Determination of Phenol with Peroxidase Immobilized on CaCO3.

Phenols are widely used in industries despite their toxicity, which requires governments to limit their concentration in water to 5 mg/L before discharge to the city sewer. Thus, it is essential to develop a rapid, simple, and low-cost detection method for phenol. This study explored two pathways of peroxidase immobilization to develop a phenol detection system: peroxidase encapsulation into polyelectrolyte microcapsules and peroxidase captured by CaCO3. The encapsulation of peroxidase decreased enzyme activity by 96%; thus, this method cannot be used for detection systems. The capturing process of peroxidase by CaCO3 microspherulites did not affect the maximum reaction rate and the Michaelis constant of peroxidase. The native peroxidase-Vmax = 109 µM/min, Km = 994 µM; CaCO3-peroxidase-Vmax = 93.5 µM/min, Km = 956 µM. Ultimately, a reusable phenol detection system based on CaCO3 microparticles with immobilized peroxidase was developed, capable of detecting phenol in the range of 700 ng/mL to 14 µg/mL, with an error not exceeding 5%, and having a relatively low cost and production time. The efficiency of the system was confirmed by determining the content of phenol in a paintwork product.

Multipoint Immobilization at Inert Center of Papain on Homo-Functional Diazo-Activated Silica Support: A Way of Restoring "Above Room-Temperature" Bio-Catalytic Sustainability.

Although enzymes play a significant role in industrial applications, their potential usage at high-level efficiency, particularly above room temperature, has not yet been fully harnessed. It brings above room-temperature catalytic sustainability of an immobilized (imm.) bio-catalyst as a long pending issue to improve enzyme stability, activity, specificity, or selectivity, particularly the enantio-selectivity over the native-enzymes. At this juncture, in a robust methodology, a heterogeneous solid phase bio-catalyst, {Si(OSi)4(H2O)1.03}n=328{OSi(CH3)2-NH-C6H4-N═N}4{papain}(H2O)251, has efficiently been prepared by immobilizing papain on homo-functionalized SG (silica-gel) via multipoint covalent attachment. The bio-catalyst is easy to be recovered and reused multiple times. The homo-functional -N═N+, which appears on the SG-surface, makes the multipoint diazo-links with the inert center of the tyrosine-moiety to couple the enzyme where all the amino, thiol, phenol, and so forth, groups of the protein, including those that belong to the active-site, remain intact. The immobilized enzyme (13.9 µmol g-1) swims in pore-water within the pore-channel, remains stable up to 70 ± 5 °C, and exhibits wider temperature adaptability in performing its hydrolyzing activities. The relative activity, 78 ± 2% at 27 °C, remains quantitative for 60 days and can be reused for 60 cycles with 53% activity at room-temperature. The thermal (relative activity: 87%; incubated at 70 ± 5 °C for 24 h) and mechanical (relative activity: 92%; incubated at 2500 rpm for 2 h at 27 °C) stability was outstanding.

Effect of Fenton oxidized lignin support on immobilized ß-glucosidase activity.

In this study, the Fenton oxidized lignin was prepared to investigate the effect of Fenton oxidation modification on the activity of lignin immobilized ß-glucosidase (ß-GL). The results demonstrated that Fenton oxidation could significantly improve the activity and stability of immobilized ß-GL. This is because the Fenton oxidation increased the electrostatic, hydrogen bonding, and hydrophobic forces between lignin and ß-GL, resulting in increased lignin adsorption onto ß-GL. The Fenton oxidation also changed the chemical structure of lignin, altering the lignin-ß-GL binding site and reducing the negative effect of lignin on the ß-GL catalytic domain. This research will improve understanding of the effect of Fenton lignin oxidation on immobilized ß-GL activity and expand the use of lignin in enzyme immobilization.

Effective biodegradation of chlorophenols, sulfonamides, and their mixtures by bacterial laccase immobilized on chitin.

Coexisting multi-pollutants like sulfonamides (SAs) and chlorophenols (CPs) in the ecological environment pose a potential risk to living organisms. The development of a strategy for the effective removal of multiple pollutants has become an urgent need. Herein, we systematically investigated the potential of immobilized bacterial laccase to remove chlorophenols (CPs), sulfonamides (SAs), and their mixtures. Laccase from Bacillus pumilus ZB1 was efficiently immobilized on chitin and its thermal stability, pH stability, and affinity to substrates were improved. Reusability assessment showed the immobilized laccase retained 75.5% of its initial activity after five cycles. The removal efficiency of CPs and SAs by immobilized laccase was significantly improved compared with that of free laccase. In particular, the removal of 2,4-dichlorophenol and 2,4,6-trichlorophenol reached 96.9% and 89.3% respectively within 8 h. The immobilized laccase could remove 63.70% of 2,4-dichlorophenol after four cycles. The degradation pathways of 2,4-dichlorophenol and sulfamethazine were proposed via LC/MS analysis. When the co-pollutants containing 2,4,6-trichlorophenol and sulfamethoxazole, immobilized laccase showed 100% removal of 2,4,6-trichlorophenol and 38.71% removal of sulfamethoxazole simultaneously. Cytotoxicity and phytotoxicity tests indicated that immobilized laccase can alleviate the toxicity of co-pollutants. The results demonstrate that chitin-based laccase immobilization can be an effective strategy for the removal of SAs, CPs, and their co-pollutants.

ZIF-8 as support for enhanced stability of immobilized lipase used with a thermoresponsive switchable solvent to simplify the microalgae-to-biodiesel process.

The zeolitic imidazole framework (ZIF)- 8 was tested as a support to enhance the stability of immobilized lipase. The lipase immobilized on ZIF-8, through surface attachment and encapsulation, was used for the simultaneous cell disruption and oil extraction from untreated, wet microalgal paste. The successful attachment of the enzyme to ZIF-8 was confirmed via Fourier-transform infrared spectroscopy. The attachment of the enzyme did not significantly affect the crystallinity or morphology of ZIF-8 crystals. The encapsulated lipase@ZIF-8 system showed higher stability than the adsorbed system, due to its reduced vulnerability to leaching. After five cycles, the encapsulated lipase@ZIF-8 retained 32% of its initial activity, whereas, for the adsorbed lipase@ZIF-8, it reduced to 21%. An increase in methanol amount greater than 0.2 mL was shown to have a negative effect on enzyme activity. The fatty acid methyl ester yield increased significantly with an increase in the extraction- duration (up to 3 h), after which the effect faded until 5 h, after which the equilibrium yield was reached. Changing the composition of the thermoresponsive switchable solvent (TSS) showed that a higher FAME yield could be achieved by increasing the percentages of Ionic Liquid (IL) and polypropylene glycol and reducing the water percentage. Further studies are needed to optimize the TSS composition and its effects on the process.

Performance enhancement of white rot fungi extracellular enzymes via new hydrogel microenvironments for remediation of benzo[a]pyrene contaminated soil.

Organic pollutants with low solubility and high ecotoxicity, mutagenicity, and carcinogenicity, are rapidly entering and accumulating in soil, resulting in soil pollution. Several methods have been investigated for remediation of organic contaminated soil, including enzymatic remediation approach. However, free enzymes are easily deactivated, which hinders their practical application in soil remediation. Immobilization of enzyme improves its stability and catalytic performance, but the immobilized material itself becomes secondary pollutants in soil. In this study, Trametes versicolor extracellular enzyme was immobilized on the degradable calcium alginate hydrogel microspheres. The laccase maintained a high activity. In addition, the addition of cellulose improved the strength of the carrier. Hydrogel microspheres solved the problems of easy inactivation of free enzyme and secondary contamination of immobilized materials. By a novel combination of extracellular enzymes and hydrogel microenvironments, immobilized enzymes not only demonstrate outstanding performance in thermal stability and pH adaptability, but also achieves a significant improvement in biocatalytic activity for benzo[a]pyrene contaminated soil. The thermal stability of immobilized enzyme was much higher than that of free enzyme. When the temperature increased to 50 °C, the activity of immobilized enzyme remained at 93.15% of the maximum enzyme activity, while the activity of free enzyme decreased to 63.76%. At pH 8, the immobilized enzyme activity maintained 74.84% of the maximum enzyme activity, while the free enzyme activity was only 11.86%. Immobilized enzymes can effectively remove 91.40% of benzo[a]pyrene from soil within 96 h. Furthermore, the catalytic oxidation of benzo[a]pyrene by enzymes that have been immobilized ultimately results in the production of 6,12-benzo[a]pyrene-dione. Molecular dynamics simulation showed that the catalytic degradation of benzo[a]pyrene was mainly through the interaction of amino acid residues PRO-391 with the Pi-alkyl of benzo[a]pyrene. This study presents an innovative strategy for designing and developing immobilized enzymes for use in biocatalytic applications related to eco-remediation of soil.

Spatially Segregated MOF Bioreactor Enables Versatile Modular Glycoenzyme Assembly for Hierarchical Glycan Library Construction.

The multienzyme cascade has received growing attention to obtain structurally defined glycans in vitro. However, due to poor enzyme stability and low compatibility between glycoenzymes, artificially designed multienzyme pathways to access glycans are often inefficient. Herein, based on the strategy "Modular-Enzymes Assembly by Spatial Segregation" (MASS), we developed a universal immobilization platform to assemble multiple glycoenzymes in compartmentalized MOF particles, inside and outside, significantly reducing the undesired interference and cross-inhibitions. By changing the enzyme modules, a series of glycosyl donor, disaccharides, oligosaccharides, and polysaccharides bearing cofactor regeneration were efficiently prepared. This bioreactor was further successfully applied to the reaction system with high substrate concentration to demonstrate its industrial potential. This robust multienzyme immobilization platform should serve to promote the enzymatic synthesis of more complex glycans.

Co-Immobilization of Lipases with Different Specificities for Efficient and Recyclable Biodiesel Production from Waste Oils: Optimization Using Response Surface Methodology.

Lipase-catalyzed transesterification is a promising and sustainable approach to producing biodiesel. To achieve highly efficient conversion of heterogeneous oils, combining the specificities and advantages of different lipases is an attractive strategy. To this end, highly active Thermomyces lanuginosus lipase (1,3-specific) and stable Burkholderia cepacia lipase (non-specific) were covalently co-immobilized on 3-glycidyloxypropyltrimethoxysilane (3-GPTMS) modified Fe3O4 magnetic nanoparticles (co-BCL-TLL@Fe3O4). The co-immobilization process was optimized using response surface methodology (RSM). The obtained co-BCL-TLL@Fe3O4 exhibited a significant improvement in activity and reaction rate compared with mono and combined-use lipases, achieving 92.9% yield after 6 h under optimal conditions, while individually immobilized TLL, immobilized BCL and their combinations exhibited yields of 63.3%, 74.2% and 70.6%, respectively. Notably, co-BCL-TLL@Fe3O4 achieved 90-98% biodiesel yields after 12 h using six different feedstocks, demonstrating the perfect synergistic effect of BCL and TLL remarkably motivated in co-immobilization. Furthermore, co-BCL-TLL@Fe3O4 could maintain 77% of initial activity after nine cycles by removing methanol and glycerol from catalyst surface, accomplished by washing with t-butanol. The high catalytic efficiency, wide substrate adaptability and favorable reusability of co-BCL-TLL@Fe3O4 suggest that it will be an economical and effective biocatalyst for further applications.

Seed-mediated strategy for synthesis of enzyme-encapsulated metal-organic frameworks with enhanced enzyme activity.

Encapsulation of enzymes into metal-organic frameworks (enzyme@MOF) can improve the stability of enzymes. Most present synthesis methods of enzyme@MOF rely on the complex modification of enzymes or the natural negative surface charge of enzymes to promote the synthesis of enzyme@MOF. Despite extensive efforts, it remains challenging to develop a surface charge-independent and convenient strategy to encapsulate various enzymes into MOF efficiently. In this study, we proposed a convenient seed-mediated strategy for efficient synthesis of enzyme@MOF from the perspective of MOF formation. The seed, acting as nuclei, makes the slow nucleation stage skipped, leading to the efficient synthesis of enzyme@MOF. The successful encapsulation of several proteins demonstrated the feasibility and advantages of the seed-mediated strategy. Moreover, the resulting composite, cytochrome (Cyt c) encapsulated in ZIF-8, exhibited a 5.6-fold increase in bioactivity compared to free Cyt c. The seed-mediated strategy provides an efficient, enzyme surface charge-independent, and non-modified method for the synthesis of enzyme@MOF biomaterials, which warrants further exploration and application in diverse fields.

Inside Out Computational Redesign of Cavities for Improving Thermostability and Catalytic Activity of Rhizomucor Miehei Lipase.

Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and catalytic activity of the enzyme; however, the synergistic effect of cavities remains unclear. In this study, Rhizomucor miehei lipase (RML) was used as a model to explore volume fluctuation and spatial distribution changes of the internal cavities, which could reveal the roles of internal cavities in the thermostability and catalytic activity. We present an inside out cavity engineering (CE) strategy based on computational techniques to explore how changes in the volumes and spatial distribution of cavities affect the thermostability and catalytic activity of the enzyme. We obtained 12 single-point mutants, among which the melting temperatures (Tm) of 8 mutants showed an increase of more than 2°C. Sixteen multipoint mutations were further designed by spatial distribution rearrangement of internal cavities. The Tm of the most stable triple variant, with mutations including T21V (a change of T to V at position 21), S27A, and T198L (T21V/S27A/T198L), was elevated by 11.0°C, together with a 28.7-fold increase in the half-life at 65°C and a specific activity increase of 9.9-fold (up to 5,828 U mg-1), one of the highest lipase activities reported. The possible mechanism of decreased volumes and spatial rearrangement of the internal cavities improved the stability of the enzyme, optimizing the outer substrate tunnel to improve the catalytic efficiency. Overall, the inside out computational redesign of cavities method could help to deeply understand the effect of cavities on enzymatic stability and activity, which would be beneficial for protein engineering efforts to optimize natural enzymes. IMPORTANCE In the present study, R. miehei lipase, which is widely used in various industries, provides an opportunity to explore the effects of internal cavities on the thermostability and catalytic activity of enzymes. Here, we execute high hydrostatic pressure molecular dynamics (HP-MD) simulations to screen the critical internal cavity and reshape the internal cavities through site-directed mutation. We show that as the global internal cavity volume decreases, cavity rearrangement can improve the stability of the protein while optimizing the substrate channel to improve the catalytic efficiency. Our results provide significant insights into understanding the mechanism of action of the internal cavity. Our strategy is expected to be applied to other enzymes to promote increases in thermostability and catalytic activity.

Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes.

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1-24 h.

Continuous Production of Dietetic Structured Lipids Using Crude Acidic Olive Pomace Oils.

Crude olive pomace oil (OPO) is a by-product of olive oil extraction. In this study, low-calorie structured triacylglycerols (TAGs) were produced by acidolysis of crude OPO with medium-chain fatty acids (caprylic, C8:0; capric, C10:0) or interesterification with their ethyl ester forms (C8EE, C10EE). These new TAGs present long-chain fatty acids (L) at position sn-2 and medium-chain fatty acids (M) at positions sn-1,3 (MLM). Crude OPO exhibited a high acidity (12.05-28.75% free fatty acids), and high contents of chlorophylls and oxidation products. Reactions were carried out continuously in a packed-bed bioreactor for 70 h, using sn-1,3 regioselective commercial immobilized lipases (Thermomyces lanuginosus lipase, Lipozyme TL IM; and Rhizomucor miehei lipase, Lipozyme RM IM), in solvent-free media at 40 °C. Lipozyme RM IM presented a higher affinity for C10:0 and C10EE. Lipozyme TL IM preferred C10:0 over C8:0 but C8EE over C10EE. Both biocatalysts showed a high activity and operational stability and were not affected by OPO acidity. The New TAG yields ranged 30-60 and the specific productivity ranged 0.96-1.87 g NewTAG/h.g biocatalyst. Lipozyme RM IM cost is more than seven-fold the Lipozyme TL IM cost. Therefore, using Lipozyme TL IM and crude acidic OPO in a continuous bioreactor will contribute to process sustainability for structured lipid production by lowering the cost of the biocatalyst and avoiding oil refining.

Immobilization of lipase in chitosan-mesoporous silica material and pore size adjustment.

Mesoporous silica MCM-41 was modified by carboxyl groups and assembled with chitosan to produce a novel nanocarrier for the immobilization of lipase. The prepared composite was grafted with silane coupling agent KH560 to decrease the pore size of the mesoporous material and prevent the loss of shed lipase molecules. According to the characterization of the material before and after modification and determination of related parameters, the residual activity of the lipase fixed in the CTS-MCM-41 carrier was 85 % after seven repeated use cycles. The grafting rate of MCM-41 and shrinkage effect were maximized when the concentration of KH560 was 5.0 %, and the modification was performed at 4 h at 70 °C. Using glutaraldehyde as the crosslinking agent, the optimal conditions for enzyme immobilization involved a mass ratio of the carrier to enzyme of 4:1, glutaraldehyde solution volume of 3 %, reaction time of 3 h, and reaction temperature of 45 °C. Overall, the proposed innovative carrier for the fixation of lipase is stable and can physically control the free enzyme in the pore. Moreover, the efficient miniature lipase reactor can promote large-scale industrial production.

Enzyme Immobilization.

The development of enzyme immobilization started in the middle of the previous century as a potential answer to the problem of the enzyme recovery and reuse [...].

Electrochemical detection of lactate produced by foodborne presumptive lactic acid bacteria.

The detection of lactate is an important indicator of the freshness, stability, and storage stability of products as well as the degree of fermentation in the food industry. In addition, it can be used as a diagnostic tool in patients' healthcare since it is known that the lactate level in blood increases in some pathological conditions. Thus, the determination of lactate level plays an important role in not only the food industry but also in health fields. As a result, biosensor technologies, which are quick, cheap, and easy to use, have become important for lactate detection. In the current study, amperometric lactate biosensors based on lactate oxidase immobilization (with Nafion 5% wt) were designed and the limit of detection, linear range, and sensitivity values were determined to be 31 µM, 50-350 µM, and 0.04 µA µM-1 cm-2, respectively. Then, it was used for the measurement of lactic acid that produced by six different and morphologically identified presumptive lactic acid bacteria (LAB) which are isolated from different naturally fermented cheese samples. The biosensors were then used to successfully perform lactate measurements within 3 min for each sample, even though a few of them were out of the limit of detection. Thus, electrochemical biosensors should be used as an alternative and quick solutions for the measurement of lactate metabolites rather than the traditional methods which require long working hours. This is the first study to use a biosensor to measure lactate produced by foodborne LAB in a real sample.

Photosystem II as a chemiluminescence-induced photosensitizer for photoelectrochemical biofuel cell-type biosensing system.

Herein, photosystem II (PSII), extracted from spinach, is used for the first time as an efficient and green sensitizer for a photobioanode in a photoelectrochemical glucose biofuel cell (PBFC) setup. The concept is based on the formation of hemin-catalyzed luminol chemiluminescence (CL) after the enzymatic oxidation of glucose and the simultaneous production of hydrogen peroxide by glucose oxidase. The photosynthetic enzyme PSII, combined with an osmium polymer serving as mediator and photosensitizer, is immobilized and wired on microporous carbonaceous material (MC) for the chemiluminescence-induced oxidation of water to O2 at the photobioanode (GCE|MC|Os polymer|PSII). Also, bilirubin oxidase immobilized on multiwalled carbon nanotubes (MWCNTs) coated electrode (GCE|MWCNT|BOx) serves as a biocathode. The photoelectrochemical biofuel cell (PBFC) is applied to a biosensor model system to validate the appropriateness of such a bioanode operating in a self-powered mode. Os redox polymer attached to MCs provides abundant PSII immobilization and a reliable electron transfer pathway. The well-matching energy levels of photosensitive entities reduce recombination phenomena while MC enhances the charge collection. Substantial photocatalytic water oxidation was observed under CL due to the well-matched CL emission and PSII absorption. The electrode is rationally designed to gain the maximum luminol CL power for the photobioanode. The open circuit potential of PBFC linearly increased with the CL power intensity and, in turn, glucose concentrations in the range of 0-6 mmol L-1. The PBFC yielded an OCP of 0.531 V in 30 mmolL-1 glucose. The study may open a new horizon to the green and pioneering PEC biosensing realm.

Novel Approach for the Isolation and Immobilization of a Recombinant Transaminase: Applying an Advanced Nanocomposite System.

The increasing application of recombinant enzymes demands not only effective and sustainable fermentation, but also highly efficient downstream processing and further stabilization of the enzymes by immobilization. In this study, a novel approach for the isolation and immobilization of His-tagged transaminase from Chromobacterium violaceum (CvTA) has been developed. A recombinant of CvTA was simultaneously isolated and immobilized by binding on silica nanoparticles (SNPs) with metal affinity linkers and additionally within poly(lactic acid) (PLA) nanofibers. The linker length and the nature of the metal ion significantly affected the enzyme binding efficiency and biocatalytic activity of CvTA-SNPs. The formation of PLA nanofibers by electrospinning enabled rapid embedding of CvTA-SNPs biocatalysts and ensured enhanced stability and activity. The developed advanced immobilization method reduces the time required for enzyme isolation, purification and immobilization by more than fourfold compared to a classical stepwise technique.

MOF-Immobilized Two-in-One Engineered Enzymes Enhancing Activity of Biocatalytic Cascade for Tumor Therapy.

Biocatalytic systems based on enzyme cascade reactions have attracted growing interest in the field of biocatalytic medicine. However, it is a major challenge to reasonably construct enzyme cascade reactions with high stability, selectivity, and catalytic efficiency for the in vivo biocatalytic application. Herein, two-in-one engineered glucose oxidase (GOx-Fe0 ) is fabricated by a biomineralization strategy, through which a nanozyme (Fe0 NP) is anchored within the inner cavity of GOx. Then, GOx-Fe0 is immobilized in a pH-sensitive metal-organic framework (MOF) zeolitic imidazolate framework-8 (ZIF-8) to establish a stable and effective MOF-immobilized two-in-one engineered enzyme, GOx-Fe0 @ZIF-8. In vitro studies show that GOx-Fe0 @ZIF-8 exhibits excellent stability and high pH/glucose selectivity, and the shorter spacing between cascade enzymes can increase the cascade throughput and effectively improve the reaction efficiency of the enzyme cascade. In vivo experiments exhibit that GOx-Fe0 @ZIF-8 solves the instability and systemic toxicity of free enzymes, and achieves deep tumor penetration and significant chemodynamic therapeutic efficacy through a pH/glucose-selective enzyme cascade reaction in tumor site. Taken together, such an orchestrated enzyme engineering strategy can effectively improve enzyme stability, selectivity, and enzyme cascade reaction efficiency via chemical transformations, and also provide a promising strategy for the application of biocatalytic cascade reactions in vivo.

Laccase: A potential biocatalyst for pollutant degradation.

In the continual march to a predominantly urbanized civilization, anthropogenic activities have increased scrupulously, industrialization have occurred, economic growth has increased, and natural resources are being exploited, causing huge waste management problems, disposal issues, and the evolution of several pollutants. In order to have a sustainable environment, these pollutants need to be removed and degraded. Bioremediation employing microorganisms or enzymes can be used to treat the pollutants by degrading and/or transforming the pollutants into different form which is less or non-toxic to the environment. Laccase is a diverse enzyme/biocatalyst belonging to the oxidoreductase group of enzymes produced by microorganisms. Due to its low substrate specificity and monoelectronic oxidation of substrates in a wide range of complexes, it is most commonly used to degrade chemical pollutants. For degradation of emerging pollutants, laccase can be efficiently employed; however, large-scale application needs reusability, thermostability, and operational stability which necessitated strategies like immobilization and engineering of robust laccase possessing desirable properties. Immobilization of laccase for bioremediation, and treatment of wastewater for degrading emerging pollutants have been focussed for sustainable development. Challenges of employing biocatalysts for these applications as well as engineering robust laccase have been highlighted in this study.

Hollow Mesoporous Carbon-Based Enzyme Nanoreactor for the Confined and Interfacial Biocatalytic Synthesis of Phytosterol Esters.

Rationally designing carriers to obtain efficient and stable immobilized enzymes for the production of food raw materials is always a challenge. In this work, hollow cube carbon (HMC) as a carrier of Candida rugosa lipase (CRL) was prepared to construct a Pickering interfacial biocatalysis system, which was applied to biphasic biocatalysis. For comparison, the nonporous carbon (HC) and porous MoS2 (HMoS2) were also designed. On these grounds, p-NPP and linolenic acid were selected as the representative substrates for hydrolysis and esterification reactions. Under the optimal conditions, the protein loading amount, specific activity, and expressed activity of CRL immobilized on HMC (HMC@CRL) were 167.2 mg g-1, 5.41 U mg-1, and 32.34 U/mg protein, respectively. In the "oil-water" biphase, the relative hydrolytic activity of HMC@CRL was higher than that of HC@CRL, HMoS2@CRL, and CRL by 50, 68, and 80%, respectively, as well as itself in one phase. Compared to other reports (1.13%), HMC@CRL demonstrated a satisfactory hydrolysis rate (3.02%) and was the fastest among all other biocatalysts in the biphase. Moreover, compared with the free CRL in one-phase system, the Pickering interfacial biphasic biocatalyst, HMC@CRL, exhibited a higher esterification rate (85%, 2.7-fold enhancement). Therefore, the HMC@CRL nanoreactors had more optimal performance in the field of biomanufacturing and food industry.