Production, biochemical characterization and application of fungal L-asparaginase

Orientador: Helia Harumi SatoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: L-Asparaginases são enzimas que catalisam a hidrólise da L-asparagina a ácido L-aspártico e amônia. As L-asparaginases microbianas têm recebido atenção devido ao seu papel no tratamento da leucemia linfoblástica aguda e devido à sua crescente aplicação na indústria de alimentos visando à redução da formação de acrilamida durante o processamento de alimentos. Um número crescente de pesquisas tem estudado a aplicação dessa enzima em diferentes matrizes alimentares comprovando seus efeitos na redução de acrilamida. Neste trabalho, foi estudada a produção de L-asparaginase por oito linhagens de fungos Aspergillus. Entre as 8 linhagens testadas, A. niger LBA 02, A. oryzae CCT 3940, A. oryzae LBA 01 apresentaram maior produção de L-asparaginase, 26,0 U mL-1; 20,6 U mL-1 e 19,1 U mL-1, respectivamente. A linhagem de A. oryzae CCT 3940 foi escolhida para os estudos de produção de L-asparaginase considerando-se as características bioquímicas da enzima (estabilidade em pH 6-8 e 40-50ºC e não inibição na presença de NaCl) para a aplicação potencial em alimentos. Uma estratégia de otimização sequencial foi utilizada para determinar o efeito de diferentes fontes de carbono, fontes de nitrogênio, concentração de indutor (L-asparagina), concentração de inóculo, pH inicial do meio, temperatura e agitção na produção de L-asparaginase de A. oryzae CCT 3940 em fermentação submersa. Na fermentação pelo micro-organismo a 30 °C em meio Czapek modificado composto de prolina (2%), glicose (0,5%), presença do indutor L-asparagina (0,2%), extrato de levedura (0,5%), KH2PO4 (0,152%), KCl (0,052%), MgSO4.7H2O (0,052%), CuNO3.3H2O (0,0001%), ZnSO4.7H2O (0,0001%) e FeSO4.7H2O (0,0001%) ajustado a pH 8,0, concentração de 3 ×107 esporos mL-1, 72 horas de fermentação a 150 rpm, a atividade de L-asparaginase foi de 67,49 U mL-1, o que representa um aumento de 225% em relação às condições iniciais não-otimizadas. A L-asparaginase de A. oryzae CCT 3940 foi purificada em um processo de quatro etapas e a massa molecular foi estimada em 115 kDa. A enzima purificada apresentou atividade ótima na faixa de pH de 7,0-8,0 a 40-50 °C. A enzima purificada apresentou atividade anti-proliferativa contra várias linhagens de células tumorais humanas, sem, contudo, inibir completamente o crescimento da linhagem não tumoral. Para verificar a eficácia da enzima e da preparação comercial de L-asparaginase em reduzir acrilamida em alimentos, batata em cubos (2 cm de aresta) foram submetidas a imersão por 30 minutos a 50 °C em três tratamentos: (i) em água destilada; (ii) água destilada com L-asparaginase purificada e (iii) água destilada com enzima comercial. A L-asparaginase purificada promoveu uma redução de 72% na formação de acrilamida enquanto que a enzima comercial apresentou 92% de redução em comparação com o sistema controle. Os resultados obtidos nessa pesquisa, indicam que a enzima L-asparaginase de A. oryzae CCT 3940 possui características bioquímicas e atividade biológica atrativas para a indústria de alimentos e para a indústria farmacêuticaAbstract: L-asparaginases are enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Microbial L-asparaginases have received attention due to its role in the treatment of acute lymphoblastic leukemia and due to its increasing use in the food industry aimed at reducing acrylamide formation during food processing. An increasing number of studies has made the application of this enzyme in different food matrices and proven its effects on the reduction of acrylamide. In this study, the production of L-asparaginase by eight strains of Aspergillus fungi was assessed. Among the eight tested strains, A. niger LBA 02, A. oryzae CCT 3940 and A. oryzae LBA 01 showed higher production of L-asparaginase, 26,0 U mL-1; 20,6 U mL-1 e 19,1 U mL-1, respectively. The strain of A. oryzae CCT 3940 was selected for the production of L-asparaginase studies considering the biochemical characteristics of the enzyme (stability at pH 6-8 and 40-50 ° C, and no inhibition in the presence of NaCl) for potential application in food. A sequential optimization strategy was used to determine the effect of different carbon sources, nitrogen sources, inductor concentration (L-asparagine), inoculum concentration, initial pH of the medium, temperature and agitation in the production of L-asparaginase from A. oryzae CCT 3940 in submerged fermentation. In the fermentation of the microorganism at 30 °C, in modified Czapeck medium consisting of proline (2%), glucose (0.5%), L-asparagine (0.2%), yeast extract (0.5%), KH2PO4 (0.152%), KCl (0.052%), MgSO4.7H2O (0.052%), CuNO3.3H2O (0.0001%), ZnSO4.7H2O (0.0001%) and FeSO4.7H2O (0,0001%) pH adjusted to pH 8.0, inoculum concentration of 3 × 107 spores mL-1, 72 hours of fermentation at 150 rpm, L-asparaginase activity was 67.49 U mL-1. This represents an increase of 225% compared to non-optimized initial conditions. The L-asparaginase from A. oryzae CCT 3940 was purified in a four-step process, and the molecular mass was estimated as 115 kDa. The purified L-asparaginase from A. oryzae CCT 3940 exhibited optimal activity at pH 7.0-8.0 at 40-50 °C. The purified enzyme showed anti-proliferative activity against various human tumor cell lines without, however, completely inhibit the growth of non-tumor cell line. In order to verify the effectiveness of the enzyme and of the commercial preparation of L-asparaginase on reducing acrylamide in foods, potato cubes (2 cm edge) were subjected to soaking for 30 minutes at 50 °C in three treatments: (i) distilled water; (ii) distilled water with purified L-asparaginase and (iii) distilled water with commercial enzyme. The purified L-asparaginase promoted a 72% reduction in acrylamide formation whereas the commercial enzyme showed 92% reduction when compared to the control system. The results obtained in this study indicate that L-asparaginase enzyme from A. oryzae CCT 3940 has attractive characteristics and interesting biological activity for the food industry and the pharmaceutical industriesDoutoradoCiência de AlimentosDoutora em Ciência de Alimentos140273/2012-32012/24046-7CNPQFAPES

Screening of Supports for the Immobilization of β-Glucosidase

A set of supports were screened for the immobilization of a partially purified extract of β-glucosidase from Aspergillus sp. These supports, namely, Eupergit, Amberlite, alginate, gelatin, polyvinyl alcohol- (PVA-) based matrices (Lentikats), and sol-gel, have proved effective for the implementation of some other enzyme-based processes. The initial criterion for selection of promising supports prior to further characterization relied on the retention of the catalytic activity following immobilization. Based on such criterion, where immobilization in sol-gel and in Lentikats outmatched the remaining approaches, those two systems were further characterized. Immobilization did not alter the pH/activity profile, whereas the temperature/activity profile was improved when sol-gel support was assayed. Both thermal and pH stability were improved as a result of immobilization. An increase in the apparent KM (Michaelis constant) was observed following immobilization, suggesting diffusion limitations

L-Asparaginase from Aspergillus spp. : production based on kinetics, thermal stability and biochemical characterization

This study describes the production of native L-asparaginases by submerged fermentation from Aspergillus strains and provides the biochemical characterization, kinetic and thermodynamic parameters of the three ones that stood out for high L-asparaginase production. For comparison, the commercial fungal L-asparaginase was also studied. Both commercial and L-asparaginase from Aspergillus oryzae CCT 3940 showed optimum activity and stability in the pH range from 5 to 8 and the asparaginase from Aspergillus niger LBA 02 was stable in a more alkaline pH range. About the kinetic parameters, the denaturation constant increased with the heating temperature for all L-asparaginases, indicating that the L-asparaginase activity decreased at higher temperatures, especially above 60 °C. Moreover, L-asparaginase from A. oryzae CCT 3940 remained stable after 60 min at 50 °C. None of the L-asparaginases were inhibited by high NaCl concentrations, which are highly desirable for food industry application. The catalytic activities of all the L-asparaginases were enhanced by the presence of Mn2+ and inhibited by p-chloromercuribenzoate and iodoacetamide. The L-asparaginase from the Aspergillus strains and the commercial enzyme had similar Km when L-asparagine was used as substrate. None of the L-asparaginases, except the L-asparaginase from A. niger LBA 02, could hydrolyze the substrate L-glutamine, which is of interest for medical proposes, since the glutaminase activity is usually related to adverse reaction during the leukemia treatment. This study showed that these new three non-recombinant L-asparaginases studied have potential application in the food and pharmaceutical industries, especially due to their good thermostability97CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloNão tem2012/24046-

Purification, characterization and antiproliferative activity of l-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity

Objective: To explore the anti-proliferative activity of purified l-asparaginase from Aspergillus oryzae CCT 3940 (A. oryzae). Methods: l-asparaginase was produced by submerged fermentation and purified to electrophoresis homogeneity by ionic exchanged chromatography in a fast protein liquid chromatographic system. The purified enzyme was characterized and used for the antiproliferative assay against nine tumor cell lines and one non-tumor cell line. Results: The free glutaminase l-asparaginase was purified 28.6 fold. l-asparaginase showed high stability under physiological condition, remaining stable in the pH range 7.0–8.0 after 1 h incubation at temperature range 30–45 °C. The Km and Vmax values of purified l-asparaginase were estimated as 0.66 mmol/L and 313 IU/mL, respectively. The purified enzyme could inhibit the growth of a broad range of human tumor cell lines at the concentrations studied. Also, the enzyme from A. oryzae CCT 3940 could inhibit tumor growth of leukemia cell line (K562) with a total growth inhibition value of (3.2 ± 2.5) IU/mL and did not inhibit the non-carcinogenic human cell line growth at the concentrations studied. Conclusions: The sensitivity of the cells lines to purified l-asparaginase from A. oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial l-asparaginase from Escherichia coli. The l-asparaginase from A. oryzae CCT 3940 has a high potential for pharmaceutical exploitation in the treatment of leukemia

Acrylamide mitigation in french fries using native l-asparaginase from aspergillus oryzae CCT 3940

The Maillard reaction is responsible for color and flavor formation in fried, roasted and baked foods. However, this reaction can also promote the formation of acrylamide, a potential human carcinogen. The application of l-asparaginase represents a promising method for acrylamide mitigation in heated products by the direct conversion of the precursor l-asparagine to l-aspartic acid and ammonia, which does not lead to additional acrylamide formation. Our research group produced and purified an l-asparaginase from native Aspergillus oryzae CCT 3940 with highly potential for acrylamide mitigation. To verify the enzymatic effectiveness, we compared the native l-asparaginase with the commercial recombinant enzyme. For this purpose, a GC-MS/MS method was developed, optimized and validated for the assessment of acrylamide in French fries. Our results indicate a relationship between the l-asparagine content and acrylamide formation in French fries. The acrylamide concentration of the fried potato treated with the l-asparaginase from A. oryzae CCT 3940 and treated with commercial enzyme reduced 72% and 92%, respectively compared to control sample. Moreover, the l-asparaginase from A. oryzae CCT 3940 showed no l-glutaminase activity, while commercial enzyme promoted a decrease in the l-glutamine content (25%). Our results suggest that l-asparaginase from A. oryzae CCT 3940 may be of great value for acrylamide mitigation76B222229FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2012/24046-711th Latin American Symposium on Food Science (SLACA)2015-11-08São Paulo, Brasi

Purification, characterization and antiproliferative activity of l-asparaginase from aspergillus oryzae CCT 3940 with no glutaminase activity

To explore the anti-proliferative activity of purified l-asparaginase from Aspergillus oryzae CCT 3940 (A. oryzae). Methods l-asparaginase was produced by submerged fermentation and purified to electrophoresis homogeneity by ionic exchanged chromatography in a fast protein liquid chromatographic system. The purified enzyme was characterized and used for the antiproliferative assay against nine tumor cell lines and one non-tumor cell line. Results The free glutaminase l-asparaginase was purified 28.6 fold. l-asparaginase showed high stability under physiological condition, remaining stable in the pH range 7.0–8.0 after 1 h incubation at temperature range 30–45 °C. The Km and Vmax values of purified l-asparaginase were estimated as 0.66 mmol/L and 313 IU/mL, respectively. The purified enzyme could inhibit the growth of a broad range of human tumor cell lines at the concentrations studied. Also, the enzyme from A. oryzae CCT 3940 could inhibit tumor growth of leukemia cell line (K562) with a total growth inhibition value of (3.2 ± 2.5) IU/mL and did not inhibit the non-carcinogenic human cell line growth at the concentrations studied. Conclusions The sensitivity of the cells lines to purified l-asparaginase from A. oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial l-asparaginase from Escherichia coli. The l-asparaginase from A. oryzae CCT 3940 has a high potential for pharmaceutical exploitation in the treatment of leukemia69785794FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2012/24046-

Scaling up the Two-Stage Countercurrent Extraction of Oil and Protein from Green Coffee Beans: Impact of Proteolysis on Extractability, Protein Functionality, and Oil Recovery

Green coffee processing has been hindered by low oil extraction yields from mechanical pressing and the need of using flammable and hazardous solvents for defatting the protein-rich cake before subsequent protein extraction. To replace the use of flammable solvents and enable the simultaneous extraction of lipids and proteins from green coffee beans at reduced water usage, a multistage countercurrent extraction process was scaled up from 0.05 to 1.14 kg and evaluated regarding protein and oil extractability, physicochemical and functional properties of the extracted protein, and oil recovery. Enzymatic extraction increased protein extractability by ~13% while achieving similar oil extractability when not using enzymes (55%). Proteolysis resulted in the release of smaller proteins with reduced surface hydrophobicity and higher solubility at acidic pH (3.0–5.0). The physicochemical changes observed due to proteolysis resulted in the formation of emulsions with reduced resistance against enzymatic and chemical demulsification strategies, enhancing the recovery of the extracted oil (48.6–51.0%). Proteolysis did not alter the high in vitro digestibility of green coffee proteins (up to 99%) or their emulsifying properties at most pH values evaluated. However, proteolysis did reduce the foaming properties of the hydrolysates compared with larger molecular weight proteins. These findings revealed the impact of extraction conditions on the extractability and structural modifications altering the functionality of green coffee proteins and the synergistic impact of extraction and demulsification strategies on the recovery of the extracted oil, paving the way for the development of structure–function processes to effectively produce green coffee proteins with desired functionality

Early detection of lipid oxidation in infant milk formula by measuring free oxylipins—Comparison with hydroperoxide value and thiobarbituric acid reactive substance methods

Infant milk formula was used as a model food to compare the sensitivity of thiobarbituric acid reactive substances (TBARS) and hydroperoxide methods to UPLC-MS/MS oxylipin analysis for detecting early lipid oxidation. Two different infant milk formulas were tested during 21 days of storage at 4°C. Formulas 1 and 2 contained canola oil and canola oil + 1% docosahexaenoic acid ethyl ester, respectively. Formulas were sampled up to 21 days of storage. Formula 2 had higher peroxide values than Formula 1 across all time points. However, no significant differences over time in TBARS and peroxide values in either formula were observed. Several oxylipins increased in both formulas starting on day 7 (linoleic acid and alpha-linolenic acid-derived oxylipins in Formula 1 and DHA-derived oxylipins in Formula 2). These results indicate that free oxylipins are effective in detecting early lipid oxidation and distinguishing between formulations containing different fatty acids. PRACTICAL APPLICATION: We have recently shown that primary oxidation products known as oxylipins can be measured in their free form by ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to detect early lipid oxidation. However, a head-to-head comparison of the sensitivity of this approach to conventional spectrophotometric methods has not been evaluated. Our results indicate that free oxylipin measurements are better than conventional methods in detecting early lipid oxidation in milk infant formula  distinguishing between different formulations

A multicomponent system based on a blend of agroindustrial wastes for the simultaneous production of industrially applicable enzymes by solid-state fermentation

<div><p>Abstract This study reports the use of statistical mixture design as a tool for the simultaneous production of lipase, CMCase, α-amylase, and β-glucosidase by Aspergillus niger under solid-state fermentation. Wheat bran, soybean meal, cottonseed meal, and orange peel were used as substrates, either individually or combined in different formulations, to study their synergistic or antagonistic effects on production of the enzymes. The highest lipase (323 U g-1) and CMCase (10 U g-1) activities were detected after 48 h, while the maximum activities of α-amylase (18 U g-1) and β-glucosidase (15 U g-1) occurred at 72 and 96 h, respectively. Considering the substrate formulation, the ternary mixture of wheat bran (1/3), soybean meal (1/3), and cottonseed meal (1/3) was the most versatile, showing production of CMCase (>5 U g-1) and α-amylase (>8 U g-1) at 24 h, lipase (>320 U g-1) at 72 h, and β-glucosidase (>10 U g-1) at 48 h.</p></div