Biosynthesis and Characterization of Laccase Catalyzed Poly(Catechol)

Enzymatic polymerization of catechol was conducted batch-wise using laccase enzyme produced by the culture Trametes versicolor (ATCC 200801). The polymerization reaction was carried out in 1:1 (v/v) aqueous-acetone solution, buffered at pH 5.0 with sodium acetate (50 mM) in a sealed, temperature-controlled reactor at 25°C. The molecular weight of the produced polymer was determined with GPC. FT-IR, DSC, and TGA were employed to investigate the structure and thermal behavior of synthesized poly(catechol). It was found that catechol units were linked together with ether bonds and thermal stability of the catechol increased in the poly(catechol) polymeric structure effectively. The number average molecular weight of poly(catechol) was found as 813 ± 3 Da with a very narrow polydispersity value of 1.17 showing selective polymerization of catechol by the enzyme.     

不同共代谢基质对白腐菌降解吲哚的作用研究

选用氨氮、苯酚和喹啉作为吲哚的共代谢基质,通过白腐菌BP对共基质体系的降解研究了白腐菌在秸秆滤出液培养基中对不同共基质体系的代谢过程,并进行了动力学分析,考察了不同共代谢基质物质对白腐菌漆酶分泌和吲哚降解的影响.结果显示,不同降解体系中的白腐菌都可去除99%以上的吲哚.充分的氮源可提高白腐菌的活性和漆酶酶活的峰值;共基质苯酚和喹啉可以增加白腐菌漆酶产量,为吲哚的降解提供较多的电子,同时苯酚和喹啉也能得到较高的去除.在秸秆滤出液中,白腐菌在pH为6～8之间对吲哚都具有较强的降解能力.吲哚在白腐菌的代谢过程中,可能首先在吡啶环的2和3位发生一步羰基化.     

Quantification of Polymerisation Processes During The Oxidative Degradation of 14C-Labelled Chlorophenols

In experiments employing the lignocellulose-decaying basidiomycetes Trametes versicolor and Stropharia rugosoannulata degrading uniformly¹⁴C-labelled 2,4-dichlorophenol and pentachlorophenol, acombination of size exclusion chromatography (SEC),fractionation, and -scintillation counting wasapplied to quantify polymerisation products formed duringchlorophenol degradation. Time-dependent mass balances weregenerated by analysis of ¹⁴C in polymerisation products,CO₂, as well as monomer non-polar and polar metabolites.Approximately 30% of the chlorophenols were found to bepolymerised. A major fraction of the polymerised productscorresponded to a molecular weight range from 0.24 – 40 kDa.Only a minor fraction could be attributed to a molecularweight >40 kDa. This method proved to be useful inquantification of polymerisation products and kinetics of thepolymerisation processes, whereas UV/Vis detection ofpolymerisation products separated by SEC led to false positiveresults. The SEC-¹⁴C method could also be applied forother complex processes where polymerisation ordepolymerisation occurs (humification, degradation oflignocellulose, formation of bound residues from xenobioticssuch as polycyclic aromatic hydrocarbons or 2,4,6-trinitrotoluene) and where spectrophotometric determinationsare difficult or impossible.     

Trametes sp.LS-10C产漆酶发酵培养基优化及其漆酶对偶氮染料的脱色性能

为了提高菌株Trametes sp.LS-10C的漆酶产量并初步研究该酶对偶氮染料的脱色性能,本文通过单因素实验及均匀设计对菌株Trametes sp.LS-10C产漆酶培养基进行了优化,获得最优培养基配方为:葡萄糖14.4 g·L~(-1)、豆渣10.1 g·L~(-1)、麸皮100 g·L~(-1)、NH_4Cl_3g·L~(-1)、KH2PO41.4g·L~(-1)、CuSO_4’5H2O 1 g·L~(-1)、NaCl 1 g·L~(-1)、MgSO_40.8 g·L~(-1)、CaCl_20.5 g·L~(-1).菌株发酵至8 d时漆酶产量为595.15 U·m L~(-1),约是优化前的35.66倍.进一步研究表明,该漆酶-介体系统对酸性红GR、酸性蓝40和酸性铬蓝K等3种偶氮染料脱色10 h后脱色率分别高达96.86%、91.28%和86.31%.相比公开发表的文献,本研究所报道的Trametes sp.LS-10C利用优化培养基发酵产漆酶达到了较高水平,具有酶活力高和发酵时间短等特征,且该漆酶在处理偶氮染料废水脱色领域中具有进一步研发和应用价值.     

重组大肠杆菌产CotA漆酶的发酵条件优化

CotA漆酶在环境保护、食品工业和纸浆漂白等工业中具有重要的应用价值.将重组表达载体pET-22b/CotA转入大肠杆菌BL21(DE3),得到工程菌株,采用单因素实验和正交实验相结合的方法,研究诱导表达条件和发酵培养基对重组大肠杆菌产CotA漆酶量的影响.结果表明,初始pH 7.5的培养基中,添加0.6 mmol L-1 Cu2+,1 g L-1葡萄糖作碳源、15 g L-1蛋白胨和2 g L-1硫酸铵作氮源,以10%的接种量,37℃、200 r/min,直到菌液的D600 nm值为1.0,加入终浓度为1.0 mmol L-1的IPTG,25℃诱导12 h,漆酶的产量最高.优化前发酵液的粗提液的漆酶活性仅为1 190 U mL-1,优化后达到3 526 U mL-1,正交实验优化后漆酶活性提高了2.96倍.纯化的CotA漆酶最适反应温度为45℃,最适pH值为7.2.CotA漆酶对RBBR的脱色率在90%以上,此CotA漆酶在短时间内对染料能够有效地脱色,能够成为有潜力的工业用酶.图6表3参17     

杂色云芝漆酶和发酵液降解对苯二酚研究

测定和分析了杂色云芝漆酶和杂色云芝发酵液对对苯二酚的降解反应.通过对不同底物质量浓度下杂色云芝漆酶酶学反应动力学特征分析,求得漆酶Lac A对对苯二酚的酶反应动力学方程V=1.52Cs/(16.55 Cs)和底物抑制反应速率方程γs=1.02/(1 12.86/Cs Cs/137.17).发酵液对对苯二酚的降解反应进程符合一级反应动力学方程lnC=-kt 6,发酵液对对苯二酚的降解反应动力学基本符合Michelis-Menten酶学反应动力学方程Vf=1.77Cs/(3.63 Cs),研究表明Lac A与发酵液对对苯二酚的酶学反应动力学具有较大的相似性,也存在着底物抑制现象,其底物抑制的动力学方程为γs'=1.40/(1 5.75/Cs Cs/278.26).比较漆酶纯酶Lac A和发酵液对对苯二酚反应的Km和Vmax数值,发现杂色云芝发酵液Km较小而Vmax数值较大,说明发酵液较漆酶纯酶具有更高的底物亲和力和催化效率.     

Differential fate of metabolism of a disperse dye by microorganisms Galactomyces geotrichum and Brevibacillus laterosporus and their consortium GG-BL

The present work aims to evaluate Brown 3 REL degrading potential of developed microbial consortium GG-BL using two microbial cultures, Galactomyces geotrichum MTCC 1360 (GG) and Brevibacillus laterosporus MTCC 2298 (BL). Microbial consortium GG-BL showed 100% decolorization of a dye Brown 3 REL, while individually G. geotrichum MTCC 1360 and B. laterosporus MTCC 2298 showed 26% and 86% decolorization under aerobic condition (shaking) respectively. Measurements of biochemical oxygen demand (BOD) (76%) and chemical oxygen demand (COD) (68%) were done after decolorization by consortium GG-BL. No induction in activities of oxidoreductive enzymes found in G. geotrichum while B. laterosporus showed induction of veratryl alcohol oxidase, Nicotineamide adenine dinucleotide-dichlorophenol indophenol (NADH-DCIP) reductase and riboflavin reductase indicating their role in dye metabolism. Consortium GG-BL showed induction in the activities of laccase, veratryl alcohol oxidase, tyrosinase, NADH-DCIP reductase and riboflavin reductase. Two different sets of induced enzymes from G. geotrichum and B. laterosporus work together in consortium GG-BL resulting in faster degradation of dye. The degradation of Brown 3 REL was analyzed using high performance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FT-IR) and gas chromatography mass spectroscopy (GC-MS). Phytotoxicity study revealed that metabolites formed after degradation was significantly less toxic in nature.     

漆酶降解阴离子型聚丙烯酰胺/聚丙烯酸酯活性的理性设计

为探究漆酶降解聚丙烯酸酯（PAA）/阴离子型聚丙烯酰胺（HPAM）的微观机理,采用对接模拟了其结构模型与枯草芽孢杆菌漆酶（B. subtilis laccase）的结合.根据-CDOCKER_Energy score打分最高的原则,对获得的最佳结合构象进行分析.然后基于亲和力虚拟氨基酸突变进行丙氨酸（ALA）扫描和饱和突变.结合模式分析表明,HPAM比PAA可更深地埋入活性口袋,B. subtilis laccase对HPAM的亲和力和结合能皆高于PAA.相互作用分析表明,疏水相互作用可能对B. subtilis laccase与底物的结合起到促进作用,而氢键作用会阻碍该酶与底物的结合.通过ALA扫描进一步得知,ARG487、GLY486和TYR133是B. subtilis laccase降解HPAM的关键氨基酸残基,而ASP113和TYR133是B. subtilis laccase降解PAA的关键氨基酸残基.通过饱和突变表明,ASP113>ARG可以提高B. subtilis laccase降解PAA的活性,这些数据为理性设计增强活性的B. subtilis laccase突变体提供了理论参考.     

偏肿拟栓菌共代谢降解芘条件的优化

为提高中国东北土著白腐真菌偏肿拟栓菌(Pseudotrametes gibbosa)对高分子量多环芳烃芘的共代谢降解效果,通过正交试验研究了共代谢基质、接种量、装液量及2,2￠-连氮-二(3-乙基苯并噻唑-6-磺酸)(ABTS)的最佳配比,同时采用浓度梯度法考察芘的初始浓度对偏肿拟栓菌共代谢降解芘的影响.结果表明,偏肿拟栓菌Pseudotrametes gibbosa共代谢降解芘的最佳培养基为:麸皮浓度为20g/L,接种量为3片直径为10mm的菌片,装液量为50mL,不加ABTS,在该培养条件下,酶活可达53.41U/mL,对芘的降解率达到88.8%.初始浓度小于10mg/L的芘对菌体产酶有一定的促进作用,大于10mg/L时抑制漆酶的分泌,同时菌体对初始浓度小于90mg/L芘的降解率在22.24%~93.54%之间.     

杂色云芝漆酶对溴酚蓝的高效降解

以杂色云芝菌(Coriolus versicolor)发酵所产漆酶为催化剂,对溴酚蓝溶液进行脱色处理。探讨了反应温度、反应体系pH、反应时间、酶加入量、溴酚蓝质量浓度及金属离子和小分子介质的添加等因素对溴酚蓝溶液脱色率的影响。实验结果表明:在反应温度为40℃、反应体系pH为4.5、反应时间为240 min、酶加入量为60U/L、溴酚蓝质量浓度为40 mg/L的条件下,溴酚蓝溶液的脱色率达到94.5%;小分子物质2,2′-连氮-双(3-乙基苯并噻吡咯啉-6-磺酸)(ABTS)或金属离子的添加对溴酚蓝溶液的脱色效果没有明显的促进作用。