Glucose‐6‐phosphate dehydrogenase encapsulated in silica‐based hydrogels for operation in a microreactor

The future of hydrogen as fuel strongly depends on the possibility to produce it in an economic and clean way. Hydrogen can be produced from carbohydrates and water under mild conditions by means of a multistep synthetic pathway (13 enzymes) with very high yield. Crossover inhibitions and different optimal conditions of involved enzymes hinder the use of one‐pot approach. Immobilization of enzymes in coupled individual reactors may avoid this problem. This work deals with the immobilization in silica‐based hydrogels of one key enzyme of this pathway: glucose 6‐phosphate dehydrogenase from Leuconostoc mesenteroides. The carriers were prepared with an ethylene glycol‐modified silane, two polymers (polyethylene oxide and Pluronic®) and amino groups created by 3‐aminopropyltriethoxysilane. These parameters influenced the enzymatic activity after immobilization. Gels prepared by addition of polyethylene oxide gave the best results and were used as monoliths in microreactors with two different geometries. The systems showed a high operational stability but a low effective enzyme activity. Enzyme leaching and a nonideal flow pattern may account for the low activity observed. This work is possibly the first one dealing with the immobilization of glucose 6‐phosphate dehydrogenase in silica‐based gels for its application in flow‐through microreactors.     

Endo‐ and exo‐inulinases: Enzyme‐substrate interaction and rational immobilization

Three‐dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoinulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase resolved so far. Docking and molecular dynamics simulations were performed to investigate the differences between the two inulinases in terms of substrate selectivity. The analysis of the structural differences between the two inulinases provided the basis for the explanation of their different regio‐selectivity and for the understanding of enzyme‐substrate interactions. Surface analysis was performed to point out structural features that can affect the efficiency of enzymes also after immobilization. The computational analysis of the three‐dimensional models proved to be an effective tool for acquiring information and allowed to formulate an optimal immobilized biocatalyst even more active that the native one, thus enabling the full exploitation of the catalytic potential of these enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Vancomycin‐based chiral stationary phases for micro‐column liquid chromatography

Vancomycin selectively immobilized to silica via either one of its two amino groups has been investigated and compared with columns made from native vancomycin. The chemical modification of vancomycin prior to immobilization involved protection of one amino group as a 9‐fluorenylmethyl carbamate. The immobilization and the subsequent cleavage of the protecting group was performed on‐column. The types of compounds that can be separated with the vancomycin chiral stationary phases resemble those separated previously by capillary electrophoresis and thin‐layer chromatography. The protected chiral stationary phases were also investigated and in some cases very high enantioselectivity were obtained. One example of this is a separation of thalidomide with an α‐value as high as 5.4. The soft immobilization procedure preserves the structure of native vancomycin, in contrast to other approaches. Good repeatability and stability of the columns have also been obtained. Chirality 11:121–128, 1999. © 1999 Wiley‐Liss, Inc.     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

Ultra‐stable phosphoglucose isomerase through immobilization of cellulose‐binding module‐tagged thermophilic enzyme on low‐cost high‐capacity cellulosic adsorbent

One‐step enzyme purification and immobilization were developed based on simple adsorption of a family 3 cellulose‐binding module (CBM)‐tagged protein on the external surface of high‐capacity regenerated amorphous cellulose (RAC). An open reading frame (ORF) Cthe0217 encoding a putative phosphoglucose isomerase (PGI, EC 5.3.1.9) from a thermophilic bacterium Clostridium thermocellum was cloned and the recombinant proteins with or without CBM were over‐expressed in Escherichia coli. The rate constant (k_cat) and Michaelis–Menten constant (K_m) of CBM‐free PGI at 60°C were 2,765 s^?1 and 2.89 mM, respectively. PGI was stable at a high protein concentration of 0.1 g/L but deactivated rapidly at low concentrations. Immobilized CBM (iCBM)‐PGI on RAC was extremely stable at ～60°C, nearly independent of its mass concentration in bulk solution, because its local concentration on the solid support was constant. iCBM‐PGI at a low concentration of 0.001 g/L had a half‐life time of 190 h, approximately 80‐fold of that of free PGI. Total turn‐over number of iCBM‐PGI was as high as 1.1 × 10⁹ mole of product per mole of enzyme at 60°C. These results suggest that a combination of low‐cost enzyme immobilization and thermoenzyme led to an ultra‐stable enzyme building block suitable for cell‐free synthetic pathway biotransformation that can implement complicated biochemical reactions in vitro. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

Enzymatic synthesis of β‐glucosylglycerol using a continuous‐flow microreactor containing thermostable β‐glycoside hydrolase CelB immobilized on coated microchannel walls

β‐Glucosylglycerol (βGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable β‐glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of βGG under conditions of continuous flow at 70°C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 µL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2‐nitrophenyl‐β‐D ‐glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25–1.0 M), and the average residence time (0.2–90 s). Glycerol caused a concentration‐dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of βGG were ≥80% and ≈60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (≥80%), giving about 120 mM (30 g/L) of βGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1‐O‐β‐D ‐glucopyranosyl‐rac‐glycerol (79%) and 2‐O‐β‐D ‐glucopyranosyl‐sn‐glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme. Biotechnol. Bioeng. 2009;103: 865–872. © 2009 Wiley Periodicals, Inc.     

A two‐enzyme immobilization approach using carbon nanotubes/silica as support

Multiple enzyme mixtures are attractive for the production of many compounds at an industrial level. We report a practical and novel approach for coimmobilization of two enzymes. The system consists of a silica microsphere core coated with two layers of individually immobilized enzymes. The model enzymes α‐amylase (AA) and glucoamylase (GluA) were individually immobilized on carbon nanotubes (CNTs). A CNT‐GluA layer was formed by adsorbing CNT‐GluA onto silica microsphere. A sol‐gel layer with entrapped CNT‐AA was then formed outside the CNT‐GluA/silica microsphere conjugate. The coimmobilized α‐amylase and glucoamylase exhibited 95.1% of the activity of the mixture of free α‐amylase and glucoamylase. The consecutive use exhibited a good stability of the coimmobilized enzymes. The developed approach demonstrates advantages, including controlling the ratio of coimmobilized enzymes in an easy way, facilitating diffusion of small molecules in and out of the matrix, and preventing the leaching of enzymes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:42–47, 2015     

Novel grafted agar disks for the covalent immobilization of β‐D‐galactosidase

Novel grafted agar disks were prepared for the covalent immobilization of β‐D‐galactosidase (β‐gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β‐gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45–55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6–4.6) after its immobilization. Additionally, the Michaelis‐Menten constant (K_m) increased for the immobilized β‐gal as compared to its free counterpart whereas the maximum reaction rate (V_max) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 675–684, 2015.     

Novel Magnetic Cross‐Linked Lipase Aggregates for Improving the Resolution of (R,S)‐2‐octanol

Novel magnetic cross‐linked lipase aggregates were fabricated by immobilizing the cross‐linked lipase aggregates onto magnetic particles with a high number of ‐NH₂ terminal groups using p‐benzoquinone as the cross‐linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross‐linked lipase aggregates were achieved. The magnetic cross‐linked lipase aggregates were able to efficiently resolve (R, S)‐2‐octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross‐linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross‐linking. These results provide a great potential for industrial applications of the magnetic cross‐linked lipase aggregates. Chirality 27:199–204, 2015. © 2014 Wiley Periodicals, Inc.     

Chemical Immobilization and Conversion of Active Polysulfides Directly by Copper Current Collector: A New Approach to Enabling Stable Room‐Temperature Li‐S and Na‐S Batteries

Room‐temperature Li/Na‐S batteries are promising energy storage solutions, but unfortunately suffer from serious cycling problems rooted in their polysulfide intermediates. The conventional strategy to tackle this issue is to design host materials for trapping polysulfides via weak physical confinement and interfacial chemical interactions. Even though beneficial, their capability for the polysulfide immobilization is still limited. Herein, the unique sulfiphilic nature of metallic Cu is revisited. Upon the exposure to polysulfide in aqueous or aprotic solution, the surface sulfidization rapidly takes place, resulting in the formation of Cu₂S nanoflake arrays with tunable texture. When the sulfidized Cu current collector is directly used as the sulfur‐equivalent cathode, it enables high‐performance Li/Na‐S batteries at room temperature with reasonable high sulfur loading. Specific capacities up to ≈1200 mAh g^?1 for Li‐S and ≈400 mAh g^?1 for Na‐S are measured when normalized to the amount of equivalent sulfur, and can be readily sustained for >1000 cycles.     

Efficient preparation and site‐directed immobilization of VHH antibodies by genetic fusion of poly(methylmethacrylate)‐binding peptide (PMMA‐Tag)

A PMMA‐binding peptide (PMMA‐tag) was genetically fused with the C‐terminal region of an anti‐human chorionic gonadotropin (hCG) single‐domain antibody (VHH). It was over‐expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one‐step IMAC purification. Monomeric and denatured PMMA‐tag‐fused VHH (VHH‐PM) was successfully prepared via the reduction and oxidation of VHH‐PM at a concentration less than 1 mg/mL in the presence of 8 M of urea. Furthermore, the VHH‐PM was refolded with a recovery of more than 95% by dialysis against 50 mM TAPS at pH 8.5, because the genetic fusion of PMMA‐tag resulted in a decrease in the apparent isoelectric point (pI) of the fusion protein, and its solubility at weak alkaline pH was considerably increased. The antigen‐binding activities of VHH‐PM in the adsorptive state were 10‐fold higher than that of VHH without a PMMA‐tag. The density of VHH‐PM on a PMMA plate was twice that of VHH, indicating that the site‐directed attachment of a PMMA‐tag resulted in positive effects to the adsorption amount as well as to the orientation of VHH‐PM in its adsorptive state. The preparation and immobilization methods for VHH‐PM against hCG developed in the present study were further applied to VHH‐PMs against four different antigens, and consequently, those antigens with the concentrations lower than 1 ng/mL were detected by the sandwich ELISA. Thus, the VHH‐PMs developed in the present study are useful for preparation of high‐performance and economical immunosorbent for detection of biomarkers. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1563–1570, 2015     

Photonic immobilization of high‐density protein arrays using Fourier optics

The technique of UV‐light‐assisted immobilization of disulfide containing proteins has been combined with the Fourier‐transforming properties of lenses as well as with a simple millimeter scale feature size spatial mask. The result is a new simple and inexpensive way of creating high‐density protein arrays with feature sizes down to a few hundred nanometers, which represents an improvement of tenfold over existing commercially available high‐density protein arraying methods.     

Noncovalent Immobilization of Ionic‐tagged Box‐Cu(OAc)2 Complex and Its Application in Asymmetric Henry Reaction

Immobilized Cu(OAc)₂‐bis(oxazolines) via hydrogen bonding by SBA‐15 was applied to asymmetric Henry reaction, and good enantioselectivities were obtained (up to 83% ee) between 2‐methoxybenzaldehyde and CH₃NO₂ in isopropyl alcohol (iPrOH). The catalyst could be reused seven times without any obvious loss in enantioselectivity. For the first time, this facile and clean immobilization method is applied to the use of bis(oxazolines) complexes. Chirality 24:1092‐1095, 2012. © 2012 Wiley Periodicals, Inc.     

Spore‐displayed enzyme cascade with tunable stoichiometry

Taking the advantages of inert and stable nature of endospores, we developed a biocatalysis platform for multiple enzyme immobilization on Bacillus subtilis spore surface. Among B. subtilis outer coat proteins, CotG mediated a high expression level of Clostridium thermocellum cohesin (CtCoh) with a functional display capability of ～10⁴ molecules per spore of xylose reductase‐C. thermocellum dockerin fusion protein (XR‐CtDoc). By co‐immobilization of phosphite dehydrogenase (PTDH) on spore surface via Ruminococcus flavefaciens cohesin‐dockerin modules, regeneration of NADPH was achieved. Both xylose reductase (XR) and PTDH exhibited enhanced stability upon spore surface display. More importantly, by altering the copy numbers of CtCoh and RfCoh fused with CotG, the molar ratio between immobilized enzymes was adjusted in a controllable manner. Optimization of spore‐displayed XR/PTDH stoichiometry resulted in increased yields of xylitol. In conclusion, endospore surface display presents a novel approach for enzyme cascade immobilization with improved stability and tunable stoichiometry. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:383–389, 2017     

Characterization of cross‐linked immobilized arylesterase from Gluconobacter oxydans 621H with activity toward cephalosporin C and 7‐aminocephalosporanic acid

Cross‐linked enzyme aggregates (CLEAs) were prepared from several precipitant agents using glutaraldehyde as a cross‐linking agent with and without BSA, finally choosing a 40% saturation of ammonium sulfate and 25 mM of glutaraldehyde. The CLEAs obtained under optimum conditions were biochemically characterized. The immobilized enzyme showed higher thermal activity and a broader range of pH and organic solvent tolerance than the free enzyme. Arylesterase from Gluconobacter oxydans showed activity toward cephalosporin C and 7‐aminocephalosporanic acid. The CLEAs had a Kcat/K_M of 0.9 M^?1/S^?1 for 7‐ACA (7‐aminocephalosporanic acid) and 0.1 M^?1/S^?1 for CPC (cephalosporin c), whereas free enzyme did not show a typical Michaelis–Menten kinetics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:36–42, 2016