Michal Rosenberg

Immobilization in biotechnology and biorecognition: from macro- to nanoscale systems

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.

Biocatalysis with immobilized Escherichia coli

Immobilization is one of the great tools for developing economically and ecologically available biocatalysts and can be applied for both enzymes and whole cells. Much research dealing with the immobilization of Escherichia coli has been published in the past two decades. E. coli in the form of immobilized biocatalyst catalyzes many interesting reactions and has been used mainly in laboratories, but also on an industrial scale, leading to the production of valuable substances. It has the potential to be applied in many fields of modern biotechnology. This paper aims to give a general overview of immobilization techniques and matrices suitable mostly for entrapment, encapsulation, and adsorption, which have been most frequently used for the immobilization of E. coli. An extensive analysis reviewing the history and current state of immobilized E. coli catalyzing different types of biotransformations is provided. The review is organized according to the enzymes expressed in immobilized E. coli, which were grouped into main enzyme classes. The industrial applications of immobilized E. coli biocatalyst are also discussed.

Fermentation of lactic acid with Rhizopus arrhizus in a stirred tank reactor with a periodical bleed and feed operation

Semicontinuous fungal fermentation of lactic acid (LA) by Rhizopus arrhizus was performed using a periodical bleed and feed (PBF) operation in a stirred tank bioreactor with a volume of 4.5 dm3. LA productivity up to 2.91 kg m−3 h−1 and yield up to 75.3% were achieved. Using PBF cycles, fermentation was prolonged to 240 h with an average LA productivity of 2.31 kg m−3 h−1 and a total yield of 67.3% at a mean pH of 5.30. The most effective production of LA required filamentous morphology of the biomass. When such morphology changed to pellets biomass growth stopped, the LA productivity decreased to 1.63 kg m−3 h−1 and the yield decreased to 62.6%. The filamentous morphology was successfully maintained for 152 h when the inoculation was with spores. CaCO3 initially used for pH neutralisation did not dissolve well since it was entrapped by biomass. It was successfully replaced by 25% ammonia. Long-time operation below pH 5 decreased LA productivity. Accumulating biomass in the fermenter did not increase the LA productivity. With an increase in the biomass growth rate, the LA productivity increased. The on-line extraction or pertraction of LA by trialkylamines demands a pH value of feed below 4. This can be achieved for a short time by interruption neutralisation at the end of each PBF cycle without a decrease in LA productivity after the restoration of the optimal pH value.

Butanol production by immobilised Clostridium acetobutylicum in repeated batch, fed-batch, and continuous modes of fermentation

Clostridium acetobutylicum immobilised in polyvinylalcohol, lens-shaped hydrogel capsules (LentiKats(®)) was studied for production of butanol and other products of acetone-butanol-ethanol fermentation. After optimising the immobilisation protocol for anaerobic bacteria, continuous, repeated batch, and fed-batch fermentations in repeated batch mode were performed. Using glucose as a substrate, butanol productivity of 0.41 g/L/h and solvent productivity of 0.63 g/L/h were observed at a dilution rate of 0.05 h(-1) during continuous fermentation with a concentrated substrate (60 g/L). Through the process of repeated batch fermentation, the duration of fermentation was reduced from 27.8h (free-cell fermentation) to 3.3h (immobilised cells) with a solvent productivity of 0.77 g/L/h (butanol 0.57 g/L/h). The highest butanol and solvent productivities of 1.21 and 1.91 g/L/h were observed during fed-batch fermentation operated in repeated batch mode with yields of butanol (0.15 g/g) and solvents (0.24 g/g), respectively, produced per gram of glucose.

Immobilisation of Clostridium spp. for production of solvents and organic acids

This review summarises the high potential of immobilised cells systems for the fermentative production of compounds, mainly produced by representatives of the Clostridium genus. Microorganisms of Clostridium species are recognised as good producers of a wide range of chemicals in almost every sector of industry. The combination of this microorganism with its immobilisation opens up new possibilities and renders the fermentation process more sophisticated than in a free-cell system. This review provides a comprehensive summary of techniques used in immobilisation of Clostridium species with regard to specific products and types of fermentation. In addition, comparisons of particular types of immobilisation techniques used in fermentation processes are summarised by specific products.

Immobilization of cells and enzymes to LentiKats

Biocatalyst immobilization is one of the techniques, which can improve whole cells or enzyme applications. This method, based on the fixation of the biocatalyst into or onto various materials, may increase robustness of the biocatalyst, allows its reuse, or improves the product yield. In recent decades, a number of immobilization techniques have been developed. They can be divided according to the used natural or synthetic material and principle of biocatalyst fixation in the particle. One option, based on the entrapment of cells or enzymes into a synthetic polyvinyl alcohol lens with original shape, is LentiKats® immobilization. This review describes the preparation principle of these particles and summarizes existing successful LentiKats® immobilizations. In addition, examples are compared with other immobilization techniques or free biocatalysts, pointing to the advantages and disadvantages of LentiKats®.

Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris MutS strain

Pichia pastoris is currently one of the most preferred microorganisms for recombinant enzyme production due to its efficient expression system. The advantages include the production of high amounts of recombinant proteins containing the appropriate posttranslational modifications and easy cultivation conditions. α-L-Rhamnosidase is a biotechnologically important enzyme in food and pharmaceutical industry, used for example in debittering of citrus fruit juices, rhamnose pruning from naringin, or enhancement of wine aromas, creating a demand for the production of an active and stable enzyme. The production of recombinant α-L-rhamnosidase cloned in the MutS strain of P. pastoris KM71H was optimized. The encoding gene is located under the control of the AOX promoter, which is induced by methanol whose concentration is instrumental for these strain types. Fermentation was upscaled in bioreactors employing various media and several methanol-feeding strategies. It was found that fed batch with BSM media was more effective compared to BMMH (Buffered Methanol-complex Medium) media due to lower cost and improved biomass formation. In BSM (Basal Salt Medium) medium, the dry cell weight reached approximately 60 g/L, while in BMMH it was only 8.3 g/L, without additional glycerol, which positively influenced the amount of enzyme produced. New methanol feeding strategy, based on the level of dissolved oxygen was developed in this study. This protocol that is entirely independent on methanol monitoring was up scaled to a 19.5-L fermenter with 10-L working volume with the productivity of 13.34 mgprot/L/h and specific activity of α-L-rhamnosidase of 82 U/mg. The simplified fermentation protocol was developed for easy and effective fermentation of P. pastoris MutS based on dissolved oxygen monitoring in the induction phase of an enzyme production.

Recombinant α-L-rhamnosidase of Aspergillus terreus immobilization in polyvinylalcohol hydrogel and its application in rutin derhamnosylation

Abstract Recombinant α-L-rhamnosidase from Aspergillus terreus expressed in Pichia pastoris was immobilized in LentiKats® lens-shaped polyvinylalcohol (PVA) capsules with an activity of 7 U g− 1, which was 21% of its original activity. Immobilization did not significantly affect the pH and temperature profile of α-L-rhamnosidase, KM increased by a factor of 3.4 whereas Vmax decreased more than 10-fold. No decrease in activity was observed after 27 repeated batch runs of rutin derhamnosylation. The enzyme proved to have an excellent storage stability (136 days) in 60 g L− 1 ethanol with no change in its activity.

Improvement of paper strength via surface application of sugar beet pectin

The influence of surface-applied original and enzymatically-modified sugar beet pectin on strength properties of fluting, coating base paper, and core board was investigated. The effect was compared with the application of commercial strength-increasing agents. With increasing the polymer uptake, measured strength of paper increased. Original sugar beet pectin increased the strength properties of papers, at the same uptake of polymer, to a higher extent than oxidised potato starch or modified grain flour, while the effect of enzymatically-modified sugar beet pectin was the lowest. For the same increase of paper strength, a several times higher uptake of enzymatically-modified sugar beet pectin was required when compared with the original pectin, oxidised potato starch, or modified grain flour.

Semicontinual synthesis of alkyl galactosides using β-galactosidase entrapped in polyvinylalcohol hydrogel

Abstract Fungal β-galactosidase from Aspergillus oryzae was immobilized into polyvinylalcohol (PVA) hydrogel by LentiKats® technology and used for the production of short-chain alkyl glycosides. Ethyl- and propyl-β-d-galactopyranosides were prepared from lactose (100 g/L) and varying initial amounts of alcohol (10–30% v/v) at 40 °C and pH 4.5. The entrapped β-galactosidase preserved 50% of the initial transgalactosylation activity after 25 repeated cycles in the production of ethyl β-d-galactopyranoside. When 5% (v/v) propanol was used as an acceptor, the enzyme activity (30–32 U/g immobilized enzyme) remained constant for 25 repeated batch runs. These findings suggest that entrapped β-galactosidase into LentiKats® has a great potential to be one effective, reusable and easy producible biocatalyst for the production of alkyl glycosides in a large scale.