Self-immobilized recombinant Acetobacter xylinum for biotransformation

Biofilms have been successfully applied for biotransformation for decades, especially in the area of bioremediation due to the feature of harsh reaction condition resistance. Acetobacter xylinum is known for its cellulose pellicle forming capability. Like biofilm, A. xylinum cells are immobilized by simultaneously produced cellulose nanofibers in the pellicle. A recombinant A. xylinum was constructed with the expectation that the cells could be self-immobilized and achieve a desired and stable biotransformation. d-Amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as the model enzyme to be expressed in the recombinant A. xylinum. The constructed recombinant A. xylinum not only successfully produced DAAO activity but also self-immobilized by cellulose nanofibers in both the static and shaken culture. Although self-immobilized cells demonstrated a DAAO activity approximately 10% of the cell crude extract activity, it provided the benefits of improved thermal stability, operational stability, and easy retrieval for repeated use.     

A rapid in situ immobilization of d-amino acid oxidase based on immobilized metal affinity chromatography

A rapid in situ immobilization process was developed based on conventional separation technique of immobilized metal affinity chromatography (IMAC) and was studied in the case of d-amino acid oxidase (DAAO) with binding–enhancing Heli-tag (His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly). A recombinant Escherichia coli strain JM105 (Δase)/pGEMK-R-DAAO-Heli was successfully constructed to synthesize chimeric protein DAAO-Heli. Without additional purification procedure, the tagged enzyme DAAO-Heli could be directly immobilized to EP-IDA-Ni²⁺ support with purity of 90 % and DAAO activity of over 70 U/g support. Experimental results showed that the immobilized DAAO-Heli was 73 times more thermally stable than free enzyme. Besides, it remained 67 % of initial activity after 100 cycles of batch catalysis and its operational stability was improved 36 times than that of the previously IMAC-immobilized DAAO-His. Furthermore, the epoxy (EP) support could be easily recovered and repeatedly used with simple steps, which could reduce the immobilization costs significantly.     

Three-dimensional immobilization of beta-galactosidase on a silicon surface

Many alternative strategies to immobilize and stabilize enzymes have been investigated in recent years for applications in biosensors. The entrapment of enzymes within silica-based nanospheres formed through silicification reactions provides high loading capacities for enzyme immobilization, resulting in high volumetric activity and enhanced mechanical stability. Here we report a strategy for chemically associating silica nanospheres containing entrapped enzyme to a silicon support. beta-galactosidase from E. coli was used as a model enzyme due to its versatility as a biosensor for lactose. The immobilization strategy resulted in a three-dimensional network of silica attached directly at the silicon surface, providing a significant increase in surface area and a corresponding 3.5-fold increase in enzyme loading compared to enzyme attached directly at the surface. The maximum activity recovered for a silicon square sample of 0.5 x 0.5 cm was 0.045 IU using the direct attachment of the enzyme through glutaraldehyde and 0.16 IU when using silica nanospheres. The immobilized beta-galactosidase prepared by silica deposition was stable and retained more than 80% of its initial activity after 10 days at 24 degrees C. The ability to generate three-dimensional structures with enhanced loading capacity for biosensing molecules offers the potential to substantially amplify biosensor sensitivity.     

Evaluation of a silica-coated magnetic nanoparticle for the immobilization of a His-tagged lipase

Magnetic particles of size 10 nm have been coated with silica to a mean diameter of 40 nm and charged with Cu²⁺ ions via a multidentate ligand, iminodiacetic acid (IDA), for the immobilization of His-tagged Bacillus stearothermopilus L1 lipase. Microporous (average pore diameter of 60 Å) silica gel with a mean particle diameter of 115 µm has been used as a comparative support material. The molar ratio of Cu²⁺ to IDA was found to be 1:1.14 and 1:1.99 in the silica gel and the silica-coated magnetic nanoparticles (SiMNs), respectively. The specific activity of the immobilized enzyme was found to conform to the following order: Cu²⁺-charged SiMN>SiMN>Cu²⁺-charged silica gel>silica gel. When it was immobilized on the Cu²⁺-charged SiMNs, over 70% of the initial activity of the lipase remained after it had been reused five times. However, only 20% of the initial activity remained after the enzyme immobilized on the Cu²⁺-charged silica gel had been reused five times. For the enzyme immobilized on supports without Cu²⁺ cations, all activity was lost after threefold reuse. The differences in the specific activities and the efficiencies of reuse of the enzymes immobilized on the various support materials are discussed in terms of immobilization mechanisms (physical adsorption vs. coordination bonding), mass transfer of a substrate and a product of the enzyme reaction, and the status of the Cu (Cu bound to the IDA on the silica layer vs. Cu directly adsorbed on the silica layer).     

In situ immobilization of β‐galactosidase from Bacillus circulans in silica by sol‐gel process: Application in prebiotic synthesis

The enzyme encapsulation is a very well‐known stabilization pathway. However, there are some challenges in order to avoid the enzyme denaturation under encapsulation conditions. The β‐galactosidase from Bacillus circulans was immobilized through sol‐gel encapsulation route assisted by Triton X‐100 surfactant and sugars. The effects of sugar presence in the immobilization process and the gelation time on the biocatalyst activity/stability were explained taking into account the characteristics of the formed silica matrix and the changes of the enzyme environment. The enzyme was effectively immobilized by this strategy, with high immobilization yield in terms of activity (29%) and expressed activity (47 IU/g). The immobilization through silica sol‐gel in the presence of 1×10^?3 M Triton X‐100 and fructose conferred 28.4‐fold higher stability to the enzyme compared with the soluble form. This is an advantage for its use in the synthesis of the galacto‐oligosaccharides at 50ºC. The total lactose conversion to galacto‐oligosaccharides was 26%wt, which is comparable with that reported in the literature. The obtained biocatalyst is useful for the synthesis of galacto‐oligosaccharides and its catalytic behavior is rationalized in this work.     

Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli

To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml⁻¹ was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l⁻¹. A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml⁻¹ and GL-7-ACA acylase activity of 950 U l⁻¹ were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly.     

Immobilization of enzymes with polyaziridine: II. D-amino acid oxidase

Summary A novel method of enzyme immobilization using a tri-functional aziridine to immobilize enzymes was used to immobilize D-amino acid oxidase (DAAO) with good retention of enzymatic activity (62%–89%). The stability of the immobilized DAAO in a fixed bed reactor with continuous operation using D-phenylalanine as substrate yielded a projected half-life of 69 days which is far superior to other methods of immobilization of DAAO.     

Purification of recombinant α-amylase with immuno-affinity chromatography using monoclonal antibody

A monoclonal antibody against recombinant thermostable α-amylase produced by Escherichia coli was isolated from serum-free medium and immobilized on Sepharose 4B. The adsorption equilibrium between α-amylase and the immobilized immuno-adsorbent showed a Langmuir type isotherm. The breakthrough curve calculated numerically using the averaged volumetric coefficient coincided well with the experimental data. More than 90% of the activity of bound α-amylase could be recovered by eluting with glycine-HCl buffer (pH 2.5). The elution profile at pH 2.5 became sharper with increasing temperature. By using an immuno-affinity column, the recombinant α-amylase produced by E. coli could be purified homogeneously from crude extract enzyme solution with two-step elution.     

Biocatalysis of silica gel-immobilized chlorophyllase in a ternary micellar system

A partially purified enzymic extract from Phaeodactylum tricornutum was immobilized on silica gel and the specific activity of chlorophyllase in its free and immobilized states were compared in a ternary micellar system. The storage stability of the free and immobilized chlorophyllase extracts, maintained at temperatures ranging from 4 to 35°C for a period of 0–20 h, was temperature-dependent. The results also showed that the specific activity of the free and immobilized chlorophyllase extracts was highest at 30°C for long-term incubation, using chlorophyll and pheophytin as substrates and that a three-fold increase in the specific activity of the immobilized chlorophyllase was observed in comparison to that obtained with the free counterpart. The findings indicated that when free and immobilized chlorophyllase extracts were recovered and reused with both substrates, the immobilized chlorophyllase extract could be recycled for longer periods of time, while the free enzyme extract showed no activity after the first cycle.     

Porcine pancreatic lipase hydrophobically adsorbed on octyl-silica: A robust biocatalyst for syntheses of xylose fatty acid esters

This work describes the immobilization of porcine pancreatic lipase (PPL), obtained from crude extract, on silica coated with octyl groups (OS) by interfacial adsorption, a simple and low-cost immobilization protocol. The biocatalyst PPL-OS was employed to the enzymatic preparation of fatty acid esters of d-xylose, a product used especially in the field of cosmetics and pharmacy, especially dermatology, improving the functionality of epidermal cells. The yields of the immobilization in terms of enzymatic activity and protein concentration (98% and 75%, respectively) suggested that PPL present in the crude extract was selectively immobilized on the octyl-silica support, which allowed the hyperactivation of the biocatalyst (recovered activity, 144%), a phenomenon that may be attributed to the interfacial activation of the enzyme on hydrophobic surfaces. The biocatalyst PPL-OS showed to be very robust in organic medium and at high temperature, which is an extremely important characteristic to produce sugar fatty acid esters from the industrial point of view. The syntheses of xylose fatty esters (oleate, caprylate and butyrate) yielded conversions around 70% after short reaction period (2?h) at 60?°C in tert-butyl alcohol. The biocatalyst, even after incubation at 60?°C for 24?h, could be reused in four esterification cycles of 2-h reaction at 60?°C, maintaining 100% of its catalytic activity.     

Enzyme immobilization via silaffin‐mediated autoencapsulation in a biosilica support

Enzymes and other biomolecules are often immobilized in a matrix to improve their stability or to improve their ability to be reused. Performing a polycondensation reaction in the presence of a biomolecule of interest relies on random entrapment events during polymerization and may not ensure efficient, homogeneous, or complete biomolecule encapsulation. To overcome these limitations, we have developed a method of incorporating autosilification activity into proteins without affecting enzymatic functionality. The unmodified R5 silaffin peptide from Cylindrotheca fusiformis is capable of initiating silica polycondensation in vitro at ambient temperatures and pressures in aqueous solution. In this study, translational fusion proteins between R5 and various functional proteins (phosphodiesterase, organophosphate hydrolase, and green fluorescent protein) were produced in Escherichia coli. Each of the fusion proteins initiated silica polycondensation, and enzymatic activity (or fluorescence) was retained in the resulting silica spheres. Under certain circumstances, the enzymatically‐active biosilica displayed improved stability relative to free enzyme at elevated temperatures. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.     

Construction and application of fusion proteins of d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid

Two fusion proteins of D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GLA) were designed to simplify the bioconversion process of cephalosporin C to 7-aminocephalosporanic acid (7-ACA), which is conventionally produced in a two-step enzymatic process. Two recombinant plasmids, pET-DLA and pET-ALD, were constructed to express fusion proteins of DAAO-linker-GLA (DLA) and GLA-linker-DAAO (ALD), respectively. When the recombinant plasmids were expressed in E. coli, the fusion protein DLA was not correctly folded and only DAAO activity could be detected. ALD, however, possessed activities of both DAAO and GLA, which directly catalyze the conversion of cephalosporin C into 7-ACA.     

Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.     

Activation of immobilized lipase in non-aqueous systems by hydrophobic poly-DL-tryptophan tethers

Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule ("tether") between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most "long" linkers (e.g., polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker-enzyme conjugate in polar environments, but this provides little benefit in non-polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non-porous, functionalized 1-microm silica microspheres, with and without an intervening hydrophobic poly-DL-tryptophan tether (n approximately 78). The polytryptophan-tethered enzyme exhibited 35 times greater esterification of n-propanol with lauric acid in the organic phase and five times the hydrolytic activity against p-nitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface-immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed.     

Toward cell‐free biofuel production: Stable immobilization of oligomeric enzymes

To overcome the main challenges facing alcohol‐based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell‐free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, β‐galactosidase, and an alcohol‐producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long‐term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH with keto‐acid decarboxylase free in solution. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:324–331, 2014     

Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources

Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, d-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and d-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.     

Simple enzymatic procedure for preparation of 15N-labeled l-glutamic acid

The application of a number of immobilized Procion dyes to the purification of inosine 5′-monophosphate dehydrogenase (EC 1.2.1.14) from Escherichia coli has been investigated. The enzyme is adsorbed to a number of these immobilized dyes and can be eluted by salt gradients with very substantial increases in specific activity. For example, adsorption of the enzyme from a crude cell-free extract of E. coli to immobilized Procion yellow MX-8G in the presence of 15% ($\text{v}\text{v}$) ethylene glycol and subsequent elution with a salt gradient yields an enzyme preparation approximately 90% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is quantitatively recovered with an overall increase in specific activity of 14-fold compared to the enzyme in the cell-free extract.     

High expression of Trigonopsis variabilis -amino acid oxidase in Pichia pastoris

To explore a new approach of high expression of -amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis (TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 (his⁻mut⁺). By means of MM and MD plates and PCR, the recombinant P. pastoris strains (his⁺mut⁺) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction.     

Numerical simulation and deacidification of nanomagnetic enzyme conjugate in a liquid–solid magnetic fluidized bed

The conventional deacidification method is difficult to achieve a better refining effect due to the high acid value in the rice bran crude oil, and the enzymatic esterification deacidification method can effectively reduce the acid value without generating chemical waste. In this study, the free lipase was immobilized on a magnetic polymer carrier Fe₃O₄/SiOx-g-P (GMA: glycidyl methacrylate) to obtain a immobilized lipase with a particle size of 105.30 ± 1.1 nm and an enzyme activity of 6580 ± 9.6 PLU/g (PLU: enzyme activity unit). Based on the batch deacidification process parameters, a multi-stage magnetic fluidized bed continuous circulation deacidification system was designed, and then the motion law of nanomagnetic immobilized lipase particles in liquid–solid magnetic fluidized bed was simulated by computer. When the iterative step was 5 × 10⁻⁵ s, the open porosity of the porous plate was 35.0%, the rice bran oil flow rate was 3.0 mm/s, and the magnetic field strength was 25.0 mT, which was beneficial to the deacidification reaction of rice bran oil. Under the conditions of magnetic immobilized lipase dosage of 4.0%, the phytosterol dosage of 22.0%, the molecular sieve dosage of 10%, the esterification temperature of 78.0 °C and the FFA (free fatty acid) content in rice bran oil decreased to 1.5%, after 48 h of reaction. The conversion rate is 92.8%, which provides a theoretical basis for the subsequent guidance of magnetic fluidized bed enzymatic continuous deacidification.