Sensitive chemical sensor array based on nanozymes for discrimination of metal ions and teas.

Tea, originating from China, is an important part of Chinese traditional culture. There are different qualities of and producing areas for tea on the market, therefore it is necessary to discriminate between teas in a fast and accurate way. In this study, a chemical sensor array based on nanozymes was developed to discriminate between different metal ions and teas. The indicators for the sensor array are three kinds of nanozymes mimicking laccase (Cu-ATP, Cu-ADP, Cu-AMP). The as-developed sensor array successfully discriminated 12 metal ions and the detection limit was as low as 0.01 µM. The as-developed sensor array was also able to discriminate tea samples. Different kinds of tea samples appeared in different areas in the canonical score plot with different response patterns. Furthermore, in a blind experiment, we successfully discriminated 12 samples with a 100% accuracy. This sensor array integrates chemistry and food science together, realizing the simultaneous detection of several kinds of teas using a sensitive method. The as-developed sensor array would have an application in the tea market and provide a fast and easy method to discriminate between teas.

Synthesis of novel laccase-biotitania biocatalysts for malachite green decolorization.

Biomimetic mineralization has emerged as a novel tool for generating excellent supports for enzyme stabilization. In this work, protamine was used to induce titanium (IV) bis(ammonium lactato) dihydroxide (Ti-BALDH) into titania nanoparticles. This biomimetic titanification process was adopted for laccase immobilization. Laccase-biotitania biocatalyst was prepared and the effect of different parameters (buffer solution, titania precursor concentration, protamine concentration, and enzyme loading) on the encapsulation efficiency and recovery of laccase were evaluated. Compared with free laccase, the thermal and pH stability of immobilized laccase were improved significantly. In addition, laccase loaded on titania was effective at enhancing its storage stability. After seven consecutive cycles, the immobilized laccase still retained 51% of its original activity. Finally, laccase-biotitania biocatalysts showed good performance on decolorization of malachite green (MG), which can be attributed to an adsorption and degradation effect. The intermediates of the MG degradation were identified by gas chromatography-mass spectrometry (GC-MS) analysis, and the most probable degradation pathway was proposed. This study provides deeper understanding of the laccase-biotitania particles as a fast biocatalyst for MG decolorization.

Produção de lacase de Pycnoporus sanguineus em meio de cultivo à base de melaço soja / Laccase production by Pycnoporus sanguineus in culture media with soybean molasses / Producción de lacasa de Pycnoporus sanguineus en medio de cultivo con melaza de soja

Lacases são polifenol oxidases que utilizam a capacidade redox de íons cobre para reduzir oxigênio a água e oxidar um substrato fenólico. A síntese e secreção de lacases de basidiomicetos dependem de vários fatores como os nutrientes presentes no meio de cultura. Visando à produção de lacase, Pycnoporus sanguineus foi cultivado em meio contendo melaço de soja como única fonte de carbono, ureia como fonte de nitrogênio suplementar em diferentes concentrações (0,6; 1,2; 2,4; 4,8 e 9,6 g/L de nitrogênio) e diferentes concentrações de CuSO4 (0, 150, 200, 250 e 300 µM). O extrato enzimático produzido nas melhores condições de cultivo foi utilizado para a descoloração dos corantes remazol azul brilhante R (antraquinona), amarelo 145, preto 5, vermelho 195 (azo) e verde malaquita (trifenilmetano). As concentrações de nitrogênio não afetaram a produção de lacase, exceto a maior concentração (9,6 g/L) que reduziu a atividade enzimática. A adição de cobre ao meio de cultivo (150 µM) aumentou a atividade de lacase em 112%. A maior atividade de lacase (~34300 U/L) promoveu a descoloração dos corantes remazol azul brilhante R (67,5%) e verde malaquita (28,3%) em 24h, sendo os corantes azo descoloridos apenas parcialmente. Concluiu-se que o melaço de soja é um resíduo agroindustrial adequado para produção de lacase de P. sanguineus com potencial para degradação de corantes.

Laccases are multicopper oxidases using the redox ability from copper ions to reduce oxygen to water, while oxidizing a phenolic substrate. Laccase synthesis and secretion in basidiomycetes depend on the conditions provided and on the nutrients present in the culture medium. Pycnoporus sanguineus was cultivated in medium containing soybean molasses as the sole carbon source, with urea as the source of supplemental nitrogen at different concentrations (0.6, 1.2, 2.4, 4.8 and 9.6 g/L nitrogen), and different CuSO4 concentrations (0, 150, 200, 250 and 300 µM). The enzymatic extract produced under the best cultivation conditions was used for the depigmentation of remazole brilliant blue R (anthraquinone), yellow 145, black 5, red 195 (azo) and malachite green (triphenylmethane). Nitrogen concentrations did not affect laccase production, except for the higher concentration (9.6 g/L) reducing enzymatic activity. The addition of copper to the culture medium (150 µM) increased laccase activity by 112%. The highest laccase activity (~34300 U/L) promoted the depigmentation of remazol brilliant blue R (67.5%) and malachite green (28.3%) dyes in 24 hours. Azo dyes were only partially discolored. Therefore, it can be considered that soybean molasses is an agro-industrial byproduct suitable for the production of P. sanguineus laccase with potential for dye degradation.

Lacasas son polifenoles oxidasas que utilizan la capacidad redox de iones de cobre para reducir el oxígeno del agua y oxidar un sustrato fenólico. La síntesis y secreción de lacasas de basidiomicetos dependen de las condiciones como los nutrientes presentes en el medio de cultura. Buscando la producción de lacasa, se cultivó Pycnoporus sanguineus en medio que contenía melaza de soja como única fuente de carbono, urea como fuente de nitrógeno suplementar a diferentes concentraciones (0,6, 1,2, 2,4, 4,8 y 9,6 g/L de nitrógeno) y diferentes concentraciones de CuSO4 (0, 150, 200, 250 y 300 µM). El extrato enzimático producido en mejores condiciones de cultivo ha sido utilizado para la decoloración de los colorantes remazol azul brillante R (antraquinona), amarillo 145, negro 5, rojo 195 (azoico) y verde malaquita (trifenilmetano). Las concentraciones de nitrógeno no afectaron la producción de lacasa, excepto la mayor concentración (9,6 g/L) que redujo la actividad enzimática. La adición de cobre al medio de cultivo (150 µM) aumentó la actividad de la lacasa en un 112%. La mayor actividad de lacasa (~34300 U/L) promovió la decoloración de los colorantes remazol azul brillante R (67,5%) y verde malaquita (28,3%) en 24h, siendo que los colorantes azoicos fueran parcialmente decolorados. Se concluye que la melaza de soja es un desecho agroindustrial adecuado para la producción de lacasa de P. sanguineus con potencial para degradación de colorantes.

Preparation and characterization of porous cross linked laccase aggregates for the decolorization of triphenyl methane and reactive dyes.

The production of porous cross-linked enzyme aggregates (p-CLEAs) is a simple and effective methodology for laccase immobilization. A three-phase partitioning technique was applied to co-precipitate laccase and starch, followed by cross-linking with glutaraldehyde and removal of starch by α-amylase to create pores in the CLEAs. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. The half-life of free laccase at 55°C was calculated to be 1.3h, while p-CLEAs did not lose any activity even after 14 h. p-CLEAs also exhibited improved storage stability, catalytic efficiency and could be recycled 15 times with 60% loss of activity. The catalysts decolorized triphenylmethane and reactive dyes by 60-70% at initial dye concentrations of 2 and 0.5 g L(-1), respectively, without any mediators. These results suggest the potential of CLEA technology in waste-water treatment.