Comparison of kinetic characteristics of xylanases from Aspergillus niger and Trichoderma sp. with pH and temperature baking process parameters

Arabinoxylans are the predominant non-starch polysaccharides of the cell walls of wheat grain, and can contribute up to 3% of the total polysaccharide content of the flour. Endo-(1-4)-β-xylanase is able to hydrolyze the glycosidic bonds between two xylose units in the xylan backbone during baking process. The use of xylanases in the baking process leads to changes in the rheology of the dough. The aim of this work was to establish the best enzymatic hydrolysis conditions. The results suggest that the pure xylanase from Aspergillus niger needs less substrate to achieve maximum velocity (Vmax). However the xylanase from Trichoderma sp. has a higher Vmax apparent which means that it needs less time to convert the substrate into products. The xylanase from A. niger presented 50°C as its optimum temperature, 40% residual activity at 25°C and 80% at 36°C. The xylanase from Trichoderma sp. presented 50°C as its optimum temperature, 20% residual activity at 25°C and 60% at 36°C, which could be evidence that this enzyme is less active than the xylanase from A. niger. Moreover, the xylanase from Trichoderma sp. presented 90% residual activity in the baking process pH range, indicating that this enzyme can be more effective in the dough making process. The catalytic reactions of both enzymes are endothermic due to positive enthalpy and they are favourable process because the both Gibbs free energy were negative.Keywords: Xylanase, baking, enzyme, Trichoderma sp., Aspergillus nige

The influence of pH, polyethylene glycol and polyacrylic acid on the stability of stem bromelain

Enzyme stability is critical in biotechnology, pharmaceutical and cosmetic industries. Investigations on this subject have drawn attention because of its practical application. Bromelain is a thiol-endopeptidase, obtained from pineapple (Ananas comosus), known for its clinical and therapeutic applications, particularly to selective burn debridement and improvement of antibiotic action and anti-inflammatory activities. To date, the use of bromelain in pharmacological or industrial applications is limited, due to commercial availability, costs, and sensitivity to pH and temperature. Therefore, a better understanding of enzyme stability would be of great interest. The aim of this study was to evaluate bromelain activity and stability in several pH (2.0 to 8.0) and in polyethylene glycol and polyacrylic acid solutions. We observed that bromelain was able to maintain its stability at pH 5.0 for the temperatures studied. PEG solutions increased bromelain stability, but PAA solutions had the opposite effect.Estabilidade de enzimas é uma questão fundamental em indústrias biotecnológicas, farmacêuticas e cosméticas. As investigações sobre o assunto têm chamado a atenção por sua aplicação prática. A bromelina é uma tiol-endopeptidase, obtida a partir do abacaxi (Ananas comosus). É conhecida por suas aplicações clínicas e terapêuticas, especialmente para desbridamento seletivo de queimaduras, melhoria de ações antibiótica e de atividades anti-inflamatórias. Até o momento, a utilização da bromelina em aplicações farmacológicas industriais é limitada, devido à disponibilidade comercial, os custos, a sensibilidade ao pH e temperatura. Portanto, a maior compreensão da estabilidade desta enzima seria de grande interesse. O objetivo deste estudo foi avaliar a estabilidade da atividade da bromelina em vários pH (2,0 a 8,0) e em soluções de polietilenoglicol e de ácido poliacrílico. Observamos que a bromelina foi capaz de manter a sua estabilidade em pH 5.0, em todas as temperaturas estudadas. Soluções de PEG aumentaram a estabilidade da bromelina, enquanto que soluções de PAA obtiveram efeito oposto

Partial purification of glucose-6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol)/phosphate systems

Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme used in biochemical and medical studies and in several analytical methods that have industrial and commercial application. This work evaluated the extraction of G6PDH in aqueous two-phase system (ATPS) of poly(ethyleneglycol) (PEG)/phosphate buffer, using as enzyme source a medium prepared through commercial baker’s yeast disruption. Firstly, the effects of PEG molar mass on the enzyme partition and of homogenization and rest on the system equilibrium were investigated. Afterwards, several ATPS were prepared using statistical analysis (22 factorial design). The results, including kinetic and thermodynamic parameters for the G6PDH activity, showed partial purification of this enzyme in ATPS composed of 17.5% (w/w) PEG400 and 15.0% (w/w) phosphate. A high enzymatic recovery value (97.7%), a high partition coefficient (351), and an acceptable purification factor (2.28 times higher than in cell homogenate) were attained from the top phase. So, it was possible to attain an effective enzyme pre-purification by separating some contaminants with a simple method such as liquid-liquid extraction in aqueous two-phase systems (ATPS).FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo / Brasil); CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico / Brasil); CAPES (Coordenação para Aperfeiçoamento do Ensino Superior / Brasil)

A stable liquid–liquid extraction system for clavulanic acid using polymer-based aqueous two-phase systems

The partitioning of Clavulanic Acid (CA) in a novel inexpensive and stable aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The aqueous two-phase systems are formed by mixing both polymers with a salt (NaCl or Na2SO4) and an aqueous solution of CA. The stability of CA on the presence of both polymers was investigated and it was observed that these polymers do not degrade the biomolecule. The effect of PEG-molecular size, polymer concentrations on the commercial CA partitioning has been studied, at 25 C. The data showed that commercial CA was preferentially partitioned for the PEG-rich phase with a partition coefficient (KCA) between 1 and 12 in the PEG/NaPA aqueous two phase systems supplemented with NaCl and Na2SO4. The partition to the PEG phase was increased in the systems with high polymer concentrations. Furthermore, Na2SO4 caused higher CA preference for the PEG-phase than NaCl. The systems having a composition with 10 wt.% of PEG4000, 20 wt.% of NaPA8000 and 6 wt.% of Na2SO4 were selected as the optimal ones in terms of recovery of CA from fermented broth of Streptomyces clavuligerus. The partitioning results (KCA = 9.15 ± 1.06) are competitive with commercial extraction methods of CA (KCA = 11.91 ± 2.08) which emphasizes that the system PEG/NaPA/Na2SO4 can be used as a new process to CA purification/concentrationFinancial support is gratefully acknowledged from FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Sao Paulo, Brazil) and from CNPq (National Council for Scientific and Technological Development, Brasilia, Brazil) and CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)

Aqueous two-phase micellar systems in an oscillatory flow micro-reactor: Study of perspectives and experimental performance

Aqueous two-phase micellar systems (ATPMS) are micellar surfactant solutions with physical properties that make them very efficient for the extraction/concentration of biological products. In this work the main proposal that has been discussed is the possible applicability and importance of a novel oscillatory flow micro-reactor (micro-OFR) envisaged for parallel screening and/or development of industrial bioprocesses in ATPMS. Based on the technology of oscillatory flow mixing (OFM), this batch or continuous micro-reactor has been presented as a new small-scale alternative for biological or physical-chemical applications. RESULTS: ATPMS experiments were carried out in different OFM conditions (times, temperatures, oscillation frequencies and amplitudes) for the extraction of glucose-6-phosphate dehydrogenase (G6PD) in Triton X-114/buffer with Cibacron Blue as affinity ligand. CONCLUSION: The results suggest the potential use of OFR, considering this process a promising and new alternative for the purification or pre-concentration of bioproducts. Despite the applied homogenization and extraction conditions have presented no improvements in the partitioning selectivity of the target enzyme, when at rest temperature they have influenced the partitioning behavior in Triton X-114 ATPMS.The authors acknowledge the financial support from FCT (Fundacao para a Ciencia e Tecnologia/Portugal, SFRH/BPD/26108/2005), as well as from FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo/Brasil), CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico/Brasil), and CAPES (Coordenacao para Aperfeicoamento do Ensino Superior/Brasil)

Liquid-liquid extraction in the presence of electrolytes of nisin and green fluorescent protein (GFPuv)

In the biotechnology field, it has been suggested that extractions in two-phase aqueous complex-fluid systems can possibly be used instead of, or as complementary processes to, the more typical chromatographic operations, to reduce the cost of the downstream processing of many biological products (Lam et al., 2004; Mazzola et al., 2006). This method offer attractive conditions to be applied in this study, thereby two-phase systems can be exploited in separation science for the extraction/purification of desired biomolecules directly on the culture medium (Mazzola et al., 2008). This study aimed to evaluate the aqueous two phase system (ATPS) composed by a nonionic surfactant, Triton X-114 (TX), in presence or absence of electrolytes, to separate two interesting biomolecules: nisin and recombinant green fluorescent protein (GFP). Results indicated that nisin partitions preferentially to the micelle rich-phase, with significant antimicrobial activity increase (up to 10-fold). GFP partitioned evenly between the phases in TX system without electrolytes.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP

Processing of byproducts to improve nisin production by Lactococcus lactis

In the last years, disposal from dairy industries have received a special attention due its polluting power in the environment. For this reason, studies have obtained a positive support to develop different alternatives to recycle milk whey components. One of them is its utilization as culture media, aiming to produce biomolecules with noble applications. Nisin is an extracellular peptide, produced by Lactococcus lactis, this peptide has been applied as a natural additive once it presents broad antibacterial activity. Applications of this bacteriocin include dental care products, pharmaceutical products such as stomach ulcers and colon infection treatment and potential birth control. In batch cultures, L. lactis was performed in two different groups of assays. The first group milk whey was prepared in distilled water in four different concentrations: 100 g/l (S100); 50 g/l (S50); 30 g/l (S30); 10 g/l (S10). In the second group of assays, two supplements were added in milk whey with concentration 100 g/l (S100): (1) 5 g/l yeast extract (A1); (2) 5 g/l yeast extract and 10 ml (v/v) tomato extract. Nisin activity was assayed through agar diffusion utilizing Lactobacillus sakei. The results show that the utilization of powder milk whey with concentration of 100 g/l can be used as a culture medium with supplementation. This media is favorable to develop L. lactis cells and nisin production, reaching an activity of about 4 logAU. Biological processing of milk byproduct can be considered as one of the profitable utilization alternatives, generating high-value bioproducts and stimulates researches for its use.The authors wish to thank the Brazilian Committees for the Scientific Technology Research (CNPq, FAPESP, CAPES), for financial support and scholarship

Nisin biotechnological production : evaluation and perspectives in the development of new applications

Nisin is a commercially available bacteriocin of the lantibiotic group produced by Lactococcus lactis ATCC 11454. The importance of nisin as an effective preservative is the fact that its spectrum of inhibitory activity includes almost all Gram-positive, Gram-negative and spoilage bacteria. With several favorable characteristics, this compound has been widely used as a natural food biopreservative. On the other hand, biotechnological production of fuel ethanol from different raw materials (sucrose-containing feedstocks - mainly sugar cane, starchy materials and lignocellulosic biomass) has led to the increase in the researches with trends for improving the bio-ethanol production. During these processes of bio-ethanol production antibiotic agents are added to the pre-fermented broth to prevent unwanted microbial growth. Thus, different questions have been made in relation to performance of the application of the nisin as a natural antibiotic to the ethanol production, as well as their beneficial effects about the yield and productivity in this process. However, the solution to this problematic depends firstly on how the development and implementation of technologies based on nisin production will be undertaken. Perspectives towards the development of modern procedures of nisin production are still needed. In this work the main proposal that has been discussed is the applicability of a novel micro-reactor operated under oscillatory flow mixing envisaged for parallel screening and/or development of industrial bioprocesses in the nisin production. For this reason, initially the objective of this study was to investigate the milk whey, a byproduct from dairy industries, in nisin production aiming to evaluate the growth conditions for L. lactis. In batch cultures, L. lactis was performed in raw milk whey and the nisin activity was assayed through agar diffusion utilizing Lactobacillus sakei ATCC 15521. The results showed that milk whey media is propitious to develop L. lactis cells and produce nisin, reaching activity around 105 AU.mL-1. Biological processing of milk sub-products (milk whey) can be considered one of profitable utilization alternatives, generating high-value bioproducts and contributing to decrease rivers disposal. The use of these sub-products as substrate together with further studies related to application of batch or continuous oscillatory flow reactor would take the more efficient nisin production and finally to a possibility of its evaluation and use in bio-ethanol production.Fundação para a Ciência e a Tecnologia (FCT)FAPESP/BrasilCAPES/Brasi

Biological and physicochemical stability of ceftazidime and aminophylline on glucose parenteral solution

Ceftazidima é um antimicrobiano administrado por via parenteral, que apresenta amplo espectro de ação, principalmente contra Pseudomonas aeruginosa. O tempo em que a concentração sérica de ceftazidima permanece acima da concentração mínima inibitória (MIC) é um importante parâmetro farmacodinâmico para a determinação da eficácia antimicrobiana e pode ser potencializado através da utilização de infusão contínua em soluções parenterais (PS). Este artigo visa a avaliar a estabilidade da ceftazidima em solução de glicose 5%, na presença e na ausência do fármaco aminofilina, através de cromatografia líquida de alta eficiência HPLC e MIC durante o período de 24 horas. Os microorganismos selecionados para a determinação do MIC foram Escherichia coli e Pseudomonas aeruginosa. Os ensaios em cromatógrafo líquido confirmaram a estabilidade dos fármacos ceftazidima e aminofilina quando são individualmente associados em PS, enquanto os valores de MIC ficaram maiores que os valores encontrados na literatura. Quando ambos os fármacos foram associados na mesma solução parenteral a concentração de ceftazidima obtida por HPLC diminuiu 25% depois de 24 horas. Os valores de MIC mostraram maior decaimento da atividade antimicrobiana neste mesmo período e também valores de MIC alterados nas soluções preparadas no tempo zero, decaimento este que não foi detectado em HPLC. Os resultados indicaram incompatibilidade na associação dos fármacos em PS, enfatizando a importância dos resultados de MIC para interações de fármacos.Ceftazidime is a broad spectrum antibiotic administered mainly by the parenteral route, and it is especially effective against Pseudomonas aeruginosa. The period of time in which serum levels exceed the Minimum Inhibitory Concentration (MIC) is an important pharmacodynamic parameter for its efficacy. One of the forms to extend this period is to administer the antibiotic by continuous infusion, after prior dilution in a Parenteral Solution (PS). The present work assessed the stability of ceftazidime in 5% glucose PS for 24 hours, combined or not with aminophylline, through High Performance Liquid Chromatography (HPLC). The physicochemical evaluation was accompanied by in vitro antimicrobial activity compared MIC test in the 24-hour period. Escherichia coli and Pseudomonas aeruginosa were the microorganisms chosen for the MIC comparison. The HPLC analysis confirmed ceftazidime and aminophylline individual stability on PS, while the MIC values were slightly higher than the mean described in the literature. When both drugs were associated in the same PS, the ceftazidime concentration by HPLC decreased 25% after 24 hours. Not only did the MIC values show high loss of antibiotic activity within the same period, but also altered MIC values immediately after the preparation, which was not detected by HPLC. Our results indicate that this drug combination is not compatible, even if used right away, and that PS might not be the best vehicle for ceftazidime, emphasizing the importance of the MIC evaluation for drug interactions