Daniela de Araújo Viana Marques

Different types of aqueous two‐phase systems for biomolecule and bioparticle extraction and purification

Upstream improvements have led to significant advances in the productivity of biomolecules and bioparticles. Today, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from previous years. Current purification platforms will reach their physical limits at some point, indicating the need for new approaches. This article reviews an alternative method to extract and purify biomolecules/bioparticles named aqueous two‐phase system (ATPS). Biocompatibility and readiness to scale up are some of the ATPS characteristics. We also discuss some of ATPS applications in the biotechnology field.

Fermentation Medium for Collagenase Production by Penicillium aurantiogriseum URM4622

Medium composition and culture conditions for maximal collagenase production by Penicillium aurantiogriseum URM4622 were optimized using a response surface approach. A full two‐level design on three factors (initial medium pH, soybean flour concentration, and temperature) was employed to identify the most significant fermentation parameters for collagenase production, and a subsequent central composite design (CCD) was used to find the optimal levels of the two most significant factors (initial medium pH and soybean flour concentration). The design results indicated that the initial medium pH and the temperature had significant negative main effects, whereas the substrate concentration had a positive effect on the collagenase production. The maximum collagenolytic activity predicted by the fitted response surface was expected to occur at pH 7.21, 1.645% soybean flour concentration and 24°C. Three replicate experiments were run at these conditions and yielded an activity response of 283.36 ± 1.33 U, which not only is the highest obtained in this study but also represents a 5‐fold increase over the lowest response observed in the initial design. Since all experiments were carried out with an inexpensive substrate, the final results point out to a cost‐effective medium for collagenase production with potential industrial‐scale applications.

Optimization of clavulanic acid production by Streptomyces daufpe 3060 by response surface methodology

Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 22 central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.

Screening of wild type Streptomyces isolates able to overproduce clavulanic acid.

The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

Effect of Aeration and Agitation on Extractive Fermentation of Clavulanic Acid by Using Aqueous Two‐Phase System

In this work, the effects of agitation and aeration rates on aqueous two‐phase system (ATPS)‐based extractive fermentation of clavulanic acid (CA) by Streptomyces variabilis DAUFPE 3060 were investigated through a 22 full factorial design, where oxygen transfer rate (OTR) and oxygen uptake rate (OUR) were selected as the responses. Aeration rates significantly influenced cell growth, OUR, and CA yield, while OTR was practically the same in all the runs. Under the intermediate agitation (950 rpm) and aeration conditions (3.5 vvm) of the central point runs, it was achieved OTR of 1.617 ± 0.049 mmol L−1 h−1, OUR of 0.132 ± 0.030 mmol L−1 h−1, maximum CA production of 434 ± 4 mg L−1, oxygen mass transfer coefficient of 33.40 ± 2.01 s−1, partition coefficient of 66.5 ± 1.5, CA yield in the top and bottom phases of 75% ± 2% and 19% ± 1%, respectively, mass balance of 95% ± 4% and purification factor of 3.8 ± 0.1. These results not only confirmed the paramount role of O2 supply, broth composition and operational conditions in CA ATPS‐extractive fermentation, but also demonstrated the possibility of effectively using this technology as a cheap tool to simultaneously produce and recover CA.

Cultivation of Pichia pastoris carrying the scFv anti LDL (−) antibody fragment. Effect of preculture carbon source

Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (−) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

Production of β-Lactamase Inhibitors by Streptomyces Species

β-Lactamase inhibitors have emerged as an effective alternative to reduce the effects of resistance against β-lactam antibiotics. The Streptomyces genus is known for being an exceptional natural source of antimicrobials and β-lactamase inhibitors such as clavulanic acid, which is largely applied in clinical practice. To protect against the increasing prevalence of multidrug-resistant bacterial strains, new antibiotics and β-lactamase inhibitors need to be discovered and developed. This review will cover an update about the main β-lactamase inhibitors producers belonging to the Streptomyces genus; advanced methods, such as genetic and metabolic engineering, to enhance inhibitor production compared with wild-type strains; and fermentation and purification processes. Moreover, clinical practice and commercial issues are discussed. The commitment of companies and governments to develop innovative strategies and methods to improve the access to new, efficient, and potentially cost-effective microbial products to combat the antimicrobial resistance is also highlighted.

Application of aqueous two‐phase micellar system to improve extraction of adenoviral particles from cell lysate

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two‐phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK‐293) cells to a concentration of 1.4 × 1010 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X‐114 in a 23 full factorial design with center points. The systems were formed with Triton X‐114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37 °C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X‐114, a cell lysate pH of 7.0, and an incubation temperature at 33 °C, yielding 3.51 × 1010 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3‐fold, purifying it by 2.2‐fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two‐phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.

Optimization of Penicillium aurantiogriseum protease immobilization on magnetic nanoparticles for antioxidant peptides’ obtainment

ABSTRACT This work reports an optimization of protease from Penicillium aurantiogriseum immobilization on polyaniline-coated magnetic nanoparticles for antioxidant peptides’ obtainment derived from bovine casein. Immobilization process was optimized using a full two-level factorial design (24) followed by a response surface methodology. Using the derivative, casein was hydrolyzed uncovering its peptides that were sequenced and had antioxidant properties tested through (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS) radical scavenging and hydrogen peroxide scavenging assays. Optimal conditions for immobilization were 2 hr of immobilization, offered protein amount of 200 µg/mL, immobilization pH of 6.3 and 7.3 hr of activation. Derivative keeps over 74% of its original activity after reused five times. Free and immobilized enzyme casein hydrolysates presented similar peptide mass fingerprints, and prevalent peptides could be sequenced. Hydrolysates presented more than 2.5× higher ROS scavenging activity than nonhydrolyzed casein, which validates the immobilized protease capacity to develop casein-derived natural ingredients with potential for functional foods.