Chitosan-mediated formation of biomimetic silica nanoparticles: an effective method for manganese peroxidase immobilization and stabilization.

Our work here, for the first time, reported the use of chitosan-mediated biomimetic silica nanoparticles in enzyme immobilization. In order to make clear the relationship among silicification process, silica nanoparticle structure and immobilized enzyme activity, a mechanism of chitosan-mediated silicification using sodium silicate as the silica source was primarily evaluated. Chitosan was demonstrated effectively to promote the silicification not only in accelerating the aggregation rate of sodium silicate, but also in templating the formation of silica nanoparticles. Although the whole biomimetic silicification process contained polycondensation-aggregation-precipitation three stages, the elemental unit in precipitated silica was confirmed to be nanoparticles with 100 nm diameter regardless of the chitosan and silicate concentration used. Furthermore, the effect of enzyme on silicification process was also investigated. The introducing of manganese peroxidase (MnP) to silica precursor solution had no obvious effect on the silicification rate and nanoparticle morphology. The residual activity and embedding rate of immobilized MnP were 64.2% and 36.4% respectively under the optimum conditions. In addition, compared to native MnP, the MnP embedded in chitosan/silica nanoparticles exhibited improved stability against organic solvent and ultrasonic wave. After ultrasonic treatment for 20 min, 77% of the initial activity was remained due to the protective effect of chitosan/silica nanoparticles, while native MnP lost almost all of its original activity.

Hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase from Rhodotorula glutinis.

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

LiCl-induced improvement of multilayer nanofibrous lipase for biodiesel synthesis.

A unique method that applied a multilayer-immobilization strategy was developed to prepare nanofibrous enzymes for biosynthesis. LiCl co-electrospun with polyurethane nanofibers enabled strong physical adsorption of bovine serum albumin (BSA), forming the first layer of protein on the nanofibers; lipase AK was subsequently crosslinked to BSA as an outer layer of enzyme. The content of LiCl in nanofibers was found to be a sensitive factor affecting the activity and stability of the immobilized lipase. For biodiesel synthesis from soybean oil and methanol in isooctane, the reaction rate catalyzed by nanofibrious lipase carrying 5 wt% LiCl was 6.6-fold higher than fibers without LiCl, with a conversion of 91% was achieved within 2 h. LiCl also induced much improved enzyme stability. The nanofibrous lipase with 5% LiCl could be repeatedly used for 42 cycles without apparent activity loss, while the immobilized lipase without LiCl lost over 90% activity within 13 reuse cycles.

Sucrose monolaurate synthesis with Protex 6L immobilized on electrospun TiO2 nanofiber.

TiO(2) nanofibers with uniform diameter about 125 nm were prepared based on sol-gel process and electrospinning technology. Protex 6L, an industrial alkaline protease, was covalently immobilized on TiO(2) nanofiber through γ-aminopropyltriethoxysilane modification and glutaraldehyde crosslinking. With 2 (v/v)% glutaraldehyde as crosslinker, the enzyme loading is about 201 mg (g nanofiber membrane)(-1), and the specific activity of the immobilized Protex 6L is 2.45 µmol h(-1) ml(-1) mg(-1) protein for synthesis of sucrose monolaurate from sucrose and vinyl laurate. The optimal condition for sucrose monolaurate production is 5% (v/v) water content in DMSO/2-methyl-2-butanol solvent mixture and 50 °C. Under this condition, 97% conversion was achieved within 36 h by nanofibrous Protex 6L, which is corresponding to a productivity 34 times higher than that of most widely used Novozym 435. After 10 cycles reuse, nanofibrous Protex 6L retained 52.4% of its original activity.

Simultaneous production of 1,3-dihydroxyacetone and xylitol from glycerol and xylose using a nanoparticle-supported multi-enzyme system with in situ cofactor regeneration.

Cofactor-dependent biotransformations often require consumption of a secondary substrate for cofactor regeneration. Alternatively, two synthetic reactions may be coupled together through cofactor regeneration cycles. Simultaneous production of value-added products from glycerol and xylose was realized in this work through an enzymatic NAD(H) regeneration cycle involving two enzymes. Glycerol dehydrogenase (GDH) catalyzed the production of 1,3-dihydroxyacetone (DHA) from glycerol, while xylose reductase (XR) enabled the reduction of xylose to xylitol using the protons released from glycerol. Both enzymes were immobilized with P(MMA-EDMA-MAA) nanoparticles. Interestingly, the immobilized multi-enzyme system showed much improved productivity and stability as compared to native enzymes, such that the total turnover number (TTN) reached 82 for cofactor regeneration while the yield reached 160g/g-immobilized GDH for DHA production.

Salt induced irreversible protein adsorption with extremely high loadings on electrospun nanofibers.

LiCl is a kosmotrope that generally promotes protein salvation in aqueous solutions. Herein we report that LiCl embedded in electrospun polymeric nanofibers interestingly induced an abnormal protein adsorption and substantially augmented the adsorption capacity of the fibers. As a result, equilibrium protein loadings reached over 64% (w/w) of the dry mass of fibers, 9-fold higher than that observed in the absence of the salt. The adsorption appeared to be irreversible such that little protein loss was observed even after washing the fibers vigorously with fresh buffer solutions. We further examined the application of such intensified protein adsorption for enzyme immobilization. Proteins including bovine serum albumin (BSA) and protamine were first adsorbed, followed by covalent attachment of an outer layer of an enzyme, α-chymotrypsin. Such a multilayer-structured nanofibrous enzyme exhibited extremely high stability with no obvious activity loss even after being incubated for 8 months at 4 °C in aqueous buffer solution. The LiCl induced irreversible protein adsorption, which has been largely ignored in previous studies with electrospun materials, rendering an interesting scenario of interfacial protein-material interactions. It also reveals a new mechanism in controlling and fabricating molecular interactions at interfaces for development of a broad range of biomaterials.

[Study on the changes of the T-lymphocyte subsets and their significance in influenza A (H1N1) virus patients].

OBJECTIVE: To study the changes of T-Lymphocyte and activated T-Lymphocyte subsets in influenza A (H1N1) virus patients. METHODS: The percentages of the subsets of Lymphocyte were detected by flow cytometry in influenza A (H1N1) virus patients (n = 144) and normal controls (n = 41). Furthermore, the subsets of T-Lymphocyte and activated T-lymphocyte were analyzed in 83 among those patients before and after treatment. RESULTS: Compared with the control group, the counts of Lymphocyte in patients with influenza A (H1N1) virus was significantly discreased, the counts of Lymphocyte in patients with influenza A virus concurrent pneumonia was significantly discreased those of no concurrent pneumonia; Compared with the control group, the percentage of T-lymphocyte in patients with influenza A virus concurrent pneumonia was significantly discreased. The counts and percentage of CD3 and CD8 cells was significantly discreased in patients (n = 83) before treatment; The counts of CD4 cells was significantly discreased before treatment. The percentage of HLA-DR+ CD+, HLA-DR+ CD4+ and HLA-DR+ CD8+ cells was significantly discreased in patients (n = 83) before treatment. CONCLUSIONS: To understand the expression of the T-Lymphocyte and activated T-Lymphocyte subsets in influenza A (H1N1) virus patients may help to evaluate the patients' cellular immune status, but also be a guideline of early diagnosis of Influenza A (H1N1) virus.

[The expression of CD95 and activated antigens in peripheral blood lymphocyte subsets in patients with hand foot and mouth disease (HFMD)].

OBJECTIVE: To investigate the expression of CD95 and special marker for activation of peripheral blood lymphocytes in patients with hand foot and mouth disease (HFMD) and its significance. METHODS: Immunofluorescent two-color flow cytometry was used to study the expression of CD95 and HLA-DR on lymphocytes in 58 patients with HFMD and 34 normal controls. RESULTS: Expression of CD3+ T cells was significantly lower in patients (63.82 +/- 7.74)% than that in controls (P < 0.001), meanwhile the expression of CD4+ T cells was (34.29 +/- 7.33)%, significantly lower than that of the controls (P < 0.005). The percentage of lymphocytes expressing HLA-DR in patients was (23.77 +/- 5.78)%, significantly higher than that of the controls (P < 0.005). Significant difference was observed in the expression of HLA- DR on CD8+ T cells in patients (1.34 +/- 1.12)% as compared with controls (P < 0.005). No significant difference in the expression of CD95 on lymphocytes was observed between patients and the controls (P > 0.05). CONCLUSION: The findings support that cellular immunodeficiency exists in patients and that lymphocytes were abnormally activated in the patients. The activation of peripheral blood T lymphocytes in patients mainly involves CD8 subset and it may play an important role in the immune response to antiviral infection.