Marcelo Fernández-Lahore

Aspartic proteinases from Mucor spp. in cheese manufacturing

Filamentous fungi belonging to the order of Mucorales are well known as producers of aspartic proteinases depicting milk-clotting activity. The biosynthesis level, the biochemical characteristics, and the technological properties of the resulting proteinases are affected by the producer strain and the mode of cultivation. While the milk-clotting enzymes produced by the Rhizomucor spp. have been extensively studied in the past, much less is known on the properties and potential applications of the aspartic proteinases obtained for Mucor spp. Indeed, several Mucor spp. strains have been reported as a potential source of milk-clotting enzymes having unique technological properties. Both submerged fermentation and solid substrate cultivation are proven alternatives for the production of Mucor spp. aspartic proteinases. This review provides an overview on the bioprocessing routes to obtain large amounts of these enzymes, on their structural characteristics as related to their functional properties, and on their industrial applications with focus on cheese manufacturing.

Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification

Megaporous cryogels with metal-ion affinity functionality, which possess enhanced protein-binding ability, were synthesized and their properties were investigated. These highly porous materials (pore sizes up to 100 μm) allowed the direct capture of a recombinant His(6)-tagged protein from a partially clarified extract. The total ligand density of the material was found to be 770 μmol/g. Application of a partially clarified cell extract in order to recover a His(6)-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) yielded 12 mg of highly purified recombinant product per gram of adsorbent. Increased dynamic binding capacities were observed upon larger degrees of grafting, although some reduction in the quality of the system hydrodynamics was also observed. Nevertheless, these immobilized metal-ion affinity cryogels show potential for a convenient single-step purification of recombinant proteins from raw cell extracts without the need for laborious pre-chromatographic sample clean-up procedures.

Synthesis and performance of 3D-Megaporous structures for enzyme immobilization and protein capture

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H+/g, 1.4 mmol H+/g, and 17 mmol H+/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ≅7.2 mg bovine serum albumin/g, ≅7.4 hen egg‐white lysozyme (HEWL) mg/g, and ≅108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work.

Physico-Mechanical and Degradation Properties of Gamma-Irradiated Biocomposites of Jute Fabric-Reinforced Poly(caprolactone)

Jute fabric-reinforced poly(caprolactone) biocomposites (30–70% jute) were fabricated by compression molding. Tensile strength, tensile modulus, bending strength, bending modulus and impact strength of the non-irradiated composites (50% jute) were found to be 65 MPa, 0.75 GPa, 75 MPa, 4.2 GPa and 6.8 kJ/m2, respectively. The composites were irradiated with gamma radiation at different doses (50–1000 krad) at a dose rate of 232 krad/hr and mechanical properties were investigated. The irradiated composites containing 50% jute showed improved physico-mechanical properties. The degradation properties of the composites were observed. The morphology was evaluated by scanning electron microscope.

Development of a transformation system for Aspergillus sojae based on the Agrobacterium tumefaciens-mediated approach

BackgroundAspergillus sojae has been an important filamentous fungus in Biotechnology due to its use in diverse fermentative processes for the production of various food products. Furthermore, this fungus is a common expression system for the production of enzymes and other metabolites. The availability of molecular genetic tools to explore its biology is thus of big interest. In this study, an Agrobacterium tumefaciens-mediated transformation (ATMT) system for A. sojae was developed and its applicability evaluated.ResultsThe donor plasmid named pRM-eGFP was constructed for ATMT of A. sojae. This plasmid contains the ble and egfp genes in its transfer DNA element (T-DNA) to confer phleomycin resistance and express the enhanced green fluorescent protein (EGFP) in A. sojae, respectively. Agrobacterium tumefaciens (LBA4404) harboring the donor plasmid and A. sojae (ATCC 20235) were co-cultured under diverse conditions to achieve ATMT. The maximum number of transformed fungi was obtained after three days of co-culturing at 28°C, and selection with 50 μg/ml phleomycin. Polymerase chain reaction (PCR), fluorescence microscopy and Western Blot analysis for EGFP expression confirmed successful genomic integration of the T-DNA element in A. sojae. The T-DNA was mitotically stable in approximately 40% of the fungal transformants after four generations of sub-culturing under phleomycin pressure.ConclusionWe successfully established a new ATMT protocol for A. sojae. This transformation system should enable further protein expression studies on this filamentous fungus.

High capacity cryogel-type adsorbents for protein purification

Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100μm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties.

Effect of chemical additives on biomass deposition onto beaded adsorbents.

Common limitations encountered during the direct recovery of bioproducts from an unclarified feedstock are related to the presence of biomass in such processing systems. Biomass-related effects can be described as biomass-to-support deposition and cell-to-cell aggregation. In this work, a number of chemical additives were screened for their ability to inhibit either biomass deposition, cell aggregation, or a combination of both effects. Several interacting pairs were screened. These were composed of (i) a commercial chromatographic matrix harbouring a variety of ligand types and (ii) intact yeast cells - as a model biomass type. Studies were performed based on partitioning tests, colloid deposition experiments, and sorption performance studies in expanded beds. Results indicated that the coating of anion-exchanger beads with the synthetic polymer PVP 360 alleviated biomass deposition and consequently restored EBA process performance. This behaviour correlated well with calculations performed according to the XDLVO approach: the secondary (interaction) free energy pockets decreased from -230 kT to -100 kT in the absence and in the presence of PVP 360, respectively. Experiments performed in parallel demonstrated that total binding capacity for the model protein (BSA) - under dynamic conditions - remained almost constant (≈ 55.7 kg m(-3)). Other combinations of additives and adsorbents were tested. However, no solution chemistry was able to inhibit biomass deposition onto strong (composite) ion exchangers. Moreover, yeast cell deposition was only marginally decreased when hydrophobic interaction and pseudo-affinity supports were explored. The utilization of non-toxic polymers could help to avoid detrimental biomass deposition during expanded bed adsorption of bioproducts and other direct contact sequestration methods.

Probing the dynamic reversibility and generation of dynamic combinatorial libraries in the presence of bacterial model oligopeptides as templating guests of tetra‐carbohydrazide macrocycles using electrospray mass spectrometry

RATIONALE Over the past few decades, bacterial resistance to antibiotics has emerged as a real threat to human health. Accordingly, there is an urgent demand for the development of innovative strategies for discovering new antibiotics. We present the first use of tetra-carbohydrazide cyclophane macrocycles in dynamic combinatorial chemistry (DCC) and molecular recognition as chiral hosts binding oligopeptides, which mimic bacterial cell wall. This study introduces an innovative application of electrospray ionisation time-of-flight mass spectrometry (ESI-TOF MS) to oligopeptides recognition using DCC. METHODS A small dynamic library composed of eight functionalised macrocycles has been generated in solution and all members were characterised by ESI-TOF MS. We also probed the dynamic reversibility and mechanism of formation of tetra-carbohydrazide cyclophanes in real-time using ESI-TOF MS. RESULTS Dynamic reversibility of tetra-carbohydrazide cyclophanes is favored under thermodynamic control. The mechanism of formation of tetra-carbohydrazide cyclophanes involves key dialdehyde intermediates, which have been detected and assigned according to their high-resolution m/z values. Three members of the dynamic library bind efficiently in the gas phase to a selection of oligopeptides, unique to bacteria, allowing observation of host/guest complex ions in the gas phase. CONCLUSIONS We probed the mechanism of the [2+2]-cyclocondensation reaction forming library members, proved dynamic reversibility of tetra-carbohydrazide cyclophanes and showed that complex ions formed between library members and hosts can be observed in the gas phase, allowing the solution of an important problem of biological interest.

A novel pectin-degrading enzyme complex from Aspergillus sojae ATCC 20235 mutants

BACKGROUND In the food industry, the use of pectinase preparations with high pectin esterase (PE) activity leads to the release of methanol, which is strictly regulated in food products. Herein, a pectin-degrading enzyme (PDE) complex exhibiting low PE activity of three Aspergillus sojae ATCC 20235 mutants (M3, DH56 and Guserbiot 2.230) was investigated. Production of exo-/endo-polygalacturonase (PG), exo-polymethylgalacturonase (PMG) and pectin lyase (PL) by mutant M3 and A. sojae using two different carbon sources was evaluated in solid-state fermentation. Finally, experimental preparations obtained from the mutants and commercial pectinases standardized to the same potency were screened for PDEs. RESULTS Mutant M3 grown on sugar beet was found to be the best producer of exo-PG, endo-PG, exo-PMG and PL, with maximum yields of 1111, 449, 130 and 123 U g(-1), respectively. All experimental preparations exhibited low PE activity, at least 21.5 times less than commercial pectinases, and higher endo-PG (40 U mL(-1)). CONCLUSION Mutant M3 was the best PDE producer using sugar beet. Mutant strains presented a PDE complex featuring high endo-PG and very low PE activities. This novel complex with low de-esterifying activity can be exploited in the food industry to degrade pectin without releasing methanol.

A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags.

In recent years, affinity fusion-tag systems have become a popular technique for the purification of recombinant proteins from crude extracts. However, several drawbacks including the high expense and low stability of ligands, their leakage during operation, and difficulties in immobilization, make it important to further develop the method. The present work is concerned with the utilization of a ceramic fluorapatite (CFT)-based chromatographic matrix to overcome these drawbacks. A heptapeptide library exhibiting a range of properties have been synthesized and subjected to ceramic fluorapatite (CFT) chromatography to characterize their retention behavior as a function of pH and composition of the binding buffer. The specific binding and elution behavior demonstrates the possible application of CFT-binding peptides as tags for enhancing the selective recovery of proteins by CFT chromatography. To materialize this strategy, a phage-derived CFT-specific sequence KPRSVSG (Tag1) with/without a consecutive hexalysine sequence, KKKKKKKPRSVSG (Tag2), were fused at the C-terminus of an enhanced green fluorescent protein (eGFP). The resulting gene constructs H-eGFP, H-eGFP-Tag1 and H-eGFP-Tag2 were expressed in Escherichia coli strain BL-21, and the clarified cell lysate was applied to the CFT column equilibrated with binding buffer (20-50mM sodium phosphate, pH 6-8.4). Sodium phosphate (500mM) or 1M NaCl in the respective binding buffer was used to elute the fused proteins, and the chromatographic fractions were analyzed by gel electrophoresis. Both the yield and purity were over 90%, demonstrating the potential application of the present strategy.