Biodegradation of phenolic environmental pollutants by a surfactant-laccase complex in organic media

Oxidative degradation of phenolic environmental pollutants in organic media was investigated using a laccase complexed with surfactants. The catalytic activity of the surfactant-laccase complex in isooctane was markedly enhanced by appropriately adjusting the water content of the reaction medium using reverse micelles. The surfactant-laccase complex showed little activity towards the oxidative reaction of bisphenol A in water-saturated isooctane (i.e., 0.0055% [v/v] water) while effectively catalyzed the same reaction in isooctane containing 4% (v/v) water, which is over the maximum water solubility. The latter system was homogeneous and was only achieved by the aid of reverse micelles. With respect to the oxidation of bisphenol A, two products, 4-isopropylphenol and 4-isopropenylphenol, were identified by gas chromatography-mass spectrometry (GC-MS) analyses, indicating the oxidative degradation of the bis-phenolic structure of bisphenol A. We also found that the surfactant-laccase complex turned out to handle other environmental pollutants, chlorophenols, by the simultaneous addition of water and a redox mediator into the reaction medium using reverse micelles.     

Hydrolysis of Milk Fat with Lipase in Reversed Micelles

Enzymatic hydrolysis of milk fat was studied in a reversed-micelle system consisting of lecithin, water (1% total volume) and butteroil. Candida cylindracea lipase entrapped in the water pools of reversed micelles hydrolyzed surrounding triglycerides. The optimum temperature for enzyme activity was 55°C with an activation energy of 15.4 kcal/mole, while maximum enzyme activity was observed between pH 4 to 6. The molar ratio of water to surfactant (R value) in the system influenced enzyme activity with maximum activity at R = 10. Enzyme activity also increased with increasing surfactant or enzyme concentration.     

漆酶催化二氢杨梅素氧化聚合

系统考察漆酶催化条件对二氢杨梅素衍生物的颜色、α-葡萄糖苷酶抑制及抗氧化活性的影响,并运用波谱手段对产物结构进行表征.结果表明漆酶可用于二氢杨梅素的氧化聚合,且漆酶浓度、pH值、反应时间及温度对衍生物的颜色有规律性影响.所得二氢杨梅素衍生物的α-葡萄糖苷酶抑制活性(IC506.2μg/mL)显著高于二氢杨梅素(IC50...     

漆酶及其在食品工业中的应用

漆酶来源广泛、催化性能优异,在食品工业中具有应用价值。本文介绍了漆酶的来源与一般催化特性,就漆酶在食品工业中的研究与应用进展予以重点综述,并对其应用前景予以展望。     

Lyophilization of lipase with cyclodextrins for efficient catalysis in ionic liquids

Lipase was lyophilized with cyclodextrins to prepare lipase formulation suitable for the efficient resolution of allethrolone in ionic liquids. The effects of the type and amount of cyclodextrin used on lipase preparation were evaluated, and the properties of lyophilized lipase such as thermostability and pH sensitivity were investigated and compared with those of native lipase. The results showed that lipase lyophilized with cyclodextrins can achieve a higher conversion rate than the native one, and that lipase lyophilized with inorganic salts cannot improve the conversion rate of the resolution reaction. The catalytic behavior of the lyophilized lipase was strongly dependent on cyclodextrin type and reaction media. The activity of the lyophilized lipase increased as the amount of added cyclodextrins increased. The activity of the lipase lyophilized with cyclodextrins was optimum at pH 7 and 40 degrees C, which was similar to that of the native one, but the half-life of the lyophilized lipase was low compared with that of the native one.     

Enhanced enzyme activity and enantioselectivity of lipases in organic solvents by crown ethers and cyclodextrins

Lipases from Candida rugosa (CRL) and Pseudomonas cepacia (PCL) were co-lyophilized with cyclic oligoethers including four crown ethers and nine cyclodextrins (CyDs), and their transesterification activity and enantioselectivity in organic solvents were evaluated. The PCL co-lyophilized with each additive showed simultaneously enhanced enzyme activity and enantioselectivity when compared to the native lipase lyophilized from buffer alone; in contrast, such enhancement was not observed for the co-lyophilized CRL. Among the cyclic oligoethers examined, permethylated betaCyD (Me1.78betaCyD), as the most suitable additive, was used for the optimization of both the co-lyophilized PCL preparation and reaction conditions by determining the effects of varying the additive/lipase ratio, aqueous pH, the nature of organic solvents, and temperature. The initial rate determined for the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in diisopropyl ether at 30 degrees C increased by up to 17-fold and the enantioselectivity represented by E could be doubled. While there was an inverse correlation between temperature and enantioselectivity, with the Me1.78betaCyD-PCL co-lyophilizate, the reaction rate even at 0 degrees C was much higher than that at higher temperatures in the native PCL-catalyzed reaction. Hence, this method seems to be of practical use for the large-scale production of optically active compounds.     

韦伯灵芝漆酶的分离纯化及其性质

采用硫酸铵分级盐析、透析、聚乙二醇浓缩和高效液相色谱柱层析从韦伯灵芝TZC-1 发酵液中分离纯化得到电泳纯漆酶,其纯化倍数为37.1 倍,酶活性回收率为21.3%。活性-PAGE(聚丙烯酰胺凝胶电泳)结果表明该漆酶由一种同工酶组成,纯化漆酶经SDS-PAGE 检测显示为单一条带,其分子质量约为40kD。该漆酶催化底物ABTS 的最适反应温度为50～60℃,最适反应pH 值为4.6,在60℃以下和pH3.0～5.0 范围内保持稳定,以ABTS为底物的表观Km 值为13.8μmol/L。Fe2+ 完全抑制酶活,Al3+ 和Mn2+ 对该漆酶也有明显抑制作用,Hg+、Cu2+ 和Mg2+ 对该酶有明显激活作用,而Zn2+、Ba2+ 及K+ 对该酶活性影响不大。     

Treatment of nonylphenol with laccase in a rotating reactor

The effects of operation conditions on reaction rate in the enzymatic oxidative treatment of nonylphenol with laccase in a rotating reactor were studied. Sea sand that adsorbed nonylphenol was used as a polluted soil model. Nonylphenol concentration decreased with laccase treatment five times faster at 40 degrees C than at 10 degrees C and the apparent activation energy of the enzyme reaction was 39 kJ mol(-1), which was in the range of the values reported for similar laccase reactions. Reaction rate increased when the angle of the axis of the rotating reactor from the vertical line increased and when the speed of revolution was increased to 10 rpm at different volumes of the enzyme solution. Thus, mixing is important for the oxidation of nonylphenol with laccase. The nonylphenol released from the sand into the enzyme solution in the initial stage of the treatment was easily oxidized. However, the nonylphenol adsorbed on the sand reacted slowly. Reaction rate increased nearly proportional to the square root of enzyme concentration, which suggests that the nonylphenol radical reacted with unoxidized nonylphenol (nonenzymatic propagation) during the enzymatic oxidative treatment. The dependence of reaction rate on the nonylphenol concentration was similar to the Michaelis-Menten type. The residual estrogenic activity of the treated sand was measured by medaka vitellogenin assay. The estrogenic activity decreased to 1/6-1/90 after 24 h of the treatment.     

Isolation of a novel alkaline-induced laccase from Flammulina velutipes and its application for hair coloring

Laccase is a member of the multi-copper oxidase family and a promising for hair coloring. In this study, we isolated a novel alkaline-induced laccase from the white-rot fungus Flammulina velutipes and studied the possibility to apply the enzyme for hair coloring. Laccase activity detected in the culture supernatant of F. velutipes was found to significantly increase when exchanging the medium to laccase inducing one whose pH was adjusted to 9.0. Three isozymes were detected by activity staining on non-denaturing SDS-PAGE. The major isozyme, Flac1, was purified from the culture supernatant after being induced at pH 9.0 by ion-exchange column chromatography. The N-terminal peptide sequence of Flac1 was determined, revealing clear homology with laccases from other white-rot fungi. Optimum pH of oxidation was found to be around pH 5.0-6.5 regardless of several different substrates used. Oxidation activities of Flac1 to several hair dye agents as substrate showed the higher activity at pH 6.5 than that at pH 9.0. Oxidation activity was also detected at pH 9.0 which was suitable for hair coloring. When the purified Flac1 was applied for hair coloring system without using hydrogen peroxide, effective coloring was observed at the protein amount of 0.25mg/1g of hair used. These results indicated that this alkaline-induced novel laccase isolated from the culture supernatant of F. velutipes might be a useful enzyme for hair color.     

真菌液态发酵产漆酶的培养条件优化研究进展

漆酶是一类可降解木质素的含铜多酚氧化酶,存在于多种植物、真菌、少数昆虫和细菌中,被广泛应用于废水处理、染整工艺及食品工业等领域。同其它来源的漆酶相比,真菌漆酶具有更好的热稳定性及更高的催化效率。液态发酵是目前人们用来生产真菌漆酶比较常用的方法。研究综述了近几年在真菌液态发酵产漆酶培养条件优化方面的研究进展。     

Production and Gelatin Entrapment of Laccase from Trametes versicolor and its Application to Quantitative Determination of Phenolic Contents of Commercial Fruit Juices

Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is a particularly promising enzyme for several industrial fields, including food industries, since this enzyme catalyzes the oxidation of ortho and para-diphenols, amino-phenols, polyphenols, polyamines, lignins, and aryl diamines as well as some inorganic ions coupled to the reduction of molecular dioxygen to water. In this study, laccase was produced from one of the best laccase-producing organisms, Trametes versicolor. For this purpose, several phenolic acids were tested as laccase inducers. Caffeic acid and ferulic acid were determined to be the best inducers among the tested phenolic acids. Also, it was shown that laccase activity could be determined by using caffeic acid and ferulic acid as phenolic substrates by measuring the rates of oxygen consumption. Laccase was immobilized in gelatin under optimized conditions. Kinetic constants K _m and V _max for immobilized enzyme were estimated to be 74.758 μM and 0.744 μmol.ΔO₂/ml.min for caffeic acid and 0.999 μM and 57.80μ mol.ΔO₂/ml.min for ferulic acid, respectively. The immobilized enzyme exhibited the maximal activity at pH 4.5, and at 35°C. Immobilized enzymes were used for the determination phenolic contents of commercially prepared fruit juices. Caffeic acid contents of black cherry, apricot, and peach juice were determined to be 1640±33, 679±24 and 408±29 mg/L, and their ferulic acid contents were determined to be 1786±28, 800±30, and 444±28 mg/L, respectively.     

A SPECTROPHOTOMETRIC ASSAY FOR LACCASE USING o-TOLIDINE

A spectrophotometric method for measuring laccase activity using o-tolidine has been developed. Oxidation of o-tolidine by laccase to a blue colored product corresponded with increases in absorbancies at 366 and 630 nm. This oxidation reaction and increases in absorbance at 366 and 630 nm could also be mimicked using hypochlorite, periodate and UV light in place of laccase. After a lag period, the assay was linear in absorbance with time, although the duration of linear region appeared to be affected by the pH. When assayed from 0.025 to 7 mM tolidine, maximum oxidation of substrate occurred using 3 mM o-tolidine. Oxidation of o-tolidine exhibited a pH dependency and showed an apparent pH optimum at approximately 5.0. The utility of this assay was shown by determining laccase activity in various fungal extracts.     

Protein refolding in nanostructured reversed micelles including a molecular chaperone

Reversed micelles including the molecular chaperone GroEL were applied to the protein refolding of denatured RNase A. The molecular chaperone was successfully functionalized in the water pools of the reversed micelles sharing a structural size of 15-25 nm. The refolding yield of RNase A in the reversed-micelle/GroEL system was much greater than that of spontaneous renaturation. The refolding yield mediated by GroEL in the reversed micelles was strongly dependent on the presence of ATP or Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active in the micelles. Under the optimum conditions, this novel refolding technique could completely renature the denatured RNase A in 1 h.     

漆酶接枝反应在植物纤维改性中的应用

漆酶能够催化氧化还原反应,与其他氧化还原体系相比,漆酶偶合接枝反应条件温和,反应过程对环境友好。目前在造纸、纺织、食品、制药和污水处理等领域得到广泛应用。本文主要论述通过漆酶对植物纤维的改性,从而提高纸张的强度、湿强度、憎水性能、抗菌性能等。     

漆酶对不同漂段纸浆催化氧化木素的效果研究

以未漂硫酸盐桉木浆(Sp浆)、氧脱木素桉木浆(O浆)、H_2O_2漂白桉木浆(P浆)作为不同漂段原料,探讨漆酶-谷氨酸体系对不同漂段纸浆的催化效果。结果表明,漆酶催化总体上可使纸浆的卡伯值增大,纸浆的白度随之下降;漆酶协同谷氨酸可催化氧化纤维表面木素与相邻纤维上的木素发生自由基偶合反应,同时使纤维表面的微细纤维暴露出来,两者共同作用使相邻纤维形成更好的结合力,使纸浆的强度性能大幅度提升;谷氨酸可通过漆酶催化氧化作用连接到木素上,增加纤维原料中的羧基含量,Sp浆、O浆、P浆羧基含量分别较未处理纸浆提高20%、50%、43%。     

白腐菌漆酶固定化及其在工业中的应用

对漆酶的几种定性及定量检测方法、漆酶固定化及其应用以及漆酶/介体系统在造纸工业中的应用进行了综述.     

响应面法优化Coriolus versicolor ZZH-2产漆酶培养基

采用响应面法对Coriolus versicolor ZZH-2产漆酶培养基进行优化,首先用Plackett-Burman（PB）法筛选出3个影响较大的重要因素,分别为酵母膏、羧甲基纤维素钠和L-天冬氨酸。在此基础上,用最陡爬坡实验逼近最大响应区域,并利用中心组合实验以及响应面分析确定了主要影响因素的最佳条件,即酵母膏13.82g/L、羧甲基纤维素钠13.29g/L和L-天冬氨酸1.44g/L,在优化培养基后漆酶酶活达到9693U/mL,比优化前提高了1.7倍,其实验值与理论值基本相符。      

Stabilization of oil-in-water emulsions by enzyme catalyzed oxidative gelation of sugar beet pectin

Enzyme catalyzed oxidative cross-linking of feruloyl groups can promote gelation of sugar beet pectin (SBP). It is uncertain how the enzyme kinetics of this cross-linking reaction are affected in emulsion systems and whether the gelation affects emulsion stability. In this study, SBP (2.5% w/v) was mixed into an oil-in-water emulsion system (4.4% w/w oil, 0.22% w/w whey protein, pH 4.5). Two separate, identically composed, emulsion systems were prepared by different methods of preparation. The emulsions prepared separately and subsequently mixed with SBP (referred as Mix A) produced significantly larger average particle sizes than the emulsions in which the SBP was homogenized into the emulsion system during emulsion preparation (referred as Mix B). Mix B type emulsions were stable. Enzyme catalyzed oxidative gelation of SBP helped stabilize the emulsions in Mix A. The kinetics of the enzyme catalyzed oxidative gelation of SBP was evaluated by small angle oscillatory measurements for horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2) catalysis, respectively. HRP catalyzed gelation rates, determined from the slopes of the increase of elastic modulus (G′) with time, were higher (P < 0.05) than the corresponding laccase catalyzed rates, but the final G′ values were higher for laccase catalyzed gels, regardless of the presence of emulsions or type of emulsion preparation (Mix A or Mix B). For both enzymes, rates of gelation in Mix A were higher (P < 0.05) than in Mix B, and higher stress was needed to break the gels in Mix A than in Mix B at similar enzyme dosage levels. These differences may be related to a lower availability of the feruloyl groups for cross-linking when the SBP was homogenized into the emulsion system during preparation.     

真菌漆酶高级结构研究进展

白腐菌分泌的木质素降解酶主要有木质素过氧化物酶、锰依赖过氧化物酶和漆酶3种。漆酶属于蓝色多铜氧化酶家族,可以催化氧化多种有机化合物,伴随着分子氧还原成水,但不同来源的漆酶降解能力差异很大,而此恰与漆酶的结构密切相关。作者综述了漆酶尤其是真菌漆酶结构特征、催化活性中心和高级结构等方面的研究进展。     

离子液体中脂肪酶活性调控对硬脂酸淀粉酯合成的影响

主要研究了离子液体中脂肪酶活性的调控对硬脂酸淀粉酯合成的影响。结果表明:在离子液体中添加不同水活度(Aw)的水合盐对对脂肪酶催化活性有显著影响,水合盐对为Na_2HPO_4·7H_2O和Na_2HPO_4·2H_2O的混合物,即Aw为0.61,添加量为80%(水合盐对淀粉质量比)时达到最佳的脂肪酶催化效果;冻干酶的pH值、添加量及冻干过程中冻干保护剂的存在对硬脂酸淀粉酯合成过程中脂肪酶活性同样影响显著,冻干酶催化反应的最适pH值为7.1,最适添加量为10.4%(冻干酶淀粉质量比),脂肪酶和有机化合物等冻干保护剂的加入能保护冻干酶的催化活性,提高反应转化率,其中以聚乙二醇效果最佳。