Enzymes are the highly versatile bio-catalysts having the potential for being employed in biotechnological and industrial sectors to catalyze biosynthetic reactions over a commercial point of view. Immobilization of enzymes has improved catalytic properties, retention activities, thermal and storage stabilities as well as reusabilities of enzymes in synthetic environments that have enthralled significant attention over the past few years. Dreadful efforts have been emphasized on the renewable and synthetic supports/composite materials to reserve their inherent characteristics such as biocompatibility, non-toxicity, accessibility of numerous reactive sites for profitable immobilization of biological molecules that often serve diverse applications in the pharmaceutical, environmental, and energy sectors. Supports should be endowed with unique physicochemical properties including high specific surface area, hydrophobicity, hydrophilicity, enantioselectivities, multivalent functionalization which professed them as competent carriers for enzyme immobilization. Organic, inorganic, and nano-based platforms are more potent, stable, highly recovered even after used for continuous catalytic processes, broadly renders the enzymes to get efficiently immobilized to develop an inherent bio-catalytic system that displays higher activities as compared to free-counter parts. This review highlights the recent advances or developments on renewable and synthetic matrices that are utilized for the immobilization of enzymes to deliver emerging applications around the globe.Copyright © 2020 Elsevier B.V. All rights reserved.

Many different micro and nano sized materials have been used for enzymes immobilization in order to increase their catalytic activity and stability. Generally, immobilized enzymes with conventional immobilization techniques exhibit improved stability while their activity is lowered compared to free enzymes. Recently, an elegant immobilization approach was discovered in synthesis of flower-like organic-inorganic hybrid nanostructures with extraordinary catalytic activity and stability. In this novel immobilization strategy, proteins (enzymes) and metal ions acted as organic and inorganic components, respectively to form hybrid nanoflowers (hNFs). It is demonstrated that the hNFs highly enhanced catalytic activities and stability in a wide range of experimental conditions (pHs, temperatures and salt concentration, etc.) compared to free and conventionally immobilized enzymes. This review mainly discussed the synthesis, characterization, development and applications of organic-inorganic hybrid nanoflowers formed of various enzymes and metal ions and explained potential mechanism underlying enhanced catalytic activity and stability.Copyright © 2016 Elsevier Inc. All rights reserved.

Nanotechnology has transformed the science behind many biotechnological sectors, and applied bio-catalysis is not the exception. In 2017, the enzyme industry was valued at more than 7 billion USD and projected to 10.5 billion by 2024. The laccase enzyme is an oxidoreductase capable of oxidizing phenolic and non-phenolic compounds that have been considered an essential tool in the fields currently known as white biotechnology and green chemistry. Laccase is one of the most robust biocatalysts due to its wide applications in different environmental processes such as detecting and treating chemical pollutants and dyes and pharmaceutical removal. However, these biocatalytic processes are usually limited by the lack of stability of the enzyme, the half-life time, and the application feasibility at an industrial scale. Physical or chemical approaches have performed different laccase's immobilization methods to improve its catalytic properties and reuse. Emerging technologies have been proven to reduce the manufacturing process cost and increase application feasibility while looking for ecological and economical materials that can be used as support. Therefore, this review discusses the trends of enzyme immobilization recently studied, analyzing biomaterials and agro-industrial waste used for that intention, their advantages, and disadvantages. Finally, the work also highlights the performance obtained with these materials and current challenges and potential alternatives.Copyright © 2018. Published by Elsevier B.V.

Several new types of carriers and technologies have been implemented in the recent past to improve traditional enzyme immobilization which aimed to enhance enzyme loading, activity and stability to decrease the enzyme biocatalyst cost in industrial biotechnology. These include cross-linked enzyme aggregates, microwave-assisted immobilization, click chemistry technology, mesoporous supports and most recently nanoparticle-based immobilization of enzymes. The union of the specific physical, chemical, optical and electrical properties of nanoparticles with the specific recognition or catalytic properties of biomolecules has led to their appearance in myriad novel biotechnological applications. They have been applied time and again for immobilization of industrially important enzymes with improved characteristics. The high surface-to-volume ratio offered by nanoparticles resulted in the concentration of the immobilized entity being considerably higher than that afforded by experimental protocols based on immobilization on planar 2-D surfaces. Enzymes immobilized on nanoparticles showed a broader working pH and temperature range and higher thermal stability than the native enzymes. Compared with the conventional immobilization methods, nanoparticle based immobilization served three important features; (i) nano-enzyme particles are easy to synthesize in high solid content without using surfactants and toxic reagents, (ii) homogeneous and well defined core-shell nanoparticles with a thick enzyme shell can be obtained, and (iii) particle size can be conveniently tailored within utility limits. In addition, with the growing attention paid to cascade enzymatic reaction and in vitro synthetic biology, it is possible that co-immobilization of multi-enzymes could be achieved on these nanoparticles.Copyright © 2011 Elsevier Inc. All rights reserved.

Laccases of fungi attract considerable attention due to their possible involvement in the transformation of a wide variety of phenolic compounds including the polymeric lignin and humic substances. So far, more than a 100 enzymes have been purified from fungal cultures and characterized in terms of their biochemical and catalytic properties. Most ligninolytic fungal species produce constitutively at least one laccase isoenzyme and laccases are also dominant among ligninolytic enzymes in the soil environment. The fact that they only require molecular oxygen for catalysis makes them suitable for biotechnological applications for the transformation or immobilization of xenobiotic compounds.

Lipase-catalyzed synthesis of sugar esters, as lactulose palmitate, requires harsh conditions, making it necessary to immobilize the enzyme. Therefore, a study was conducted to evaluate the effect of different chemical surfaces of hierarchical meso-macroporous silica in the immobilization of two lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), which exhibit esterase activity. Porosity and chemical surface of silica supports, before and after functionalization and after immobilization, were characterized by gas adsorption and Fourier transform infrared (FTIR) spectroscopy. PsL and AsL were immobilized in octyl (OS), glyoxyl (GS), and octyl-glyoxyl silica (OGS). Hydrolytic activity, thermal and solvent stability, and sugar ester synthesis were evaluated with those catalysts. The best support in terms of expressed activity was OS in the case of PsL (100 IU g(-1)), while OS and OGS were the best for AsL with quite similar expressed activities (60 and 58 IU g(-1), respectively). At 60 °C in aqueous media the more stable biocatalysts were GS-PsL and OGS-AsL (half-lives of 566 and 248 h, respectively), showing the advantage of a heterofunctional support in the latter case. Lactulose palmitate synthesis was carried out in acetone medium (with 4% of equilibrium moisture) at 40 °C obtaining palmitic acid conversions higher than 20% for all biocatalysts, being the highest of those obtained with OGS-AsL and OS-PsL. Therefore, screening of different chemical surfaces on porous silica used as supports for lipase immobilization allowed obtaining active and stable biocatalyst to be employed in the novel synthesis of lactulose palmitate.

Flower-shaped inorganic nanocrystals have been used for applications in catalysis and analytical science, but so far there have been no reports of 'nanoflowers' made of organic components. Here, we report a method for creating hybrid organic-inorganic nanoflowers using copper (II) ions as the inorganic component and various proteins as the organic component. The protein molecules form complexes with the copper ions, and these complexes become nucleation sites for primary crystals of copper phosphate. Interaction between the protein and copper ions then leads to the growth of micrometre-sized particles that have nanoscale features and that are shaped like flower petals. When an enzyme is used as the protein component of the hybrid nanoflower, it exhibits enhanced enzymatic activity and stability compared with the free enzyme. This is attributed to the high surface area and confinement of the enzymes in the nanoflowers.

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60 degrees C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.

Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

A purified papaya laccase was immobilized in chitosan beads using entrapment approach and its physico-chemical properties were investigated and compared with that of free enzyme. Increase in properties of the laccase such as optimum temperature (by 10 °C), thermostability (by 3-folds) and optimum pH (from 8.0 to 10.0) was observed after immobilization. Immobilization led to increased tolerance of enzyme to a number of metal ions (including heavy metals) and organic solvents namely, ethanol, isopropanol, methanol, benzene and DMF. The catalytic efficiency (Kcat/Km) of the immobilized enzyme was found to increase more than ten folds, in comparison to that of the free enzyme, with hydroquinone as substrate. Immobilization of laccase also led to improvement in dye decolorization such that the synthetic dye indigo carmine (50 μg/ml) was completely decolorized within 8h of incubation as compared to that of the free laccase which decolorized the same dye to only 56% under similar conditions. Thus, immobilization of laccase into chitosan beads led to tremendous improvement in various useful attributes of this enzyme thereby making it more versatile for its industrial exploitation.Copyright © 2016 Elsevier B.V. All rights reserved.

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize various substrates, and those which are tolerant to and even activated by salts have attracted a lot of attention due to their application potential in certain industries. The mechanism of the salt activation of laccases is awaiting to be elucidated yet. Our previous study (Li, Xie et al. 2018) supposed that the salt activation of marine laccase Lac15 might be attributed to Cl ion specifically binding to some local sites to interfere substrate binding and/or electron transfer. In this study, we found two sites whose mutations resulted in elimination of the salt activation of Lac15's activity towards catechol and dopamine respectively, and revealed that the mutations affected the activity by altering both E and k, demonstrating the supposed mechanism. A model for the salt activation of laccases was accordingly proposed, albeit some details are to be elucidated.Copyright © 2019 Elsevier Inc. All rights reserved.

Due to the increasing quantities of phenolic compounds present in wastewater, the use of enzymatic degradation with the laccase has attracted much attention as a green option for their removal. In this work, we developed a novel immobilization technology using 3D bioprinting for laccase immobilization. The hydrogel mechanism properties were optimized by experimenting with different component ratios of sodium alginate (SA), acrylamide (AM), and hydroxyapatite (HA). The improved mechanism properties were validated by morphology pictures and rheology characteristics. The optimal AM:HA:SA ratio was determined to be 4:1.2:1. We then employed an extrusion-based bioprinting technique to prepare the immobilized laccase. The substrate conversion was increased with the addition of HA, which improved the permeability of the matrix, and proved to be suitable for immobilization. The resulting immobilized laccase was used for the biodegradation of p-chlorophenol. The effects of the initial substrate concentration, pH, and temperature were evaluated. The immobilized laccase exhibited good storage stability and reusability, retaining over 80% of its initial activity after 72 h of storage, and was able to be reused for seven batches. These results highlight that the immobilized laccase prepared by 3D bioprinting has great potential for use in the biodegradation of phenolic compounds.Copyright © 2020. Published by Elsevier B.V.

Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate "green catalysts". This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts. Copyright © 2014 Elsevier Inc. All rights reserved.

A novel method of laccase immobilization on epoxy-functionalized silica particles was developed. Laccase from Myceliophthora thermophila was covalently immobilized onto epoxy-functionalized matrix by nucleophilic attack of amino groups of laccase to epoxy groups of the support. The enzyme loading on the support was about 30 mg/g under the optimum conditions (pH 4.5, 24 h). The effect of pH, temperature and organic solvent on immobilized enzyme activity was determined and compared with those of free enzyme. In general the immobilized enzyme was found to be stabilized compared to the free enzyme. Lineweaver-Burk plots were used to calculate kinetic parameters for ABTS oxidation. K values were 24.0 and 25.3 μM while v values were 10.0 and 1.6 μM min for free and immobilized laccase, respectively. The performance of the biocatalyst was evaluated by the degradation of phenolic compounds including phenol, p-chlorophenol and catechol. The removal efficiency of catechol by immobilized laccase was about 95% after 2 h.Copyright © 2017 Elsevier B.V. All rights reserved.

This work describes the preparation of an electrochemical biosensor for polyphenols determination in propolis samples. The biosensing scheme is based on a nanocomposite film of laccase enzyme (Lac) immobilized on gold nanoparticles (AuNPs) electrodeposited in a screen-printed carbon electrode (SPCE) modified with polypyrrole (Ppy) through an in-situ electropolymerization. The electrodeposition of the AuNPs increases the available area for Lac immobilization. The nanocomposite film (Ppy/Lac/AuNPs/SPCE) was characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy and cyclic voltammetry. Polyphenols were detected in ethanolic extracts of propolis (EEP), where in presence of the Lac oxidized to the polyphenols, and so they can be reduced on the Ppy/Lac/AuNPs/SPCE by amperometry at -450 mV vs Ag/AgCl. The calibration plot showed a linear response in the concentration range from 1 to 250 mu M expressed as caffeic acid, with a limit of detection of 0.83 mu M. The time required for analysis was 15 min, compared to the time (85 min) by spectrophotometric methods, especially the so-called Folin-Ciocalteu method. The method exhibited good selectivity, stability and reproducibility for detecting polyphenols in propolis samples.

Response surface methodology was employed for the optimization of different nutritional and physical parameters for the production of laccase by the filamentous bacteria Streptomyces psammoticus MTCC 7334 in submerged fermentation. Initial screening of production parameters was performed using a Plackett - Burman design and the variables with statistically significant effects on laccase production were identified. Incubation temperature, incubation period, agitation rate, concentrations of yeast extract, MgSO(4)7H(2)O, and trace elements were found to influence laccase production significantly. These variables were selected for further optimization studies using a Box-Behnken design. The statistical optimization by response surface methodology resulted in a three-fold increase in the production of laccase by S. psammoticus MTCC 7334.

Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet.

Laccases are blue multicopper oxidases that catalyse the four-electron reduction of O(2) to water coupled with the oxidation of small organic substrates. Secreted basidiomycete white-rot fungal laccases orchestrate this with high thermodynamic efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-of-play regarding expression of high reduction potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biology, particularly in unmasking structure-function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases.2009 Elsevier Ltd. All rights reserved.

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

Melanin is major cause of dark skin, which is regarded as social status in eastern Asia. As a result, researchers in cosmetic industries are developing skin whitening agents. Melanin can be decolorized by many oxidative enzymes. Laccase (CueO) from Escherichia coli and dye-decolorizing peroxidase (DyP) from Bacillus subtilis were merged with the dockerin domain of endoglucanase B from Clostridium cellulovorans. Scaffoldin has great potential to exert structural benefits that enable complementary enzyme effects. The carbohydrate binding module (CBM) in scaffoldin was replaced with the melanin binding peptide (MBP) to increase melanin binding and thereby enhance melanin degradation. The modified scaffoldin exhibits a nearly 64% increase in specific binding to melanin over that of the native scaffoldin. Laccase was used to degrade melanin via the production of hydrogen peroxide, which produced synergistic activity with peroxidase. The activity of the optimized complex was approximately 6.4-fold greater than that of laccase alone. This enzyme complex can also reduce the number of melanin granules in corneocytes. Based on these results, a recombinant enzyme complex is suitable for use in melanin degradation by next generation whitening agents in the skin cosmetics industry.Copyright © 2019 Elsevier B.V. All rights reserved.

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.

Novel magnetic cross-linked enzyme aggregates of alpha amylase were prepared by chemical cross-linking of enzyme aggregates with amino functionalized magnetite nanoparticles which can be separated from reaction mixture using magnetic field. Of the initially applied alpha amylase activity 100% was recovered in magnetic CLEAs, whereas only 45% was recovered in CLEAs due to the low content of Lys residues in alpha amylase. Scanning electron microscopy analysis showed that CLEAs and magnetic CLEAs were spherical structures. The CLEAs and magnetic CLEAs displayed a shift in optimal pH towards the acidic side, whereas optimal temperature of magnetic CLEAs was improved compared to free enzyme and CLEAs. Although V(max) of enzyme in CLEAs and magnetic CLEAs did not change, substrate affinity of the enzyme increased. The magnetic CLEAs also enhanced the thermal stability and storage stability. Moreover, the magnetic CLEAs retained 100% initial activity even after 6 cycles of reuse.Copyright © 2012 Elsevier Ltd. All rights reserved.

Wood-inhabiting fungi are one of the most important parts of forest ecosystem,and play an important role in degrading the wood in forest ecosystem.The major species of these fungi include the groups of Aphyllophorales (Basidiomycota),Discomycetes (Ascomycota) and some imperfect fungi.They have the ability to degrade cellulose,hemicelluloses and lignin of wood.Three type of wood decaying have been found,i.e.,white rot,brown rot and soft rot.Many other organisms of forest ecosystem have symbiosis relationship with wood-decaying fungi.Wood-inhabiting fungi could offer the nutrition for many insects and birds,and spores of many wood-rotting species are spread by some insects.The high biodiversity of wood-decaying fungi is one of the important factors for the health of forest ecosystem.

The successful incorporation of enzymes into materials through multipoint covalent immobilization (MPCI) has served as the foundation for numerous advances in diverse fields, including biocatalysis, biosensing, and chemical weapons defense. Despite this success, a mechanistic understanding of the impact of this approach on enzyme stability has remained elusive, which is critical for realizing the full potential of MPCI. Here, we showed that the stabilization of lipase upon MPCI to polymer brush surfaces resulted from the rigidification of the enzyme with an increase in the number of enzyme-brush attachments. This was evident by a 10-fold decrease in the rates of enzyme unfolding and refolding as well as a reduction of the intrinsic fluctuations of the folded and unfolded states, which was measured by single-molecule (SM) Förster Resonance Energy Transfer imaging. Moreover, our results illuminate an important trade-off between stability and activity as a function of this decrease in structural dynamics of the immobilized lipase. Notably, as the thermal stability of lipase increased, as indicated by the temperature optimum for activity of the enzyme, the specific activity of lipase decreased. This decrease in activity was attributed to a reduction in the essential motions of the folded state that are required for catalytic turnover of substrate. These results provide direct evidence of this effect, which has long been a matter of speculation. Furthermore, our findings suggest that the retention of activity and stabilization of an enzyme may be balanced by tuning the extent of enzyme attachment.

Bentonite is a natural and environmentally clay mineral, and bentonite-derived mesoporous materials (BDMMs) were obtained conveniently from the alkali and acid treatment of bentonite. In the present study, BDMMs were explored for immobilization of laccase obtained from Trametes versicolor. As a result, bentonite-derived mesoporous materials-Laccase (BDMMs-Lac) was developed for the removal of tetracycline (TC). The enzyme immobilization process was carried out through physical adsorption contact (ion exchange adsorption, hydrogen bond adsorption, and Van der waals adsorption) between the BDMMs and laccase. The process of immobilization remarkably increased its operating temperature. The BDMMs-Lac exhibited over 60% removal efficiency for TC within 3 h in the presence of 1-hydroxybenzotriazole (HBT). In conclusion, BDMMs-Lac showed more promising potential than free laccase for practical continuous applications.Copyright © 2019 Elsevier Ltd. All rights reserved.

Lignin is the most abundant renewable source of aromatic polymer in nature, and its decomposition is indispensable for carbon recycling. It is chemically recalcitrant to breakdown by most organisms because of the complex, heterogeneous structure. The white-rot fungi produce an array of extracellular oxidative enzymes that synergistically and efficiently degrade lignin. The major groups of ligninolytic enzymes include lignin peroxidases, manganese peroxidases, versatile peroxidases, and laccases. The peroxidases are heme-containing enzymes with catalytic cycles that involve the activation by H2O2 and substrate reduction of compound I and compound II intermediates. Lignin peroxidases have the unique ability to catalyze oxidative cleavage of C-C bonds and ether (C-O-C) bonds in non-phenolic aromatic substrates of high redox potential. Manganese peroxidases oxidize Mn(II) to Mn(III), which facilitates the degradation of phenolic compounds or, in turn, oxidizes a second mediator for the breakdown of non-phenolic compounds. Versatile peroxidases are hybrids of lignin peroxidase and manganese peroxidase with a bifunctional characteristic. Laccases are multi-copper-containing proteins that catalyze the oxidation of phenolic substrates with concomitant reduction of molecular oxygen to water. This review covers the chemical nature of lignin substrates and focuses on the biochemical properties, molecular structures, reaction mechanisms, and related structures/functions of these enzymes.

Magnetic cross-linked enzyme aggregates (M-CLEAs) were prepared for Cerrena laccase and used in antibiotic treatment. Of the seven antibiotics examined in this study, Cerrena laccase M-CLEAs were most effective in degradation of tetracycline (TC) and oxytetracycline (OTC), followed by ampicillin, sulfamethoxazole and erythromycin. The redox mediator ABTS was not able to improve efficiencies of degradation of TC and OTC. Cerrena laccase at 40U/mL eliminated 100μg/mL TC at pH 6 and 25°C in 48h in the absence of a redox mediator, with over 80% degradation occurring within the first 12h. Laccase treatment also significantly suppressed the antimicrobial activity of TC and OTC. Three TC transformation products, the levels of which initially increased and subsequently decreased during laccase treatment were identified by using LC-TOF MS. A mechanism of laccase-mediated TC oxidation was proposed based on the identified intermediates.Copyright © 2016 Elsevier B.V. All rights reserved.

In proteomics research, the screening and monitoring of disease biomarkers is still a major challenge, mainly due to their low concentration in biological samples. However, the universal enrichment of intact proteins has not been further studied. In this work, we developed a FeO-chitosan@graphene (FeO-CS@G) core-shell composite to enrich low-abundance proteins from biological samples. FeO-CS@G composite holds chitosan layer decorated FeO core, which improves the hydrophilicity of materials greatly. Meanwhile, the graphene nanosheets shell formed via electrostatic assembly endows the composite with huge surface area (178m/g). The good water dispersibility ensures the sufficient contact opportunities between graphene composites and proteins, and the large surface area provides enough adsorption sites for the enrichment of proteins. Using FeO-CS@G, four standard proteins Cyt-c, BSA, Myo and OVA were enriched with better adsorption capacity and recovery rate, compared with previously reported magnetic graphene composites. Additionally, the mechanism of compared to" is corrected into "compared with". proteins adsorption on FeO-CS@G was further studied, which indicates that hydrophobic and electrostatic interaction work together to facilitate the universal and efficient enrichment of proteins. Human plasma sample was employed to further evaluate the enrichment performance of FeO-CS@G. Eventually, 123 proteins were identified from one of SAX fractions of human plasma, which is much better than commercial Sep-pak C18 enrichment column (39 proteins). All these outstanding performances suggest that FeO-CS@G is an ideal platform for the enrichment of low-abundance intact proteins and thus holds great potential to facilitate the identification of biomarkers from biological samples in proteomics research.Copyright © 2017 Elsevier B.V. All rights reserved.

A novel laccase (Tolacc-T) from white rot fungus Trametes orientalis was enriched to apparent homogeneity with a specific activity of 20.667U/mg protein and recovery yield of 47.33%. The SDS-PAGE gave a single band indicating that Tolacc-T appears as a monomeric protein with a molecular mass of 44.0kDa. Domain structure analysis revealed that Tolacc-T contained a typical copper II binding domain and shared three potential N-glycosylation sites, but had no copper I binding domain, demonstrating that the enzyme is really a laccase, but a novel laccase. Optimal pH and temperature of Tolacc-T was 4.0 and 80°C, respectively, and it retained more than 80% of its original activity after 2h incubation at 10°C to 50°C. The enzyme exhibited strict substrate specificity towards ABTS but showed no or trace activities towards other substrates. Among the metals tested, Mn was proved to be the best activator for enhancing the laccase activity. A strongly inhibiting effect was found when NaN, -cysteine, and DTT were added to the enzyme. However, Tolacc-T activity was little bit inhibited in the presence of chelator EDTA. Furthermore, the enzyme was capable of degrading structurally different synthetic dyes in the absence of a redox mediator.Copyright © 2017 Elsevier B.V. All rights reserved.

我国有机污染废水排放总量巨大,其释放到生态环境中对野生动物和人群健康构成严重威胁。目前,生物修复技术因其绿色环保、经济高效而备受关注。漆酶能够催化不同类型有机污染物的氧化耦合和分解,该反应具有催化速率高、简单可控、底物广谱和生态友好等特点。鉴于游离态漆酶性能不稳定且难以回收利用,而酶固定化技术可显著改善游离态漆酶的稳定性、增加其循环利用次数和催化功效,有望实现漆酶在环境生物修复中的大规模应用。综述了真菌漆酶的分子结构、底物谱和催化性能,对比了漆酶的4种常规固定化方法(吸附、包埋、共价结合和交联)的优缺点。重点总结了固定化漆酶介导雌激素、抗生素、多环芳烃、个人护理产品、合成染料和磺胺类药物等有毒有害污染物的自由基耦合和氧化分解机理。固定化漆酶以分子氧作为电子受体,催化有机污染物氧化生成活性自由基或醌类中间体。这些活性中间体既能以共价耦合的途径形成聚合物,也可通过自由基相互攻击形成分解产物,进而显著降低母体化合物的生态毒性。固定化漆酶在净化有机废水、消除环境污染和维持生态健康等方面已表现出巨大的应用潜力,未来需要进一步筛选廉价固定化载体和提高固定化漆酶循环利用效率。

漆酶是一种多酚氧化酶,可催化氧化多种难降解有机污染物,在环境污染防治领域具有良好的应用前景。总结了植物漆酶、动物漆酶以及微生物漆酶的研究现状,详细讨论了漆酶固定化载体的研究进展,进一步指出了目前漆酶研究存在的问题,并提出未来的研究方向,旨在为漆酶的开发与应用研究提供参考。

漆酶是一种含有铜离子的多酚氧化酶,其作用底物广泛,能够催化多种酚类化合物和芳香族化合物氧化,在食品工业等众多领域中发挥着重要作用。在此基础上,论述了漆酶催化氧化作为一种新型生物处理方法,不仅可以改善果汁、葡萄酒、啤酒等饮品的风味和澄清度,还可以提高面包的品质等,为食品的安全与卫生提供了保障,并对漆酶在环境保护、造纸工业、生物检测、农业和医药等领域进行了综述;同时对漆酶在各行各业上的研究与发展等进行了展望,期望可以发挥漆酶的优点,避免漆酶的缺点,使漆酶能够更好地发挥自身作用。

磁性介孔二氧化硅复合材料作为酶固定化载体具有优异的酶固定化性能和良好的磁分离性能，受到国内外学术界广泛关注。本文在自制的β-FeOOH空心微球表面上包覆致密的SiO₂保护层，在酸性条件下以P123为模板剂，十六烷基三甲基溴化铵（CTAB）为辅助导向剂成功制备出了磁性β-FeOOH@SiO₂@介孔SiO₂空心复合微球，最后在还原气氛下煅烧得到Fe₃O₄@SiO₂@介孔SiO₂空心微球。结果表明，所制备的Fe₃O₄@SiO₂@介孔SiO₂微球空心结构未坍塌，具有规整的球形结构，介孔SiO₂壳层（平均厚度约为11nm）均匀地包覆在β-FeOOH@SiO₂中空微球表面。伴随着CTAB量的增加，微球的最可几孔径由4.30nm减小到3.19nm，比表面积从376m²/g升高到640m²/g，孔容从0.36cm³/g升高到0.56cm³/g。复合微球的饱和磁化强度为11.3emu/g，矫顽力为111.5Oe，外加磁场作用下可以实现样品的快速分离，且样品的再分散性良好。当介孔孔径为4.30nm时，Fe₃O₄@SiO₂@介孔SiO₂空心复合微球漆酶固定量高达234mg/g。固定化漆酶在不同pH、温度下的活性显著优于游离酶。

漆酶是一种天然的绿色催化剂,由于具有催化效率高、底物特异性广、对辅因子和特定环境条件要求少、无毒等优点,因而在制浆造纸、生物合成、改善纤维性能、食品加工、生物传感器制造、农林废弃物的生物转化和炼制,特别是环境污染物的生物降解和生物修复等领域具有巨大的应用潜力。但游离漆酶稳定性差且成本较高,固定化方法成为解决该问题的有效手段,其能够有效增强酶的热稳定性及对极端环境的耐受力、使酶易与产物分离以提高酶回收率。本研究基于前期筛选获得的一株高产漆酶白腐真菌菌株东方栓孔菌（Trametes orientalis）所分泌的漆酶（Tolacc-T）,对其纯化酶蛋白以壳聚糖作为载体、戊二醛作为交联剂进行了固定化处理,命名为Tolacc-T@Chit@GA,并优化了固定化条件为戊二醛浓度0.7%（V/V）、交联时间4 h、给酶量6.0 mL、固定化时间6 h。与Tolacc-T相比,Tolacc-T@Chit@GA的pH适应性、耐热变性能力及贮存稳定性均显著增强。循环使用时的稳定性和耐久性也十分明显,循环使用7次后,Tolacc-T@Chit@GA的相对活性仍可保持在80%以上。此外,Tolacc-T@Chit@GA还可使不同类型的染料脱色,尤其对金属络合染料萘酚绿B的脱色效果最佳,气相色谱-质谱联用（gas chromatography-mass spectroscopy,GC-MS）检测确定该染料部分代谢产物为1-萘胺、2-萘酚、1-氨基-2-萘酚、1-亚硝基-2-萘酚、1-亚硝基-2-萘酚-6-磺酸。上述结果证明,东方栓孔菌固定化漆酶Tolacc-T@Chit@GA具有较好的稳定性和可重复利用性,存在广阔的应用前景。

木材腐朽菌是森林生态系统的重要组成部分,在森林生态系统中起着极为重要的降解还原作用,主要包括担子菌门非褶菌目、子囊菌门盘菌纲和半知菌类的部分真菌,能全部或部分降解木材中的木质素、纤维素和半纤维素,其降解机制有3种:白色腐朽、褐色腐朽和软腐朽.木材腐朽菌与生态系统中其它生物关系密切,为很多昆虫、鸟类提供营养,有些昆虫也能使木腐菌得到传播.保护木材腐朽菌的生物多样性是保护森林生态系统、维护生态系统健康的重要因素.

真菌漆酶是一种性质优良的多酚氧化酶,由于在分子氧的协助下可将酚类、芳胺类化合物等多种底物氧化,最终得到水及其终产物,符合当代环保工业要求,因而在纸浆漂白、环境治理、生物检测、有机合成等领域有着巨大的应用潜力。就漆酶的生物学性质、生产、纯化、固定化等研究进展和现状进行了介绍和总结,同时对其今后的发展方向进行了展望。

漆酶是一种含铜离子的多酚氧化酶，广泛存在于植物及真菌中。漆酶含特有的铜离子，其功能为传递结构中的电子，使漆酶具有了较强的氧化还原能力，能与木质素、胺类化合物、芳香化合物等底物发生作用，且大多数反应的唯一产物为水。目前，漆酶在降解多种有毒物质和有害污染物方面表现出高效、成本较低的特性，如白腐真菌所产的高水平漆酶已广泛成熟应用在工业废水处理等生物整治和修复领域。近年来最新研究利用载体固定化酶的技术使漆酶能够在使用后回收反复利用且更具有稳定性，这降低成本的同时还保持了漆酶催化氧化的特性，克服了不少漆酶在解决环境污染中出现的问题。利用介体的介导作用解决了漆酶氧化还原电势较低的问题，大量增多了可降解底物的种类，使其在废水处理、污染物降解、土壤修复、工业染料漂白等领域的应用前景更广阔。对现有漆酶应用于各领域进行研究总结，综述了降解各领域中的有害污染物等底物种类，提出了利用漆酶的降解过程中的现有不足和改进方向，以期为生物法降解环境污染物的研究提供参考。