Telma Teixeira Franco

Feasibility of acrylic acid production by fermentation

Acrylic acid might become an important target for fermentative production from sugars on bulk industrial scale, as an alternative to its current production from petrochemicals. Metabolic engineering approaches will be required to develop a host microorganism that may enable such a fermentation process. Hypothetical metabolic pathways for insertion into a host organism are discussed. The pathway should have plausible mass and redox balances, plausible biochemistry, and plausible energetics, while giving the theoretically maximum yield of acrylate on glucose without the use of aeration or added electron acceptors. Candidate metabolic pathways that might lead to the theoretically maximum yield proceed via β-alanine, methylcitrate, or methylmalonate-CoA. The energetics and enzymology of these pathways, including product excretion, should be studied in more detail to confirm this. Expression of the selected pathway in a host organism will require extensive genetic engineering. A 100,000-tons/year fermentation process for acrylic acid production, including product recovery, was conceptually designed based on the supposition that an efficient host organism for acrylic acid production can indeed be developed. The designed process is economically competitive when compared to the current petrochemical process for acrylic acid. Although the designed process is highly speculative, it provides a clear incentive for development of the required microbial host, especially considering the environmental sustainability of the designed process.

Partitioning of whey proteins, bovine serum albumin and porcine insulin in aqueous two-phase systems.

Partitioning of the proteins from cheese whey, bovine serum albumin and porcine insulin were analysed using aqueous two-phase systems (ATPS) prepared with PEG-phosphate, PEG-citrate and PEG-maltodextrin (MD). Proteins were quantified through one of the following methods: FPLC, Bradford and spectrophotometry at 280 nm. Results showed that whey proteins partitioned unevenly on the phases of the systems used, with alpha-lactoalbumin (alpha-La) concentrated in the upper phase and beta-lactoglobulin (beta-Lg) in the lower. Albumin in PEG-MD systems concentrated in the MD-rich lower phase. Porcine insulin showed great affinity with the PEG-rich phase, its partition coefficient was always over 10 and increases with PEG molecular mass.

Effect of feeding strategies on lipid production by Lipomyces starkeyi.

The aim of this study was to produce microbial oil from Lipomyces starkeyi DSM 70296 grown in hemicellulose hydrolysate (H-H). Glucose and xylose were used for batch, fed-batch, repeated fed-batch, and continuous cultures, and H-H was tested at continuous culture. The highest cell and lipid concentrations of 85.4 and 41.8g/L, respectively, were obtained using repeated fed-batch strategy. Continuous culture with dilution rate of 0.03h(-1) presented the highest overall cell (0.443g/g) and lipid yields (0.236g/g). At 0.06h(-1) were obtained the highest cell and lipid productivities. Continuous cultivation using H-H at 0.03h(-1) resulted in higher cell productivity than that obtained using glucose:xylose. Gas chromatography analysis of the esterified lipids indicated that the major constituents of this complex are palmitic acid, stearic acid, oleic acid, and linoleic acid with an estimated cetane number (approximately 61) similar to that of palm biodiesel, which is important for biofuel production.

Chitosan/pectin polyelectrolyte complex as a pH indicator.

A polyelectrolyte complex (PEC) matrix formed between chitosan and pectin was developed to entrap a bioactive compound (anthocyanin), obtaining an useful pH indicator device. Polysaccharides of opposite charges such as chitosan and pectin can have a very strong intermolecular interaction. The innovation lies in obtaining a new system based on natural and biodegradable compounds, which is simple to manufacture, to indicate variation in pH by visual changes in colour. This device has potential applications in food packaging. The PEC was studied using chitosan and pectin solutions at different pHs values (3.0, 4.0, 5.0 and 5.5) and pectin/chitosan molar ratios (1.0 to 10/1.0 to 5.0). PEC films were homogeneous and showed the highest yield (60.0%) at pH 5.5. Diffusion tests indicated efficient bioactive compound entrapment in the PEC matrix. Thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy indicate the compatibility between the polymers and bioactive compound.

Purification of soybean peroxidase (Glycine max) by metal affinity partitioning in aqueous two-phase systems.

Combining two concepts in downstream processing, this work investigates the partitioning of a crude soybean peroxidase (Glycine max) in an aqueous two-phase system by metal affinity. A liquid-liquid extraction process using metal ligands was developed in two steps with the aim of purifying the enzyme peroxidase. PEG 4000 was activated using thionyl chloride, covalently linked to iminodiacetic acid (IDA), and the specific metal ligand Cu2+ was attached to the PEG 4000-IDA. In the first step, the system was composed of 14% (w/w) PEG 4000-IDA-Cu2+ and 8% (w/w) Na2SO4, and the peroxidase partitioned mainly to the top phase (K = 24). In the second step, a system formed by 14% PEG 4000 and 10% phosphate was used to revert the value of the partition coefficient of peroxidase to the bottom salt-rich phase (K = 0.05), thereby achieving a recovery of 64% of the purified enzyme.

Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening

In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor. The evaluation of the fermentative performance of mutants was carried out using xylose as sole carbon source; the fermentation of wild-type strain was used as reference. Using this strategy it was possible to identify one mutant (termed A1) presenting a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain. A1 mutant was further studied in bioreactor using the same fermentation parameters optimized for L. starkeyi lipid production from a mixed carbon source (xylose:glucose), as previously determined by other studies in our laboratory. A1 presented a productivity increase of 15.1% in biomass and 30.7% in lipid productivity when compared to the wild-type strain with a similar fatty acid composition, despite a slight increase (approx. 7%) on the unsaturated fraction. Our work demonstrates the feasibility of the random mutagenesis strategy coupled with mutant selection based on cerulenin screening for the genetic improvement of the oleaginous yeast L. starkeyi.

Characterization of alkaline xylanases from Bacillus pumilus

Alkaline xylanases produced by four different strains of Bacillus pumilus were characterized. The optimal pH and temperature were pH 9.0 and 60oC for strain 13a, and pH 8.0 and 55oC for strains 52, 514, and 4a. Under these conditions the following activities were found after 10 min in the presence of 1% xylan (birchwood): 328 U.ml-1, 131 U.ml-1, 90 U.ml-1, and 167 U.ml-1, respectively, for the four strains. The enzymes were stable at 40oC, with 40% of the xylanase activity remaining after 2 hours for the enzymes of strain 52 and 60% for the other three strains. Stability at 50oC was improved by addition of glycerol. Taking into account the conditions under which kraft pulps are bleached during the manufacture of paper, xylanases from B. pumilus exhibit favorable potential for application to bleaching in the paper making process.

Preparative chromatography of xylanase using expanded bed adsorption

Expanded bed adsorption was used to purify a marketable xylanase often used in the kraft pulp bleaching process. Experiments in packed and expanded beds were carried out mainly to study the adsorption of xylanase on to a cationic adsorbent (Streamline SP) in the presence of cells. In order to study the presence of cells, a Bacillus pumilus mass (5% wet mass) was mixed with the enzyme extract and submitted to an expanded bed adsorption system. One xylanase was purified to homogeneity in the packed bed. However, the 5% cell content hampered purification.

Aqueous two-phase system formed by thermoreactive vinyl imidazole/vinyl caprolactam copolymer and dextran for partitioning of a protein with a polyhistidine tail

A new type of aqueous two-phase system (ATPS) has been developed for application combining two attractive concepts in downstream processing: the immobilised metal affinity partitioning and the use of thermoprecipitating polymers. ATPS consisting of the thermoprecipitating copolymer of N-vinyl caprolactam/1-vinyl imidazole loaded with Cu ions (Cu-poly-VI-VCL) in the top phase and dextran T70 in the bottom phase was used for purification of recombinant lactate dehydrogenase carrying an affinity tag of 6 histidine residues (His-LDH ) from a crude E. coli extract. The enzyme partitioned preferentially into the top Cu-poly-VI-VCL-rich phase. After phase separation, the latter was mixed with EDTA. Temperature increase to 45°C resulted in thermoprecipitation of VCL/VI-polymer, which could subsequently be recycled. His-LDH remained solubilized in the aqueous phase resulting in 8-fold purification and 80 % recovery in a single step.

Production and purification of alkaline xylanases

Abstract This work investigated more than 500 colonies with xylanolytic activity which were able to grow in a medium containing corn xylan as the only carbon source. Out of the 500 colonies, 22 microorganisms were also able to grow in birchwood xylan and were cellulase-free producers. The xylanase activity was studied at pH 10.0 and pH 5.0. It was observed that the three best producers of alkaline xylanase yielded enzyme levels in the range of 2.6 to 4.0 U/ml. Enzyme levels of 1.0 to 1.25 U/ml were achieved by four other microorganisms. Conversely, there were three microorganisms that produced a xylanase which was mostly active at pH 5.0. There was just one microorganism able to produce an enzyme active at pH 10.0. The alkaline xylanases from crude fermentation broth were extracted in aqueous two-phase systems (ATPS) composed of 16% polyethyleneglycol (PEG 6000) and 8% phosphate salt. A purification factor of 57 and a 41% yield of enzyme activity were achieved for the system containing 16% PEG 6000, 8% K 2 HPO 4 and 12% NaCl.