Immobilized Cells

Three basic types of immobilization (i.e. without carrier, entrapment and immobilization on the carrier surface) of microbial cells, nonmicrobial cell populations and subcellular organelles are reviewed. These are further developed into a number of combined and less frequently used techniques of immobilization and application of cell biocatalysts for industrial biotransformations in pharmacy, food industry and agriculture, including novel approached and some unpublished authors’ results.     

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.     

Candida parapsilosis: A versatile biocatalyst for organic oxidation-reduction reactions

This review highlights the importance of the biocatalyst, Candida parapsilosis for oxidation and reduction reactions of organic compounds and establishes its versatility to generate a variety of chiral synthons. Appropriately designed reactions using C. parapsilosis effect efficient catalysis of organic transformations such as deracemization, enantioselective reduction of prochiral ketones, imines, and kinetic resolution of racemic alcohols via selective oxidation. This review includes the details of these biotransformations, catalyzed by whole cells (wild type and recombinant strains), purified enzymes (oxidoreductases) and immobilized whole cells of C. parapsilosis. The review presents a bioorganic perspective as it discusses the chemo, regio and stereoselectivity of the biocatalyst along with the structure of the substrates and optical purity of the products. Fermentation scale biocatalysis using whole cells of C. parapsilosis for several biotransformations to synthesize important chiral synthons/industrial chemicals is included. A comparison of C. parapsilosis with other whole cell biocatalysts for biocatalytic deracemization and asymmetric reduction of carbonyl and imine groups in the synthesis of a variety of enantiopure products is presented which will provide a basis for the choice of a biocatalyst for a desired organic transformation. Thus, a wholesome perspective on the present status of C. parapsilosis mediated organic transformations and design of new reactions which can be considered for large scale operations is provided. Taken together, C. parapsilosis can now be considered a ‘reagent’ for the organic transformations discussed here.     

Camphor-grown Pseudomonas putida,a multifunctional biocatalyst for undertaking Baeyer-Villiger monooxygenase-dependent biotransformations

Summary Both NADH- and NADPH-dependent Baeyer-Villiger monooxygenase activities with potential uses as biocatalysts for biotransformations are present to different extents throughout the growth of Pseudomonas putida NCIMB 10007 on (+)-camphor. The two activities give a different pattern of stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Aspects for the future of biotransformations

Biotransformations have gained extensive importance in practical use as a support for chemical synthesis or in the conversion of natural products. Biotransformations may present an enlargement, a sequential degradation or a specific modification of synthetic or natural compounds. The tools for biotransformations are principally mammalian, plant or microbial cells and their cell-free enzymes. In technical practice the biocatalysts are so far limited to the use of microorganisms and some cell-free enzymes of low cost. Although numerous microbial or enzymatical reactions were already developed for industrial processes, the capacities of biotransformations offer a broad field of inexhaustible possibilities for the future.

     

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions¹. Microalgae not only capture solar energy more efficiently than plants³, but also synthesize advanced biofuels^2-4. Generally, atmospheric CO₂ is not a sufficient source for supporting maximal algal growth⁵. On the other hand, the high concentrations of CO₂ in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient “flue gas to algae” system. Researchers have proposed different photobioreactor configurations^4,6 and cultivation strategies^7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO₂; 2) how we can find optimal CO₂ concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.     

On the kinetics of the enzyme-catalyzed hydrolysis of axial chiral alkyl allenecarboxylates: preparation of optically active R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid and its optically pure S-(+)-methylester

Pig liver esterase (PLE) was used for the preparation of optically active alkyl allenecarboxylates with axial chirality. Free and immobilized enzymes were used as biocatalysts for the kinetic resolution of racemic ester substrates. Whereas the biotransformations using the free biocatalyst resulted in moderately to high enantiomeric ratios, the immobilization significantly decreased the E-value. The reaction conditions were optimized with respect to the enantiomeric ratio and scaled up. The enantiomeric ratio (E-value) was thereby enhanced by a factor of four to E=60. Under optimized conditions (free enzyme, addition of acetone as a cosolvent and Triton X-100 as an emulgator) in a preparative scale biotransformation, 282 mg of optically pure S-(+)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid methylester (96% ee, 82% yield) and 257 mg of R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid (83% ee, 80% yield) could be synthesized from the racemic substrate.     

Disentangling the roles of spatial and environmental variables in shaping benthic algal assemblages in rivers of central and northern China

Benthic algae were collected from central and northern Chinese rivers to test the hypothesis that geographic location has significant contributions in shaping algal assemblages. We used Moran’s eigenvector maps (MEM) to model spatial components and variation partitioning to quantify the influences of spatial and environmental variables on regional patterns of algal richness and community composition, respectively. We found that variation in algal richness was attributed to MEM component 2, 8, and 9 and the quadratic term of N–NO₃. Regarding abundance data, latitude, longitude, and MEM component 1, 2, and 7 were important spatial variables. Although P–PO₄, pH, and annual mean temperature were significant environmental variables influencing algal community composition, they were all spatially structured. Among the total explained variance in both algal metrics, spatial proportions were higher than that of environmental variables. We also found that abundant species of Achnanthidium minutissimum, Cocconeis placentula, Cymbella delicatula, Cymbella affinis, Cymbella turgidula, and Synedra ulna displayed clear spatially related patterns. In conclusion, the contributions of spatial and environmental variables to regional variation of algal assemblages are scale-dependent. As for our study scale (~1,000 km), spatial control may be more important. Since spatial effects could obscure local environmental impacts on algal communities, appropriate study scale and statistical methods should be taken into account in algal bioassessment. We recommend inclusion of both algal richness and community composition in study of algal biogeography, due to their different relationships with spatial and environmental variables.     

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG₂ and PGH₂. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.     

Non-water miscible ionic liquid improves biocatalytic production of geranyl glucoside with <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing a glucosyltransferase

Whole cells of Escherichia coli overexpressing a glucosyltransferase from Vitis vinifera were used for the glucosylation of geraniol to geranyl glucoside. A high cell density cultivation process for the production of whole-cell biocatalysts was developed, gaining a dry cell mass concentration of up to 67.6 ± 1.2 g L^?1 and a glucosyltransferase concentration of up to 2.7 ± 0.1 g protein L^?1 within a process time of 48 h. Whole-cell batch biotransformations in milliliter-scale stirred-tank bioreactors showed highest conversion of geraniol at pH 7.0 although the pH optimum of the purified glucosyltransferase was at pH 8.5. The biocatalytic batch process performance was improved significantly by the addition of a water-immiscible ionic liquid (N-hexylpyridinium bis(trifluoromethylsulfonyl)imid) for in situ substrate supply. The so far highest final geranyl glucoside concentration (291 ± 9 mg L^?1) and conversion (71 ± 2 %) reported for whole-cell biotransformations of geraniol were achieved with 5 % (v/v) of the ionic liquid.     

Free Ammonia Inhibition of Algal Photosynthesis in Intensive Cultures

The effect of free NH₃ inhibition on short-term photosynthesis was investigated in three microalgal species: the freshwater chlorophyte Scenedesmus obliquus, the marine diatom Phaeodactylum tricornutum and the marine chlorophyte Dunaliella tertiolecta. By performing a series of assays at various concentrations of added NH₄Cl and culture pH, we demonstrated that the inhibitory compound was free NH₃ and that pH played no role in determining the magnitude of inhibition, other than in establishing the degree of dissociation of nontoxic NH₄⁺ to toxic NH₃. When corrections were made for pH, all three species displayed the same sigmoidal response curve to free NH₃ concentration; 1.2 mM NH₃ led to 50% reduction in photoassimilation of ¹⁴C. Based on literature values, some marine phytoplankton appear to be significantly more sensitive to free NH₃ than were the test species, which are noted for their excellent growth characteristics. However, the combination of low algal biomass and strong pH buffering commonly found in most marine and many freshwater environments probably limits the possibilities for NH₃ toxicity to low alkalinity freshwaters and intensive algal cultures in which NH₄⁺ is the main source of N. Such conditions occur commonly in algal wastewater treatment systems.     

Layer-by-Layer coated tyrosinase: An efficient and selective synthesis of catechols

Agaricus bisporous tyrosinase was immobilized on commercial available epoxy-resin Eupergit®C250L and then coated by the Layer-by-Layer method (LbL). The two novel heterogeneous biocatalysts were characterized for their morphology, pH and storage stability, kinetic properties (K_m, V_max, V_max/K_m) and reusability. These biocatalysts were used for the efficient and selective synthesis of bioactive catechols under mild and environmental friendly experimental conditions. Ascorbic acid was added in the reaction medium to inhibit the formation of ortho-quinones, thus avoiding the known enzyme suicide inactivation process. Catechols were obtained mostly in quantitative yields and conversion of substrate. Tyrosinase immobilized on Eupergit®C250L and coated by the LbL method showed better catalytic activities, higher pH and storage stability, and reusability with respect to immobilized uncoated tyrosinase. Since chemical procedures to synthesize catechols are often expensive and with high environmental impact, the use of immobilized tyrosinase represents an efficient alternative for the preparation of this family of bioactive compounds.     

Direct and indirect effects of high pCO2 on algal grazing by coral reef herbivores from the Gulf of Aqaba (Red Sea)

Grazing on marine macroalgae is a key structuring process for coral reef communities. However, ocean acidification from rising atmospheric CO₂ concentrations is predicted to adversely affect many marine animals, while seaweed communities may benefit and prosper. We tested how exposure to different pCO₂ (400, 1,800 and 4,000 μatm) may affect grazing on the green alga Ulva lactuca by herbivorous fish and sea urchins from the coral reefs in the northern Gulf of Aqaba (Red Sea), either directly, by changing herbivore behaviour, or indirectly via changes in algal palatability. We also determined the effects of pCO₂ on algal tissue concentrations of protein and the grazing-deterrent secondary metabolite dimethylsulfoniopropionate (DMSP). Grazing preferences and overall consumption were tested in a series of multiple-choice feeding experiments in the laboratory and in situ following exposure for 14 d (algae) and 28 d (herbivores). 4,000 μatm had a significant effect on the biochemical composition and palatability of U. lactuca. No effects were observed at 1,800 relative to 400 μatm (control). Exposure of U. lactuca to 4,000 μatm resulted in a significant decrease in protein and increase in DMSP concentration. This coincided with a reduced preference for these algae by the sea urchin Tripneustes gratilla and different herbivorous fish species in situ (Acanthuridae, Siganidae and Pomacanthidae). No feeding preferences were observed for the rabbitfish Siganus rivulatus under laboratory conditions. Exposure to elevated pCO₂ had no direct effect on the overall algal consumption by T. gratilla and S. rivulatus. Our results show that CO₂ has the potential to alter algal palatability to different herbivores which could have important implications for algal abundance and coral community structure. The fact that pCO₂ effects were observed only at a pCO₂ of 4,000 μatm, however, indicates that algal-grazer interactions may be resistant to predicted pCO₂ concentrations in the near future.     

Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V_MAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml⁻¹. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V_MAX(app) of 491 mU ml⁻¹ was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD_578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.     

Yeast cell factories for fine chemical and API production

This review gives an overview of different yeast strains and enzyme classes involved in yeast whole-cell biotransformations. A focus was put on the synthesis of compounds for fine chemical and API (= active pharmaceutical ingredient) production employing single or only few-step enzymatic reactions. Accounting for recent success stories in metabolic engineering, the construction and use of synthetic pathways was also highlighted. Examples from academia and industry and advances in the field of designed yeast strain construction demonstrate the broad significance of yeast whole-cell applications. In addition to Saccharomyces cerevisiae, alternative yeast whole-cell biocatalysts are discussed such as Candida sp., Cryptococcus sp., Geotrichum sp., Issatchenkia sp., Kloeckera sp., Kluyveromyces sp., Pichia sp. (including Hansenula polymorpha = P. angusta), Rhodotorula sp., Rhodosporidium sp., alternative Saccharomyces sp., Schizosaccharomyces pombe, Torulopsis sp., Trichosporon sp., Trigonopsis variabilis, Yarrowia lipolytica and Zygosaccharomyces rouxii.     

Photosynthetic CO2-use efficiency in lichens and their isolated photobionts: the possible role of a CO2-concentrating mechanism

The CO₂ dependence of net CO₂ assimilation was examined in a number of green algal and cyanobacterial lichens with the aim of screening for the algal/cyanobacterial CO₂-concentrating mechanism (CCM) in these symbiotic organisms. For the lichens Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and P. neopolydactyla (Gyeln.) Gyeln., the photosynthetic performance was also compared between intact thalli and their respective photobionts, the green alga Coccomyxa PA, isolated from Peltigera aphthosa and the cyanobacterium Nostoc PC, isolated from Peltigera canina. More direct evidence for the operation of a CCM was obtained by monitoring the effects of the carbonic-anhydrase inhibitors acetazolamide and ethoxyzolamide on the photosynthetic CO₂use efficiency of the photobionts. The results strongly indicate the operation of a CCM in all cyanobacterial lichens investigated and in cultured cells of Nostoc PC, similar to that described for free-living species of cyanobacteria. The green algal lichens were divided into two groups, one with a low and the other with a higher CO₂-use efficiency, indicative of the absence of a CCM in the former. The absence of a CCM in the low-affinity lichens was related to the photobiont, because free-living cells of Coccomyxa PA also apparently lacked a CCM. As a result of the postulated CCM, cyanobacterial Peltigera lichens have higher rates of net photosynthesis at normal CO₂ compared with Peltigera aphthosa. It is proposed that this increased photosynthetic capacity may result in a higher production potential, provided that photosynthesis is limited by CO₂ under natural conditions.     

Characterization of in vivo biotransformations for trastuzumab emtansine by high-resolution accurate-mass mass spectrometry

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) designed for the treatment of HER2-positive cancers. T-DM1 is composed of the humanized monoclonal antibody trastuzumab connected to a maytansine derivative cytotoxic drug, via a nonreducible thioether linker at random lysine residues, and therefore has a very complex molecular structure. It was anticipated that T-DM1 undergoes biotransformations in circulation. However, there was limited knowledge on these structural changes due to bioanalytical challenges. Here, we have investigated the in vivo biotransformations of T-DM1 using a high-resolution accurate-mass (HR/AM) mass spectrometry approach. Three types of biotransformations were characterized for T-DM1 in circulation in tumor-bearing mice, including cysteine or glutathione adduct formation via maleimide exchange, loss of maytansinol via ester hydrolysis, as well as addition of H₂O via linker-drug hydrolysis. These results provide new insights into in vivo catabolism of T-DM1.     

Influence of inoculum to substrate ratios (ISRs) on the performance of anaerobic digestion of algal residues

With increasing concerns of microalgal-biodiesel, algal residues after lipid extraction are raising great attention for energy production. A batch test of 15 days under mesophilic condition was conducted to evaluate the effects of inoculum to substrate ratios (ISRs) on the methane production by anaerobic digestion of Chlorella sp. residue. The stability and progress of the reaction from algal residue to methane were monitored by measuring the pH, volatile fatty acids (VFAs), total ammoniacal nitrogen (TAN), methane volume on a daily basis. The results indicated that the values obtained were 26.6, 191.6, 195.6 and 210.6 ml CH₄/g volatile solid (VS) for ISRs of 1:2, 1:1, 2:1 and 3:1. The methane production was significantly decreased as the ISR was lower than 1:1, which was resulting from the poor methanogenesis inhibited by NH₄ ⁺-N. It would be of great importance that determination of ISRs might provide useful information on how to initialize a batch digester with algal residue as material.     

Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei

In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.     

Dried alginate-entrapped enzymes (DALGEEs) and their application to the production of fructooligosaccharides

A modification of the classical calcium alginate enzyme entrapment technique is described aiming to overcome some of the limitations of the former gel-based biocatalysts. Dried alginate entrapped enzymes (DALGEEs) were obtained dehydrating calcium alginate gel beads containing entrapped enzymes. A fructosyltransferase from Aspergillus aculeatus, present in Pectinex Ultra SP-L, was entrapped using this technique. The resulting DALGEEs were successfully tested both operating batchwise and in a continuous fixed-bed reactor for fructooligosaccharides (FOS) synthesis from sucrose. Interestingly, DALGEEs did not re-swell upon incubation in concentrated (600 g/L) sucrose solutions, probably due to the lowered water activity (a_w) of such media. Confocal laser scanning microscopy of DALGEEs revealed that the enzyme molecules accumulated preferably in the shell of the particles. DALGEEs showed an approximately 30-fold higher volumetric activity (300 U/mL) compared with the calcium alginate gel beads. Moreover, a significant enhancement (40-fold) of the space-time-yield of fixed-bed bioreactors was observed when using DALGEEs as biocatalyst compared with gel beads (4030 g/day L of FOS vs. 103 g/day L). The operational stability of fixed-bed reactors packed with DALGEEs was extraordinary, providing a nearly constant FOS composition of the outlet during at least 700 h. It was also noticeable their resistance against microbial attack, even after long periods of storage at room temperature. The DALGEEs immobilisation strategy may also be useful for other biotransformations, in particular when they take place in low a_w media.