No abstract

Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular components in an immobilized form are extensively used in biotechnology, in biorecognition and in many medicinal applications. This review provides a comprehensive summary of the developments in new immobilization materials, techniques, and their practical applications previously developed by the authors. A detailed overview of several immobilization materials and technologies is given here, including bead cellulose, encapsulation in ionotropic gels and polyelectrolyte complexes, and various immobilization protocols applied onto surfaces. In addition, the review summarises the screening and design of an immobilization protocol, practical applications of immobilized biocatalysts in the industrial production of metabolites, monitoring, and control of fermentation processes, preparation of electrochemical/optical biosensors and biofuel cells.

Direct comparison of key physical and chemical-engineering properties of two representative matrices for multipurpose immobilisations was performed for the first time. Polyvinyl alcohol lens-shaped particles LentiKats® and polyelectrolyte complex microcapsules were characterised by advanced techniques with respect to the size distribution of the particles, their inner morphology as revealed by fluorescent probe staining, mechanical resistance, size-exclusion properties, determination of effective diffusion coefficient and environmental scanning electron microscope imaging. While spherical polyelectrolyte complex microcapsules composed of a rigid semipermeable membrane and a liquid core are almost uniform in shape and size (diameter of 0.82 mm; RSD = 5.6 %), lens-shaped LentiKats® are characterised by wider size distribution (diameter of 3.65 mm; RSD = 10.3 % and height of 0.341 mm; RSD = 32.3 %) and showed the same porous structure throughout their whole volume at the mesoscopic (micrometre) level. Despite differences in their inner structure and surface properties, the pore diameter of ∼ 2.75 nm for regular polyelectrolyte complex microcapsules and ∼ 1.89 nm for LentiKats® were similar. These results were used for mathematical modelling, which provided the estimates of the effective diffusion coefficient of sucrose. This value was 1.67 × 10(-10) m(2) s(-1) for polyelectrolyte complex microcapsules and 0.36 × 10(-10) m(2) s(-1) for LentiKats®. Recombinant cells Escherichia coli-overexpressing enzyme cyclopentanone monooxygenase were immobilised in polyelectrolyte complex microcapsules and LentiKats® for comparison of their operational stability using model Baeyer-Villiger oxidation of (±)-cis-bicyclo [3.2.0] hept-2-en-6-one to regioisomeric lactones as important chiral synthons for potential pharmaceuticals. Both immobilisation matrices rendered high operational stability for whole-cell biocatalyst with no reduction in the biooxidation rate over 18 repeated reaction cycles.

Recombinant Escherichia coli cells, over-expressing cyclopentanone monooxygenase activity, were immobilized in polyelectrolyte complex capsules, made of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl(2) and NaCl. More than 90% of the cell viability was preserved during the encapsulation process. Moreover, the initial enzyme activity was fully maintained within encapsulated cells while it halved in free cells. Both encapsulated and free cells reached the end point of the Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one to 4,9-dioxabicyclo[4.2.1]non-7-en-3-one at the same time (48 h). Similarly, the enantiomeric excess above 94% was identical for encapsulated and free cells.

Novel and selective microbial amperometric biosensors that use Gluconobacter oxydans cells to monitor the bacterial bioconversion of glycerol (Gly) to 1,3-propanediol (1,3-PD) are described. Two different mediators, ferricyanide and flexible polyvinylimidazole osmium functionalized polymer (Os-polymer), were employed to prepare two different microbial biosensors, both of which gave high detection performance. The good operational stabilities of both types of biosensor were underlined by the ability to detect 1,3-PD throughout 140 h of continuous operation. Both microbial biosensor systems showed excellent selectivity for 1,3-PD in the presence of a high excess of glycerol [selectivity ratios (1,3-PD/Gly) of 118 or 245 for the ferricyanide and Os-polymer systems, respectively]. Further, the robustness of each microbial biosensor was highlighted by the high reliability of 1,3-PD detection achieved (average RSD of standards<2%, and well below 4% for samples). The biosensor implementing the Os-polymer mediator exhibited high selectivity towards 1,3-PD detection and allowed moderate sample throughput (up to 12 h-1) when integrated into a flow system. This system was used to monitor the concentration of 1,3-PD during a real bioprocess. Results from biosensor assays of 1,3-PD in bioprocess samples taken throughout the fermentation were in a very good agreement with results obtained from reference HPLC assays (R2=0.999).