This study reported a novel and facile method for the spontaneous synthesis of flowerlike cobalt phosphate nanocrystals (Co3(PO4)2 nanoflowers) without adding any templates, and the growth mechanism was further studied. Subsequently, an excellent nanobiocatalyst system was established via the biomimetic mineralization of cobalt phosphate with Co-type nitrile hydratase (NHase). Because of the interactions between Co ions in the microenvironment of cobalt phosphate and enzyme active site, the encapsulated NHase (NHase@Co3(PO4)2) exhibited high catalytic efficiencies and desirable stabilities. The enzymatic activity of encapsulated NHase was 238.4 U/g and the protein loading amount was 210.73 mg/g. Compared with free NHase, the optimum temperature of NHase@Co3(PO4)2 was 40 °C, which was higher than that of the free NHase (30 °C). The thermal, pH, antiproteolytic, and storage stability of NHase@Co3(PO4)2 were all improved. Furthermore, NHase@Co3(PO4)2 could be applied in the production of nicotinamide (NAM) with a satisfying yield and it could be reused seven times. All these results in this work clearly confirmed that the cobalt phosphate nanocrystals, as an original nanocarrier, would have promising applications for constructing nanobiocatalyst systems.

•Monodisperse core-shell magnetic organosilica nanoflowers were prepared.•CALB was immobilized on the nanoflowers via covalent bonding method.•CALB@nanoflowers showed improved stabilities than CALB@MMPS.•CALB@nanoflowers showed improved catalytic performance in the synthesis of alkyl levulinates.The monodisperse core-shell magnetic organosilica nanoflowers were successfully synthesized by a method based on the bicontinuous microemulsion phase of the Winsor III system. The obtained nanoflowers with magnetic core and flower-like organosilica shell possess radial-wrinkle, tunable perpendicular channels, and high magnetization (62 emu/g). The nanoflowers were employed as support for Candida antarctica lipase B (CALB) covalent immobilization. The maximum amount of CALB immobilized on the nanoflowers was 93 mg/gsupport, while the specific hydrolytic activity was 22,700 U/gsupport. Meanwhile, the CALB@nanoflowers showed better pH stability than free CALB and CALB@MMPS, which can remain 65% of initial activity after incubated in cyclohexane at 60 °C for 96 h, and remain 90% of initial activity after stored in room temperature for 42 days. In addition, the CALB@nanoflowers could catalyze the synthesis of alkyl levulinates through the esterification of levulinic acid alcohols with different chain length (n-butyl alcohol, n-caprylic alcohol, n-lauryl alcohol) in a solvent-free system effectively. After recycling 10 times, the CALB@nanoflowers retained more than 85% of its initial activity in catalyzing the synthesis of butyl-levulinates and more than 96% of the initial activity in catalyzing the synthesis of long chain alkyl levulinates, which was much higher than commercial N435. With these desired characteristics, the design of hydrophobic nanoflowers with tunable wrinkle channels for lipases immobilization opens up a new way to improve the catalytic activity and stability of the lipases, and may have potential applications in various lipase-based industrial processes.Download high-res image (69KB)Download full-size image

•Biocatalytic colloidosome with solid core were prepared.•LP@colloidosome showed improved stabilities than free lipase and N435.•LP@colloidosome showed improved activity when applied in esterification and transesterification.•LP@colloidosome was a promising biocatalyst for enzymatic synthesis of GlyC.The synthesis of silica-based colloidosome with a magnetic solid core via Pickering emulsification and its use as an immobilized enzyme system is described in this work. Lipase B from Candida antarctica (CALB) was physically adsorbed on the material to form the immobilized enzyme (LP@colloidosome) and the enzyme loading was 166.3 mg/gsupport. The specific hydrolytic activity of LP@colloidosome was 209.6 U/gsupport and its immobilization yield was 22.08%. The stability test indicated that LP@colloidosome can retain 49% of original activity after incubation at 50 °C for 6 h and 105.6% of original activity after incubation in t-butanol for 96 h, while the free lipase lost most the initial activity under the same condition. These findings showed that LP@colloidosome had the enhanced thermal stability and tolerance in organic solvents. Meanwhile, LP@colloidosome can be easily recovered by magnet due to the addition of modified Fe3O4 nanoparticles in the solid core. LP@colloidosome was successfully used to catalyze the esterification of fatty acids and alcohols with different length in organic solvent. The yields of the corresponding esters that catalyzed by LP@colloidosome were significantly higher than those catalyzed by free lipase. Additionally, the versatility of LP@colloidosome was also verified in the synthesis of glycerol carbonate through transesterification. The results indicated that biosynthesis of glycerol carbonate from glycerol using the LP@colloidosome (6.1 wt%) as catalyst afforded 85.20% of conversion rate at 60 °C. This work offers insight into the design of colloidosome for efficient immobilization of lipase as well as improvement of the lipase’s properties.Download high-res image (156KB)Download full-size image

A novel immobilized lipase of Burkholderia cepacia lipase (BCL) was prepared for the first time through the combination of bioimprinting and cross-linked protein-coated microcrystal technology (CLPCMC). p-Benzoquinone was used as the cross-linker for preparing the bioimprinted CLPCMC (denoted as IM-CLPCMC). The morphology and the properties of the IM-CLPCMC were investigated. The IM-CLPCMC could retain ca. 84% of initial activity after being incubated in n-hexane/ethanol system at 80 °C for 4 h, and retain 98% of initial activity after being stored in n-hexane/ethanol for 120 h. These results indicated that IM-CLPCMC exhibited improved thermal stability and long-term stability in organic solvents. Furthermore, IM-CLPCMC was used as biocatalysts for producing biodiesel from Jatropha curcas L. oil. Under the optimal conditions, the maximum yield of biodiesel obtained via transesterification could reach 95.0%. After seven cycles, more than 54.7% of biodiesel yield can be retained. Such results demonstrated that IM-CLPCMC is a promising catalyst for biotechnological applications.

In this study, lipase from Candida sp. 99-125 was immobilized on glutaraldehyde-activated APTES-functionalized or epoxy-functionalized porous ceramic monoliths. The activity and stability of the immobilized lipases were investigated. The results indicated that the stabilities of immobilized lipases were improved significantly compared to native lipase. The immobilized lipases were used as catalysts for the production of oil gelling agent by using mannitol and fatty acid vinyl ester as substrates. Take Man-8 as an example, the yield of this oil gelling agent was more than 78% after 48 h reaction that catalyzed by the immobilized lipases. After the 5th batch reaction, more than 50% of yield can be maintained. Furthermore, the results of gelation tests indicated that the oil gelling agent can gel various hydrocarbons and oils. When the oil gelling agent was used to recover diesel from a biphasic mixture, more than 90% of the initial diesel can be recovered.

Nitrile hydratase cross-linked enzyme aggregates (CLEAs) were formed in mesoporous onion-like silica (NHase-CLEAs@MOS) by using macromolecular dextran polyaldehyde as a cross-linker through the carrier-bound CLEAs method. The effect of preparation parameters on the recovery of enzyme activity was investigated. The properties such as pH, thermal and storage stability, and kinetic parameters of NHase-CLEAs@MOS were also studied. The maximum amount of NHase absorbed in MOS was 535 mg/g. Under optimized conditions, the maximum activity recovery of NHase-CLEAs@MOS was 48.2%. The stabilities of NHase-CLEAs@MOS were improved significantly compared to the NHase@MOS prepared by physical adsorption and free NHase. This work demonstrated that the mesoporous onion-like silica can be efficiently employed as host materials for NHase immobilization, and the carrier-bound CLEAs method can lead to enhanced activity and stability of the immobilized enzymes.

A bienzyme system aiming at removal of glucose from isomaltooligosaccharide (IMO) was developed in this study. In this bienzymatic system, the first enzyme (glucose oxidase, GOD) catalyzed glucose to generate H2O2 and gluconic acid, while the second one (catalase, CAT) broke down the undesired H2O2 to water and oxygen, which can reduce the peroxide-induced degradation of GOD. For improving the catalytic activity, realizing the recyclability, and simplifying the separation operations of the enzymes, a Pickering emulsion stabilized by GOD- and CAT-containing silica particles was constructed. The optimal reaction temperature and pH were identified as 45 °C and 6.0, respectively. Under such conditions, the removal efficiency of glucose could be maintained at ca. 73.57%, even after five cycles in repeated batch operations. It is believed that the proposed process of glucose removal with biocatalytic Pickering emulsion is promising for the purification of IMO on an industrial scale.

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.

Mesoporous silica (MPS) was synthesized and used as a support for lipase Candida sp. 99–125 immobilization. The immobilization procedure was simple and effective: lipase Candida sp. 99–125 was first immobilized in the MPS by adsorption (named ADL@MPS), then chemical crosslinking was conducted for stabilizing the lipase and inhibiting leakage, and cross-linked enzyme aggregates (CLEAs) of Candida sp. 99–125 lipase in the MPS were obtained (named CLL@MPS). The stability of ADL@MPS and CLL@MPS was investigated. Compared with ADL@MPS and native lipase, CLL@MPS showed outstanding stability under vigorous shaking conditions and the thermal stability of CLL@MPS in the presence of organic solvents was also improved. Additionally, CLL@MPS exhibited high catalytic performance in hydrolysis, esterification, and transesterification reactions with increased stability and recyclability.

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.

•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

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

•Tannic-acid-templated magnetic mesoporous silica nanoparticles are synthesized.•Immobilized NHase is prepared by adsorption and cross-linking method.•Immobilized NHase exhibits improved thermal, pH, mechanical and storage stability.•Yield of nicotinamide can reach more than 98% by using immobilized NHase as catalyst.Tannic-acid-templated magnetic mesoporous silica nanoparticles (TA-MMSNs) were synthesized for the first time. The TA-MMSNs were monodisperse spherical particles with a diameter of around 250 nm and a magnetization saturation value of 35.26 emu/g. The specific surface area of TA-MMSNs was 423.4 m2/g, and the diameter and cumulative volume of the pores were 9.349 nm and 1.071 cm3/g, respectively. The TA-MMSNs were used to prepare immobilized NHase (CLNHAs@TA-MMSNs) by forming cross-linked nitrile hydratase aggregates (CLNHAs) in pores of TA-MMSNs using glutaraldehyde as a cross-linker. CLNHAs@TA-MMSNs and free NHase had the same optimum pH (pH 7), and the optimum temperature of CLNHAs@TA-MMSNs (40 °C) was higher than that of free NHase (30 °C). Compared with free NHase, CLNHAs@TA-MMSNs exhibited improved thermal, pH, mechanical and storage stability. Furthermore, CLNHAs@TA-MMSNs was applied in production of nicotinamide, and yield of nicotinamide could reach more than 98%. The tolerance of CLNHAs@TA-MMSNs to high concentration of substrate was better than that of free NHase, and yield of nicotinamide could still reach 29.74% after seven cycles of reaction. The kinetic parameters were investigated and the results indicated a lower substrate affinity and catalytic efficiency of CLNHAs@TA-MMSNs in comparison with free NHase. This work demonstrated that TA-MMSNs could be efficiently employed as supports for enzyme immobilization.

Mesoporous silica (MPS) was synthesized and used as a support for lipase Candida sp. 99–125 immobilization. The immobilization procedure was simple and effective: lipase Candida sp. 99–125 was first immobilized in the MPS by adsorption (named ADL@MPS), then chemical crosslinking was conducted for stabilizing the lipase and inhibiting leakage, and cross-linked enzyme aggregates (CLEAs) of Candida sp. 99–125 lipase in the MPS were obtained (named CLL@MPS). The stability of ADL@MPS and CLL@MPS was investigated. Compared with ADL@MPS and native lipase, CLL@MPS showed outstanding stability under vigorous shaking conditions and the thermal stability of CLL@MPS in the presence of organic solvents was also improved. Additionally, CLL@MPS exhibited high catalytic performance in hydrolysis, esterification, and transesterification reactions with increased stability and recyclability.