility at rPr-MnP3 active site due to disruption of the haem-linked hydrogen-bonding network in the distal haem pocket. Calcium ions were observed to significantly stabilised the enzyme’s spectral features and reduce the loss of activity during the alkaline pH transition. Calcium ions enhance the recovery of the initial activity but cannot prevent the final time-dependent irreversible denaturation and aggregation.

Synergistic effects of cellobiose dehydrogenase and manganese-dependent peroxidases during lignin degradation

The synergistic effects of cellobiose dehydro-genase (CDH) and manganese-dependent peroxidases (MnP) on the degradation of kraft pulp cellulolytic enzyme lignin (CEL) were investigated. Addition of CDH significantly increased the amount of water-soluble products reduced from CEL by MnP. CDH facilitated the reduction of the contents of methoxyl, carboxyl, phenolic hydroxyl and total hydroxyl groups of CEL by MnP. 1H-NMR analysis showed that addition of CDH also decreased further the amount of protons of CEL degraded by MnP. The results proved for the first time that CDH could promote degradation of lignin by MnP and suggest that CDH could not only promote degradation of cellulose but also is an important part of the lignin biodeg-radation system.

愈创木酚法测定锰过氧化物酶活力

拟定了用愈创木酚测定锰过氧化物酶活力的方法。本方法通过测定465nm 处吸光度的变化,能直接反映愈创木酚被酶氧化的过程,而且,在吸收谱线的直线范围内,可以比较精确地计算出酶的活力。本方法的最适条件为:0.4mmol/L 愈创木酚,0.1mmol/L H_2O_2,0.2mmol/LMnS0O_和50mmol/L 琥珀酸钠缓冲液,pH4.0—4.5。

白腐真菌F2的生长及产木质素降解酶特性的研究

考察了从我国生物资源中筛分出的白腐真菌F2的生长特性,采用多因素试验法研究了F2菌产木质素降解酶的特性;用统计学方法对试验数据进行了误差分析,并分析了多种影响因素对F2菌产木质素降解酶的影响.实验结果表明:F2菌在PDA培养基平板上和Tien＆Kirk培养基中都生长良好且生长速率较高;在自由悬浮振荡培养(不通氧气)条件下,F2菌产LiP的最高酶活可达60 5U·L-1,MnP的最高酶活可达263 4U·L-1,其产酶能力超过了黄孢原毛平革菌在自由悬浮振荡培养(不另外通入氧气)条件下的产酶能力.本研究还发现向培养基中添加的Cu2+浓度超过1μmol·L-1时会抑制F2菌产LiP,以往的文献未报道过Cu2+的这种抑制作用.

锂离子动力电池热老化的路径依赖性研究

以北京奥运会纯电动公交车用90A·h锰酸锂电池为研究对象,将电池放置于温箱中从25℃开始,依次经历0℃、25℃、40℃、60℃和25℃的'热漂移',在每个温度下对电池依次进行C/20、C/3、C/2、2C/3和1C充放电的基准性能测试,用于量化电池容量、倍率特性和其他性能变化。'热漂移'引起电池容量衰减的根本原因包括活性材料损失、反应动力学衰退和欧姆电阻增加。利用容量增量分析法对实验结果进行分析,结果表明:电池经历0~40℃的'热漂移'已经出现容量衰退,而暴露在60℃下会导致不可逆的容量衰退,其主要原因是活性材料损失和反应动力学衰退。实验方法和结论有助于电动汽车在实际应用条件下锂离子动力电池衰退的路径依赖性研究。

白腐真菌产锰过氧化物酶的研究

以锰过氧化物酶活性为评价指标,研究各种因子对白腐真菌生长及产锰过氧化物酶的影响。结果表明:藜芦醇、苯甲醇和吐温80的添加有利于促进白腐真菌的生长及产锰过氧化物酶活性,在浓度分别为150mg·L-1、150mg·L-1、14mg·L-1时对白腐真菌生长及产锰过氧化物酶促进作用最好;Ca2+、Fe2+对白腐真菌生长及产锰过氧化物酶活性的影响是一致的,均表现为"先促进,后抑制"的作用;Cu2+浓度小于0.4mg·L-1时能略微促进白腐真菌的生长及产锰过氧化物酶活性,大于该浓度时具有较明显的抑制作用。

活性红M-3BE脱色真菌的筛选及脱色性能研究

从自然环境中分离到2株对染料活性红M-3BE具有明显脱色效果的真菌，经形态学和26SrDNA序列分析，将其鉴定为Fusarium oxysporum和Geosmithia viridis。采用初始浓度50mg／LM-3BE的液体培养基同步脱色培养，F．oxysporum在24h内对M-3BE的脱色率为96％，G．viridis在36h内的脱色率为82％。对脱色酶系的检测结果表明，脱色过程中F．oxysporum能产生LiP和MnP，G．viridis则仅产生LiP。此外，F．oxysporu和G．viridis对另外7种染料的脱色率也可分别达到16％～100％和83．3％～100％。

污水处理系统中染料脱色酵母菌的筛选及脱色性能

从某印染厂和制药厂的污水处理系统中筛选了21株脱色酵母菌,测定了各菌株对染料RB5,M-3BE、KNR的脱色能力以及锰依赖过氧化物酶(MnP)的产生情况.结果表明,培养72h时有18株酵母菌对RB5,M-3BE的脱色率超过90%,且检测到MnP活性.26SrDNA序列分析结果表明,脱色酵母菌主要属于Rhodotorula mucilaginosa,Dipodascus capitatus,Torulaspora delbrueckii,Candida parapsilosis,Candida zeylanoides和Debaryomyces hansenii.

活性黑对黄孢原毛平革菌锰过氧化物酶的影响

观察了活性黑KN-B(Reactive Black KN-B,RB KN-B)对黄孢原毛平革菌(Phanerochaete chrysosporium)锰过氧化物酶(MnP)酶活力和菌丝超微结构的影响以及黄孢原毛平革菌对RB KN-B的降解。于P.chrysosporium培养液MnP酶活达最高前,分别加入质量浓度为50 mg/L,200 mg/L,350 mg/L和500 mg/L的RB KN-B。分光光度法检测培养液MnP酶活,电镜观察菌丝超微结构的影响,紫外可见光谱法检测培养液中RB KN-B的降解。结果显示,1)与对照组相比,50 mg/L RBKN-B组的MnP酶活力增强,200 mg/L3、50 mg/L和500 mg/L组的MnP酶活力均显著低于对照组;2)电镜观察显示,经RB KN-B作用后,菌丝细胞膜受损,细胞内含物减少,胞质浓缩,出现质壁分离现象,500mg/L组有大量细胞解体;3)紫外可见光谱扫描显示,RB KN-B经黄孢原毛平革菌降解,可见光波段最大吸收峰由598 nm移至525 nm和556 nm,峰值减小,紫外波段的吸收峰由315 nm移至352 nm。结果显示,黄孢原毛平革菌对RB KN-B的反应类似机体对不良环境因子的应激反应,经历了诱导、抑制及衰退的过程;RB KN-B对黄孢原毛平革菌菌丝细胞超微结构的损伤随RB KN-B浓度增高而增强,表明RBKN-B对MnP酶活的抑制与黄孢原毛平革菌结构受损密切相关;黄孢原毛平革菌对RB KN-B具有一定的降解能力,其中MnP作为关键酶起了重要的作用。

锰过氧化物酶的生物信息学分析

用生物信息学的分析方法,系统地研究了锰过氧化物酶的核苷酸序列和蛋白质氨基酸序列,用DAMBE软件计算了基因长度、碱基组成、GC含量,蛋白质长度、氨基酸组成,找出了基因和蛋白质的相似片段,并分别绘制了每种同工酶的基因序列和蛋白质序列的系统进化树。

锰依赖过氧化物酶研究进展

介绍了锰过氧化物酶的产生菌、催化机理、基因克隆及其性质等方面的研究成果,并对其可能带来的工业应用前景以及今后的研究方向进行了展望。

木素酶在制浆造纸工业中的应用研究进展

简述了近年来木素酶在制浆造纸工业中应用研究的最新进展 ,包括木素酶法漂白、木素酶法处理改善纸浆性能。

分光光度法测定酵母菌脱色酶系的研究

利用分光光度法测定一株对染料活性黑5(Reactive Black 5)具有高效降解活性的酵母菌——Debary-omyces polymorphus的脱色酶活性。结果表明,脱色过程中该菌株可产生高达1200U/L的锰依赖过氧化物酶(MnP),而木质素过氧化物酶(LiP)、漆酶(Laccase)以及细胞色素P450酶系活性均未测出。

利用国外锰矿安全评价与对策

如何提高我国利用国外高品位锰矿资源的安全性是关系我国钢铁工业发展的大问题。通过对国外锰矿资源、生产、国际市场贸易以及我国锰矿进口等方面进行系统研究,探讨我国利用国外高品位锰矿资源所面临的严峻形势与问题,进而分别从资源不可再生角度和比较利益原则等方面综合考虑并提出化解这些不利因素的若干应对措施。

甲烷厌氧氧化及其微生物特性研究进展

甲烷是一种温室气体,其引起的温室效应是CO2的20倍。微生物通过甲烷厌氧氧化(AOM)可以减少自然环境中该温室气体的排放,对缓解全球温室效应具有重大作用。根据耦联反应的不同,可将AOM分为3类,包括硫酸盐还原型、反硝化型以及铁锰依赖型。系统地介绍了AOM的类型及参与的微生物的特性,并对未来更多的AOM微生物的发现以及机理的研究提出展望,以期为甲烷排放控制和管理提供依据。

选择压力对染料脱色酵母菌活性影响的研究

一株对染料活性黑5(Reactive Black 5,RB5)具有高效降解活性的酵母菌—Debaryomyces polymor-phus,在传代培养过程中脱色能力及产锰依赖过氧化物酶(MnP)活性逐步降低。研究结果表明,采用向培养体系中反复投加新鲜染料的方法,可以对脱色酵母菌的生长形成持续性选择压力,从而使菌株的脱色和产MnP活性得以显著提高。

富锂锰基正极材料在不同温度下的极化行为

富锂锰基正极材料作为极具潜力的下一代锂离子动力电池正极材料,在不同温度下电化学性能表现出很大差异,严重限制了其在实际环境中的应用。采用多种电化学测试表征了富锂锰基材料在5~45℃温度范围内电化学性能的差异,从极化的角度分析了材料性能与温度依赖关系的影响因素。结果表明:富锂锰基材料的充放电容量随着温度的降低而降低,主要源于高电压和低电压区间内氧/锰离子反应随温度降低极化显著增大,造成其贡献的容量显著降低。这主要是因为氧/锰离子本征动力学性能差使电荷转移过程具有较高的表观活化能。此外,氧/锰离子参与电荷补偿反应使材料结构发生较大变化,一方面诱发界面膜成分发生变化,增加了低电压区间界面锂离子传输表观活化能,另一方面造成充放电末期锂离子固相扩散具有较高的表观活化能。因此,改善富锂锰基材料氧/锰离子反应动力学是提高其环境适应性的主要措施。

Effects of glucose on the decolorization of Reactive Black 5 by yeast isolates

The cometabolic roles of glucose were investigated in decolorization of an azo dye, Reactive Black 5, by yeast isolates, Debaryomyces polymorphus and Candida tropicalis. The results indicated that the dye degradation by the two yeasts was highly associated with the yeast growth process and glucose presence in the medium. Color removal of 200 mg dye/L was increased from 76.4% to 92.7% within 60 h to 100% within 18-24 h with the increase of glucose from 5 to 10 g/L, although the activity of manganese dependent peroxidase （MnP） decreased by 2-8 times in this case. Hydrogen peroxide of 233.3 μg/L was detected in 6 h in D. polymorphus culture. The cometabolic functions of glucose and hydrogen peroxide could be also confirmed by the further color removals of 95.8% or 78,9% in the second cycle of decolorization tests in which 7 g glucose/L or 250 μg H202/L was superadded respectively together with 200 mg dye/L.

Effect of nitrogen concentration in culture mediums on growth and enzyme production of Phanerochaete chrysosporium

Effect of different nitrogen concentration in the mediums on growth and enzyme production of Phanerochaete chrysosporium was studied when glucose concentration was 10 g/L. The results showed that the medium contained 0.8 g/L ammonium tartrate is the best. It not only supply abundant nutrients for the growth of Phanerochaete chrysosporium, which make mycelia the best grow compared with the other medium, but also produce higher manganese-dependent peroxidase(Mnp) and laccase(Lac) activity. In addition, it is observed that the variation of mycelia surface is related to ligninolytic enzyme secreted by Phanerochaete chrysosporium. When the surface of mycelium pellets appeared burs, it predicts secondary metabolism begin. This experimentation demonstrated that when the ratio of carbon and nitrogen in nitrogen limited medium is equal to 100∶8, growth and enzyme production of Phanerochaete chrysosporium is the best, it could achieve the maximum Mnp and Lac activity.