Charles Junghanns

Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: Production, partial characterization and application for the elimination of endocrine disruptors

Versatile peroxidase (VP) from Bjerkandera adusta was insolubilized in the form of cross-linked enzyme aggregates (CLEA®s). Of the initially applied activity 67% was recovered as CLEA®s. Co-aggregation of VP with glucose oxidase from Aspergillus niger led to an increased activity recovery of 89%. The combined CLEA®s showed higher stability against H(2)O(2) and exerted VP activity upon glucose addition. The elimination of the endocrine disrupting chemicals bisphenol A, nonylphenol, triclosan, 17α-ethinylestradiol and the hormone 17β-estradiol (10 mg L(-1) each) and the removal of their estrogenic activity by combined CLEA®s were tested in batch experiments. Within 10 min, the combined CLEA®s were able to remove all the endocrine disruptors except triclosan (residual concentration 74%). The removal of the estrogenic activity was higher than 55% for all compounds, except triclosan. A membrane reactor continuously operated with combined CLEA®s could almost completely remove bisphenol A (10 mg L(-1)) for 43 h.

Design-of-experiment strategy for the formulation of laccase biocatalysts and their application to degrade bisphenol A

Immobilizing enzymes can expand their applicability to continuous process operations and facilitates process intensification. An optimized formulation of immobilized biocatalysts is therefore of strategic interest in the field of industrial biotechnology. Nevertheless, biocatalyst formulation still largely relies on empirical approaches which lack effectiveness in the identification of optimum immobilization conditions. In the present study, design of experiments, multiple linear regressions and modeling were used to screen, interpret and finally optimize crucial immobilization parameters. A laccase preparation from Coriolopsis polyzona MUCL38443 was immobilized via a sequential adsorption-crosslinking process on mesoporous silica particles. As a target variable, biocatalyst activity was doubled (∼280 U g(-1)) while dramatically reducing processing time (two hours instead of 26 hours) and reagent inputs (80 mm instead of 1m glutaraldehyde (GLU)). Immobilization yield (∼50%) and thermostability (∼60% residual activity after 24 hours at 45°C) could be maintained under the optimized conditions. As an example of its application in environmental biotechnology, the optimized biocatalyst was implemented in a continuous stirred-tank membrane reactor (CSTMR) to continuously degrade the endocrine disruptor bisphenol A (BPA) in wastewater. A 90% removal of 50 μm BPA was achieved over 30 reactor volumes (hydraulic residence time (HRT) of 1.85 hours, 50 mL working volume).

Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design

The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate (CuSO4). The best conditions obtained from screening experiments in shaken flasks using tomato juice, CuSO4, and soybean oil were integrated in an experimental design. Enhanced levels of tomato juice as the medium, CuSO4 and soybean oil as inducers (36.8% (v/v), 3 mmol/L, and 1% (v/v), respectively) were determined for 10 L stirred tank bioreactor runs. This combination resulted in laccase titer of 143 000 IU/L (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), pH 3.0), which represents the highest activity so far reported for P. sanguineus in a 10-L fermentor. Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20 000 IU/L, whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times. Based on a partial characterization, the laccases of this strain are especially promising in terms of thermostability (half-life of 6.1 h at 60 °C) and activity titers.概要研究目的优化获得血红密孔菌(P. sanguineus)的最佳培养基组成, 提高耐热漆酶的产量。创新要点获得了目前文献报道的最高水平的漆酶活力。研究方法通过单因素试验研究了不同培养基(番茄汁、 麦麸、 麦芽提取物和葡萄糖细菌蛋白胨培养基) 和不同组合诱导剂(大豆油、 阿魏酸、 没食子酸、 二甲基苯胺、 酸性蓝62 和活性蓝19 分别与硫酸铜组合诱导剂)对P. sanguineus 产耐热漆酶的影响。 在此基础上采用中心组合试验设计, 进一步研究了番茄汁培养基结合硫酸铜和大豆油组合诱导剂对P. sanguineus 产耐热漆酶的影响。 利用SAS10.0 和响应面分析方法对试验结果进行了统计分析和建立回归模型。重要结论通过中心组合设计优化得出P. sanguineus 产耐热漆酶的最优培养基条件: 以36.8%番茄汁为培养基, 以3 mmol/L 硫酸铜和1%大豆油作为组合诱导剂。 该条件下在10 L 搅拌槽生物反应器中漆酶活力达到了143 000 IU/L(2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)为底物, pH 值为3.0)。

Quantification of the Influence of Extracellular Laccase and Intracellular Reactions on the Isomer-Specific Biotransformation of the Xenoestrogen Technical Nonylphenol by the Aquatic Hyphomycete Clavariopsis aquatica

ABSTRACT The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selective suppression of either laccase or intracellular reactions resulted in essentially comparable maximal removal rates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained from laccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reaction mixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomers by bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast with laccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric composition of remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficiently degraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern of the quaternary α-carbon of t-NP isomers does not seem to be very important for intracellular transformation in C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectral data of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimethylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, and the subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturation and methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.

Stimulation of laccases from Trametes pubescens: Use in dye decolorization and cotton bleaching

ABSTRACT The production of laccases from Trametes pubescens was investigated along with the role of nutrients and elicitors. Copper proved to be a fundamental inducer, although productivity yields were consistently enhanced only in the presence of additional compounds (textile dyes). Using a central composite design, the optimal culture condition was examined, by taking into consideration the three distinct variables and their combinatorial effect. The 290 U ml−1 of laccases were produced after setting nitrogen, copper, and reactive blue 19 concentration; in a bioreactor, activity recovery was lower (90 U ml−1) and pellet morphology was different. The activity of the laccase crude extract was maximal at 60°C and stable for 14 h at 50°C and for 2 months at pH 6 and room temperature. The biotechnological potential was assessed, confirming the capacity to decolorize single or mixed solutions of textile dyes and to enhance the whitening yield of raw cotton fibers, working in synergism with the conventional H2O2-based method.

Dynamic measurement of oxidase activity based on oxygen consumption in open systems

Oxidases catalyze the oxidation of a variety of substrates with the concomitant reduction of molecular oxygen as a final electron acceptor. UV‐visible spectrophotometry is a simple and high‐throughput method commonly used to measure oxidase activities. However, drawbacks such as light scattering exist especially concerning the activity assessment of enzymes immobilized on supports. Monitoring of the universal cosubstrate O2 circumvents these drawbacks. This study aimed at developing a methodology that allows activity measurement of many types of oxidases based on O2 consumption applicable to various open systems. Dissolved oxygen in the reaction medium was monitored by an O2 sensor and the reaction rate was deduced from the O2 mass balance equation correcting for atmospheric diffusion. Common activity units (μMproduct min−1 or U/L) could be subsequently derived using calibration curves. The sensitivity of the method toward temperature, atmospheric pressure, and ionic strength variations was evaluated, and made it possible to define operating windows for the simplification of the proposed methodology.