Recent advances in immobilized enzymes on nanocarriers

Recent progress in nanotechnology has provided high-performance nanomaterials for enzyme immobilization.Nanobiocatalysts combining enzymes and nanocarriers are drawing increasing attention because of their high catalytic performance,enhanced stabilities,improved enzyme-substrate affinities,and reusabilities.Many studies have been performed to investigate the efficient use of cellulose nanocrystals,polydopamine-based nanomaterials,and synthetic polymer nanogels for enzyme immobilization.Various nanobiocatalysts are highlighted in this review,with the emphasis on the design,preparation,properties,and potential applications of nanoscale enzyme carriers and nanobiocatalysts.

Effect Research of Immobilized Algae-bacteria Removal Ammonia Nitrogen of Aquaculture Wastewater and Proposed Model

Applied Immobilized algae bacteria (ABI) to remove ammonia of freshwater aquaculture wastewater. Temperature (T),PH,light intensity (I),dissolved oxygen (DO) and filling rate five factors plays important role in the process of ammonia nitrogen removal ,related data between ammonia removal and five factors was received through multi-factor orthogonal test,and established relations model between the five factor and nitrogen removal. The results show that five-factors had significant effect on AR,and the best combinations for removing AR was temperature 30 ℃,pH=7.0,light intensity 6 000 lux,dissolved oxygen 5.0 mg/L and the fill rate 10%. According to the experimental data,equation model was proposed and coefficient of determination R2 =0.864 8,P<0.05. Samples T-test was done between the model predictions and the actual measured values.Test results showed that the significant difference of overall mean value sig. (2-tailed) was 0.978 (P>0.05),it Shows that had no significant difference between model predictions and the actual measured value,and model had a high degree of fitting.

IMMOBILIZED ASPERGILLUS ORYZAE CELLS AND THEIR ACTIVITIES

Aminoacylase was immobilized on the mycelium cells of Aspergillus oryzae by using ethylenediamine, gelatin and glutylalhyde. The proper immobilized condition was studied by orthogonal experimental design. The immobilized cells with excellent activity and stability for optically resoluting N acetyl DL alanine were obtained. The effects of pH, temperature, ion concentration and substrate concentration on the reactive activity of immobilized cells were studied. The continuous optical resolution of N acetyl DL alanine was investigated respectively in an immobilized cells column (ICC) and in a novel couple immobilized cells bed & membrane reactor(CICBMR). The results indicate that the immobilized cells are suitable for industrial applications.

β-Glucuronidase在植物基因功能研究中的应用

β-Glucuronidase(GUS)是从大肠杆菌K-12菌株分离出的一种酸性水解酶,具有高稳定性、检测的灵敏性与简易性及其对转基因生物体无毒性的特点,是转基因研究中最常用的标识基因。有三种商品化的底物分别用于分光光度定量分析、荧光定量分析与组织化学定位研究。由于植物体内存在着不同程度的GUS本底表达,可以通过在反应体系中加入20%甲醇来抑制。GUS基因有弱的表达特点,可以在其前面加终止子来抑制。利用带有GUS基因的杆菌转化拟南芥具有简单易行的特点,能有效保障学习与了解植物转基因的过程与机制。

Decolorization of reactive dyes by laccase immobilized in alginate/gelatin blent with PEG

To achieve effective decolorization of reactive dyes,laccase immobilization was investigated.Laccase 0.2%(m/V)(Denilite IIS) was trapped in beads of alginate/gelatin blent with polyethylene glycol(PEG),and then the supporters were activated by cross-linking with glutaraldehyde.The results of repeated batch decolorization showed that gelatin and appropriate concentration of glutaraldehyde accelerated the decolorization of Reactive Red B-3BF(RRB);PEG had a positive effect on enzyme stability and led to an inc...

Biodegradation of phenol by free and immobilized Acinetobacter sp.strain PD12

A new phenol-degrading bacterium with high biodegradation activity and high tolerance of phenol, strain PD 12, was isolated from the activated sludge of Tianjin Jizhuangzi Wastewater Treatment Facility in China. This strain was capable of removing 500 mg phenol/L in liquid minimal medium by 99.6% within 9 h and metabolizing phenol at concentrations up to 1100 mg/L. DNA sequencing and homologous analysis of 16S rRNA gene identified PD12 to be an Acinetobacter sp. Polyvinyl alcohol （PVA） was used as a gel matrix to immobilize Acinetobacter sp. strain PDI2 by repeated freezing and thawing. The factors affecting phenol degradation of immobilized cells were investigated, and the results showed that the immobilized cells could tolerate a high phenol level and protected the bacteria against changes in temperature and pH. Storage stability and reusability tests revealed that the phenol degradation functions of immobilized cells were stable after reuse for 50 times or storing at 4℃ for 50 d. These results indicate that immobilized Acinetobacter sp. strain PD 12 possesses a good application potential in the treatment of phenol-containing wastewater.

Comparison of Di-n-methyl Phthalate Biodegradation by Free and Immobilized Microbial Cells

To compare the biodegradation of di-n-methyl pathalate by free and immobilized microbial cells. Methods The enrichment and isolation technique was used to isolate the microorganism. The PAV-entrapment method was utilized to immobilize the microorganisms. The scanning electron microscophy (SEM) was used to observe the growth and distribution of microbial cells immobilized inside the PVA bead gels. The GC/MS method was used to identify the main intermediates of DMP degradation. Results The microbial cells could grow quite well in PVA gel. The metabolic pathway did not change before and after immobilization of the microbial cells. The degradation rate of immobilized cells was higher than that of free cells. Conclusion The immobilized microbial cells possess advantages than free cells when applied to the biodegradation of toxic organic pollutants.

Biosorption characteristics of unicellular green alga Chlorella sorokiniana immobilized in loofa sponge for removal of Cr(Ⅲ)

Loofa sponge （LS） immobilized biomass of Chlorella sorokiniana （LSIBCS）, isolated from industrial wastewater, was investigated as a new biosorbent for the removal of Cr（Ⅲ） from aqueous solution. A comparison of the biosorption of Cr（Ⅲ） by LSIBCS and free biomass of C. sorokiniana （FBCS） from 10-300 mg Cr（Ⅲ）/L aqueous solutions showed an increase in uptake of 17.79% when the microalgal biomass was immobilized onto loofa sponge. Maximum biosorption capacity for LSIBCS and FBCS was found to be 69.26 and 58.80 mg Cr（Ⅲ）/g biosorbent, respectively, whereas the amount of Cr（Ⅲ） ions adsorbed onto naked LS was 4.97 mg/g. The kinetics of Cr（Ⅲ） biosorption was extremely rapid and equilibrium was established in about 15 and 20 min by LSIBCS and FBCS, respectively. The biosorption equilibrium was well defined by Langmuir adsorption isotherm model. The biosorption kinetics followed the pseudo-second order kinetic model. The biosorption was found to be pH dependent and the maximum sorption occurred at the solution pH 4.0. Desorption studies showed that 98% of the adsorbed Cr（Ⅲ） could be desorbed with 0.1 mol/L HNO3, while other desorbing agents were less effective in the order： EDTA 〉 H2SO4 〉 CH3COOH 〉 HCl. The regenerated LSIBCS retained 92.68% of the initial Cr（Ⅲ） binding capacity up to five cycles of reuse in continuous flow-fixed bed columns. The study revealed that LSIBCS could be used as an effective biosorbent for the removal of Cr（Ⅲ） from wastewater.

Comparison of four supports for adsorption of reactive dyes by immobilized Aspergillus fumigatus beads

Four materials, sodium carboxymethylcellulose （Na-CMC）, sodium alginate （SA）, polyvinyl alcohol （PVA）, and chitosan （CTS）, were prepared as supports for entrapping fungus Aspergillusfumigatus. The adsorption of synthetic dyes, Reactive Brilliant Blue KN- R, and Reactive Brilliant Red K-2BP, by these immobilized gel beads and plain gel beads was evaluated. The adsorption efficiencies of Reactive Brilliant Red K-2BP and Reactive Brilliant Blue KN-R by CTS immobilized beads were 89.1% and 93.5% in 12 h, respectively. The adsorption efficiency by Na-CMC immobilized beads was slightly lower than that of mycelial pellets. But the dye culture mediums were almost completely decolorized in 48 h using the above-mentioned two immobilized beads （exceeding 95%）. The adsorption efficiency by SA immobilized beads exceeded 92% in 48 h. PVA-SA immobilized beads showed the lowest adsorption efficiency, which was 79.8% for Reactive Brilliant Red K-2BP and 92.5% for Reactive Brilliant Blue KN-R in 48 h. Comparing the adsorption efficiency by plain gel beads, Na-CMC plain gel beads ranked next to CTS ones. SA and PVA-SA plain gel beads hardly had the ability of adsorbing dyes. Subsequently, the growth of mycelia in Na-CMC and SA immobilized beads were evaluated. The biomass increased continuously in 72 h. The adsorption capacity of Reactive Brilliant Red K-2BP and Reactive Brilliant Blue KN-R by Na-CMC immobilized beads was 78.0 and 86.7 mg/g, respectively. The SEM micrographs show that the surface structure of Na-CMC immobilized bead is loose and finely porous, which facilitates diffusion of the dyes.

Biodegradation of benzo[a]pyrene in soil by Mucor sp.SF06 and Bacillus sp.SB02 co-immobilized on vermiculite

Two indigenous microorganisms, Bacillus sp. SB02 and Mucor sp. SF06, capable of degrading polycyclic aromatic hydrocarbons （PAHs） were co-immobilized on vermiculite by physical adsorption and used to degrade benzo[a] pyrene （BaP）. The characteristics of BaP degradation by both free and co-immobilized microorganism were then investigated and compared. The removal rate using the immobilized bacterial-fungal mixed consortium was higher than that of the freely mobile mixed consortium. 95.3% of BaP was degraded using the co-immobilized system within 42 d, which was remarkably higher than the removal rate of that by the free strains. The optimal amount of inoculated co-immobilized system for BaP degradation was 2%. The immobilized bacterial-fungal mixed consortium also showed better water stability than the free strains. Kinetics of BaP biodegradation by co-immobilized SF06 and SB02 were also studied. The results demonstrated that BaP degradation could be well described by a zero-order reaction rate equation when the initial BaP concentration was in the range of 10--200 mg/kg. The scanning electronic microscope （SEM） analysis showed that the co-immobilized microstructure was suitable for the growth of SF06 and SB02. The mass transmission process of co-immobilized system in soil is discussed. The results demonstrate the potential for employing the bacterial-fungal mixed consortium, co-immobilized on vermiculite, for in situ bioremediation of BaP.

Production of laccase by Coriolus versicolor and its application in decolorization of dyestuffs: (Ⅱ) Decolorization of dyes by laccase containing fermentation broth with or without self-immobilized mycelia

The capability of decolorization for commercial dyes by Coriolus versicolor fermentation broth containing laccase with or without immobilized mycelium was evaluated. With cell free fermentation broth containing laccase, high decolorization ratio was achieved for acid orange 7, but not for the other dyes concerned. The immobilized mycelium was proved to be more efficient than the cell free system. All the four dyestuffs studied were found being decolourized with certain extent by immobilized mycelium. The repeated batch decolorization was carried out with satisfactory results. The experimental data showed that the continuous decolorization of wastewater from a printing and dyeing industry was possible by using the self immobilized C. Versicolor.

Copper biosorption on immobilized seaweed biomass:Column breakthrough characteristics

The biosorption of copper by the brown seaweed Sargassum baccularia, immobilized onto polyvinyl alcohol （PVA） gel beads, was investigated with fixed-bed experiments. Laboratory-scale column tests were performed to determine breakthrough curves with varying flow rates and feed concentrations. A theoretical fixed-bed model, known as the Bohart-Adams equation, was evaluated in simulating the experimental breakthrough curves. The Bohart-Adams model qualitatively predicted the breakthrough trends. PVA- immobilized seaweed biomass beads were amenable to efficient regeneration with aqueous solution containing the chelating agent ethylenediaminetetraacetic acid （EDTA）. The biosorbent retained most of its original uptake capacity over three cycles of use. The excellent reusability of the biosorbent could lead to the development of a viable metal remediation technology.

Removal of Nitrogen, Phosphorus, and Organic Pollutants From Water Using Seeding Type Immobilized Microorganisms

Objective To study the possibility of removing nitrogen, phosphorus, and organic pollutants using seeding type immobilized microorganisms. Methods Lakes P and M in Wuhan were chosen as the objects to study the removal of nitrogen, phosphorus, and organic pollutants with the seeding type immobilized microorganisms. Correlations between the quantity of heterotrophic bacteria and the total nitrogen （TN）, total phosphorus （TP）, and total organic carbon （TOC） in the two lakes were studied. The dominant bacteria were detected, inoculated to the sludge and acclimated by increasing nitrogen, phosphorus and decreasing carbon source in an intermittent, time-controlled and fixed-quantity way. The bacteria were then used to prepare the seeding type immobilized microorganisms, selecting diatomite as the adsorbent cartier. The ability and influence factors of removing nitrogen, phosphorus, and organic pollutant from water samples by the seeding type immobilized microorganisms were studied. Results The coefficients of the heterotrophic bacterial quantity correlated with TOC, TP, and TN were 0.9143, 0.8229, 0.7954 in Lake P and 0.9168, 0.7187, 0.6022 in Lake M. Ten strains of dominant heterotrophic bacteria belonging to Pseudomonas, Coccus, Aeromonas, Bacillus, and Enterobateriaceae, separately, were isolated. The appropriate conditions for the seeding type immobilized microorgansims in purifying the water sample were exposure time=24 h, pH=7.0-8.0, and quantity of the immobilized microorganisms=0.75-1g/50 mL. The removal rates of TOC, TP, and TN under the above conditions were 80.2%, 81.6%, and 86.8%, respectively. Conclusion The amount of heterotrophic bacteria in the two lakes was correlated with TOC, TP, and TN. These bacteria could be acclimatized and prepared for the immobilized microorganisms which could effectively remove nitrogen, phosphorus, and mixed organic pollutants in the water sample.

Repeated-Batch and Continuous Production of L-Lactic Acid by Rhizopus oryzae Immobilized in Calcium Alginat Beads:ReactorPerformance and Kinetic Model

Repeated-batch and continuous production of L-lactic acid by immobilized Rhizopusoryzae with calcium alginate entrapment method in a three-phase fluidized-bed bioreactor was stud-ied.The operation conditions were optimized.The productivity based on total reactor volume wasabout 3 times higher than that with free cells in a traditional stirred tank bioreactor.A mathemat-ical model was proposed and the model predictions were in good agreement with the experimentaldat.

Studies on Manganese Peroxidase Immobilized in Gelatin-containing Microemulsion-based Gels

The immobilized technique of manganese peroxidase(MnP) in gelatin-containing microemulsion-based gels and the effects of storage time and reuse times on its catalytic activity were studied. The results show that the MnP immobilized together with Mn 2+ and H_2O_2 could effectively oxidize syringaldazine in n-heptane. The immobilized MnP still had a high catalytic activity after one-month storage under a freezing condition. The reuse times have a relation to the amount of the immobilized H_2O_2. When the amount of the immobilized H_2O_2 is sufficient, the microemulsion-based gels containing MnP could be used many times.

Zwitterionic polymer-coated porous poly(vinyl acetate–divinyl benzene)microsphere: A new support for enhanced performance of immobilized lipase

Enzyme immobilization has attracted great attention for improving the performance of enzymes in industrial applications.This work was designed to create a new support for Candida rugosa lipase(CRL)immobilization.A porous poly(vinyl acetate–divinyl benzene)microsphere coated by a zwitterionic polymer,poly(maleic anhydride-alt-1-octadecene)and N,N-dimethylethylenediamine derivative,was developed for CRL immobilization via hydrophobic binding.The catalytic activity,reaction kinetics,stabilities and reusability of the immobilized CRL were investigated.It demonstrated the success of the zwitterionic polymer coating and subsequent CRL immobilization on the porous microsphere.The immobilized lipase(p2-MS-CRL)reached27.6 mg·g^-1 dry carrier and displayed a specific activity 1.5 times higher than free CRL.The increase of Vmax and decrease of Kmwere also observed,indicating the improvement of catalytic activity and enzyme-substrate affinity of the immobilized lipase.Besides,p2-MS-CRL exhibited significantly enhanced thermal stability and pH tolerance.The improved performance was considered due to the interfacial activation regulated by the hydrophobic interaction and stabilization effect arisen by the zwitterionic polymer coating.This study has thus proved the advantages of the zwitterionic polymer-coated porous carrier for lipase immobilization and its potential for further development in various enzyme immobilizations.

Culture of yeast cells immobilized by alginate-chitosan microcapsules in aqueous-organic solvent biphasic system

Immobilization biocatalysis is a potential technology to improve the activity and stability of biocatalysts in nonaqueous systems for efficient industrial production.Alginate-chitosan(AC)microcapsules were prepared as immobilization carriers by emulsifi cation-internal gelation and complexation reaction,and their contribution on facilitating the growth and metabolism of yeast cells were testifi ed successfully in culture medium-solvent biphasic systems.The cell growth in AC microcapsules is superior to that in alginate beads,and the cells in both immobilization carriers maintain much higher activity than free cells,which demonstrates AC microcapsules can confer yeast cells the ability to resist the adverse effect of solvent.Moreover,the performance of AC microcapsules in biphasic systems could be improved by adjusting the formation of outer polyelectrolyte complex(PEC)membrane to promote the cell growth and metabolic ability under the balance of resisting solvent toxicity and permitting substrate diffusion.Therefore,these findings are quite valuable for applying AC microcapsules as novel immobilization carriers to realize the biotransformation of value-added products in aqueous-solvent biphasic systems.

Lipase immobilized on HOOC-MCF:A highly enantioselective catalyst for transesterification resolution of (R,S)-1-phenylethanol

Pseudomonas cepacia lipase （PSL） immobilized on the carboxyl-functionalized meso-cellular foams （HOOC-MCF） was used for the transesterification resolution of （R,S）-l-phenylethanol in organic solvent. The results showed that the ee value of （S）-1- phenylethanol and （R）-1-phenylethyl acetate reached 99% with 50% conversion of 1-phenylethanol using toluene as solvent. Furthermore, it was found that PSL/HOOC-MCF exhibited high enantioselectivity in organic solvent with log P ≤ 2 such as toluene and hexane.

The research on the adsorption effect on metal ions by immobilized marine algae

The process of adsorption of Cu^2+ Cd^2+ by immobilized marine algae was investigated, it can be noted from the results that, the process for biosorption of heavy metals (copper, cadmium) by immobilized Laminaria japonica can be described by the Banerm model. According to the model, the adsorption rate constant calculated was 0.107 8 and 0.030 28 min^-1 for Cu^2+ and Cd^2+ respectively. The experimental biosorption equilibrium data for Cu^2+ and Cd^2+ were in good agreement with those calculated by the Langmuir model. The maximum uptake capacity calculated was 83.3 and 112.4 mg/g for Cu^2+ and Cd^2+ according to the Langmuir model, respectively. The appetency of Laminaria japonica to Cu^2+ was better than Cd^2+.

An efficient synthesis of ampicillin on magnetically separable immobilized penicillin G acylase

Penicillin G acylase(PGA) was immobilized on the magnetic hydrophilic polymer microspheres with average pore size of 17.1 nm,specific surface area of 128.2 m2/g and saturate magnetization of 6.4 emu/g.The 96.7%ampicillin yield with 1.60 of the synthesis/hydrolysis(S/H) ratio from 6-aminopenicillanic acid(6-APA) and D-(-)-alpha-phenylglycine methyl ester(D-PGME) can be achieved using the resultant magnetic biocatalyst in ethylene glycol,where only 82.1%yield with 1.40 of the S/H ratio was obtained using the free PGA under the identical reaction conditions.The immobilized PGA can be separated magnetically and recycled for five times without obvious loss of its catalytic activity.