Chiara Schiraldi

Trehalose production: exploiting novel approaches

Trehalose (alpha-D-glucopyranosyl alpha-D-glucopyranoside) is a unique sugar capable of protecting biomolecules against environmental stress. It is a stable, colorless, odor-free and non-reducing disaccharide, and is widespread in nature. Trehalose has a key role in the survival of some plants and insects, termed anhydrobionts, in harsh environments, even when most of their water body is removed. The properties of these types of organisms drove attention towards the study of trehalose. Since then, it proved to be an active stabilizer of enzymes, proteins, biomasses, pharmaceutical preparations and even organs for transplantation. Recently, trehalose has been accepted as a safe food ingredient by the European regulation system following approval by the US Food and Drug Administration. The wide range of applications of this sugar has increased the interest of many research groups into the development of novel and economically feasible production systems. This article provides a comprehensive review of the current achievements in the biotechnological production of trehalose.

The production of biocatalysts and biomolecules from extremophiles

The discovery of life in seemingly prohibitive environments continues to challenge conventional concepts of the growth-limiting conditions of many cellular organisms. The diversity of extremophiles has barely been tapped -estimates generally agreeing that <1% of the microorganisms in the environment have been cultivated in pure cultures to date. The production of extremophilic biomass is very important to provide sufficient material for enzyme and biomolecule isolation and characterization, eventually revealing particular features of industrial interest. Hence, special equipment and custom-tailored processes have been developed and are currently under evaluation for the improvement of fermentation productivity. Despite the remarkable opportunities that these uncommon organisms present for biotechnological applications only few instances can be reported for actual exploitation. This lack of progress from the research findings at a laboratory-scale to the actual development of pilot and large-scale production is correlated with the difficulties encountered in extremophile cultivations. Here, we report recent achievements in the production of biomass and related enzymes and biomolecules from extremophile sources, especially focusing on the application of novel fermentation strategies.

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.

Production of chondroitin sulfate and chondroitin

The production of microbial polysaccharides has recently gained much interest because of their potential biotechnological applications. Several pathogenic bacteria are known to produce capsular polysaccharides, which provide a protection barrier towards harsh environmental conditions, and towards host defences in case of invasive infections. These capsules are often composed of glycosaminoglycan-like polymers. Glycosaminoglycans are essential structural components of the mammalian extracellular matrix and they have several applications in the medical, veterinary, pharmaceutical and cosmetic field because of their peculiar properties. Most of the commercially available glycosaminoglycans have so far been extracted from animal sources, and therefore the structural similarity of microbial capsular polysaccharides to these biomolecules makes these bacteria ideal candidates as non-animal sources of glycosaminoglycan-derived products. One example is hyaluronic acid which was formerly extracted from hen crests, but is nowadays produced via Streptococci fermentations. On the other hand, no large scale biotechnological production processes for heparin and chondrotin sulfate have been developed. The larger demand of these biopolymers compared to hyaluronic acid (tons vs kilograms), due to the higher titre in the final product (grams vs milligrams/dose), and the scarce scientific effort have hampered the successful development of fermentative processes. In this paper we present an overview of the diverse applications and production methods of chondroitin reported so far in literature with a specific focus on novel microbial biotechnological approaches.

Perspectives on biotechnological applications of archaea

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.

Structural analysis of bikunin glycosaminoglycan.

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography–triple detector array system during in vitro enzymatic degradation

Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC-TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights (M(w) and M(n), respectively), polydispersity index (M(w)/M(n)), hydrodynamic radius (R(h)), and intrinsic viscosity ([eta]). The Mark-Houwink-Sakurada (MHS) curves (log[eta] versus logM(w)) were then derived directly. Fragments covering a whole range of M(w) (10-900kDa) and size (R(h)=4-81nm) and presenting a rather narrow distribution of molar masses (M(w)/M(n)=1.6-1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the M(w). HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC-TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions.

Human Ng2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid-lys scaffold fabricate a skeletal muscle tissue†‡

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross‐linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2+ ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2− ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT‐PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut‐4, and PPAR‐γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2+ ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre‐differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration. J. Cell. Physiol. 228: 1762–1773, 2013.

High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production

BackgroundThe bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L-1 K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.ResultsThe inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L-1), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw-1), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.ConclusionThe increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.

A microfiltration bioreactor to achieve high cell density in Sulfolobus solfataricus fermentation

Abstract A novel technique is proposed to achieve higher cell yield in extremophile fermentation. Because the accumulation of toxic compounds is thought to be responsible for low biomass yields, a bioreactor has been designed based on a microfiltration hollow-fiber module located inside the traditional fermentation vessel. Using the cul-tivation of the thermoacidophilic archeon Sulfolobus solfataricus Gı as a model, a biomass of 35 g l−1 dry weight was obtained which proved greater than that of 2 g l−1 obtained in batch fermentation. The bioreactor was characterized by running several fermentation experiments to check the high stability of the membrane module to sterilization cycles, high temperatures, and acidic pHs, even for prolonged periods of time. It was shown that the exhaust medium is unable to sustain growth for the presence of toxic compounds, and ultrafiltration and ion-exchange techniques were used in all the attempts to regenerate it. The results demonstrated the ability of the method to lower inhibitor concentrations and prolong the growth phase, thus achieving high cell density. Furthermore, they indicated that the toxic compounds are ionic species of less than 1kDa.