Bacterial surface display of a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis using the Bacillus subtilis spore display system

To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.     

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant ω-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, ω-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using ω-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.     

Magnetic catechol-chitosan with bioinspired adhesive surface: preparation and immobilization of ω-transaminase

The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed. Here we report a simple catechol modified protocol for preparing a novel catechol-chitosan (CCS)-iron oxide nanoparticles (IONPs) composites carrying adhesive moieties with strong surface affinity. The ω-transaminase (ω-TA) was immobilized onto this magnetic composite via nucleophilic reactions between catechol and ω-TA. Under optimal conditions, 87.5% of the available ω-TA was immobilized on the composite, yielding an enzyme loading capacity as high as 681.7 mg/g. Furthermore, the valuation of enzyme activity showed that ω-TA immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme. Importantly, the immobilized ω-TA retained more than 50% of its initial activity after 15 repeated reaction cycles using magnetic separation and 61.5% of its initial activity after storage at 4°C in phosphate buffered saline (PBS) for 15 days. The results suggested that such adhesive magnetic composites may provide an improved platform technology for bio-macromolecules immobilized.     

Bacterial Surface Display of a Co-Factor Containing Enzyme, ω-Transaminase from Vibrio fluvialis Using the Bacillus subtilis Spore Display System

To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.     

Magnetic chitosan beads for covalent immobilization of nucleoside 2′-deoxyribosyltransferase: application in nucleoside analogues synthesis

Cross-linked magnetic chitosan beads were prepared in presence of epichlorohydrin under alkaline conditions, and subsequently incubated with glutaraldehyde in order to obtain an activated support for covalent attachment of nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT). Changing the amount of magnetite (Fe₃O₄) and epichlorohydrin (EPI) led to different macroscopic beads to be used as supports for enzyme immobilization, whose morphology and properties were characterized by scanning electron microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM). Once activated with glutaraldehyde, the best support was chosen after evaluation of immobilization yield and product yield in the synthesis of thymidine from 2′-deoxyuridine and thymine. In addition, optimal conditions for highest activity of immobilized LrNDT on magnetic chitosan were determined by response surface methodology (RSM). Immobilized biocatalyst retained 50 % of its maximal activity after 56.3 h at 60 °C, whereas 100 % activity was observed after storage at 40 °C for 144 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of 2′-deoxyribonucleoside analogues as well as arabinosyl-nucleosides such as vidarabine (ara-A) and cytarabine (ara-C). Furthermore, this is the first report which describes the enzymatic synthesis of these arabinosyl-nucleosides catalyzed by an immobilized nucleoside 2′-deoxyribosyltransferase. Finally, the attached enzyme to magnetic chitosan beads could be easily recovered and recycled for 30 consecutive batch reactions with negligible loss of catalytic activity in the synthesis of 2,6-diaminopurine-2′-deoxyriboside and 5-trifluorothymidine.     

Covalent immobilization of Kluyveromyces fragilis β-galactosidase on magnetic nanosized epoxy support for synthesis of galacto-oligosaccharide

The novel magnetic nanobeads with epoxy groups on the surface were prepared from glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EGDMA) and hydroxyethyl methacrylate (HEMA) via emulsifier-free emulsion polymerisation, and they were characterized by scanning electron microscopy and vibrating sample magnetometer. The magnetic poly(GMA-EDGMA-HEMA) nanobeads were used as support for covalent immobilization of Kluyveromyces fragilis β-galactosidase, the maximum amount enzyme attached onto the support was 145.6?mg/g with activity recovery of 72.6%. The loading capacity of this novel support for K. fragilis β-galactosidase was improved 2.6-folds compared with Eupergit(?) C (commercial epoxy support). The immobilized K. fragilis β-galactosidase exhibited high catalytic activity for the reaction of galacto-oligosaccharide (GOS) synthesis, and a total of 2,240?g GOS were produced per gram of immobilized enzyme during consecutive batch reaction of 10 times. The immobilized biocatalyst retained 81.5% of its original activity after 10 reaction cycles.     

Immobilization of Escherichia coli containing ω-transaminase activity in LentiKats®

Whole Escherichia coli cells overexpressing ω‐transaminase (ω‐TA) and immobilized cells entrapped in LentiKats® were used as biocatalysts in the asymmetric synthesis of the aromatic chiral amines 1‐phenylethylamine (PEA) and 3‐amino‐1‐phenylbutane (APB). Whole cells were permeabilized with different concentrations of cetrimonium bromide (CTAB) and ethanol; the best results were obtained with CTAB 0.1% which resulted in an increase in reaction rate by 40% compared to the whole cells. The synthesis of PEA was carried out using isopropyl amine (IPA) and L ‐alanine (Ala) as amino donors. Using whole cell biocatalysis, the reaction with IPA was one order of magnitude faster than with Ala. No reaction was detected when permeabilized E. coli cells containing ω‐TA were employed using Ala as the amino donor. Additionally, the synthesis of APB from 4‐phenyl‐2‐butanone and IPA was studied. Whole and permeabilized cells containing ω‐TA and their immobilized LentiKats® counterparts showed similar initial reactions rates and yields in the reaction systems, indicating 100% of immobilization efficiency (observed activity/activity immobilized) and absence of diffusional limitations (due to the immobilization). Immobilization of whole and permeabilized cells containing ω‐TA in LentiKats® allowed improved stability as the biocatalyst was shown to be efficiently reused for five reaction cycles, retaining around 80% of original activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Immobilization of thermostable α-galactosidase from Pycnoporus cinnabarinus on chitosan beads and its application to the hydrolysis of raffinose in beet sugar molasses

α-Galactosidase (EC 3.2.1.22) from Pycnoporus cinnabarinus was immobilized on chitosan beads, BCW 1000, and crosslinked chitosan beads, BCW 3000 and 3500, of three different sizes, which were untreated or previously treated with glutaraldehyde. The activity yields of the immobilized enzymes were between 25 to 45%, except for glutaraldehyde-untreated B BCW 1000. Leakage of the enzyme with increasing ionic strength was observed in glutaraldehyde-untreated BCW 1000 and 3000. The α-galactosidases immobilized on glutaraldehyde-treated BCW 3000 and 3500 were active at pH 3–6 and at 70–80°C, and stable between pH 3 and 9, and below 70°C. The immobilized α-galactosidase was continuously used for 30 days to hydrolyze raffinose in beet sugar molases.     

Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of ω-functionalized thiols

A new efficient immobilization method that enables oriented immobilization of biologically active proteins was developed based on concepts of active site masking and kinetic control. Taq DNA polymerase was immobilized covalently on mixed self-assembled monolayers (SAMs) of ω-carboxylated thiol and ω-hydroxylated thiol through amide bonds between the protein and the carboxyl group in SAMs. Activity of the immobilized enzyme as large as 70% of solution-phase enzyme was achieved by masking the active site of the Taq DNA polymerase prior to the immobilization. In addition, the number of immobilization bonds was controlled by optimizing the carboxyl group concentration in the mixed monolayer. The maximum activity of immobilized Taq DNA polymerase was achieved at 5% of 12-mercaptododecanoic acid. The activity observed with protected immobilized enzyme was approximately 20 times higher than that observed with randomly immobilized enzyme. The maximum activity was acquired at a 1:1 DNA/enzyme masking ratio, immobilization pH 8.3, and within 10 min of reaction time. This concept of the active site masking and kinetic control of the number of covalent bonds between proteins and the surface can be generally applicable to a broad range of proteins to be immobilized on the solid surface with higher activity.     

Enzymatic synthesis of l-norephedrine by coupling recombinant pyruvate decarboxylase and ω-transaminase

l-Norephedrine, a natural plant alkaloid, possesses similar activity as ephedrine and can be used as a vicinal amino alcohol for the asymmetric synthesis of a variety of optically pure compounds, including pharmaceuticals, fine chemicals, and agrochemicals. Because of the existence of two asymmetric centers, efficient synthesis of l-norephedrine has been challenging. In the present study, an R-selective pyruvate decarboxylase from Saccharomyces cerevisiae and an S-selective ω-transaminase from Vibrio fluvialis JS17 were coupled to develop a sequential process for the stereoselective biosynthesis of l-norephedrine. After systematic optimization of the reaction conditions, a green, economic, and practical biocatalytic method to prepare l-norephedrine was established to achieve de and ee values of greater than 99.5 % and a molar yield over 60 %. The present coupling approach can facilitate the development of sequential reactions by various biocatalysts.     

Effect of immobilization on stability and properties of N-acetyl-β-d-hexosaminidase from Turbo cornutus

A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with bovine serum albumin and glutaraldehyde, and then cast as membranes. The properties of immobilized N-acetyl-β-d-hexosaminidase were studied. The recovery of N-acetyl-β-d-hexosaminidase after immobilization was unaffected by increasing the concentration of glutaraldehyde, but was decreased by increasing the bovine serum albumin concentration. The immobilized enzyme showed enhanced resistance towards proteolytic and thermal inactivation. While the pH optimum for the soluble enzyme was 4.0, a bimodal pH curve with optima at 3.4 and 5.0 was observed after insolubilization. This bimodality was abolished when the immobilized enzyme was assayed in the presence of M NaCl. The K_m values, for p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside, of the immobilized isoenzymes of N-acetyl-β-d-hexosaminidase were larger than those of their soluble counterparts. No loss of activity could be detected in the membrane after using it for 24 consecutive assays or after storage for at least 50 days at 4°.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Continuous production of galacto-oligosaccharides from lactose by Bullera singularis β-galactosidase immobilized in chitosan beads

Galacto-oligosaccharides (GalOS) were continuously produced using lactose and immobilized β-galactosidase from Bullera singularis ATCC 24193 in a packed bed reactor. Partially purified β-galactosidase was immobilized in Chitopearl BCW 3510 bead (970 GU/g resin) by simple adsorption. 55% (w/w) oligosaccharides was obtained continuously with a productivity of 4·4 g/(litre-h) from 100 g/litre lactose solution during a 15-day operation. Batch productivity was 6·5 g GalOS/(litre-h) from 300 g/litre lactose.     

Evaluation of CV2025 ω-transaminase for the bioconversion of lignin breakdown products into value-added chemicals: synthesis of vanillylamine from vanillin

Abstract

Lignin is an essential component of the cell wall of various plants and represents an abundant and renewable natural resource. Both thermo-chemical and biological pre-treatment can be applied to break down the phenylpropanoid polymer subunits present in lignin. These liberate a range of phenolic compounds which represent potential substrates for bioconversion by ω-transaminases. In this work, the CV2025 ω-transaminase (ω-TAm) from Chromobacterium violaceum DSM30191, heterologously expressed in E. coli, was explored for selective amination of lignin breakdown intermediates into value-added products. Eight potential ω-TAm substrates were initially screened using (S)-α-methylbenzylamine (MBA) as the amino donor. Vanillin was identified as the best potential substrate which is converted into vanillylamine; an intermediate in the preparation of pelargonic acid vanillylamide used as a hyperemia inducing active substance in wound dressings. At low vanillin and MBA concentrations (< 10 mM) and with an excess of the amine donor (1:4 mol/mol) 100% w/w conversion of vanillin into vanillylamine was observed within 25 min. At vanillin concentrations above 10 mM, substrate inhibition was observed decreasing the rate and yield of the bioconversion. High concentrations of the reaction product (vanillylamine) and by-product (acetophenone) also limited the conversion due to increased backward reaction rate and inhibition. Vanillylamine synthesis could be carried out by both whole cell and clarified lysate forms of the CV2025 ω-TAm while fed-batch bioconversions (feeding low concentrations of both vanillin and MBA) could help overcome substrate inhibition and double the final product concentrations obtained. These results demonstrate the potential for bioconversion of lignin breakdown products into value-added chemicals but illustrate the need for enzymes with improved substrate range and implementation of techniques to overcome product inhibition and equilibrium constraints.     

Reusable ω-transaminase sol–gel catalyst for the preparation of amine enantiomers

Heterogeneous ω-transaminase sol–gel catalysts were prepared and characterized in terms of immobilization degree, loading capacity and catalytic behavior in the kinetic resolution of racemic 1-phenylethylamine (a model compound) with sodium pyruvate in phosphate buffer (pH 7.5). The catalyst obtained when ω-transaminase from Arthrobacter sp. was encapsulated from the aqueous solution of the enzyme, isopropyl alcohol and polyvinyl alcohol in the sol–gel matrices, consisting of the 1:5 mixture of tetramethoxysilane and methyltrialkoxysilane, proved to be optimal including the reuse and storage stabilities of the catalyst. The optimized immobilizate was shown to perform well in the kinetic resolution of four structurally different aromatic primary amines in aqueous DMSO (10, v/v-%). The enzyme preparation showed synthetic potential by enabling the catalyst reuse in five consecutive preparative scale kinetic resolutions using 100 mM 1-phenylethylamine in aqueous DMSO (10, v/v-%). It was typical to fresh catalyst preparations that the kinetic resolution tended to exceed 50% before the reaction stopped leaving the (S)-amine unreacted while thereafter in reuse the reactions stopped at 50% conversion as expectable to highly enantioselective reactions.     

Activity and stability of different immobilized preparations of recombinant E. coli cells containing ω-transaminase

Production of chiral amines using ω-transaminases has been thoroughly studied in recent years. Immobilized ω-transaminases, however, have been used on relatively few occasions despite potential benefits such as reuse of enzyme and ease of product purification. In this study principally different methods including surface immobilization, entrapment and sweep flocculation using titanium oxide, Ca-alginate and chitosan respectively were evaluated for the immobilization of recombinant Escherichia coli cells. The enzyme expressed was a modified Arthrobacter citreus ω-transaminase with improved thermostability. The preparations were compared in terms of cell loading capacity, operational stability in repeated batches and storage stability using the conversion of methylbenzylamine to acetophenone.The use of chitosan for cell immobilization proved to be the method of choice since it was both very simple and effective. At a very high cell loading of 3.2 g cells/g chitosan >60% activity was observed. The preparation was reused in eight successive 1-h batches with >90% remaining activity. To further demonstrate its usability the preparation was used for asymmetric synthesis of (S)-4′-cyano-(α)-methylbenzylamine in three repeated bathes (cycle time >20 h), using isopropylamine as the amine donor. Storage stability was comparable with that of non-immobilized cells. It was concluded that the chitosan method due to its properties and simplicity would be advantageous for use also on a larger scale.     

Can immobilization of Bacillus megaterium cells in alginate beads protect them against bacteriophages?

The ability of free and alginate-immobilized cells of Bacillus megaterium to dissolve tricalcium phosphate as well as their susceptibility to phages were compared in pure liquid cultures and in pot experiment with maize. In both liquid culture and cultivated soil, alginate-immobilized cells of B. megaterium exhibited much higher efficiency in increasing the availability of phosphorus than the free cells. Bacteriophages of B. megaterium were found to be common in soil. Specific bacteriophages, in the presence of free cells of B. megaterium, completely inhibited the phosphate-dissolving activity of the bacteria in pure liquid culture and markedly decreased their number in rhizosphere of maize plants. The phosphorus content of maize plants inoculated with free cells of B. megaterium decreased in the presence of their specific bacteriophages, whereas, when alginate-immobilized cells were used as inoculum, no effect of the presence of bacteriophages on phosphate-dissolving activity of bacterial cells was detected.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan–graphene oxide beads

The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan–graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box–Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76?±?0.02% of recovered protein had yielded 7.2?±?0.04?mg?g^?1 and 211?±?0.3%?U?g^?1 of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20?hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70?rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.     

Metabolically driven equilibrium shift of asymmetric amination of ketones by ω-transaminase using alanine as an amino donor

Removal of a side product to overcome unfavorable equilibrium is a prerequisite for the asymmetric amination of ketones using ω-transaminase (ω-TA). Alanine has been preferred as an amino donor because its deamination product (i.e. pyruvate) is easily removable by several enzymatic methods. Here, we demonstrated that the removal of pyruvate by an innate metabolic pathway could afford equilibrium shift of the ω-TA reactions.