Mesoporous silica nanoparticles were synthesized by using tannic acid as a pore-forming agent, which is an environmentally friendly, cheap, and non-surfactant template. SEM and TEM images indicated that the tannic acid-templated mesoporous silica nanoparticles (TA-MSNs) are monodisperse spherical-like particles with an average diameter of 195 ± 16 nm. The Brunauer–Emmett–Teller (BET) results showed that the TA-MSNs had a relatively high surface area (447 m2/g) and large pore volume (0.91 cm3/g), and the mean pore size was ca. 10.1 nm. Burkholderia cepacia lipase was immobilized on the TA-MSNs by physical adsorption for the first time, and the properties of immobilized lipase (BCL@TA-MSNs) were investigated. The BCL@TA-MSNs exhibited satisfactory thermal stability; strong tolerance to organic solvents such as methanol, ethanol, isooctane, n-hexane, and tetrahydrofuran; and high operational reusability when BCL@TA-MSNs were applied in esterification and transesterification reactions. After recycling 15 times in the transesterification reaction for biodiesel production, over 85 % of biodiesel yield can be maintained. With these desired characteristics, the TA-MSNs may provide excellent candidates for enzyme immobilization.

This study describes a general strategy using norepinephrine (NE) as a stable anchor for the immobilization of enzyme onto three-dimensionally ordered macroporous (3DOM) silica. Penicillin G acylase (PGA) was chosen as a model enzyme. The effect of pH and temperature on the activity of PGA@PN–SiO2 (PGA immobilized on poly(norepinephrine)-modified 3DOM silica) was investigated. The operational stability, storage stability and kinetic properties of the PGA@PN–SiO2 were also examined. Compared with free PGA and PGA@SiO2 (PGA immobilized on blank 3DOM silica through physical adsorption), the stabilities of PGA@PN–SiO2 were improved significantly. PGA@PN–SiO2 retained high activity during the hydrolysis in a continuous packed-bed reactor (PBR) after 30 catalytic cycles, which demonstrated that PGA@PN–SiO2 can bear endurance of continuous catalysis. The method presented in this study has broad potential for immobilizing enzymes and other biomolecules.

A facile and promising approach was developed to fabricate enzyme-based 3D-ordered macroporous biocatalysts (enzyme-based inverse opals) by using the colloidal crystal templating method. Horseradish peroxidase- and amylase-based inverse opals were prepared, which verified that this method is suitable for various enzymes.

With the aim to provide a highly stable and active biocatalyst, cross-linked enzyme aggregates (CLEAs) of lipase Candida sp. 99-125 were prepared in three-dimensionally ordered macroporous silica materials (CLEAs-LP@3DOM-SiO2). Lipase Candida sp. 99-125 was first precipitated in the pores of 3DOM SiO2 (named EAs-LP@3DOM-SiO2), and further cross-linked by glutaraldehyde to form CLEAs-LP@3DOM-SiO2. Saturated ammonium sulfate was used as a precipitant and glutaraldehyde with a concentration of 0.25% (w/w) was employed as a cross-linker. Compared with EAs-LP@3DOM-SiO2 and native lipase, CLEAs-LP@3DOM-SiO2 exhibited excellent thermal and mechanical stability, and could maintain more than 85% of initial activity after 16 days of shaking in organic and aqueous phase. When CLEAs-LP@3DOM-SiO2 was applied in esterification and transesterification reactions, improved activity and reusability were achieved. This method can be used for the immobilization of other enzymes of interest.Keywords: biodiesel; cross-linked enzyme aggregates; enzyme immobilization; lipase; Three-dimensionally ordered macroporous materials;

In this article, a simple, versatile, and mild method was used to fabricate porous enzyme microspheres using CaCO3 microparticles as templates. The preparation process involved three steps including coprecipitation of enzyme with CaCO3, cross-linking, and template removal. The preparation process was optimized and the obtained porous horseradish peroxidase (HRP) microspheres (p-HRP) were characterized. The p-HRP exhibited improved thermal stability and was more stable under alkali conditions than its free counterpart. These features are attractive for practical applications. Compared to free HRP, the Km value of p-HRP was increased and the Vmax value was decreased. The catalytic performance of p-HRP in removing phenolic compound and dye from aqueous solution was explored by using phenol and Direct Black-38 as model substrates. The results indicated that p-HRP exhibited high removal efficiency and moderately good reusability.

Cross-linked enzyme aggregates (CLEAs) are a versatile and effective method for enzyme immobilization, which is exquisitely simple and amenable to rapid optimization. In this study, nature egg white, which is low cost, easily available, and nontoxic, was used as protein feeder to replace traditional protein feeder (bovine serum albumin, etc.) in the preparation of laccase CLEAs (CLEAs-egg). The effects of the various parameters—nature of the precipitant, temperature, glutaraldehyde concentration, and cross-linking time—on the activity recovery of the resulting CLEAs were studied. The laccase CLEAs-egg exhibited increased stability compared to the free and the laccase CLEAs without protein feeder. The thermal stability of CLEAs-egg was improved and showed 1.3- and 1.8-fold increase in activity at 40 and 60 °C after 5 h incubation, respectively. The stability of CLEAs-egg against denaturants (urea and GndHCl) and protease (trypsin) was also improved. Laccase CLEAs-egg was also demonstrated to be an active and stable biocatalyst in the removal of chlorophenol. After 30 h, 83.6 and 91.5 % of 4-chlorophenol and 2,4-dichlorophenol can be removed.

Lipase Candida sp. 99–125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99–125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.

Magnetic silica composite particles were prepared by using the biosilicification reaction, in which Fe3O4 nanoparticles were entrapped in a silica matrix. The composite particles were functionalized with 3-aminopropyltriethoxysilane (APTES). These functionalized magnetic composite particles were used to immobilize a kind of valuable bulky industrial enzyme (laccase). The incorporation of magnetic nanoparticles greatly facilitates the manipulation of the immobilized biocatalyst, since it can be easily and quickly recovered from the reaction system by the simple application of an external magnetic field. The effects of immobilization conditions were optimized and the activity of the immobilized laccase was investigated. The results showed that the highest specific activity of immobilized laccase reached to 224 U and the activity recovery was 83.9%. Compared with free laccase, the thermal, pH, operational and storage stabilities of the immobilized laccase were improved significantly. The catalytic activity of the immobilized laccase was also demonstrated by the degradation of two phenolic substances (i.e., 2,4-dichlorophenol and 4-chlorophenol). It was found that the removal rates of 2,4-dichlorophenol and 4-chlorophenol were 80.9% and 64.2% in about 12 h, respectively.

In an effort to reduce the production cost of biodiesel and realize the lipase recovery of the in situ transesterification technology, a reactor with facile catalyst separation device was developed in this study. Compared with conventional method of biodiesel production, this improved reactive extraction technology can realize not only the biodiesel synthesis in a single step, but also the separation of immobilized lipases from the oilseed residues easily. This technology may present a sustainable, economically attractive process for biodiesel production. J. curcas L. seed, methanol, Lipozyme TL IM, and cosolvent n-hexane were used in this study. The effect of various process parameters on the yield of fatty acid methyl esters (FAMEs) was investigated. The highest FAMEs yield of 90.6% could be achieved under the optimum conditions: n-hexane/seed ratio of 3.5:1 mL/g, methanol/oil molar ratio of 6:1, 15% (w/w) of Lipozyme TL IM, reaction temperature of 45 °C, water content of 1.0%, and reaction time of 12 h. The lipase could maintain more than 40% of its initial activity after 13 batches operation under optimal reaction conditions. This simple in situ transesterification technology with facile catalyst separation device can be a potential route for biodiesel production, which may greatly reduce the processing steps and costs.

Compared with conventional production of biodiesel from oilseeds, in situ reactive extraction of oilseeds with acyl acceptors is an attractive approach, which consists of sustainable, economically attractive biotechnological processes for biodiesel synthesis. In this study, the presence of lipase activity in germinating oilseeds was detected and the lipase activity increased to a maximum after 4 days of germination. At that time, the germinated seeds featured an only slightly lower oil content relative to the ungerminated seeds. As such, an environmentally friendly and low-cost in situ self-catalytic process for biodiesel production was developed. The biodiesel was prepared by reactive extraction of germinated Jatropha curcas L. seeds with methanol without any other catalyst added. In this process, n-hexane was used as reactive extraction solvent. The highest fatty acid methyl esters (FAMEs) yield of 87.6% could be achieved under the optimum conditions: a n-hexane/germinated seed ratio of 2.5 ml g−1, a methanol/germinated seed oil (contained approximately 90% FFA) molar ratio of 1.5:1, a reaction temperature of 35 °C, a germinated seed water content of 2.9% and a reaction time of 8 h. This simple reactive extraction process without additional catalyst can be a potential route for biodiesel production, which may greatly reduce the processing steps and costs.

Genipin, a natural cross-linking agent, was used for the immobilization of lipase from Candida sp. 99-125 by cross-linking to two kinds of mesoporous resins. Under optimum conditions, the activity recovery of immobilized lipase on resin NKA-9 could reach up to 96.99% when the genipin concentration was 0.5%, and it could reach up to 86.18% for S-8 with a genipin concentration of 0.25%. Compared with using glutaraldehyde as a cross-linking agent, the immobilized lipase using genipin showed better pH and thermal stability, storage stability, and reusability. The residual activity of immobilized lipase using genipin as cross-linker remained more than 60% of its initial activity after six hydrolytic cycles, whereas only about 35% activity remained by using glutaraldehyde as cross-linker.

The kinetics of lipase-catalyzed interesterification synthesis of L-ascorbyl lactate was studied. To determine the enzyme kinetic constants of the interesterification, a three-factor and five-level central composite design was used. The factors studied were ethyl lactate concentration, reaction temperature (T), and water content (w). Moreover, a statistical approach called the response surface method (RSM) was used to predict the kinetic constants. Finally, the relationships between the kinetic constants (Vmand Km) and the reaction parameters (T and w) were obtained.To assess the accuracy of the RSM approach for determining Vmand Km′detailed validation experiments were carried out by the conventional approach at four different reaction parameters (35°C, 10μL; 45°C, 20μL; 55°C, 15 μL; 65°C, 18μL). The results indicated that the RSM approach gave reasonable results for the determination of Vm and Km in the range of tested parameters.

A novel method was developed for papain immobilization through a biomimetic silicification process induced by papain. By incubating papain in a silica precursor solution, the papain-silica composite formed rapidly and papain was encapsulated. The encapsulation efficiency and the recovery activity were 82.60% and 83.09%, respectively. Compared with enzymes and biomolecules immobilized in biosilica matrix in the presence of additional silica-precipitating species, this papain encapsulation process, a biomimetic approach, realized high encapsulation efficiency by its autosilification activity under mild conditions (near-neutral pH and ambient temperature). Furthermore, the encapsulated papain exhibits enhanced thermal, pH, recycling and storage stabilities. Kinetic analysis showed that the biomimetic silica matrix did not significantly hinder the mass transport of substrate or the release of product.

•Protein-based inverse opals were prepared for the first time.•The properties of the immobilized PGA were investigated.•The immobilized PGA showed improved thermal and pH stability.•The immobilized PGA can be applied in continuous catalysis in packed-bed reactor.In this study, protein-based inverse opals were prepared for the first time by using the colloidal crystal templating method. The preparation process involved three steps including filling the templates with protein molecules, crosslinking, and template removal. The obtained inverse opals were used to immobilize Penicillin G acylase (PGA) because of its intrinsic biocompatible property. The immobilization process was optimized and the properties of the immobilized PGA (PGA@IO) were investigated. PGA@IO exhibited improved thermal and pH stability compared with its free counterpart. After reusing nine times, it retained 70% of the initial activity. Besides, the PGA@IO retained high activity during the hydrolysis reactions in continuous catalysis in packed-bed reactor (PBR) after 15 days.A facile approach was provided to prepare protein-based inverse opals for the first time and the obtained opals were used to immobilize enzyme.Download high-res image (162KB)Download full-size image

•A novel silica-based macro–mesoporous monolith (Si(HIPEs)) was synthesized.•The Si(HIPEs) was functionalized with amino or epoxy groups.•The native Si(HIPEs) and functionalized Si(HIPEs) were used to immobilized PGA.•The immobilized PGA exhibited enhanced stability compared with free PGA.A novel material labeled as Si(HIPEs) which possesses macroporosity and mesostructuration was synthesized and functionalized with amino or epoxy groups. The native Si(HIPEs) and functionalized Si(HIPEs) were employed as supports for penicillin G acylase (PGA) immobilization. The effect of pH and temperature on the activity of immobilized PGA was investigated. The reusability, operational stability, storage stability and kinetic properties of the immobilized PGA were examined. Compared with free PGA, the stabilities of immobilized enzyme were improved significantly, especially PGA immobilized on Amino-Si(HIPEs). The excellent reusability of the immobilized PGA will make it useful for potential commercial applications.Download full-size image

•3DOM/m-S was prepared and applied for lipase immobilization.•CALB@3DOM/m-S showed improved stabilities than CALB@3DOM-S.•CALB@3DOM/m-S showed improved usability when applied in esterification.Hierarchically ordered macroporous/mesoporous silica (3DOM/m-S) material was prepared through the dual templating method with polystyrene (PS) colloidal crystals as the hard template and amphiphilic triblock copolymers (P123) as the soft template. The achieved 3DOM/m-S possesses ordered macropores of 400 nm and mesopores of 5.1 nm, which provides a promising platform for enzyme immobilization. Lipase B from Candida antarctica (CALB) was employed as a model enzyme to verify the possibility and advantages of enzyme immobilized on 3DOM/m-S. The immobilized lipase shows excellent stability towards heat even at 80 °C and organic solvents for long-term incubation (288 h). Also, the immobilized CALB could be used for esterification reactions between acids and alcohols with different chain lengths, and 90% of conversion rate could be reached. In examining the reusability in esterification of oleic acid and ethanol, the conversion rate can retain 75% after 10 reaction cycles, indicating a remarkable reusability of the immobilized lipase.Download full-size image

Antifouling coatings are used extensively on vessels and underwater structures. Conventional antifouling coatings contain toxic biocides and heavy metals, which may induce unwanted adverse effects such as toxicity to non-target organisms, imposex in gastropods and increased multiresistance among bacteria. Therefore, enzyme-based coatings could be a new alternative solution. A H2O2-producing bienzyme system was developed in this study. H2O2 can be produced from starch by the cooperation of α-amylase and glucose oxidase, which promotes the hydrolysis of polymeric chain and oxidizes the glucose to produce H2O2, respectively. The encapsulated bienzyme (A-G@BS) exhibits enhanced stabilities of thermal, pH, recycling and tolerance of xylene. The A-G@BS-containing coating releases H2O2 at rates exceeding a target of 36 nmol·cm− 2·d− 1 for 90 days in a laboratory assay. The results demonstrate that the method is a promising coating technology for entrapping active enzymes, presenting an interesting avenue for enzyme-based antifouling solutions.Antifouling coatings are used extensively on vessels and underwater structures. enzyme-based coatings could be a environmental-friendly solution. A H2O2-producing bienzyme system was developed in this study, which can deter fouling organisms by the toxicity of H2O2. H2O2 can be produced from starch by the cooperation of α-amylase and glucose oxidase, which promotes the hydrolysis of polymeric chain and oxidizes the glucose to produce H2O2, respectively. The results demonstrate that the method is a promising coating technology for entrapping active enzymes, presenting an interesting avenue for enzyme-based antifouling solutions.Download full-size image

•Novel structured interlocked-microcapsules (SIMC) was synthesized.•The enzyme-immobilized SIMC presented high catalytic performance.•The enzyme-immobilized SIMC exhibited significantly enhanced stability.•Internal diffusion resistance was reduced due to the unique structure.A facile approach to prepare structured interlocked-microcapsules (SIMC) was developed, which combined the advantages of open mouthed structure, hierarchical porous nanostructure and interlocked architecture. The specific surface area of SIMC was 374.6 m2/g and the diameter of the pores was 8.707 nm. Nitrile hydratase (NHase) was immobilized on SIMC via covalent bonding to realize the easy separation of the enzyme and improve properties of enzyme such as pH tolerance and heat stability. This work demonstrated that the enzyme-immobilized SIMC presented high catalytic performance and significantly improved stability.Download high-res image (305KB)Download full-size image

•HRP was encapsulated in titania particles through biomimetic titanification process.•The stabilities of HRP were obviously improved by encapsulation in titania.•The encapsulated HRP can be used in removal of dye and phenolic compounds.•The encapsulated HRP showed better removal efficiency than free HRP.•The encapsulated HRP showed a moderately good capability of reutilization.In this study, horseradish peroxidase (HRP) was encapsulated in phospholipid-templated titania particles through the biomimetic titanification process and used for the treatment of wastewater polluted with phenolic compounds and dye. The encapsulated HRP exhibited improved thermal stability, a wide range of pH stability and high tolerance against inactivating agents. It was observed an increase in Km value for the encapsulated HRP (8.21 mM) when compared with its free counterpart. For practical applications in the removal of phenolic compounds and dye by the encapsulated HRP, the removal efficiency for phenol, 2-chlorophenol, Direct Black-38 were 92.99%, 87.97%, and 79.72%, respectively, in the first treatment cycle. Additionally, the encapsulated HRP showed better removal efficiency than free HRP and a moderately good capability of reutilization.

Candida antarctica lipase B (CALB) was immobilized on the macroporous resin by physical adsorption in organic medium. The immobilization was performed in 5 mL isooctane, and the immobilization conditions were optimized. The results were achieved with the mass ratio of lipase to support 1:80, the buffer of pH 6.0, initial addition of PBS 75 μL, and immobilization time of two hours at 30°C. Under the optimal conditions, the activity recovery was 83.3%. IM-CALB presented enhanced pH and thermal stability compared to the free lipase, and showed comparable stability with the commercial Novozym 435, after 7 times repeated use for catalyzing the synthesis of ethyl lactate, 56.9% of its initial activity was retained, and only 24.7% was retained when used for catalyzing the hydrolysis of olive oil.

•Dual-functional PA-66 nanofibrous membrane was prepared for OPs protection.•The membrane has particle filtration efficiency over 99%.•OPH immobilized on PA-66 nanofibers catalyzed OPs degradation.•Degradation efficiency for methyl parathion was about 40%.•The dual-functional membrane has good stability.In both civilian and military domains, the increasing use of highly toxic organophosphates (OPs) has created an urgent demand for developing an effective method that could protect the human body from OP poisoning. In this study, a novel dual-functional protecting material with both OP filtration and degradation functions was designed and fabricated. The filtration function was endowed by the electrospinning polyamide 66 (Nylon 66, PA-66) nanofibrous membrane, while the OP degradation function was based on the immobilization of organophosphorus hydrolase (OPH) on membrane through glutaraldehyde (GA) crosslinking. A systematic study of the relationship between the membrane structure, filtration ability and OP degradation activity revealed that PA-66 nanofibrous enzyme with 20 μm thickness exhibited perfect uniform morphology, and the particle filtration efficiency was over 99%. The nanofibrous enzyme exhibited a specific activity of 41 U/g; the enzyme activity recovery was 53.3%, and the methyl parathion (MP) degradation efficiency was 40%. Moreover, the nanofibrous enzyme expressed excellent organic solvent stability; approximately 70% of its initial activity was retained after incubation in xylene solvent for 24 h at 25 °C. The residual activity was 40% after being stored for 30 d at 25 °C. After 10 repeated uses, the residual activity of the nanofibrous membrane was 37%. This study demonstrated the application potential of dual-functional PA-66 nanofibrous enzyme in biochemical protection, aimed at various military and civilian applications.Download full-size image

Nano-materials have been applied in many fields due to their excellent characteristics, such as the high surface area-to-volume ratio, excellent physicochemical properties and biological compatibility. In this study, multi-walled carbon nanotubes (MWCNTs) were utilized to prepare MWCNTs–papain bioconjugates and then realized the immobilization of papain. MWCNTs functionalized with carboxyl- and amine- groups on their surface were used as immobilization carriers. The immobilization of papain on the functionalized MWCNTs through physical absorption was examined. The conjugates were denoted as MWCNTs–papain bioconjugates. To improve the stability, the bioconjugates were further coated by silica through the biomimetic silicification process that induced by papain (denoted as silica-coated bioconjugates). The as-prepared MWCNTs–papain bioconjugates and the silica-coated bioconjugates were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The preliminary results showed that the bioconjugates could retain most of the initial activity of papain. Compared to free papain and MWCNTs–papain bioconjugates, the silica-coated bioconjugates exhibited significantly improved thermal, pH and recycling stability. Comparisons of the kinetic parameters between MWCNTs–papain bioconjugates and the silica-coated bioconjugates revealed that the Km value of the immobilized papain experienced a slight increase after silica coating, which suggested that the silica coating did not significantly hinder papain's access to substrate or release of product.

A facile and promising approach was developed to fabricate enzyme-based 3D-ordered macroporous biocatalysts (enzyme-based inverse opals) by using the colloidal crystal templating method. Horseradish peroxidase- and amylase-based inverse opals were prepared, which verified that this method is suitable for various enzymes.